Systems and methods to detect interleukin-17 or colony-stimulating factor-1 chronic graft versus host disease from the peripheral blood of hematopoietic transplant recipients

Abstract

Systems and methods to detect immune signatures of interleukin-17 (IL-17) or colony-stimulating factor-1 (CSF-1) chronic graft versus host disease (cGVHD) from the peripheral blood of hematopoietic transplant recipients are described. The systems and methods can detect the immune signatures before clinical symptoms of cGVHD emerge and also allow directed personalized treatment for patients with these types of cGVHD.

Description

F053-0195PCT / 25-085-WO-PCTSYSTEMS AND METHODS TO DETECT INTERLEUKIN-17 OR COLONY-STIMULATING FACTOR-1 CHRONIC GRAFT VERSUS HOST DISEASE FROM THE PERIPHERAL BLOOD OF HEMATOPOIETIC TRANSPLANT RECIPIENTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 747,779 filed January 21, 2025, the entire contents of which are incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA236229, and CA118953 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The current disclosure provides systems and methods to detect immune signatures indicative of interleukin-17 (IL-17) or colony-stimulating factor-1 (CSF-1) chronic graft versus host disease (cGVHD) from the peripheral blood of hematopoietic transplant recipients. The immune signatures can arise before clinical symptoms of cGVHD emerge and also allow directed personalized treatment for patients with these types of cGVHD.BACKGROUND OF THE DISCLOSURE

[0004] The hematopoietic system refers to cells of the blood system, which includes cells of the immune system.

[0005] Hematopoietic stem cells (HSC) are pluripotent and ultimately give rise to all types of terminally differentiated blood cells. HSC can self-renew or can differentiate into more committed progenitor cells which are irreversibly determined to be ancestors of only a few types of blood cell

[0006] Hematopoietic stem progenitor cells (HSPC) can self-renew or differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and natural killer cells (NK cells).

[0007] People undergo allogeneic hematopoietic cell transplantation (HCT) to treat a variety of malignant and genetic conditions. For example, HCT can be used to treat a variety of blood cancers, primary immunodeficiencies, and blood clotting disorders. While HCT provides an important therapy for these conditions, in certain patients, the transplanted hematopoietic cells develop into immune cells that recognize the patient's own cells as foreign and begin to attack and damage the patient from within. This phenomenon is referred to as graft-versus-host disease. Graft-versus-host disease can be acute or chronic.

[0008] Chronic graft-versus-host disease (cGVHD) remains the leading cause of non-relapse morbidity and mortality after HCT. Effective therapeutic agents targeting dysregulated cytokines including IL-17 and CSF-1 have been definedF053-0195PCT / 25-085-WO-PCTin preclinical models of cGVHD, and efficacy in subsequent clinical trials has led to their recent FDA approval. Despite this, these agents are effective in only a subset of patients, expensive, difficult to access outside the US, and used in a trial-and-error fashion. Thus, the ability to readily discern druggable, dysregulated immunity in patients having cGVHD is desperately needed to facilitate the selection of appropriate treatment and to identify high-risk individuals for preemptive therapy.SUMMARY OF THE DISCLOSURE

[0009] The current disclosure provides systems and methods to detect immune signatures indicative of interleukin-17 (IL-17) or colony-stimulating factor-1 (CSF-1chronic graft versus host disease (cGVHD) from the peripheral blood of hematopoietic transplant recipients. The immune signatures can arise before clinical symptoms of cGVHD emerge and also allow directed personalized treatment for patients with these types of cGVHD.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0010] Some of the drawing submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0011] FIGs. 1A-1D. IL-17 experimental overview. (FIG. 1A) Experimental schematic of mouse transplants and serial peripheral blood collection for scRNAseq processing. (FIG. 1B) scRNAseq gene clustering of major peripheral blood cell populations. (FIG. 1C) Canonical CITE-seq protein markers and gene expression by cell type. (FIG. 1D) Representation of the major peripheral blood cell populations by donor genotype.

[0012] FIGs. 2A-2D. T cells used in IL-17 experiment. (FIG. 2A) scRNAseq gene clustering of T cell populations. (FIG. 2B) Canonical CITE-seq protein markers and gene expression by T cell cluster(s). (FIG. 2C) Proportion of peripheral blood T cells occupied by each cluster in FIG. 2A, by posttransplant timepoint and donor genotype (Wilcoxon signed rank test). (FIG. 2D) IL-17 experiment CD8+ effector memory T cells and IL-17 experiment CD8+ exhausted / effector memory T cells.

[0013] FIGs. 3A-3E. GVHD-related IL-17 signaling modifies peripheral blood monocyte differentiation, chiefly in Ly6Clo monocytes. (FIG. 3A) scRNAseq gene clustering of murine peripheral blood monocytes with RNA velocity, beginning from a Proliferating cluster, developing into an Early circulating group, and further into three Ly6Chi circulating monocyte clusters (Circulating 1, Circulating 2, Circulating 3) and a granulocyte-like monocyte cluster, culminating in Ly6Clo cells. (FIG. 3B) Proportion of peripheral blood monocytes occupied by each cluster in FIG. 3A, by donor genotype and posttransplant timepoint. (FIG. 3C) LySCIo clusters' differential gene expression by donor genotype and posttransplant timepoint of monocyte / macrophage activation markers, NF-KB and MAPK pathway-related genes, chemotactic markers, and other selected genes. (FIG. 3D) Major monocyte clusters’ differentially expressed genes (DEGs) which are implicated in IL-17-driving GVHD (p_val_adj<0.05 both for WT GVHD control vs. TCD (non-GVHD) control and for WT GVHD control vs. IL-17RC- / - recipients). (FIG. 3E) Ly6Clo monocyte expressionF053-0195PCT / 25-085-WO-PCTof IL-17-associated GVHD genes by experiment group (all timepoints combined).

[0014] FIGs. 4A-4G. IL-17 experiment monocyte markers and differentially expressed genes. (FIG. 4A) Heatmap of monocyte top 15 marker genes per cluster. Monocyte genes included in FIG. 4A from top to bottom are as follows: Stmnl, Hmgb2, Birc5, Mki67, Ccnb2, Pclaf, Cenpa, H2afv, Top2a, Ptma, Nuckl, Tubb4b, Tubb5, Hist1h2ap, Hist1h1b, Lgalsl, Ppla, H2atz, Hmgn2, Prtn3, S100a10, Tspo, Ly6c2, F13a1, Rnaseh2c, Fn1, Hsp90b1, Vcan, Tagln2, Vim, ligpl, Tppp3, Capg, Cripl, Gm4951, Gbp2, Ifi47, Pnp, Lmna, Klf2, lfl203, Slfn5, Ahnak, Sub1 , Stxbp6, Nfkbla, Mgstl, Atp2b1, Ypel3, Pdliml, Fam102b, Tlr2, Ccdc88c, Klf10, Thbsl, Cxcl2, Nfxbiz, Tnf, Gm14636, Ccl3, Ccl4, Tnfaip3, Clec4e, MarcksH, Maltl, Gadd45b, Csrnpl, Ptgs2, CxcHO, Lrg1, Wfdc21, Lcn2, Chll3, Mpm8, S100a8, Saa3, Fos, Apoc2, Apoe, S100a9, Ifitml, Ace, H2-Eb1, Slc12a2, Ly6a, Fcgt4, H2-Aa, H2-Ab1. Bcl2a1. Columns from left to right include: Proliferating, Early, Circulating 1, Circulating 2, Circulating 3, Granulocyte-like, and Ly6Cl0. (FIG. 4B) Ly6C CITE-seq, Csflr transcript abundance, and Cx3cr12 transcript abundance of monocyte clusters. (FIG. 4C) Differential gene expression of Circulating 1 vs. Circulating 2 clusters which are relatively overrepresented in the WT vs. IL-17RC KO graft recipients, respectively. (FIGs. 4D-4G) Individual mice's monocyte cluster proportions by group and timepoint (WSRT).

[0015] FIGs. 5A-5D. IL-17 neutrophil cluster assignments and individual mouse G5c cluster decrease associated with IL-17 abrogation. (FIG. 5A) Seurat clusters' expression of Xie et al. peripheral blood and bone marrow neutrophil clusters' top 10 genes (filtered on avg_log2FC>0.4, pct, 1 >0.15, and p_val_adj<0.05 and calculated by highest values of (pct.1 -pct.2) / pct.2 for which pct.1 is the indicated cluster and pct.2 is all other clusters in the combined blood / bone marrow dataset), with corresponding cluster assignment). (FIGs. 5B, 5C) Individual recipient mice’s post-HCT proportion of cluster neutrophils, by post-transplant day. Statistics shown for G5c cluster; no other statistically significant differences between WT and IL-17RC KO or IL-17A KO in other clusters (WSRT). (FIG. 5D) Csf1 expression among all peripheral blood cells.

[0016] FIGs. 6A-6E. IL-17 controls neutrophil maturation during GVHD. (FIG. 6A) scRNAseq gene clustering of murine peripheral blood neutrophils with RNA velocity, per developmental schema of mouse peripheral blood neutrophil populations (FIG. 6B) Post-transplant mouse peripheral blood neutrophil expression of cluster-identifying marker genes derived from Xie et al. (top 10 genes per cluster assigned in descending cluster specificity order using thresholds of avg_log2FC>0.4, pct. 1 >0.18, and p_val_adj<0.05). (FIG. 6C) Representation of each neutrophil transcriptional cluster as portion of total neutrophils, by day posttransplant (D+14, D+21, D+28) and donor type. (FIG. 6D) Expression of neutrophil maturation, apoptosis, and chemotaxis gene scores in clusters across combined timepoints (gene lists proposed in Xie et al.) (FIG. 6E) Neutrophil clusters’ differentially expressed genes (DEGs) which are implicated in IL-17-driven GVHD (p_val_adj<0.05 both for WT GVHD control vs. TCD (non-GVHD) control and for WT GVHD control vs. IL-17RC- / - recipients).

[0017] FIGs. 7A-7G. G-CSF expands Csf1 -expressing terminally differentiated neutrophils. Mice were treated daily with PBS (control group) or G-CSF for 6 days and blood analyzed on day 7 by scRNAseq. (FIG. 7A) UMAP of peripheralF053-0195PCT / 25-085-WO-PCTblood neutrophils with annotations per Xie et al. applied via Seurat's FindTransferAnchors functions. (FIG. 7B) Neutrophil expression of Csf3r (encoding G-CSF receptor) and Csf1. (FIG 7C) Abundance of neutrophil clusters between control and G-CSF-treated groups. (FIG. 7D) Subclustering of the terminally differentiated neutrophil populations G5c. (FIG. 7E) Csf1 expression among terminally differentiated neutrophil clusters. (FIG. 7F) Proportions of terminally differentiated neutrophils between experimental groups. (FIG. 7G) Terminally differentiated neutrophil abundance as a proportion of total blood leukocytes.

[0018] FIGs. 8A-8I. scRNAseq of donor strain mice’s peripheral blood. (FIG. 8A) scRNAseq gene clustering of major peripheral blood cell populations per 3 WT B6 and 3 IL-17RC KO mice. (FIG. 8B) Canonical CITE-seq protein markers and gene expression by cell type. (FIG. 80) Representation of major peripheral blood cell populations by donor genotype. (FIG. 8D) Gene clustering of murine peripheral blood monocyte / DC populations, labeled according to FIG.3A monocyte populations using Seurat FindTransferAnchors function. (FIG. 8E) Representation of each monocyte cluster by mouse genotype. (FIG. 8F) Differential gene expression of WT vs. IL-17RC KO in Ly6Chi aggregate monocyte clusters. Included genes have differential gene expression with p_val_adj<0.05. (FIG. 8G) Gene clustering of murine peripheral blood neutrophil populations, labeled according to FIG. 6A neutrophil populations using the Seurat FindTransferAnchors function. (FIG. 8H) Representation of each neutrophil cluster by mouse genotype. (FIG. 8I) Differential gene expression of WT vs. IL-17RC KO in dominant neutrophil clusters G5a and G5c. Included genes have differential expression with p_val_adj<0.05.

[0019] FIGs. 9A-9C. CSF-1 experiment overview. (FIG. 9A) Experiment schematic of mouse transplants, serial peripheral blood collection at days +14, +21, and +28, and downstream scRNAseq on 10x Genomics platform. (FIG.9B) scRNAseq gene clustering of major peripheral blood cell populations. (FIG. 90) Canonical CITE-seq protein markers and gene expression by cell type.

[0020] FIGs. 10A-10C. CSF-1 experiment T cells. (FIG. 10A) scRNAseq gene clustering of T cell populations. (FIG.10B) Proportion of donor-derived peripheral blood T cells occupied by each cluster in FIG. 10A, by posttransplant timepoint and experimental group. No statistically significant differences observed. (FIG. 10C) Canonical CITE-seq protein markers and gene expression by T cell cluster.

[0021] FIGs. 11A-11E. Neutrophils in CSF-1 R targeting and neutrophil cluster assignments. (FIG. 11 A) scRNAseq gene clustering of murine peripheral blood neutrophils with RNA velocity, per developmental schema of mouse peripheral blood neutrophil populations identified in Xie et at., 2020 Nat. Immunol. (FIG. 11 B) Representation of each neutrophil transcriptional cluster as portion of total neutrophils, by day posttransplant (D+14, D+21, D+28) and experimental condition (GVHD control, CSF-1 R-blocking antibody treatment, and TCD). (FIG. 11 C) Neutrophil clusters’ differentially expressed genes (DEGs) which are implicated in CSF-1 -driven GVHD (p_val_adj<0.05 both for GVHD control vs. TCD (non-GVHD) control and for GVHD control vs. anti-CSF-1R recipients). (FIG. 11 D) Seurat clusters' expression of Xie et al. peripheral blood and bone marrow neutrophil clusters' top 10 genes (filtered on avg_log2FC>0.4, pct.1 >0.15, and p_val_adj<0.05. and calculated by highest values of (pct.1-pct.2) / pct.2 for whichF053-0195PCT / 25-085-WO-PCTpct.1 is the indicated cluster and pct.2 is all other clusters in the combined blood / bone marrow dataset), with corresponding cluster assignment). (FIG. 11 E) Cluster proportions by timepoint, group, and individual animal (WSRT).

[0022] FIGs. 12A-12C. CSF-1R signaling specifically facilitates development of Ly6Clo monocytes and a highly activated Ly6Chi monocyte population. (FIG. 12A) scRNAseq gene clustering and RNA velocity of peripheral blood monocytes (control GVHD and anit-CSF-1 R-treated groups included on plot). (FIG. 12B) Proportion of peripheral blood monocytes occupied by each cluster in A, by experimental group and posttransplant timepoint. (FIG. 12C) Individual recipient mice’s post-transplant proportion of Ly6Clo and Circulating 2 cluster monocytes, by post-transplant day (WRST).

[0023] FIGs. 13A-13G. CSF-1 experiment monocyte markers and differentially expressed genes. (FIG. 13A) Heatmap of monocyte to 15 marker genes per cluster. Rows from top to bottom include: Ccnb2, Stmnl, Ptma, H2afz, Ppia, Hmgn2, H2atv, Hmgbl, S100a10, Hsp90b1, Lgalsl, Hmgb2, Lsm5, Prtn3, F13a1, Ahnak, Fn1, Cripl, Ly6c2, Sell, Calr, Rpn1, Tagln2, Ccr2, Vcan, Pdliml, Lmna, Nfkbia, Cxcl2, Nfkbiz, Tnf, Fos, Atf3, Duspl, Tnfaip3, Clec4e, Ccl3, Oer3, Egr1, Maltl, CxcHO, Apoe, Plac8, Gngt2, Samhdl, Tppp3, Itgal, Sat1, Cd74, Abi3, H2-Aa, Gbp2, Serpinb2, Stk10, Klf2, lfi205, Cbfa2t3, S100a8, Lcn2, Wfdc21, Chil3, S100a11, Mml8, Hp, S100a9, Slp1, Anxal, Wfdc17, Fpr2, Lrg1, Mki67, Pclaf, Birc5, Cenpa, Top2a, Lmnbl, Tubb4b, Nucksl, Tubb5, Hist1h2ap, Hist1h2ae, Hist1h1b, Slc12a2, H2-Eb1, Ace, H2-Ab1, Ccnd2, Adgre4, Bcl2a1b, Treml4, Fcgr4, Ly6a, and Dusp2. Columns from left to right include: Proliferating, Early, Circulating 1, Circulating 2, Circulating 3, Granulocyte-like, and TRMP. (FIG.13B) Ly6C CITE-seq of monocyte clusters. (FIG. 13C) Differential gene expression of Circulating 1 and Circulating 3 clusters by experiment group. (FIGs. 13D, 13E, 13F) Monocyte cluster proportion changes by timepoint and individual mouse (WSRT). (FIG. 13G) Marker genes displayed by post-transplant timepoint. Rows from top to bottom include: Ccnb2, Stmnl, Cdkn3, Spc24, Elane, Gcat, Rdm1, Dnah8, 2610318N02Rik, 1700097N02Rik, Mnd1, 1700006J14Rik, E330020D12Rik, Gfsfl, Gm9929, Stxbp6, Lmha, Emp1, Rfx2, Atp2b4, ArhgeflO, Gm15902, Jmid8, Enkur, Car5b, Ednrb, Reml, Smim5, Gprin3, Cxcl2, Tnfsf9, Gm14636, Ptgs2, Egr2, Olfr432, Mir155hg, Gm48302, 111a, 1110002J07Rik, Gm4065, Gm2449, Gm29946, Gngt2, Serpinb2, Rbpms, Gm5547, Scnlb, Adora2a, Cyp2ab1, GfHb, Abcbla, Gm12866, 1700061 G19Rik, Ifit3b, Gm10484, Gm12185, Rai14, S100a8, Lcn2, Wfpc21, Mmp8, S100A9, Lrg1, Fcnb, Abca13, Crispld2, Olfml2b, F730016J06Rik, Gpldl, Foxd4, Platr7, Rnf128, Aspm, Nusapl, Depdcla, Kif2c, Kif14, Nek2, Rad51ap1, Pimreg, Pbk, E2f7, Kif18b, Pif1, BC030867, Aunjp, Fcerla, Slc12a2, Ccnd2, Cd300e, Adrbl, Itgax, Adamdecl, Jade2, Pdgfb, Spic, Cd300ld3, Vegfc, DnaH12, Adgrl3, Ltga9, and Ccdc192. Columns from left to right include: Proliferating, Early, Circulating 1, Circulating 2, Circulating 3, Granulocyte-like, and Ly6Cl0. (13G) Mouse CSF-1 -dependent cluster markers. Avg_log2FC>1.0, p_val_adj<0.05, pct.1 >0.3 per Seurat FindMarkers function on CSF-1 experiment total mouse monocyte object.

[0024] FIGs. 14A-14E. Mouse-derived monocyte gene signatures may identify patients whose cGVHD is associated with IL-17 and CSF-1 dysregulation. (FIG. 14A) Canonical gene expression and cell type assignments (depicted on samples from patients at day +100 post-HCT). (FIG. 14B) IL-17 and (FIG. 14C) CSF-1 signatures were refined asF053-0195PCT / 25-085-WO-PCTdescribed in the Methods, retaining the genes for which the top quartile expression threshold of D+100 patients who subsequently developed cGVHD (n=30) was at least equal to the top quartile expression threshold of D+100 patients who did not subsequently develop cGVHD (n=15) expression. Genes were weighted equally and normalized to the maximal D+100 non-cGVHD group's value (set at 10) and averaged into composite immune pathway signature scores. A positive threshold was defined as the lesser of the D+100 non-cGVHD's group’s mean + 2 standard deviations or the maximal non-cGVHD value (points above this colored red). Signature expression values were also calculated for the respective monocyte populations from patients at the onset of cGVHD before starting systemic immunosuppression (n=10), patients 10-12 months post-HCT who had remained cGVHD-free to that point (n=7), and non-transplanted healthy volunteers (n=3). One 10-12 month non-cGVHD patient subsequently developed sclerotic cGVHD five months after the sample collection date, and another had ongoing active skin aGVHD (points colored in yellow). (FIG.14D) Characteristics of patients at day +100 post-HCT. (FIG. 14E) Clinical characteristics of patients at cGVHD onset and 10-12 months post-HCT.

[0025] FIGs. 15A-15C. Characterization of HCT patient monocytes. (FIG. 15A) Cell type in patient and volunteer samples. (FIG. 15B) Day +100 post-HCT patient coexpression of IL-17 and CSF-1 nonclassical monocyte signatures derived from respective mouse Ly6Clo monocytes. (FIG. 15C) IL-17 and CSF-1 refined signature lists.

[0026] FIG. 16. Example computer architecture for a computer capable of executing program components for implementing functionality described herein.

[0027] FIG. 17. Gene names, abbreviations, and exemplary accession numbers to publicly available sequences and information.DETAILED DESCRIPTION

[0028] Chronic GVHD (cGVHD) is the leading cause of non-relapse late morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT), affecting up to 30-50% of survivors. cGVHD represents a broad spectrum of disease manifestations, including oral and ocular Sjogren’s, lichen planus, and, most morbidly irreversible fibrosis, causing cutaneous sclerosis and pulmonary bronchiolitis obliterans.

[0029] The understanding of cGVHD has increased significantly in the last decade, principally from improved preclinical models which have been foundational for recently approved therapies. It is now clear that cGVHD pathophysiology arises from dysregulation of multiple immune pathways, including biased T-cell differentiation where enhanced IL-17-secreting CD4 / CD8 T-cell (Th / Tc17), T follicular helper (Tfh), and deficient regulatory T-cell (Treg) responses are pathologic. Aberrant downstream germinal center B cell differentiation is associated with auto- and alloantibody production, resulting in lung fibrosis. Finally, colony stimulating factor-1 receptor (CSF-1 R)-dependent macrophage sequestration and alternative differentiation in target tissue appear necessary for fibrotic skin and lung cGVHD; this macrophage-mediated fibrosis is likewise IL-17-dependent, the mechanism of which is currently unresolved.F053-0195PCT / 25-085-WO-PCT

[0030] Recently FDA-approved cGVHD therapies target many of these components. These include belumosudil (ROCK2 inhibitor preventing Th17 / Tc17 and Tfh development and accompanying IL-17 production), ruxolitinib (JAK1 / 2 inhibitor utilizing STAT1 / 3 signaling), ibrutinib (a Bruton's tyrosine kinase inhibitor), and axatilimab (CSF-1 R-blocking antibody). While these agents induce significant responses in many patients, only a minority completely or sustainably respond to any particular agent, suggesting that the predominate drivers of cGVHD immune dysregulation likely differ between patients.

[0031] Before the current disclosure, there was a lack in the ability to differentiate dominant dysregulated immune pathways in a pragmatic, patient-specific manner to rationally select targeted cGVHD therapy. Identifying active pathways before irreversible end-organ pathology develops represented a major unmet need, as detailed by the recent NIH Chronic GVHD Consensus Conference. While preclinical murine models have been highly biologically informative and have guided the development of recent cGVHD immune pathway-targeting drugs, characterization of immune responses in mouse cGVHD systems has typically been in tissue, including primary and secondary lymphoid organs. These sites are not practicably or serially clinically accessible in humans to discern whether a specific pathway is perturbed and driving a given patient's cGVHD. In contrast, peripheral blood is accessible, but sensitive or specific detection of meaningful aberrant immunity in blood during cGVHD has thus far not been possible in mice or humans. In particular, conventional flow cytometry of blood and protein-based serologic analysis have not yet met this challenge.

[0032] Given the heterogeneous nature of cGVHD pathogenesis, it was hypothesized that unbiased single cell RNA sequencing (scRNAseq) could potentially discriminate immunologic drivers of disease with high resolution by simultaneously accessing the entire transcriptome within single immune cell subsets. The current disclosure thus focused on two prominent cGVHD immune pathways that have been established in preclinical mouse systems. Sequencing technology was used to interrogate peripheral blood immune cell signatures serially in order to characterize the hitherto undefined downstream effects of these cytokines on immune reconstitution during cGVHD in blood at a molecular level.

[0033] Chronic GVHD is characterized by dysregulated monocyte differentiation so temporal changes in myeloid cells during disease development were assessed. It is important to note that there is no prior art to suggest that pathogenic cGVHD immune signatures are detectable in blood, whether in mouse or human, nor that any newly defined mouse immune signature will be detectable in patients.

[0034] An "immune signature" refers to an aspect of a subject’s immune system that correlates with or is predictive of a physiological occurrence. An immune signature can refer to one aspect of a subject’s immune system, but also can combine different aspects into correlative or predictive groups. Immune signatures can be based on aspects of a subject’s immune cells, gene expression, cytokine release, cell surface marker expression, and other relevant immune-related measures. A subject can be a human transplant recipient.

[0035] Human monocytes are typically classified based on the expression of CD 14 and CD 16. Classical monocytes express high levels of CD14 and lack CD16 expression (CD14++CD16-). These monocytes represent the majority ofF053-0195PCT / 25-085-WO-PCTmonocytes in circulation, typically making up 80-90% of the total monocyte population in healthy individuals.

[0036] Classical monocytes are heavily involved during acute infections (e.g., bacterial infections) They exhibit high expression of chemokine receptors, such as CCR2, and rapidly migrate to sites of infection or injury and differentiate into macrophages or dendritic cells that are highly efficient at engulfing pathogens and cellular debris.

[0037] Nonclassical monocytes express low levels of CD14 and high levels of CD16 (CD14+ CD16++). These monocytes represent 5-10% of circulating monocytes. In an individual with inflammation, the proportion of nonclassical monocytes may increase, suggesting a role in patrolling and resolving inflammation. Nonclassical monocytes express CX3CR1 and produce anti-inflammatory cytokines, such as IL-10 and TGFp and differentiate into tissue macrophages.

[0038] Table 1 provides a summary of salient features of classical versus nonclassical monocytes described above. Table 1. Summary of Salient Features of Classical versus Nonclassical Monocytes.

[0039] Ly6C is a surface glycoprotein that serves as an important marker for identifying different subsets of monocytes and other immune cells, particularly in mice. Ly6Chimonocytes are analogous to classical monocytes (CD14++ CD16-) in humans while Ly6'° monocytes are analogous to nonclassical monocytes (CD14+ CD16++) in humans.

[0040] Unsupervised clustering revealed seven distinctive circulating monocyte populations, with putative differentiation trajectory assigned by RNA velocity analysis, plus one macrophage and one dendritic cell cluster. These included a proliferative initiating population (Proliferating), characterized by high expression of cell cycle-related genes, and its major descendent (Early). Early gave rise to three major intermediate Ly6Chipopulations (Circulating 1, Circulating 2, and Circulating 3). Notably, Circulating 1, was characterized by relatively higher expression of "resting” genes Klf2 and Ly6a, compared to Circulating 2 which was enriched for monocyte / macrophage activation-associated genes including NF-KB pathway genes, Tlr2, Cxcl2, Tnf, and Clec4e. An additional Ly6Chicluster was characterized by the expression of S100a8, Wfdc21, Lcn2, Chil3, and Mmp8. Circulating 3 shared many gene expression similarities with Circulating 2 and was also characterized by the unique expression of Stxbp6, Ccl2, and CD177. These cells also expressed Cx3cr1 and Csflr and were Ly6Cint / l°.

[0041] The remaining Ly6Cl0monocyte cluster represented a putative tissue resident macrophage precursor population. This cluster expressed the highest levels of Csflr and Cx3cr1 RNA and was Ly6Cl0by protein analysis.

[0042] Neutrophils were clustered originating from the least mature G4, parallelly differentiating into intermediate G5aF053-0195PCT / 25-085-WO-PCTand G5b, then converging into terminal G5c. These cell clusters are described in Xie et al. (2020) Nat Immunol. 21(9): 1119-33 which is specifically incorporated by reference for its description of the same. Briefly, G4 neutrophils represent those in the final stage of neutrophil differentiation in the bone marrow, that highly express Mmp8 and Cxcl2. G5a neutrophils are a subset of mature peripheral blood neutrophils. These cells highly express Mmp8 and S100a8, genes related to neutrophil migration and inflammatory responses. G5b neutrophils are distinct for their expression of interferon-stimulated genes (ISGs), such as Ifit3 and Isg15. G5c neutrophils are the most aged and terminally differentiated subset of peripheral neutrophils. They are known for their unique transcriptional signature related to aging and apoptosis.

[0043] Analysis of these cell types resulted in the identification of immune signatures to detect interleukin-17 (IL-17) or colony-stimulating factor-1 (CSF-1) chronic graft versus host disease (cGVHD) from the peripheral blood of human hematopoietic transplant recipients. The immune signatures can arise before clinical symptoms of cGVHD emerge and also allow directed personalized treatment for patients with these types of cGVHD

[0044] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4, SOCS3, SQSTM1, TLR2, and TNFSF9.

[0045] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CCL3 and at least 1 , 2, 3, 4, or 5 other genes selected from CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0046] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CLEC4E and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0047] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of EHD1 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0048] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of EIF3J and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0049] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of FTH1 and at least 1, 2,F053-0195PCT / 25-085-WO-PCT3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. S0CS3, SQSTM1, TLR2, and TNFSF9.

[0050] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of GADD45B and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0051] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of GPX4 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0052] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of IER3 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0053] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of MAFF and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0054] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of MBNL2 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0055] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NDUFB1 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0056] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of NFKBID and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0057] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of NFKBIZ and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.F053-0195PCT / 25-085-WO-PCT

[0058] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ODC1 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

[0059] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SDC4 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SOCS3, SQSTM1, TLR2, and TNFSF9.

[0060] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SOCS3 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SQSTM1, TLR2, and TNFSF9.

[0061] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SQSTM1 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1 , NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, TLR2, and TNFSF9.

[0062] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of TLR2 and at least 1, 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, and TNFSF9.

[0063] In particular embodiments, an IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of TNFSF9 and at least 1 , 2, 3, 4, or 5 other genes selected from CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, and TLR2.

[0064] In certain examples, the panel excludes CCL3. In certain examples, the panel excludes CLEC4E. In certain examples, the panel excludes EHD1. In certain examples, the panel excludes EIF3J. In certain examples, the panel excludes FTH1. In certain examples, the panel excludes GADD45B. In certain examples, the panel excludes GPX4. In certain examples, the panel excludes IER3. In certain examples, the panel excludes MAFF. In certain examples, the panel excludes MBNL2. In certain examples, the panel excludes NDUFB1. In certain examples, the panel excludes NFKBID. In certain examples, the panel excludes NFKBIZ. In certain examples, the panel excludes 0DC1. In certain examples, the panel excludes SDC4. In certain examples, the panel excludes SOCS3. In certain examples, the panel excludes SQSTM1. In certain examples, the panel excludes TLR2. In certain examples, the panel excludes TNFSF9.

[0065] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of APOE, CCL4, CCRL2,F053-0195PCT / 25-085-WO-PCTCD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0066] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of APOE and at least 1, 2, 3, 4, or 5 other genes selected from CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0067] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CCL4 and at least 1 , 2, 3, 4, or 5 other genes selected from APOE, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0068] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CCRL2 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0069] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CD14 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0070] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CSRNP1 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0071] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CXCL2 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0072] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CXCL10 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0073] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of DUSP1 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

[0074] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detectedF053-0195PCT / 25-085-WO-PCTfrom a human transplant recipient's peripheral blood sample includes differential expression of EGR1 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0075] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of POS and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0076] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of GADD45B and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

[0077] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NFKBIZ and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NLRP3, OSM, SLC7A11, and TLR2.

[0078] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NLRP3 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, OSM, SLC7A11, and TLR2.

[0079] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of OSM and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, SLC7A11, and TLR2.

[0080] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SLC7A11 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, and TLR2.

[0081] In particular embodiments, a CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of TLR2 and at least 1, 2, 3, 4, or 5 other genes selected from APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, and SLC7A11.

[0082] In certain examples, the panel excludes APOE. In certain examples, the panel excludes CCL4. In certain examples, the panel excludes CCRL2. In certain examples, the panel excludes CD14. In certain examples, the panel excludes CSRNP1. In certain examples, the panel excludes CXCL2. In certain examples, the panel excludes CXCL10.F053-0195PCT / 25-085-WO-PCTIn certain examples, the panel excludes DUSP1. In certain examples, the panel excludes EGR1. In certain examples, the panel excludes POS. In certain examples, the panel excludes GADD45B. In certain examples, the panel excludes NFKBIZ. In certain examples, the panel excludes NLRP3. In certain examples, the panel excludes OSM. In certain examples, the panel excludes SLC7A11. In certain examples, the panel excludesTLR2.

[0083] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, D0K2, DUSP2, EGR1, EN03, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, P0T1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 (collectively, "the CSF-1 nonclassical monocyte list”).

[0084] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ABCG1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0085] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of ABHD12 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0086] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ABI3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0087] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of ACE and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0088] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ACP2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0089] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of ADCY7 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0090] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of AGPAT4 andF053-0195PCT / 25-085-WO-PCTat least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0091] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of BATF3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0092] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of BBC3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0093] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of BTG2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0094] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CBLB and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0095] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CCL3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0096] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CCL4 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0097] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CD274 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0098] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CD300C and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0099] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CD300E and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0100] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CD74 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0101] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CD82 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.F053-0195PCT / 25-085-WO-PCT

[0102] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CD9 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0103] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CDC42EP2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0104] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CEACAM1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0105] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CFB and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0106] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CHKA and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0107] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CIITA and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0108] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of CX3CR1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0109] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of CYTH3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0110] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of DOCK5 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0111] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of D0K2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0112] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of DUSP2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0113] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytesF053-0195PCT / 25-085-WO-PCTdetected from a human transplant recipient's peripheral blood sample includes differential expression of EGR1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0114] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of EN03 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0115] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ENTPD1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0116] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of FAM43A and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0117] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of FGD2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0118] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of FILIP1L and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0119] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of FYN and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0120] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of GNGT2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0121] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of HAVCR2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0122] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of HES1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0123] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of ITGA2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0124] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ITGAL and atF053-0195PCT / 25-085-WO-PCTleast 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0125] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of ITGAV and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0126] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of JUN and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0127] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of LACC1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0128] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of LDLRAP1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0129] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of LPP and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0130] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of MAN1A1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0131] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of MY018A and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0132] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NABP1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0133] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of NDST 1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0134] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NFKBID and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0135] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of NFKBIE and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.F053-0195PCT / 25-085-WO-PCT

[0136] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of NFKBIZ and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0137] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of NR4A1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0138] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PDE4B and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0139] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PILRB and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0140] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PLA2G7 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0141] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PLTP and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0142] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PMAIP1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0143] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of P0T1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0144] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PP1R15A and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0145] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of PRR5L and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0146] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of PTPN12 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0147] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytesF053-0195PCT / 25-085-WO-PCTdetected from a human transplant recipient's peripheral blood sample includes differential expression of PTPRJ and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0148] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of RAP1GAP2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0149] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of RAP2A and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0150] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of RASGRP1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0151] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of RUNX2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0152] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SDC3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0153] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SEMA4D and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0154] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SH2D1 B and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0155] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SKI L and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0156] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SLAMF8 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0157] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SLC11A1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0158] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SLC8A1 andF053-0195PCT / 25-085-WO-PCTat least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0159] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SMPDL3B and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0160] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SOCS3 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0161] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of SPN and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0162] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of SRGN and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0163] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of STAP1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0164] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of STK10 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0165] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TBC1 D2B and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0166] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of TCF7L2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0167] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TGFBR1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0168] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of TGTFBR2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0169] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TGIF1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.F053-0195PCT / 25-085-WO-PCT

[0170] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TGM2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0171] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample includes differential expression of THEMIS2 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0172] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TMEM51 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0173] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TRAF1 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0174] In particular embodiments, a CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient's peripheral blood sample includes differential expression of TREML4 and at least 1, 2, 3, 4, or 5 other genes selected from the CSF-1 nonclassical monocyte list.

[0175] In certain examples, the panel excludes ABCG1. In certain examples, the panel excludes ABHD12. In certain examples, the panel excludes ABI3. In certain examples, the panel excludes ACE. In certain examples, the panel excludes ACP2. In certain examples, the panel excludes ADCY7. In certain examples, the panel excludes AGPAT4. In certain examples, the panel excludes BATF3. In certain examples, the panel excludes BBC3. In certain examples, the panel excludes BTG2. In certain examples, the panel excludes CBLB. In certain examples, the panel excludes CCL3. In certain examples, the panel excludes CCL4. In certain examples, the panel excludes CCRL2. In certain examples, the panel excludes CD274. In certain examples, the panel excludes CD300C. In certain examples, the panel excludes CD300E. In certain examples, the panel excludes CD74. In certain examples, the panel excludes CD82. In certain examples, the panel excludes CD9. In certain examples, the panel excludes CDC42EP2. In certain examples, the panel excludes CEACAM1. In certain examples, the panel excludes CFB. In certain examples, the panel excludes CHKA. In certain examples, the panel excludes CIITA. In certain examples, the panel excludes CX3CR1. In certain examples, the panel excludes CYFIP2. In certain examples, the panel excludes CYTH3. In certain examples, the panel excludes DOCK5. In certain examples, the panel excludes D0K2. In certain examples, the panel excludes DUSP2. In certain examples, the panel excludes EGR1. In certain examples, the panel excludes EN03. In certain examples, the panel excludes ENTPD1. In certain examples, the panel excludes FAM43A. In certain examples, the panel excludes FGD2. In certain examples, the panel excludes FILIP1L. In certain examples, the panel excludes FYN. In certain examples, the panel excludes GNGT2. In certain examples, the panel excludes HAVCR2. In certain examples, the panel excludes HES1. In certain examples, the panel excludes ITGA2. In certain examples, the panel excludes ITGAL. In certain examples, the panel excludes ITGAV. In certain examples, the panel excludes JUN. In certain examples, theF053-0195PCT / 25-085-WO-PCTpanel excludes LACC1. In certain examples, the panel excludes LDLRAP1. In certain examples, the panel excludes LPP. In certain examples, the panel excludes MAN1A1. In certain examples, the panel excludes MY018A. In certain examples, the panel excludes NABP1. In certain examples, the panel excludes NDST1. In certain examples, the panel excludes NFKBID. In certain examples, the panel excludes NFKBIE. In certain examples, the panel excludes NFKBIZ. In certain examples, the panel excludes NR4A1. In certain examples, the panel excludes PDE4B. In certain examples, the panel excludes PILRB. In certain examples, the panel excludes PLA2G7. In certain examples, the panel excludes PLTP. In certain examples, the panel excludes PMAIP1. In certain examples, the panel excludes P0T1. In certain examples, the panel excludes PP1R15A. In certain examples, the panel excludes PRR5L. In certain examples, the panel excludes PTPN12. In certain examples, the panel excludes PTPRJ. In certain examples, the panel excludes RAP1GAP2. In certain examples, the panel excludes RAP2A. In certain examples, the panel excludes RASGRP1. In certain examples, the panel excludes RUNX2. In certain examples, the panel excludes SDC3. In certain examples, the panel excludes SEMA4D. In certain examples, the panel excludes SH2D1B. In certain examples, the panel excludes SKIL. In certain examples, the panel excludes SLAMF8. In certain examples, the panel excludes SLC11 A1. In certain examples, the panel excludes SLC8A1. In certain examples, the panel excludes SMPDL3B. In certain examples, the panel excludes S0CS3. In certain examples, the panel excludes SPN. In certain examples, the panel excludes SRGN. In certain examples, the panel excludes STAP1. In certain examples, the panel excludes STK10. In certain examples, the panel excludes TBC1D2B. In certain examples, the panel excludes TCF7L2. In certain examples, the panel excludes TGFBR1. In certain examples, the panel excludes TGFBR2. In certain examples, the panel excludes TGI F1. In certain examples, the panel excludes TGM2. In certain examples, the panel excludes THEMIS2. In certain examples, the panel excludes TMEM51. In certain examples, the panel excludes TRAF1. In certain examples, the panel excludes. In certain examples, the panel excludes TREML4.

[0176] As is understood by one of ordinary skill in the art, the direction of differential expression (e.g., up-regulated or down-regulated) can change based on the reference control population selected to provide a ground truth. Assuming a common reference control population as one described herein, the direction of expression of each gene would be as follows:IL- 171 nonclassical monocytes: CCL3 (present / expressed), CLEC4E (up), EHD1 (up), EIF3J (up), FTH1 (up), GADD45B (up), GPX4 (up), IER3 (up), MAFF (up), MBNL2 (up), NDUFB1 (up), NFKBID(up), NFKBIZ (up), ODC1 (up), SDC4 (up), SOCS3 (up), SQSTM1 (up), TLR2 (up), and TNFSF9 (up).Both CSF-1 monocyte immune signatures result from CSF-1 dependent cell populations. Thus, these genes are designated as present / expressed.CSF-1 / classical monocytes: APOE (present / expressed), CCL4 (present / expressed), CCRL2 (present / expressed), CD 14 (present / expressed), CSRNP1 (present / expressed), CXCL2 (present / expressed), CXCL10 (present / expressed), DUSP1 (present / expressed), EGR1 (present / expressed), POS (present / expressed), GADD45B (present / expressed), NFKBIZ (present / expressed), NLRP3 (present / expressed), OSM (present / expressed), SLC7A11F053-0195PCT / 25-085-WO-PCT(present / expressed), and TLR2 (present / expressed).CSF-1 / nonclassical monocyte: ABCG1 (present / expressed), ABHD12 (present / expressed), ABI3 (present / expressed), ACE (present / expressed), ACP2 (present / expressed), ADCY7 (present / expressed), AGPAT4 (present / expressed), BATF3 (present / expressed), BBC3(present / expressed), BTG2 (present / expressed), CBLB (present / expressed), CCL3 (present / expressed), CCL4 (present / expressed), CCRL2 (present / expressed), CD274 (present / expressed), CD300C (present / expressed), CD300E (present / expressed), CD74 (present / expressed), CD82 (present / expressed), CD9 (present / expressed), CDC42EP2 (present / expressed), CEACAM1 (present / expressed), CFB (present / expressed), CHKA (present / expressed), CIITA (present / expressed), CX3CR1 (present / expressed), CYFIP2 (present / expressed), CYTH3 (present / expressed), DOCK5 (present / expressed), DOK2 (present / expressed), DUSP2 (present / expressed), EGR1 (present / expressed), ENO3 (present / expressed), ENTPD1 (present / expressed), FAM43A (present / expressed), FGD2 (present / expressed), FILIP1L (present / expressed), FYN (present / expressed), GNGT2 (present / expressed), HAVCR2 (present / expressed), HES1 (present / expressed), ITGA2 (present / expressed), ITGAL (present / expressed), ITGAV (present / expressed), JUN (present / expressed), LACC1 (present / expressed), LDLRAP1 (present / expressed), LPP (present / expressed), MAN1A1 (present / expressed), MYO18A (present / expressed), NABP1 (present / expressed), NDST1 (present / expressed), NFKBID (present / expressed), NFKBIE (present / expressed), NFKBIZ (present / expressed), NR4A1 (present / expressed), PDE4B (present / expressed), PILRB (present / expressed), PLA2G7 (present / expressed), PLTP (present / expressed), PMAIP1 (present / expressed), POT1 (present / expressed), PP1R15A (present / expressed), PRR5L (present / expressed), PTPN12 (present / expressed), PTPRJ (present / expressed), RAP1GAP2 (present / expressed), RAP2A (present / expressed), RASGRP1 (present / expressed), RUNX2 (present / expressed), SDC3 (present / expressed), SEMA4D (present / expressed), SH2D1B (present / expressed), SKIL (present / expressed), SLAMF8 (present / expressed), SLC11A1 (present / expressed), SLC8A1 (present / expressed), SMPDL3B (present / expressed), SOCS3 (present / expressed), SPN (present / expressed), SRGN (present / expressed), STAP1 (present / expressed), STK10 (present / expressed), TBC1D2B (present / expressed), TCF7L2 (present / expressed), TGFBR1 (present / expressed), TGFBR2 (present / expressed), TGIF1 (present / expressed), TGM2 (present / expressed), THEMIS2 (present / expressed), TMEM51 (present / expressed), TRAF1 (present / expressed), and TREML4 (present / expressed).

[0177] In particular embodiments, an IL-17 cGVHD immune signature expressed by LyC6l0monocytes includes upregulated expression of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcl1, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, Marcksll, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1C0a6, S1C0a8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.

[0178] In particular embodiments, an IL-17 cGVHD immune signature expressed by LyC6bmonocytes includes downregulated expression of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.F053-0195PCT / 25-085-WO-PCT

[0179] In particular embodiments, an IL-17 cGVHD immune signature expressed by Early monocytes includes downregulated expression of Lyz2.

[0180] In particular embodiments, an IL-17 cGVHD immune signature expressed by Circulating 1 monocytes includes upregulated expression of Ier3, Maltl , Mfkbiz, and Tnf.

[0181] In particular embodiments, an IL-17 cGVHD immune signature expressed by Circulating 1 monocytes includes downregulated expression of Gm4951, Igtp, ligpl, Klf2, and Rsrpl.

[0182] In particular embodiments an IL-17 cGVHD immune signature expressed by Circulating 1 monocytes includes downregulated expression of Nfkbiz, Maltl, Ccl3, and Pde4b.

[0183] In particular embodiments an IL-17 cGVHD immune signature expressed by Circulating 1 monocytes includes upregulated expression of ligpl and Ly6a.

[0184] In particular embodiments, an IL-17 cGVHD immune signature expressed by Circulating 2 monocytes includes upregulated expression of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.

[0185] In particular embodiments, an IL-17 cGVHD immune signature expressed by Circulating 2 monocytes includes downregulated expression of Fos, Ifitm3, Klf2, and Tsc22d3

[0186] In particular embodiments an IL-17 cGVHD immune signature expressed by Circulating 2 monocytes includes downregulated expression of Gm4951, ligpl, and Ly6a.

[0187] In particular embodiments an IL-17 cGVHD immune signature expressed by Circulating 2 monocytes includes upregulated expression of Nfkbia, Tlr2, Marckssll, 111b, and Pde4b.

[0188] In particular embodiments, an IL-17 cGVHD immune signature expressed by G4 neutrophils includes upregulated expression of Cd6.

[0189] In particular embodiments an IL-17 cGVHD immune signature expressed by G4 neutrophils, includes upregulated expression of Ltf, Camp. Itgb21, Stmnl, Serpiabla, Tinagll, Adpgk, and Gm26532.

[0190] In particular embodiments an IL-17 cGVHD immune signature expressed by G4 neutrophils, includes downregulated expression of Hbb-bs, Vmp1, Sfxn5, GmS483, GM10116, Notch2, Klf2, Stfa2l1, Fosl2, B9d2, Atf3, Gad1-ps. Gm2a, and Pdcd2l.

[0191] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5a neutrophils includes upregulated expression of CxcIS, Egr1 , Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.

[0192] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5a neutrophils includes downregulated expression downregulated expression of Csf1, Fgl2, Vim, and Xpc.

[0193] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5b neutrophils includes upregulated expression of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.

[0194] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5b neutrophils includes downregulated expression downregulated expression of Ifitm2 and Vim.F053-0195PCT / 25-085-WO-PCT

[0195] In particular embodiments an IL-17 cGVHD immune signature expressed by G5b neutrophils, includes upregulated expression of Ifitl, Ifit3, Ifit3b, Usp18, Stfn8, and Xaf1.

[0196] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5c neutrophils includes upregulated expression upregulated expression Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.

[0197] In particular embodiments, an IL-17 cGVHD immune signature expressed by G5c neutrophils includes downregulated expression downregulated expression of Fgl2.

[0198] In particular embodiments an IL-17 cGVHD immune signature expressed by G5c neutrophils, includes upregulated expression of Elane, Clec4n, Csf1, Cgnt2, and Pdcd21.

[0199] In particular embodiments, a CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes includes upregulated expression of Ccl3 and F13a1.

[0200] In particular embodiments, a CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes includes downregulated expression of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.

[0201] In particular embodiments a CSF-1 cGVHD immune signature expressed by Circulating 2 monocytes includes upregulated expression of Cxcl2, Tnfsf9, Gm14636, Ptgs2, Egr2, Olfr432, Mir155hg, Cxcl1, Edn1, Gm48302, 1110002J07Rik, Gm4065, Gm2449, and Gm29946.

[0202] In particular embodiments, a CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes includes upregulated expression of H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.

[0203] In particular embodiments, a CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes includes downregulated expression of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.

[0204] In particular embodiments a CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes includes upregulated expression of Gngt2, Serpinb2, Rbpms, Gm5547, Scnlb, Adora2a, Gm12866, 1700061G19Rik, Ifit3b, Gm 10484, and Rai 14.

[0205] In particular embodiments a CSF-1 cGVHD immune signature expressed by Granulocyte-like monocytes includes upregulated expression of S100a8, Lcn2, Wfdc21, Mmp8, S100a9, Lrg1, Fcnb, Abca13, Crispld2, Olfml2b, F730016J06Rik, Gpldl, Foxd4, Platr7, Rnf128.

[0206] In particular embodiments a CSF-1 cGVHD immune signature expressed by Ly6Cl0monocytes includes upregulated expression of Slc12a2, Ccnd2, Cd300e, Adrbl, Itgax, Adamded, Jade2, Pdgfb, Spic, Cd300ld3, Vegfc, Dnahl 2, Adgrl3, Itga9m, and Ccdc192.

[0207] In particular embodiments a CSF-1 cGVHD immune signature expressed by Ly6Cl0monocytes includes downregulated expression of Stxbp6, Lmha, Rfx2, Gm15902, Enkur, Reml, Smim5, Wfdc21, and Mmp8.

[0208] In particular embodiments, a CSF-1 cGVHD immune signature expressed by G4 neutrophils includes upregulated expression of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.F053-0195PCT / 25-085-WO-PCT

[0209] In particular embodiments, a CSF-1 cGVHD immune signature expressed by G5a neutrophils includes upregulated expression of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1 , Steap4, Tnf, Tnfaip3, and Wfdc17.

[0210] In particular embodiments, a CSF-1 cGVHD immune signature expressed by G5a neutrophils includes downregulated expression downregulated expression of Tsc22d3.

[0211] In particular embodiments, a CSF-1 cGVHD immune signature expressed by G5b neutrophils includes upregulated expression of Cxcl2 and Nfkbia.

[0212] In particular embodiments, a CSF-1 cGVHD immune signature expressed by G5c neutrophils includes upregulated expression upregulated expression Cxcl2, Ifitm6, S100a8, Tnf, Wfdc17, and Wfdc21.

[0213] Aspects of the disclosure are now described with additional detail and options as follows: (i) Sample Collection & Processing; (ii) RNA Sequencing; (iii) Reference Levels and Control Populations; (iv) Kits; (v) Methods of Treatment Following Immune Signature Detection; (vi) Exemplary Embodiments; (vii) Experimental Example; and (viii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0214] (i) Sample Collection & Processing. Methods regarding collection, anti-coagulation and processing, etc. of peripheral blood samples are well known in the art. Monocytes can be collected and isolated from a sample using any appropriate technique. Appropriate collection and isolation procedures include magnetic separation; fluorescence activated cell sorting (FACS); nanosorting based on fluorophore expression; affinity chromatography; cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins; "panning" with antibody attached to a solid matrix; selective agglutination using a lectin such as soybean; immunomagnetic bead-based sorting or combinations of these techniques, etc.

[0215] In particular embodiments, it is important to remove contaminating cell populations that can interfere with isolation of monocytes, in particular red blood cells. Removing includes both biochemical and mechanical methods to remove the undesired cell populations. Examples include lysis of red blood cells using detergents, hetastarch, hetastarch with centrifugation, cell washing, cell washing with density gradient filters, and other protocols that have been used both in processing of cells for diagnostic purposes.

[0216] In particular embodiments, cell populations can be isolated and / or analyzed based on light scattering properties of the cells based on side scatter channel (SSC) brightness and forward scatter channel (FSC) brightness. Side scatter refers to the amount of light scattered orthogonally (90° from the direction of the laser source), as measured by flow cytometry. Forward scatter refers to the amount of light scattered generally less than 90° from the direction of the light source. Generally, as cell granularity increases, the side scatter increases and as cell diameter increases, the forward scatter increases.

[0217] Side scatter and forward scatter are measured as intensity of light. Those skilled in the art recognize that the amount of side scatter can be differentiated with user-defined settings. In particular embodiments, low (Io) side scatter refers to less than 50% intensity, less than 40% intensity, less than 30% intensity, or even less intensity, in the sideF053-0195PCT / 25-085-WO-PCTscatter channel of the flow cytometer. Conversely high (hi) side scatter cells are the reciprocal population of cells that are not low side scatter. Forward scatter is defined in the same manner as side scatter but the light is collected in forward scatter channel. Thus, particular embodiments include selection of cell populations based on precise combinations of cell surface markers (CD markers) and the associated light scattering properties of the cells.

[0218] Isolation of particular cell types described herein can rely on positive expression of particular CD markers and the negative expression of other CD markers. As is understood by one of ordinary skill in the art of flow cytometry, “hi’’, “int”, “Io”, “+” and refer to the intensity of a signal relative to negative or other populations. In particular embodiments, positive expression (+) means that the marker is detectable on a cell using flow cytometry. In particular embodiments, negative expression (-) means that the marker is not detectable using flow cytometry. In particular embodiments, “hi” means that the positive expression of a marker of interest is brighter as measured by fluorescence (using for example FACS) than other cells also positive for expression. In these embodiments, those of ordinary skill in the art recognize that brightness is based on a threshold of detection. Generally, one of skill in the art will analyze a negative control tube first, and set a gate (bitmap) around the population of interest by FSC and SSC and adjust the photomultiplier tube voltages and gains for fluorescence in the desired emission wavelengths, such that 97% of the cells appear unstained for the fluorescence marker with the negative control. Once these parameters are established, stained cells are analyzed and fluorescence recorded as relative to the unstained fluorescent cell population. In particular embodiments, and representative of a typical FACS plot, hi implies to the farthest right (x line) or highest top line (upper right or left) while Io implies within the left lower quadrant or in the middle between the right and left quadrant (but shifted relative to the negative population). In particular embodiments, "hi" refers to greater than 20-fold of +, greater than 30-fold of +, greater than 40-fold of +, greater than 50-fold of +, greater than 60-fold of +, greater than 70-fold of +, greater than 80-fold of +, greater than 90-fold of +, greater than 100-fold of +, or more of an increase in detectable fluorescence relative to + cells. Conversely, “Io” can refer to a reciprocal population of those defined as "hi".

[0219] (ii) RNA Sequencing. RNA-Seq is often used to identify, analyze, and quantify the expression of a multitude of genes at a certain moment in time and under certain experimental conditions. RNA-Seq can utilize one or more next generation sequencing platforms, allowing rapid analysis of various sized genomes compared to previous sequencing technologies. Typically, RNA-Seq consists of some or all of identifying a biological sample of interest that has been subjected to one or more experimental conditions, isolating RNA therefrom, obtaining RNA reads, aligning the RNA reads to a transcriptome (e.g., of a transcriptome library), and performing various downstream analyses, such as differential expression analysis.

[0220] Single-cell RNA-sequencing, (scRNA-seq) partitions RNA-Seq data into libraries with unique DNA barcodes for each RNA sample cell of origin. scRNA-Seq, as this enables profiling the transcriptomes of many cells in parallel. A typical scRNA-Seq experiment can profile millions of cells. The release of the first million-cell dataset occurred in 2017.

[0221] Different primers and / or adapters can be used to select different types of RNA for cDNA generation andF053-0195PCT / 25-085-WO-PCTsequencing. Exemplary types of RNA include small RNA such as a micro RNAs (miRNA), piwi interacting RNA (piRNA), small interfering RNA (siRNA), repeat associated siRNA (rasiRNA), trans-acting siRNA (tasiRNA), CRISPR RNA (crRNA), transfer RNA (tRNA), Promoter-associated RNA (PASR), Transcription stop site associated RNAs, signal recognition particle RNA, transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmyRNA, small Cajal Body-specific RNA (scaRNA), Guide RNA (gRNA), Spliced leader RNA, ribosomal RNA (rRNA), Telomerase RNA, Ribonuclease P, or a large RNA such as long non-coding RNAs or messenger RNAs, retrotransposons, satellite RNA, virioids, viral genomes or fragments thereof.

[0222] In certain examples, polyT primers (also known as Oligo-d(T) or Oligo-d(T)20 primers) can be selected to selectively produce cDNA from protein-encoding RNA. In certain examples, random hexamers can be used as primers, Random hexamers are random sequences of six nucleotides that anneal to complementary sites on an RNA and act as primers for cDNA synthesis. Gene-specific primers bind target sequences within an mRNA of interest, allowing amplification of only that region Particular embodiments can combine use of polyT primers, random hexamers, and / or gene-specific primers.

[0223] As is understood by one of ordinary skill in the art, adapters can also be used to target particular types of RNA for cDNA generation or to allow for labeling all types of RNA for non-selective cDNA generation. Useful RNA adapters are described in, for example, US2014 / 0357528. Adapters which provide priming sequences for both amplification and sequencing of fragments for use with the 454 Life Science GS20 sequencing system are described by F. Cheung, et al. in BMC Genomics 2006, 7:272.

[0224] Ligation of RNA adapters to RNA can be achieved using a suitable nucleic acid ligase such as T4 RNA ligase 1 (T4 Rnl1) T4 RNA ligase 2 (T4 Rnl2), T4 RNA ligase 2 truncated (also defined as T4 RNA Ligase 2 1-249) and T4 ligase 2 truncated K227Q (T4 Rnl2tr K227Q), T4 DNA ligase 2 truncated R55K, K227Q (T4 Rnl2tr KQ), T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. co / / DNA ligase, 9° N™ DNA ligase, Thermus aquaticus DNA ligase, Paramecium bursaria chlorella virus 1 (PBCV-1) ligase, Methanobacterium thermoautotrophicum RNA ligase (Mth ligase), or RtcB family ligases such as E. coli RtcB ligase or variants of these ligases (New England Biolabs, Ipswich, Mass.) that support the complete ligation reaction or at least phosphodiester bond formation between nucleic acid polymers.

[0225] Particular embodiments increase the incorporation of adapters into cDNA sequences, which can be used to add synthetic priming sites on targets of interest (to facilitate target amplification), or barcodes to aid the computational processing of resulting sequencing reads for greater accuracy.

[0226] RNA can be subjected to any form of cDNA generation or sequencing. RT are enzymes that perform reverse transcription of RNA into a first strand of cDNA. More processive RT can be used to increase sequence read lengths. In certain examples, the processivity of an RT refers to the ability of an RT to generate a complementary strand of DNA across the full-length of the template RNA. Some RT enzymes (e.g., SuperScript IV (SSIV) achieve this via multiple binding events, whereas others (e.g., MarathonRT), can do so in single binding event. RT with higher processivity synthesize longer cDNA strands than RT with lower processivity. In certain examples, an RT that adds 1,500F053-0195PCT / 25-085-WO-PCTnucleotides is considered highly processive or to have high processivity.

[0227] Any appropriate sequencing method can be used. In particular embodiments, sample partition PCR methods may be used. In sample partitioning, numerous methods can be used to divide samples into discrete partitions (e.g., droplets). Exemplary partitioning methods and systems include use of one or more of emulsification, droplet actuation, microfluidics platforms, continuous-flow microfluidics, reagent immobilization, and combinations thereof. In particular embodiments, partitioning is performed to divide a sample into a sufficient number of partitions such that each partition contains one or zero nucleic acid molecules. In particular embodiments, the number and size of partitions is based on the concentration and volume of the bulk sample

[0228] Methods and devices for partitioning a bulk volume into partitions by emulsification are described in Nakano et al. J Biotechnol 102, 117-124 (2003) and Margulies et al. Nature 437, 376-380 (2005). Systems and methods to generate "water-in-oil11droplets are described in U.S. Publication No. 2010 / 0173394. Microfluidics systems and methods to divide a bulk volume into partitions are described in U.S. Publication Nos. 2010 / 0236929; 2010 / 0311599; and 2010 / 0163412, and U.S. Patent No. 7,851,184. Microfluidic systems and methods that generate monodisperse droplets are described in Kiss et al. Anal Chem. 80(23), 8975-8981 (2008). Further microfluidics systems and methods for manipulating and / or partitioning samples using channels, valves, pumps, etc. are described in U.S. Patent No.7,842,248. Continuous-flow microfluidics systems and methods are described in Kopp et al., Science, 280, 1046-1048 (1998).

[0229] Partitioning methods can be augmented with droplet manipulation techniques, including electrical (e.g., electrostatic actuation, dielectrophoresis), magnetic, thermal (e.g., thermal Marangoni effects, thermocapillary), mechanical (e.g., surface acoustic waves, micropumping, peristaltic), optical (e.g., opto-electrowetting, optical tweezers), and chemical means (e.g., chemical gradients). In particular embodiments, a droplet microactuator is supplemented with a microfluidics platform (e.g. continuous flow components).

[0230] Particular embodiments use a droplet microactuator. A droplet microactuator can be capable of effecting droplet manipulation and / or operations, such as dispensing, splitting, transporting, merging, mixing, agitating, and the like. Droplet operation structures and manipulation techniques are described in U.S. Publication Nos. 2006 / 0194331 and 2006 / 0254933 and U.S. Patent Nos. 6,911,132; 6,773,566; and 6,565,727.

[0231] In particular embodiments, amplification can be performed by sample partition dPCR (spdPCR). An example of sample partition dPCR is Droplet Digital PCR. Droplet digital PCR (ddPCR) (e.g., Droplet Digital™ PCR (ddPCR™) (Bio-Rad Laboratories, Hercules, CA)) technology uses a combination of microfluidics and surfactant chemistry to divide PCR samples into water-in-oil droplets. Hindson et al., Anal. Chem. 83(22): 8604-8610 (2011). The droplets support PCR amplification of template molecules they contain and use reagents and workflows similar to those used for most standard Taqman probe-based assays.

[0232] Following PCR, each droplet is analyzed or read in a flow cytometer to determine the fraction of PCR-positive droplets in the original sample. These data are then analyzed using Poisson statistics to determine the targetF053-0195PCT / 25-085-WO-PCTconcentration in the original sample. See Bio-Rad Droplet Digital™ (ddPCR™) PCR Technology.

[0233] Amplification. Nucleic acids of a sample (e.g. , partitioned nucleic acids) can be amplified by any suitable PCR methodology. Exemplary PCR types include allele-specific PCR, assembly PCR, asymmetric PCR, endpoint PCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inverse PCR, linear after exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, polymerase cycling assembly, qualitative PCR, quantitative PCR, real-time PCR, single-cell PCR, solid-phase PCR, thermal asymmetric interlaced PCR, touchdown PCR, universal fast walking PCR, etc. Ligase chain reaction (LCR) may also be used

[0234] PCR may be performed with a thermostable polymerase, such as Taq DNA polymerase (e.g., wild-type enzyme, a Stoffel fragment, FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase, Vent polymerase, or a combination thereof, among others.

[0235] PCR and LCR are driven by thermal cycling. Alternative amplification reactions, which may be performed isothermally, can also be used. Exemplary isothermal techniques include branched-probe DNA assays, cascade-RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid based amplification (NASBA), nicking enzyme amplification reaction (NEAR), PAN-AC, Q-beta replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand-displacement amplification, etc.

[0236] In examples using sample partitioning, amplification reagents can be added to a sample prior to partitioning, concurrently with partitioning and / or after partitioning has occurred. In particular embodiments, all partitions are subjected to amplification conditions (e.g. reagents and thermal cycling), but amplification only occurs in partitions containing target nucleic acids (e.g. nucleic acids containing sequences complementary to primers added to the sample). The template nucleic acid can be the limiting reagent in a partitioned amplification reaction. In particular embodiments, a partition contains one or zero target (e.g. template) nucleic acid molecules.

[0237] In particular embodiments, nucleic acid targets (e.g., RNA), primers, and / or probes are immobilized to a surface, for example, a substrate, plate, array, bead, particle, etc. Immobilization of one or more reagents provides (or assists in) one or more of: partitioning of reagents (e.g. target nucleic acids, primers, probes, etc.), controlling the number of reagents per partition, and / or controlling the ratio of one reagent to another in each partition. In particular embodiments, assay reagents and / or target nucleic acids are immobilized to a surface while retaining the capability to interact and / or react with other reagents (e.g. reagent dispensed from a microfluidic platform, a droplet microactuator, etc.). In particular embodiments, reagents are immobilized on a substrate and droplets or partitioned reagents are brought into contact with the immobilized reagents. Techniques for immobilization of nucleic acids and other reagents to surfaces are well understood by those of ordinary in the art. See, for example, U.S. Patent No. 5,472,881 and Taira et al. Biotechnol. Bioeng 89(7), 835-8 (2005).

[0238] Target Sequence Detection. Detection methods can be utilized to identify sample partitions containing amplified target(s) (i.e., unique sequences). Detection can be based on one or more characteristics of a sample suchF053-0195PCT / 25-085-WO-PCTas a physical, chemical, luminescent, or electrical aspects, which correlate with amplification.

[0239] In particular embodiments, fluorescence detection methods are used to detect amplified target(s), and / or identification of samples (e.g., partitions) containing amplified target(s). Exemplary fluorescent detection reagents include TaqMan probes, SYBR Green fluorescent probes, molecular beacon probes, scorpion probes, and / or LightUp probes® (LightUp Technologies AB, Huddinge, Sweden). Additional detection reagents and methods are described in, for example, U.S. Patent Nos. 5,945,283; 5,210,015; 5,538,848; and 5,863,736; PCT Publication WO 97 / 22719; and publications: Gibson et al., Genome Research, 6, 995-1001 (1996); Heid et al., Genome Research, 6, 986-994 (1996); Holland et al., Proc. Natl. Acad Sci. USA 88, 7276-7280, (1991); Livak et al., Genome Research, 4, 357-362 (1995); Piatek et al., Nat. Biotechnol. 16, 359-63 (1998); Neri et al., Advances in Nucleic Acid and Protein Analysis, 3826, 117-125 (2000); Compton, Nature 350, 91-92 (1991); Thelwell et al., Nucleic Acids Research, 28, 3752-3761 (2000); Tyagi and Kramer, Nat. Biotechnol. 14, 303-308 (1996); Tyagi et al., Nat. Biotechnol. 16, 49-53 (1998); and Sohn et al., Proc. Natl. Acad. Sci. U.S.A. 97, 10687-10690 (2000).

[0240] In particular embodiments, detection reagents are included with amplification reagents added to a bulk or partitioned sample. In particular embodiments, amplification reagents also serve as detection reagents. In particular embodiments, detection reagents are added to partitions following amplification. In particular embodiments, measurements of the absolute copy number and the relative proportion of target nucleic acids in a sample (e.g. relative to other targets nucleic acids, relative to non-target nucleic acids, relative to total nucleic acids, etc.) can be measured based on the detection of samples (e.g., partitions) containing amplified targets.

[0241] In particular embodiments, following amplification, samples containing amplified target(s) are sorted from samples not containing amplified targets or from samples containing other amplified target(s). In particular embodiments, samples are sorted following amplification based on physical, chemical, and / or optical characteristics of the samples, the nucleic acids therein (e.g. concentration), and / or status of detection reagents. In particular embodiments, individual samples are isolated for subsequent manipulation, processing, and / or analysis of the amplified target(s) therein. In particular embodiments, samples containing similar characteristics (e.g. same fluorescent labels, similar nucleic acid concentrations, etc.) are grouped (e.g. into packets) for subsequent manipulation, processing, and / or analysis.

[0242] Particular embodiments utilize NGS. In particular embodiments, sequencing with commercially available NGS platforms may be conducted with the following steps. First, DNA sequencing libraries may be generated by clonal amplification by PCR in vitro. Second, the DNA may be sequenced by synthesis, such that the DNA sequence is determined by the addition of nucleotides to the complementary strand rather through chain-termination chemistry. Third, the spatially segregated, amplified DNA templates may be sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step. While these steps are followed in most NGS platforms, each utilizes a different strategy (see e.g., Anderson, M. W. and Schrijver, I., 2010, Genes, 1: 38-69.). Examples of NGS platforms include Oxford Nanopore Technologies, Roche 454, GS FLX Titanium, Illumina, HiSeq 2000, GenomeF053-0195PCT / 25-085-WO-PCTAnalyzer IIX, HE, IScanSQ, Life Technologies Solid 4, Helicos Biosciences Heliscope, Pacific Biosciences (PacBio) SMART and PacBio HiFi.

[0243] In particular embodiments, DNA segments can undergo an amplification as part of NGS sequencing. In embodiments where an amplification process was used to create a target-increased sample, this amplification would be a second amplification step. The second amplification can provide a stronger signal than if the second amplification was not performed.

[0244] In particular embodiments, the methods include detecting a control. A control can refer to an RNA or DNA sequence that is “spiked” into a sample at a known or otherwise specified amount. In particular embodiments, the control is spiked into the sample at a known quantity (e.g. , known copy number), which can be useful, for example, to determine the absolute quantity of an RNA or DNA sequence (e.g., a unique sequence).

[0245] In certain examples, resulting data and conclusions are provided in a report that can be transmitted. In these examples, a report generator is configured to generate a report based, at least in part, on a detected immune signature. The report, for example, includes consumable data that can inform a care provider about the predicted condition of the human transplant recipient.

[0246] In some implementations, the report indicates that a follow-up test of the human transplant recipient is indicated. For instance, in response to determining that the categorization of an immune signature indicative of a type of cGVHD of the human transplant recipient is inconclusive, the report generator may generate the report to indicate that one or more additional tests should be performed.

[0247] In various cases, the report is output to a clinical device. For example, the report generator transmits the report to the clinical device. In various implementations, the clinical device is a computing device that is operated by, owned by, or otherwise associated with the care provider. For instance, the clinical device may be a desktop computer, a laptop computer, a smart phone, or some other computing device associated with the care provider. The clinical device, in various cases, outputs the report to the care provider. In some cases, the clinical device includes a display (e.g., a screen) that visually presents the report. In various cases, the clinical device includes a speaker that outputs a sound indicative of the report. The clinical device, in various cases, may output the information in the report using one or more output mechanisms or devices.

[0248] The care provider may review the report by interacting with the clinical device. The report, in various cases, enhances the clinical decision-making of the care provider. For instance, the care provider can prepare and / or administer a therapy to the human transplant recipient based on the report. According to various implementations, the care provider may initiate the therapy and / or refer the human transplant recipient to another care provider to receive the therapy.

[0249] In various implementations, the care provider can develop a diagnosis and / or prognosis of the human transplant recipient based on the report. In various implementations, the care provider may communicate information in the report to the human transplant recipient.F053-0195PCT / 25-085-WO-PCT

[0250] FIG. 16 shows an example computer architecture for a computer 100 capable of executing program components for implementing functionality described herein. The computer architecture shown in FIG. 16 illustrates a conventional computer, workstation, desktop computer, laptop, tablet, network appliance, e-reader, smartphone, or other computing device, and can be utilized to execute any of the processes described herein.

[0251] The computer 100 includes a baseboard 102, or “motherboard," which is a printed circuit board to which a multitude of components or devices can be connected by way of a system bus or other electrical communication paths. In one illustrative configuration, one or more processing units, such as (“CPUs”) 104, GPUs, TPUs, ASICs, FPGAs, or the like, and / or threads, kernels, cores, and / or the like thereof, that may operate in conjunction with a chipset 106 The CPUs 104 can be standard programmable processors that perform arithmetic and logical operations necessary for the operation of the computer 100.

[0252] The CPUs 104 perform operations by transitioning from one discrete, physical state to the next through the manipulation of switching elements that differentiate between and change these states. Switching elements generally include electronic circuits that maintain one of two binary states, such as flip-flops, and electronic circuits that provide an output state based on the logical combination of the states of one or more other switching elements, such as logic gates. These basic switching elements can be combined to create more complex logic circuits, including registers, adders-subtractors, arithmetic logic units, floating-point units, and the like.

[0253] The chipset 106 provides an interface between the CPUs 104 and the remainder of the components and devices on the baseboard 102. The chipset 106 can provide an interface to a random-access memory (RAM) 108 or any other suitable form of memory, used as the main memory in the computer 100. The chipset 106 can further provide an interface to a computer-readable storage medium such as a read-only memory (ROM) 110 or non-volatile RAM (NVRAM) for storing basic routines that help to startup the computer 100 and to transfer information between the various components and devices. The ROM 110 or NVRAM can also store other software components necessary for the operation of the computer 100 in accordance with the configurations described herein.

[0254] The computer 100 can operate in a networked environment using logical connections to remote computing devices and computer systems through a network, such as the network 124. The chipset 106 can include functionality for providing network connectivity through a network interface controller (NIC) 112, such as a gigabit Ethernet adapter. The NIC 112 is capable of connecting the computer 100 to other computing devices over the network 124. It should be appreciated that multiple NICs 112 can be present in the computer 100, connecting the computer 100 to other types of networks and remote computer systems. In some instances, the NICs 112 may include at least on ingress port and / or at least one egress port.

[0255] The computer 100 can include an input / output (I / O), such as a controller sufficient to transmit processorexecutable instructions to or receive processor-executable instructions from a device. For example, the I / O controller include or interface with one or more user interface devices (e.g., a display, speaker, a keyboard, a mouse, a trackpad, a touchscreen), one or more servers, laboratory equipment (e.g., DNA / RNA sequencing device(s), staining and / or stainF053-0195PCT / 25-085-WO-PCTimaging device(s), probe hybridization / imaging / decoding device(s)), and / or the like. Interfacing with any of these devices may additionally or alternatively be executed by the network interface controller 112.

[0256] The computer 100 can be connected to a storage device 118 that provides non-volatile storage for the computer. The storage device 118 can store an operating system 120, programs 122, and data, which have been described in greater detail herein. The storage device 118 can be connected to the computer 100 through a storage controller 114 connected to the chipset 106. The storage device 118 can include one or more physical storage units. The storage controller 114 can interface with the physical storage units through a serial attached small computer system interface (SCSI) (SAS) interface, a serial advanced technology attachment (SATA) interface, a fiber channel (FC) interface, or other type of interface for physically connecting and transferring data between computers and physical storage units.

[0257] The computer 100 can store data on the storage device 118 by transforming the physical state of the physical storage units to reflect the information being stored. The specific transformation of physical state can depend on various factors, in different embodiments of this description. Examples of such factors include the technology used to implement the physical storage units, whether the storage device 118 is characterized as primary or secondary storage, and the like.

[0258] For example, the computer 100 can store information to the storage device 118 by issuing instructions through the storage controller 114 to alter the magnetic characteristics of a particular location within a magnetic disk drive unit, the reflective or refractive characteristics of a particular location in an optical storage unit, or the electrical characteristics of a particular capacitor, transistor, or other discrete component in a solid-state storage unit. Other transformations of physical media are possible without departing from the scope and spirit of the present description, with the foregoing examples provided only to facilitate this description. The computer 100 can further read information from the storage device 118 by detecting the physical states or characteristics of one or more particular locations within the physical storage units.

[0259] In addition to the storage device 118 described above, the computer 100 can have access to other computer-readable storage media to store and retrieve information, such as program modules, data structures, or other data. It should be appreciated by those skilled in the art that computer-readable storage media is any available media that provides for the non-transitory storage of data and that can be accessed by the computer 100. In some examples, the operations performed by any network node described herein may be supported by one or more devices similar to computer 100. Stated otherwise, some or all of the operations performed by a network node may be performed by one or more computer devices 100 operating in a cloud-based arrangement.

[0260] By way of example, computer-readable storage media can include volatile and non-volatile, removable and non-removable media implemented in any method or technology. Examples of computer-readable storage media includes RAM, ROM, erasable programmable ROM ("EPROM”), electrically-erasable programmable ROM (“EEPROM”), flash memory or other solid-state memory technology, compact disc ROM ("CD-ROM”), digital versatileF053-0195PCT / 25-085-WO-PCTdisk (“DVD"), high definition DVD (“HD-DVD”), BLU-RAY, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information in a non-transitory fashion.

[0261] As mentioned briefly above, the storage device 118 can store an operating system 120 utilized to control the operation of the computer 100. According to one embodiment, the operating system 120 includes the LINUX® operating system. According to another embodiment, the operating system 120 includes the WINDOWS SERVER® operating system from MICROSOFT Corporation of Redmond, Washington. According to further embodiments, the operating system can include the UNIX® operating system or one of its variants. It should be appreciated that other operating systems can also be utilized. The storage device 118 can store other system or application programs and data utilized by the computer 100.

[0262] In one embodiment, the storage device 118 or other computer-readable storage media includes one or more programs 122. The programs 122, for example, include computer-executable instructions which, when loaded into the computer 100, transform the computer from a general-purpose computing system into a special-purpose computer capable of implementing the embodiments described herein. These computer-executable instructions transform the computer 100 by specifying how the CPUs 104 transition between states, as described above. According to one embodiment, the computer 100 has access to computer-readable storage media storing computer-executable instructions which, when executed by the computer 100, perform the various processes described herein. The computer 100 can also include computer-readable storage media having instructions stored thereupon for performing any of the other computer-implemented operations described herein. The program(s) 122, for example, include one or more processes. The process(es) may include instructions that, when executed by the CPU(s) 104, cause the computer 100 and / or the CPU(s) 104 to perform one or more operations.

[0263] The computer 100 can also include one or more input / output controllers 126 for receiving and processing input from a number of input devices, such as a keyboard, a mouse, a touchpad, a touch screen, an electronic stylus, or other type of input device. Similarly, an input / output controller 126 can provide output to a display, such as a computer monitor, a flat-panel display, a digital projector, a printer, or other type of output device. It will be appreciated that the computer 100 might not include all of the components shown in FIG 16, can include other components that are not explicitly shown in FIG. 16, or might utilize an architecture completely different than that shown in FIG. 16.

[0264] A computing device can transmit one or more communication signals to another computing device, wherein the communication signal(s) encode at least a portion of the data, and in particular embodiments provide a report. Examples of communication signals include electromagnetic signals, optical signals, ultrasonic signals, optical signals, and electrical signals. For example, communication signals can be transmitted wirelessly and / or in a wired fashion. The communication signals, for instance, are transmitted over one or more wireless channels and / or one or more wired channels (e.g., optical cabling, electrical cabling, etc.). In various cases, the communication signal(s) are transmitted over one or more communication networks. A communication network, for instance, may be defined according to oneF053-0195PCT / 25-085-WO-PCTor more physical channels, such as one or more frequency spectra. In some cases, a communication network is defined according to one or more communication protocols and / or standards. Examples of communication networks include fiber optic networks, Institute of Electrical and Electronics Engineers (IEEE) networks (e.g., WI-FI™ networks, WiMAX networks, BLUETOOTH™ networks, etc.), cellular networks (e.g., a 3rdGeneration Partnership Project (3GPP) radio network, such as a Long Term Evolution (LTE) network, a New Radio (NR) network; or a cellular core network such as a 3rdGeneration (3G) core, a 4thGeneration (4G) core, a 5thGeneration (5G) core, etc.), ultrasonic networks, and the like. In some cases, the data is broadcasted from one device to multiple other devices. In some cases, the data is unicasted from one device to another device. For instance, various forms of data described herein may be transmitted via a peer-to-peer (P2P) connection.

[0265] In some instances, one or more components may be referred to herein as “configured to,” “configurable to," “operable / operative to,” “adapted / adaptable,” “able to,” “conformable / conformed to,” etc. Those skilled in the art will recognize that such terms (e.g., “configured to”) can generally encompass active-state components and / or inactivestate components and / or standby-state components, unless context requires otherwise.

[0266] (iii) Reference Levels and Control Populations. A “dataset” as used herein is a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition. The values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample and constructing a dataset from these measurements; or alternatively, by obtaining a dataset from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.

[0267] In certain embodiments of the present disclosure, a dataset of values is determined by measuring biomarkers from a healthy population (i.e., non-transplanted), a +100 post-HCT without systemic aGVHD therapy population, a recently diagnosed cGVHD not yet on systemic immunosuppression population, and a cGVHD negative at 10-12 months population. Datasets can be interrogated by an interpretation function to derive an IL-17 and CSF-1 cGVHD-associated signature expression, which can provide a quantitative or qualitative measure of cGVHD risk or presence in a human transplant recipient. In particular embodiments, the amount of the biomarker(s) can be measured in a sample and used to derive an expression score, which is then compared to a “reference level”. Reference levels can include “normal" or “control” levels or values, defined according to, e.g., discrimination limits or risk defining thresholds, in order to define cut-off points and / or abnormal values for cGVHD. The reference level then, in particular embodmients, is the level of one or more biomarkers or combined biomarkers indices typically found in a human transplant recipient who is not suffering from cGVHD. Other terms for “reference levels” can include “index,” “baseline," “standard," “healthy,” etc. Such normal levels can vary based on whether a biomarker is used alone or in a formula combined with other biomarkers to output a score. Alternatively, the reference level can be a database of biomarker patterns from previously tested human transplant recipients who did not develop cGVHD over a clinically relevant time period. Reference levels can also be derived from, e.g., a control subject or population whose cGVHD diagnosis is known. In some embodiments, the reference value can be derived from one or more human transplant recipients who have beenF053-0195PCT / 25-085-WO-PCTexposed to treatment for cGVHD, or from human transplant recipients who have shown improvements in cGVHD as a result of exposure to treatment. In some embodiments, the reference level can be derived from one or more human transplant recipients who have not been exposed to treatment. A reference level can also be derived from disease activity algorithms or computed indices from population studies.

[0268] In particular embodiments, a “reference level” can refer to a standardized value for IL-17 and CSF-1 signatures which represents a level not associated with any disease; a level associated with a particular stage of the disease; or a level associated with a particular human transplant recipient at the time of diagnosis, at the beginning of treatment, or at a time point during a treatment. The reference level can be a universal reference level which is useful across a variety of testing locations or can be a reference level specific for the testing location and sequencing techniques used to measure the IL-17 and CSF-1 cGVHD-associated signature expressions. Reference levels for a human transplant recipient can also be related to time points of a human transplant recipient not undergoing treatments to monitor the natural progression or regression of a disease.

[0269] (iv) Kits. The present disclosure further provides kits including one or more reagents for sequencing and / or detection assays for practicing any of the methods disclosed herein. The kits may include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of prognostics or diagnostics which notice reflects approval by the agency of the manufacture for human testing. The reagents for sequencing and / or detection assays of the kits may include any necessary or appropriate polynucleotides, polypeptides, conjugates, antibodies, expression vectors, compositions, systems, and / or useful for the detection of IL-17 and CSF-1 dysregulation signatures in peripheral blood monocyte subsets. The kits may include genomic or probe-based tests, whose sequences would be those for relevant genes (i.e., those upregulated or downregulated by IL-17 / CSF-1 ). Additionally, the kits may provide reagents to operate with either single cell or bulk sequencing of the monocyte subsets in peripheral blood.

[0270] In particular embodiments, kits include RNA annealing primers that are gene specific to a gene or a combination of genes disclosed herein. In particular embodiments, kits include RNA annealing primers, for example, polyT primers and / or those that are gene specific to a subset of a combination of genes disclosed herein. Particular embodiments of kits can also include components such as a DNA polymerase, a reverse transcriptase, a transcription buffer, a reverse transcription buffer, a ligase, an adapter, and / or detection molecules.

[0271] Persons of ordinary skill in the art will understand that certain reagents would be available at testing locations such as water, saline, etc. and that these reagents might not be included in the kits. The kits further comprise reference levels, or instructions on determining reference levels. Optionally reference levels are determined using the sequencing and / or detection kits. The kits may also include instructions for practicing any method described herein.

[0272] (v) Methods of Treatment Following Immune Signature Detection. Within the current disclosure, human transplant recipients, for example, receiving a bone marrow or stem cell transplant, can monitored for the development of an immune signature described herein. Monitoring can include obtaining blood samples from the human transplant recipients at an interval following transplant. For example, the interval could be daily, weekly, bi-weekly, or monthly.F053-0195PCT / 25-085-WO-PCTBlood samples obtained from human transplant recipients can be processed and analyzed for an immune signature.

[0273] Upon detection of an immune signature associated with IL-17-related cGVHD, the human transplant recipient can be administered an anti-IL-17 therapeutic, in certain instances before clinical symptoms of cGVHD occur. Examples of anti-IL-17 therapeutics include include Bimekizumab, Secukinumab, Ixekizumab, Brodalumab, Iguratimod, Chlorpromazine, eBio64DEC17 (Thermo Fisher), MAB3171-100 (R&D systems), 66148-1-lg (Proteintech), 210-501-B32 (Rockland), 12047-M237 (Sino Biological-IL-17A), sc-374218 (SantaCruz Biotechnology), FT17-2F3 (Ampersand Biosciences), izokibep, and LY3509754.

[0274] Liu et al. describe a linear peptide configured to inhibit IL-17 (Sci. Rep. 2016;6: 26071). Abdelraheem et al. describe a macrocyclic compound configured to inhibit IL-17 activity (ACS Med. Chem. Lett. 2022; 13( 9): 1468-71). Hwang et al. describe a K562 protein-derived peptide that exerts inhibitory effects against IL-17 (J. Microbiol. Biotechnol. 2020;30(12): 1810-1818). Various small molecule IL-17 inhibitors are described by Ramos, et al. J. Med. Chem. 2024;67(8): 6456-6494.

[0275] IL-17 inhibitors include ROCK2 inhibitors, such as belumosudil (KD025), Zelasudil (RXC007), Y-27632, H-1152, PF-04929113 (Ripasudil), AT13148, GSK269962A, BMS-247550, K-115, Ripasudil, Fasudil, GV101, GNS-3595, SR3677, H-1152 dihydrochloride (2HCI), Azaindole 1 (TC-S 7001), GSK269962A HCI, RKI-1447, GSK429286A, Y-276322HCI, Y-27632, and Hydroxyfasudil (HA-1100) HCI. Zhang et al. describe a compound with selective inhibition of ROCK2 over ROCK1 (Biochem. Biophys. Res. Commun. 2024;699).

[0276] Additional examples of IL-17 inhibitors can include JAK inhibitors (e.g., Cibinqo, Rinvoq, Jyseleca, baricitinib (Olumiant), tofacitinib (Xeljanz), and upadacitinib, filgotinib, fedratinib, tofacitinib, peficitinib, NVP-BBT594, NVP-CHZ868, deucravacitinib, LS104, ON044580, Ritlecitinib, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclcitinib, peficitinib, pacritinib, ruxolitinib, tofacitinib, and upadacitinib); IL-23 inhibitors (e.g., Ustekinumab, Briakinumab, Guselkumab, Tildrakizumab, and Risankizumab); IL-6 inhibitors (e.g., Tocilizumab, Sarilumab, and Siltuximab); IL-1 inhibitors (e.g., Anakinra, Rilonacept, Canakinumab, Dapansutrile, Gevokizumab, Diacerein, Inzomelid, and MCC950); inhibitors of the ERK / MAPK pathway (e.g., LTT462, VX-11e, PD325901, Serum thymic factor acetate, Temuterkib (LY3214996), PD98059, SCH772984, Tauroursodeoxycholate, Ulixertinib (BVD-523; VRT752271), RMC-7977, Tauroursodeoxycholate, Piperlongumine, Ravoxertinib (GDC-0994), Patritumab, RMC-6291, Lidocaine, Gamma-linolenic acid, FR 180204, HH2710, PD98059, PD184352, U0126, and PD0325901); TBK1 inhibitors, such as GSK8612, and BX795); and STAT3 inhibitors (e.g., STX-0119, Stattic, S3I-201, WP1066, HJC0123, Sorafenib, and TTI-101)

[0277] Upon detection of an immune signature associated with CSF-1 -related cGVHD, the human transplant recipient can be administered an anti- CSF-1 therapeutic, in certain instances before clinical symptoms of cGVHD occur. Examples of anti- CSF-1 therapeutics include Axatilimab, Pexidartinib (PLX3397, PLX108-01), Edicotinib (JNJ-40346527), Sotuletinib (BLZ945), Emactuzumab (RG7155), Cabiralizumab (FPA008), AMG820, IMC-CS4 (LY3022855), Chiauranib (CS2164), Dovitinib, Sorafenib, Dasatinib, PLX7486, ARRY-382, AMG820, IMC-CS4,F053-0195PCT / 25-085-WO-PCTBPR1R024, PD173074, PLX5622, GW2580, RG7155, BLZ945, DCC3014, 25949-1-AP (Proteintech), mAb #67455(Cell Signaling Technology), MCS110, PD-0360324, AF416 (R&D systems), #3152 (Cell Signaling Technology), CF806567 (ThermoFisher), (-)-kusunokinin, BPR1K871, SNDX6532, Vatalanib, Dovitinib, and OSI-930.

[0278] Additional examples of CSF-1 inhibitors can include c-Kit inhibitors (e.g., Imatinib, Pazopanib, Sunitinib, Regorafenib, Midostaurin, Ripretinib, and Avapritinib); IL-34 inhibitors (e.g., AMG820, ARRY382, FPA008, and IMCCS4; and transforming growth factor 1 (TGF 1) inhibitors (e.g., Vactosertib (TEW-7197), Bintrafusp alfa (M7824), Luspatercept, Trabedersen (AP 12009), Fresolimumab (GC1008), Galunisertib (LY2157299), AVID200, ABBV-151, and SRK-181

[0279] Upon detection of an immune signature associated with IL-17-related cGVHD and an immune signature associated with CSF-1 -related cGVHD, the human transplant recipient can be administered an anti-IL-17 therapeutic and an anti- CSF-1 therapeutic.

[0280] In some situations, a human transplant recipient may develop clinical symptoms of cGVHD and an IL-17 or CSF-1 -related immune signature has not been detected. In this clinical scenario, the human transplant recipient’s therapy should begin with an anti-cGVHD therapeutic that is not an anti-1 L-17 therapeutic or an anti-CSF-1 therapeutic, for example the FDA-approved agents ruxolitinib or ibrutinib.

[0281] The actual dose amount administered to a particular human transplant recipient can be determined by a treating physician taking into account parameters such as physical and physiological factors including target, body weight, severity of cGVHD, days since transplant, previous or concurrent therapeutic interventions, idiopathy of the human transplant recipient and route of administration.

[0282] Useful doses of therapeutics can range from, for example, 0.1 to 5 pig / kg or from 0.5 to 1 pig / kg. In other examples, a dose can include 1 pig / kg, 15 pig / kg, 30 pig / kg, 50 pig / kg, 55 pig / kg, 70 pjg / kg, 90 pig / kg, 150 pig / kg, 350 pig / kg, 500 pig / kg, 750 pig / kg, 1000 pig / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0283] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0284] The compositions and formulations described herein can be administered by, for example, injection or infusion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intraperitoneal, intraparenteral, intramuscular, subcutaneous, and / or sublingual administration.

[0285] Therapeutics described herein can be administered on top of a current standard of care for cGVHD transplant recipients, or in combination or alternation with any other compound or therapy that a healthcare provider deemsF053-0195PCT / 25-085-WO-PCTbeneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative. In particular embodiments, the therapeutics described herein can be administered with a secondary treatment. In particular embodiments, the therapeutics described herein can be administered at the same time or at a different time as a secondary treatment.

[0286] The Exemplary Embodiments and Examples below are included to demonstrate particular, non-limiting embodiments of the disclosure. Those of ordinary skill in the art will recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0287] (vi) Exemplary Embodiments.1. An IL-17 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1 , NFKBID, NFKBIZ, ODC1, SDC4. S0CS3, SQSTM1, TLR2, and TNFSF9.2. The IL-17 cGVHD immune signature of embodiment 1, including, the immune signature including differential expression of CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.3. A CSF-1 cGVHD immune signature expressed by classical monocytes detected from a human transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, PGS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.4. The CSF-1 cGVHD immune signature of embodiment 3, including, the immune signature including differential expression of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.5. A CSF-1 cGVHD immune signature expressed by nonclassical monocytes detected from a human transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1 , 2, 3, 4, or 5 of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, D0K2, DUSP2, EGR1, EN03, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, P0T1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.6. The CSF-1 cGVHD immune signature of embodiment 5, including differential expression of ABCG1, ABHD12,F053-0195PCT / 25-085-WO-PCTABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, D0CK5, D0K2, DUSP2, EGR1, EN03, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, P0T1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, S0CS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.7. An IL-17 cGVHD immune signature expressed by LyC6lo monocytes from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl , Mapk6, Marcksll, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl , Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1C0a8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, TnfaipS, Tnfsfp, Wfdc17, and Zfp91.8. The IL-17 cGVHD immune signature of embodiment 7, including differential expression of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, Marcksll, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1C0a4, S1C0a6, S1COa8, S1C0a9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.9. An IL-17 cGVHD immune signature expressed by LyCSIo from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl , Themis2, and Tsc22d3.10. The IL-17 cGVHD immune signature embodiment 9, including differential expression of at least 1, 2, 3, 4, or 5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3. 11. An IL-17 cGVHD immune signature expressed by Early monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of Lyz2.12. An IL-17 cGVHD immune signature expressed by Circulating 1 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, or 4 of Ier3, Maltl, Mfkbiz, and Tnf.13. The IL-17 cGVHD immune signature of embodiment 12, including differential expression of Ier3, Maltl, Mfkbiz, and Tnf.14. An IL-17 cGVHD immune signature expressed by Circulating 1 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1 , 2, 3, 4, or 5 of Gm4951 , Igtp, I igp 1 , Klf2, and Rsrpl15. The IL-17 cGVHD immune signature of embodiment 14, including differential expression of Gm4951, Igtp, ligpl, Klf2, and Rsrpl.F053-0195PCT / 25-085-WO-PCT16. An IL-17 cGVHD immune signature expressed by Circulating 2 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Bhlhe40, Btg1, Clec4e, Maltl , and Tnf.17. The IL-17 cGVHD immune signature of embodiment 16, including differential expression of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.18. An IL-17 cGVHD immune signature expressed by Circulating 2 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, or 4 of Fos, Ifitm3, Klf2, and Tsc22d3.19. The IL-17 cGVHD immune signature of embodiment 18, including differential expression of Fos, Ifitm3, Klf2, and Tsc22d3.20. An IL-17 cGVHD immune signature expressed by G4 neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of Cd6.21. An IL-17 cGVHD immune signature expressed by G5a neutrophils from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or5 of Cxcl6, Egr1 , Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.22. The IL-17 cGVHD immune signature of embodiment 21, including differential expression of Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.23. An IL-17 cGVHD immune signature expressed by G5a neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, or 4 of Csf1, Fgl2, Vim, and Xpc.24 The IL-17 cGVHD immune signature of embodiment 23, including differential expression of Csf1, Fgl2, Vim, and Xpc.25. An IL-17 cGVHD immune signature expressed by G5b neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.26. The IL-17 cGVHD immune signature of embodiment 25, including differential expression of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.27. An IL-17 cGVHD immune signature expressed by G5b neutrophils from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1 or 2 of Ifitm2 and Vim.28. The IL-17 cGVHD immune signature of embodiment 27, including differential expression of Ifitm2 and Vim. 29. An IL-17 cGVHD immune signature expressed by G5c neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1 , 2, 3, 4, or 5 of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.30. The IL-17 cGVHD immune signature of embodiment 29, including differential expression of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.F053-0195PCT / 25-085-WO-PCT31. An IL-17 cGVHD immune signature expressed by G5c neutrophils from a transplant recipient's peripheral blood sample, the immune signature including differential expression of Fgl2.32. A CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1 or 2 of Ccl3 and F13a1.33. The CSF-1 cGVHD immune of embodiment 32, including differential expression of Ccl3 and F13a1.34. A CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Wfdcl 7, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.35. The CSF-1 cGVHD immune signature of embodiment 34, including differential expression of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.36. A CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.37 The CSF-1 cGVHD immune signature of embodiment 36, including differential expression of H2-Ab1 , H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.38. A CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.39. The CSF-1 cGVHD immune signature of embodiment 38, including differential expression of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.40. A CSF-1 cGVHD immune signature expressed by G4 neutrophils from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdcl 7, and Wfdc21.41. The CSF-1 cGVHD immune signature of embodiment 40, including differential expression of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.42 A CSF-1 cGVHD immune signature expressed by G5a neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.43. The CSF-1 cGVHD immune signature of embodiment 42, including differential expression of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.44. A CSF-1 cGVHD immune signature expressed by G5a neutrophils from a transplant recipient’s peripheral blood sample, the immune signature including differential expression of Tsc22d3.45. A CSF-1 cGVHD immune signature expressed by G5b neutrophils from a transplant recipient's peripheral blood sample, the immune signature including differential expression of at least 1 or 2 of Cxcl2 and Nfkbia.F053-0195PCT / 25-085-WO-PCT46. The CSF-1 cGVHD immune signature of embodiment 45, including differential expression of Cxcl2 and Nfkbia.47 A method including detecting an immune signature of any of embodiments 1-46 from a sample of a transplant recipient's peripheral blood, wherein the immune signature is indicative of IL-17 or CSF-1 related cGVHD.48. The method of embodiment 47, wherein the transplant recipient is a human transplant recipient.49. The method of embodiment 47 or 48, wherein the method further includes treating the human transplant recipient for cGVHD before symptoms emerge.50. The method of embodiment 49, wherein the treating is for anti-IL-17 cGVHD.51 The method of embodiment 50, wherein the treating includes administering belumosudil.52. The method of embodiment 49, wherein the treating is for anti-CSF-1 cGVHD.53. The method of embodiment 52, wherein the treating includes administering Axatilimab.54. A method of determining that a human transplant recipient has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the method includingObtaining monocytes from a peripheral blood sample obtained from the human transplant recipient;Processing the monocytes to detect an expression level ofat least one or all of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes;at least one or all of APOE, CCL4, CCRL2, CD 14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, PCS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 by classical monocytes; and / orat least one or all of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by non-classical monocytes;wherein differential expression of at least one or all of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop IL-17 related cGVHD;wherein differential expression of at least one or all of at least one of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, PCS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 by classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop CSF-1 related cGVHD; and / orF053-0195PCT / 25-085-WO-PCTwherein differential expression of at least one or all of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CEB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1 , POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, S0CS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by non-classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop CSF-1 related cGVHD.55. The method of embodiment 54, wherein the processing the monocytes to detect an expression level includes bulk monocyte processing.56. The method of embodiment 54, wherein the processing the monocytes to detect an expression level includes single cell monocyte processing.57 The method of any of embodiments 54-56, wherein the processing the monocytes to detect an expression level includes RNA sequencing.58. The method of embodiment 57, wherein the RNA sequencing includes bulk RNA sequencing.59. The method of embodiment 57, wherein the RNA sequencing includes single cell RNA sequencing.60. The method of any of embodiments 54-59, including detecting differential expression of at least 1, 2, 3, 4, or 5 of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by nonclassical monocytes from a transplant recipient's peripheral blood.61. The method of any of embodiments 54-60, including detecting differential expression of CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.62. The method of any of embodiments 54-61, including detecting differential expression of at least 1, 2, 3, 4, or 5 of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, PGS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 A CSF-1 cGVHD by classical monocytes from a transplant recipient's peripheral blood.63. The method of any of embodiments 54-62, including detecting differential expression of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 by classical monocytes from a transplant recipient’s peripheral blood.64. The method of any of embodiments 54-63, including detecting differential expression of at least 1, 2, 3, 4, or 5 of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2,F053-0195PCT / 25-085-WO-PCTITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, P0T1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, S0CS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by nonclassical monocytes from a transplant recipient’s peripheral blood.65. The method of any of embodiments 54-64, including detecting differential expression of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.66 The method of any of embodiments 54-65, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Bcl2l11 , Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl , Mapk6, MarcksH, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl , Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1C0a4, S1C0a6, S1C0a8, S1C0a9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdcl 7, and Zfp91 by LyC6lo monocytes from a transplant recipient’s peripheral blood.67. The method of any of embodiments 54-66, including detecting differential expression of Acodl, Bcl2l11 , Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1 , Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, MarcksH, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1C0a6, S1C0a8, S1C0a9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.68. The method of any of embodiments 54-67, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3 by LyC6lo monocytes from a transplant recipient’s peripheral blood.69 The method of any of embodiments 54-68, including detecting differential expression of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.70. The method of any of embodiments 54-69, including detecting differential expression of Lyz2 by Early monocytes from a transplant recipient’s peripheral blood.71. The method of any of embodiments 54-70, including detecting differential expression of at least 1, 2, 3, or 4 of Ier3, Maltl, Mfkbiz, and Tnf by Circulating 1 monocytes from a transplant recipient’s peripheral blood.72 The method of any of embodiments 54-71, including detecting differential expression of Ier3, Maltl, Mfkbiz, and Tnf.73. The method of any of embodiments 54-72, including detecting differential expression of at least 1, 2, 3, 4, or 5 ofF053-0195PCT / 25-085-WO-PCTGm4951, Igtp, ligpl, Klf2, and Rsrpl by Circulating 1 monocytes from a transplant recipient's peripheral blood. 74 The method of any of embodiments 54-73, including detecting differential expression of Gm4951 , Igtp, ligpl , Klf2, and Rsrpl.75. The method of any of embodiments 54-74, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf by Circulating 2 monocytes from a transplant recipient’s peripheral blood. 76. The method of any of embodiments 54-75, including detecting differential expression of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.77 The method of any of embodiments 54-76, including detecting differential expression of at least 1, 2, 3, or 4 of Fos, Ifitm3, Klf2, and Tsc22d3 by Circulating 2 monocytes from a transplant recipient's peripheral blood.78. The method of any of embodiments 54-77, including detecting differential expression of Fos, Ifitm3, Klf2, and Tsc22d3.79. The method of any of embodiments 54-78, including detecting differential expression of Cd6 by G4 neutrophils from a transplant recipient’s peripheral blood.80 The method of any of embodiments 54-79, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip by G5a neutrophils from a transplant recipient's peripheral blood.81. The method of any of embodiments 54-80, including detecting differential expression of Cxcl6, Egr1 , Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.82. The method of any of embodiments 54-81, including detecting differential expression of at least 1, 2, 3, or 4 of Csf1, Fgl2, Vim, and Xpc by G5a neutrophils from a transplant recipient's peripheral blood.83. The method of any of embodiments 54-82, including detecting differential expression of Csf1, Fgl2, Vim, and Xpc.84. The method of any of embodiments 54-83, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36 by G5b neutrophils from a transplant recipient’s peripheral blood.85. The method of embodiment 84, including detecting differential expression of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.86 The method of any of embodiments 54-85, including detecting differential expression of at least 1 or 2 of Ifitm2 and Vim by G5b neutrophils from a transplant recipient's peripheral blood.87. The method of any of embodiments 54-86, including detecting differential expression of Ifitm2 and Vim.88. The method of any of embodiments 54-87, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, and Traf by G5c neutrophils from a transplant recipient’s peripheral blood.89 The method of any of embodiments 54-88, including detecting differential expression of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf. 90. The method of any of embodiments 54-89, including detecting differential expression of Fgl2 by G5c neutrophilsF053-0195PCT / 25-085-WO-PCTfrom a transplant recipient’s peripheral blood.91 The method of any of embodiments 54-90, including detecting differential expression of at least 1 or 2 of Cel 3 and F13a1 by Circulating 1 monocytes from a transplant recipient's peripheral blood.92. The method of any of embodiments 54-91, including detecting differential expression of Ccl3 and F13a1.93. The method of any of embodiments 54-92, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21 by Circulating 1 monocytes from a transplant recipient’s peripheral blood.94 The method of any of embodiments 54-93, including detecting differential expression of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.95. The method of any of embodiments 54-94, including detecting differential expression of at least 1, 2, 3, 4, or 5 of H2-Ab1, H2-Eb1, H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1 by Circulating 3 monocytes from a transplant recipient’s peripheral blood.96. The method of any of embodiments 54-95, including detecting differential expression of H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth197. The method of any of embodiments 54-96, including detecting differential expression of at least 1, 2, 3, 4, or 5 of TTmem71, PI16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21 by Circulating 3 monocytes from a transplant recipient’s peripheral blood.98. The method of any of embodiments 54-97, including detecting differential expression of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.99 The method of any of embodiments 54-98, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21 by G4 neutrophils from a transplant recipient's peripheral blood.100. The method of any of embodiments 54-99, including detecting differential expression of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.101. The method of any of embodiments 54-100, including detecting differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17 by G5a neutrophils from a transplant recipient's peripheral blood.102. The method of any of embodiments 54-101, including detecting differential expression of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.103.The method of any of embodiments 54-102, including detecting differential expression of Tsc22d3 by G5a neutrophils from a transplant recipient’s peripheral blood.104.The method of any of embodiments 54-103, including detecting differential expression of at least 1 or 2 of Cxcl2 and Nfkbia by G5b neutrophils from a transplant recipient’s peripheral blood.105.The method of any of embodiments 54-104, including detecting differential expression of Cxcl2 and Nfkbia.F053-0195PCT / 25-085-WO-PCT106. The method of any of embodiments 54-105, wherein the transplant recipient is a human transplant recipient. 107. The method of any of embodiments 54-106, including initiating a treatment for cGVHD based on the detected differential expression before symptoms emerge.108.The method of embodiment 107, wherein the treatment is for anti-IL-17 cGVHD.109. The method of embodiment 108, wherein the treatment includes belumosudil.110. The method of embodiment 107, wherein the treatment is for anti-CSF-1 cGVHD.111. The method of embodiment 110, wherein the treatment includes Axatilimab.112. A kit for determining that a human transplant recipient has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the kit includingat least one RNA annealing primer that binds CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, or TNFSF9;at least one RNA annealing primer that binds APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, or TLR2;at least one RNA annealing primer that binds ABCG1 , ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, or TREML4;at least one RNA annealing primer that binds Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl , Mapk6, MarcksH, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl , Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdcl 7, or Zfp91;at least one RNA annealing primer that binds Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, or Tsc22d3;at least one RNA annealing primer that binds Lyz2;at least one RNA annealing primer that binds Ier3, Maltl, Mfkbiz, or Tnf;at least one RNA annealing primer that binds Gm4951, Igtp, ligpl, Klf2, or Rsrpl;at least one RNA annealing primer that binds Bhlhe40, Btg1, Clec4e, Maltl, or Tnf;at least one RNA annealing primer that binds Fos, Ifitm3, Klf2, or Tsc22d3;at least one RNA annealing primer that binds Cd6;at least one RNA annealing primer that binds Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, orTxnip;F053-0195PCT / 25-085-WO-PCTat least one RNA annealing primer that binds Csf 1 , Fgl2, Vim, or Xpc;at least one RNA annealing primer that binds Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, or Zfp36;at least one RNA annealing primer that binds Ifitm2 or Vim;at least one RNA annealing primer that binds Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, or Traf;at least one RNA annealing primer that binds Fgl2;at least one RNA annealing primer that binds Ccl3 or F13a1;at least one RNA annealing primer that binds Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, or Wfdc21;at least one RNA annealing primer that binds Klf2, Rsrpl, Tsc22d3, Ifitml, or Wfdc21;at least one RNA annealing primer that binds H2-Ab1, H2-Eb1, H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1 , or Fth1 ;at least one RNA annealing primer that binds TTmem71, Pi16, Bachl , Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, or Wfdc21;at least one RNA annealing primer that binds Cstdc5, Cxcl2, 111 b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, or Wfdc21;at least one RNA annealing primer that binds Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1 , Steap4, Tnf, Tnfaip3, or Wfdc17;at least one RNA annealing primer that binds Tsc22d3; and / orat least one RNA annealing primer that binds Cxcl2 or Nfkbia.113. The kit of embodiment 112, including RNA annealing primers that bind CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.114.The kit of embodiment 112 or 113, including RNA annealing primers that bind APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.115. The kit of any of embodiments 112-114, including RNA annealing primers that bind ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.116. The kit of any of embodiments 112-115, including RNA annealing primersthat bind Acodl, Bcl2l11, Bhlhe40, Ccl2,F053-0195PCT / 25-085-WO-PCTCdkml a, Clec4e, Crrnpl , Cxcll, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, MarcksH, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.117. The kit of any of embodiments 112-116, including RNA annealing primers that bind Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.118.The kit of any of embodiments 112-117, including RNA annealing primers that bind Lyz2.119. The kit of any of embodiments 112-118, including RNA annealing primers that bind Ier3, Maltl, Mfkbiz, and Tnf.120. The kit of any of embodiments 112-119, including RNA annealing primers that bind Gm4951, Igtp, ligpl, Klf2, and Rsrpl .121. The kit of any of embodiments 112-120, including RNA annealing primers that bind Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.122. The kit of any of embodiments 112-121, including RNA annealing primers that bind Fos, Ifitm3, Klf2, and Tsc22d3.123.The kit of any of embodiments 112-122, including RNA annealing primers that bind Cd6.124.The kit of any of embodiments 112-123, including RNA annealing primers that bind Cxcl6, Egr1 , Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.125.The kit of any of embodiments 112-124, including RNA annealing primers that bind Csf1, Fgl2, Vim, and Xpc. 126. The kit of any of embodiments 112-125, including RNA annealing primers that bind Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.127. The kit of any of embodiments 112-126, including RNA annealing primers that bind Ifitm2 and Vim.128.The kit of any of embodiments 112-127, including RNA annealing primers that bind Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.129. The kit of any of embodiments 112-128, including RNA annealing primers that bind Fgl2.130. The kit of any of embodiments 112-129, including RNA annealing primers that bind Ccl3 and F13a1.131. The kit of any of embodiments 112-130, including RNA annealing primers that bind Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.132. The kit of any of embodiments 112-131, including RNA annealing primers that bind H2-Ab1, H2-Eb1, H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.133. The kit of any of embodiments 112-132, including RNA annealing primers that bind TT mem71 , Pi 16, Bach 1 , Rsrpl , Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.134.The kit of any of embodiments 112-133, including RNA annealing primers that bind Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.135.The kit of any of embodiments 112-134, including RNA annealing primers that bind Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1 , Steap4, Tnf, Tnfaip3, and Wfdc17.136. The kit of any of embodiments 112-135, including RNA annealing primers that bind Tsc22d3.F053-0195PCT / 25-085-WO-PCT137. The kit of any of embodiments 112-136, including RNA annealing primers that bind Cxcl2 and Nfkbia.138.The kit of any of embodiments 112-137, wherein the RNA-annealing primer is a polyT primer or a gene-specific primer.139. The kit of any of embodiments 112-138, wherein the kit further includes a DNA polymerase, a reverse transcriptase, a transcription buffer, a reverse transcription buffer, a ligase, an adapter, and / or a detection molecule.140. A method of treating a human transplant recipient that has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the method including:obtaining a report indicating that the human transplant recipient has an immune signature associated with IL-17 and / or CSF-1 related (cGVHD); andadministering an anti-IL-17 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with IL-17 related cGVHD;administering an anti-CSF-1 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with CSF-1 related cGVHD; or administering an anti-IL-17 cGVHD therapeutic and an anti-CSF-1 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with IL-17 related cGVHD and an immune signature associated with CSF-1 related cGVHD.141. The method of embodiment 140, wherein the anti-IL-17 cGVHD therapeutic includes belumosudil.142. The method of embodiment 140 or 141, wherein the anti-CSF-1 cGVHD therapeutic includes Axatilimab.

[0288] (vii) Experimental Example. Defining pathogenic IL-17 and CSF-1 gene expression signatures in chronic graft-versus-host disease

[0289] Methods. Female B6 (H-2b) mice were purchased from JAX laboratories. B6D2F1 (H-2b / d) mice were bred at the Fred Hutchinson Cancer Center (FHCC). IL-17A'AB6 mice were supplied by the University of Tokyo. 11-17RC+B6 mice were supplied by Amgen Inc. (Thousand Oaks, CA). (Varelias et al. (2017) Blood). All B6 mince were backcrossed at least 10 generations. Mice were transplanted using sclerodermatous GVHD models. (Hill et al. (2010) Blood 116(5): 819-28; Alexander et al. (2014) J Clin Invest. 124(10): 4266-80). On day -1, recipient B6D2F1 (H-2b / d) mice received 1100 cGy total body irradiation, split into two doses separated by three hours. In the IL-17 investigations, B6 (H-2b) recipients received wild-type (WT) T-cell depleted bone marrow (TOD BM) alone or BM and purified splenic T-cells, both from WT, IL-17A'-, or IL-17RC '- donors (Hill et al. (2010) Blood 116(5): 819-28; Alexander et al. (2014) J Clin Invest. 124(10): 4266-80). In the CSF-1R investigation, B6 mice were transplanted with TCD BM alone or BM and purified splenic T-cells from WT BG mice. Mice received M279 rat IgG' (Amgen) or control antibody (400 pig three times per week from day 7) (MacDonald et al. (2017) Blood 129(1): 13-21). The same four mice per group per experiment were bled at days 14, 21, and 28 after transplant, with blood processing. Three B6 mice were injected subcutaneously with 10 pig G-CSF daily for six days. A control group of three B6 mice were given PBS. On day 7, mice were bled and analyzed by scRNAseq analysis.F053-0195PCT / 25-085-WO-PCT

[0290] Mouse peripheral blood preparation and single cell captures. Erythrocyte lysis was performed using Gey’s lysis buffer. Cells were treated with 2.4G2 Fc block. For transplantation experiments and naive animal profiling, cells were labeled with TotalSeq™-B hashtag antibodies for downstream sample identification and TotalSeq™-B CITE-seq antibodies for cell lineage identification (CD4, CD8, CD25, CD90.2, l-A / l-E, CD11 b, CD11c, F4 / 80, CCR2, Ly6C, Ly6G, and B220; BioLegend). For the G-CSF mobilization experiment, cells were labeled with TotalSeq™-C hashtag antibodies for downstream sample identification and TotalSeq™-C Ly6C CITE-seq antibody for monocyte identification (BioLegend). Cells were labeled with anti-CD45.2-PE and anti-TER-119-FITC antibodies (BioLegend), and live (7-AADne9) CD45.2+TER-119- leukocytes were FACS-sorted to avoid erythrocyte fragment contamination in downstream processing. Sorted cells were captured used a 3' single index platform (mouse transplant and naive animal profiling; 10X Genomics, Pleasanton, CA) or 5' GEM-X platform (G-CSF experiment; 10X Genomics, Pleasanton, CA), and cDNA production and library construction performed per the manufacturer's instructions. Libraries were sequenced on a NovaSeq 6000 or NextSeq 2000 targeting 20,000 raw reads / cell for gene expression libraries and 5,00 raw reads / cell for feature barcode libraries. Cell Ranger was used to generate gene expression matrices and hashtag and CITE-seq antibody count matrices. Seurat was used to calculate quality control metrics, filter, and normalize cells for analysis, with dying, doublet, or contaminating cell type fragments excluded from analysis. Gene expression profiles were visualized in a low dimensional embedding using Seurat’s implementation of the UMAP algorithm. Differential gene expression was calculated using Seurat’s default non-parametric Wilcoxon rank-sum test. Gene set scores were calculated using Seurat. Additional estimates of RNA velocity were calculated using the command line velocyto tool and visualized using scVelo.

[0291] Human PBMC fixed RNA profiling and analysis. HCT patient's and healthy control donors' PBMCs were previously obtained on FHCC Institutional Review Board protocols and analyzed via 10X Genomics fixed RNA profiling platforms. A refined IL-17 GVHD signature from the mouse Ly6Cl0monocyte DEGs and CSF-1 signatures from the mouse Ly6ChlCirculating 2 and Ly6Cl0monocyte clusters were applied, setting inclusion thresholds based on semiequivalence of the top expression quartiles among reference groups of patients +100 days post-HCT who did or did not subsequently develop cGVHD. Single cell capture of unselected PBMCs was performed using either singleplex or multiplex hybridized probe approaches (both with and without Total Seq™-C hashtag antibodies for further multiplexing, BioLegend), and downstream library construction was performed per the manufacturer's instructions. Gene expression libraries were sequenced on a NextSeq 2000 to a depth of 10,000 reads / cell. Cell Ranger was used to generate gene expression matrices and Seurat was used for quality control filtering and gene expression profiling as above in the mouse. An IL-17 GVHD signature from the mouse Ly6C° monocytes was queried by starting with the DEGs (multiple comparisons-adjusted p-value <0.05 both between WT GVHD vs. IL-17RC7’ and WT GVHD vs. non-GVHD TCD groups). Likewise, a CSF-1 signature was applied using the cluster marker genes from the mouse Circulating 2 cluster having avg_logFC>1 and adjusted p-value <0.05 and expressed in at least 30% of this mouse cluster's cells. Mouse genes were converted to human gene orthologs using babelgene and manually verified. For each starting list, aF053-0195PCT / 25-085-WO-PCTmonocyte-based signature was refined among monocyte populations from recipient PBMCs collected at day +100 post-HCT by selecting the genes for which the top quartile expression threshold was at least the top quartile expression threshold of recipients who did not develop cGVHD through at least 18 months post-HCT (GraphPad Prism 10.2.3 and RStudio 2023.12.1). Gene expression values were normalized to each gene’s maximal expression value in the referent day +100 non-cGVHD group (arbitrarily set to 10). Each gene’snormalized value was equally weighted into a composite immune signature expression score. A positive threshold was defined as the lesser of the D+100 non-cGVHD’s group’s mean + 2 standard deviation or the maximal non-cGVHD value.

[0292] Results. scRNAseq of murine peripheral blood after transplantation. Preclinical models to define immune dysregulation and identify clinically effective therapeutics were used to define specific complex immune signatures in the blood of transplant recipients. By defining the specific complex immune signatures, the identification of specific pathways of alloimmunity before or at cGVHD aids in the selection of an appropriate therapy. Utilizing a well-described preclinical model of cGVHD whereby IL-17- and CSF-1R-dependent scleroderma develops at 3-4 weeks after HCT, B6D2f1 mice were lethally irradiated and transplanted with T-cell-depleted (TCD) BM with (GVHD) or without (non-GVHD control) purified T-cells from B6 wild type (B6.WT), B6. IL=17RC / , or B6. IL-17AAmice as described in Methods. scRNAseq was performed on erythrocyte-lysed blood collected longitudinally at 14, 21, and 28 days after HCT from the same transplant recipients (FIG. 1A). Dimensionality reduction of single cell gene expression profiles with canonical surface proteins (CITE-seq) and gene expression identified major peripheral blood immune cell types (FIGs. 1B, 1C). Profound B cell lymphopenia was noted in mice with GVHD (WT GVHD vs. non-GVHD control p = 0.0235 at Day +28), without major quantitative difference between the GVHD groups (FIG. 1D).

[0293] Peripheral blood T-cell subsets are not quantitatively altered by absence of IL-17 signaling. Transcriptional analysis of the peripheral donor T-cells was done. These profiles could identify naive CD4 and CD8 T-cells (CD44ne9CD62L+CCR7+), CD25+Foxp3+Tregs (including CD62L+and CD62L subsets), together with two effector memory (Tern) subsets characterized as CD44+CD62L CCR7n°9 including one population also expressing high levels of exhaustion-related genes Tox, Pdcdl, Tigit, Lag3, a T-cell central memory population (Tern) characterized as CD44+CD62L+CCR7+, and a population including both CD4+and CD8+cells with high expression of cell cycle / proliferation genes (Cycling T-cells) (FIGs. 2A, 2B). Major quantitative difference in most subsets was not seen between the GVHD groups compared by post-HCT timepoint (FIG. 2C), although compared to the TCD group there was increased CD8+ effector memory T cells (FIG. 2D).

[0294] GVHD-related IL-17 signaling modifies peripheral blood monocyte differentiation. Because chronic GVHD in these systems was characterized by dysregulated monocyte differentiation, the temporal changes in myeloid cells during disease development was a focus. Unsupervised clustering revealed seven distinct circulating monocyte populations, with putative differentiation trajectory assigned by RNA vector analysis, plus one macrophage and one dendritic cell cluster (FIG. 3A). These included a proliferative initiating population (Proliferating), characterized by high expression of cell cycle-related genes (FIG. 4A), and its major descendent (Early), both of which were someone moreF053-0195PCT / 25-085-WO-PCTabundant by timepoint in the GVHD groups compared to non-GVHD TOD controls (FIGs. 3B, 4D-4G). This gave rise to three major intermediate Ly6Chipopulations (Circulating 1, Circulating 2, Circulating 3). Notably, Circulating 1, which was most abundant in IL-17RC7' and IL-17A7’ graft recipients, was characterized by relatively higher expression of “resting” genes Klf2 and Ly6a, compared to Circulating 2 (comparatively more abundant in WT and TCD recipients) was enriched for monocypte / macrophage activation-associated genes including NF-KB pathway genes, Tlr2, Cxcl2, Tnf, and Clec4e- genes which were also differentially expressed in tissue resident macrophage precursors (described below) between WT vs. IL-17RC'- recipients (FIG. 4C). An additional Ly6Chlcluster was characterized by the expression of S100a8, Wfdc21, Lcn2, Chil3, and Mmp8 (FIG. 4A) and has been described as “granulocyte-like” elsewhere (Yanez et al. (2017) Immunity 47(5): 890-902). This cluster was expanded in some IL-17RC recipients at latter time points. In absence of GVHD, there was relative enrichment of the Circulating 3 cluster which shared many gene expression similarities with Circulating 2 and was also characterized by the unique expression of Stxbp6, Ccl2, and CD177. These cells also expressed Cx3cr1 and Csflr and were Ly6Cint / l° (FIG. 4A-4B).

[0295] The remaining Ly6Cl0monocyte cluster represented a putative tissue resident macrophage precursor population, associated with fibrogenic macrophage presence in GVHD target tissue (Alexander et al. (2014) J Clin Invest. 124(10): 4266-80). This cluster expressed the highest levels of Csflr and Cx3cr1 RNA and was Ly6Cl0by protein analysis (FIGs. 4B, 4C). Differential gene expression (aggregating all three timepoints for maximal quantitative power) was more penetrant in WT vs. IL-17RCArelative to WT vs. IL-17AA, reflecting loss of predominant IL-17 signaling in the receptor-deficient donor cells (FIG. 3C). These expression differences emerged at day +21 and persisted at day +28. Ly6Cl0clusters in WT recipient mice demonstrated high expression of activation markers (Tlr2, Cd69, Tnf, Acodl), chemotaxis-related (Ccl3, Cxcl2, Gadd45b), and NF-KB and MAPK-activating genes (FIG. 3C). Interestingly, Ly6Cl0cells from IL-17RC7’ recipients showed elevated expression of the MAPK pathway inhibitor Duspl, which has been associated with decreased monocyte / macrophage chemotaxis (Grimshaw et al. (2001) Eur J Immunol.31(2): 480-9) or M2 macrophage polarization (Ying et al. (2015) J Immunol. 194(3): 1239-51). Ly6Cl0cells from WT graft recipients showed increased expression of paracaspase / protease Maltl , which potentiates NF-KB signaling in lymphoid cells (Ruland et al. (2019) Nature review Immunol. 19(2): 118-134), though this activity has not previously been characterized in monocytes and / or GVHD. Maltl has also been implicated in signaling be damage-associated molecular pattern receptors including Mincle, encoded by Clec4e (Unterreiner et al. (2019) J Vis Exp. 143) which was also upregulated in recipients of WT-cells in comparison to IL-17RCA

[0296] Key transcription factors in this cluster associated with IL-17 signaling included Klf2, which is associated with a resting monocyte phenotype and maintenance of the Ly6Cl0phenotype (Kurotaki et al. (2017) International Immunol.29(3): 97-107), was enriched in Ly6Cl0monocytes of recipient of IL-17RC / - grafts. Conversely, transcription factors and regulators Bhlhe40, Id3, and Prdml, which have been implicated in monocyte-to-macrophage differentiation and / or maintenance of tissue macrophages (Kurotaki et al. (2017) International Immunol. 29(3): 97-107; Jarjour et al. (2019) Nat Immunol. 20(6): 687-700), were increased in WT compared to IL-17RC '- donors (FIGs. 3C, 3D). To generate geneF053-0195PCT / 25-085-WO-PCTexpression signatures of IL-17-dependent GVHD, a comparison between major monocyte clusters' DEGs from these WT vs. IL-17RC7- T-cell replete analyses to DEGs in the WT T-cell replete (GVHD) vs. TCD (non-GVHD) recipients was done (FIG. 3D). Ly6Cl0monocytes displayed the highest number of GVHD-related gens which were IL-17-associated, with 47 genes more highly expressed in the WT GVHD control and 14 genes with reduced expression in the WT GVHD control, measured as significant as <0.05 with adjustment for multiple comparisons. Previously identified IL-17-dependent genes, Bhlhe40, Maltl, and Ehd1 were increased in an IL-17 and GVHD-specific fashion, whereas Klf2 was reduced. This integrated into a composite IL-17 signature most penetrant in an I L- 17RC-oriented fashion (FIG.3E). A smaller number of IL-17-dependent GVHD genes were differentially expressed in the Ly6Chiclusters (FIG. 3D). These data suggest that IL-17 modulates GVHD-related peripheral blood monocyte development via changes in activation, chemotaxis, and differentiation. Furthermore, IL-17-dependent GVHD is characterized by differential gene expression primarily within the Ly6Cl0cluster, putatively prior to homing and egress into GVHD target tissue to become fibrogenic macrophages.

[0297] IL-17 controls neutrophil differentiation to modulate CSF-1 production during GVHD. Four clusters were assigned per published classifications for homeostatic bone marrow and peripheral blood neutrophils (FIG. 5A) (Xie et al. (2020) Nat Immunol. 21(9): 1119-3340), originating from the least mature G4, parallelly differentiating into intermediate G5a and G5b, then converging into terminal G5c (FIG. 6A). Over the experimental time course, recipients of IL-17RC '- grafts had reductions in the terminally differentiated G5c abundance compared to WT or TCD recipients (FIGs. 6B, 5B, 5C). In order to determine the likely cause of decreased terminally differentiated neutrophiles, gene sets used in prior homeostatic mouse neutrophil classification were interrogated. Compared to WT recipients, IL-17AAand IL=17RC / - recipients displayed lower maturation gene expression scores (as per neutrophil maturation gene set used by Xie et al. (Xie et al. (2020) Nat Immunol. 21(9): 1119-33)) in all for neutrophil clusters, suggesting that 11-17 signaling influences neutrophil differentiation, including the progression to G5c terminal differentiation (FIG. 6D). Neutrophils from IL-17 and IL-17RCArecipients did not show increased apoptosis or chemotaxis scores (respective GO terms likewise used by Xie - Positive regulation of apoptotic process (GC:0043065) and Chemotaxis (GC:0030593)), suggesting that the reduction in G5c differentiation does not arise from enhanced cell death or egress from peripheral circulation.

[0298] Interestingly, a highly specific marker gene of the G5c cluster in both our transplant experiment and the referenced homeostatic neutrophil dataset (Xie et al. (2020) Nat Immunol. 21 (9): 1119-33) was Csf 1 (FIG. 6C), which encodes CSF-1 and is a major mediator of cGVHD target tissue fibrosis by stimulating monocyte differentiation into tissue macrophages (Alexander et al. (2014) J Clin Invest. 124(10): 4266-80). Neutrophil Csf1 expression was markedly higher than that of other major blood cells in the data set (FIG. 5D), except for basophils; however, basophils were outnumbered 28-fold by neutrophils. Mature neutrophils thus represent a hitherto unappreciated source of CSF-1 in cGVHD and a therapeutic target. This G5c cluster also expressed the highest transcript levels of Csf3r encoding the G-CSF receptor. Since transplantation of G-CSF mobilized stem cells is known to be a major risk factor for cGVHD,F053-0195PCT / 25-085-WO-PCTwhich is itself highly CSF-1 dependent, the murine peripheral blood neutrophils were profiled after G-CSF mobilization. Cluster designations were applied from the Xie et al. dataset using Seurat's FindTransferAnchor function and confirmed localization of Csf1 and Csf3r to the terminally differentiated population (FIG. 7A-7B). G-CSF administration increased abundance of all granulocyte subsets, with an increased portion of less mature neutrophil forms in the blood (FIG. 7C). This was accompanied by increased numbers of a Csf1 -expressing subpopulations of more terminally differentiated neutrophils (termed G5c-1 and G5c-2) (FIG. 7C-7G).

[0299] Additional analysis was performed on IL-17-dependent GVHD effects by determining DEG overlap in neutrophil clusters from WT vs. IL-17RCZ- (FIG. 6D) and WT (GVHD) vs. TCD (non-GVHD) recipients. This demonstrated IL-17 dependent enhancement of Cxcl2 (promoting neutrophil extravasation / migration), G0sa2 (an inducer of cell cycle progression), inflammatory early response genes (Ier2, Junb, Fos), and Osginl (an oxidative stress response gene) during GVHD. Vim, which encodes from the intermediate filament vimentin, and Fgl2, which encodes fibrinogen-like protein 2, were the only DEGs consistently identified across multiple clusters as downregulated by IL-17 during GVHD. Interestingly, vimentin has recently been described as a PKC-theta target and inhibition of this pathway including vimentin, results in enhanced regulatory T-cell function (McDonald-Hyman et al. (2018) J Clin Invest.128(10): 4604-21). Vimentin is involved in NETosis and autoantigen generation (Khandpur et al. (2013) Science translational medicine 5(178)) and also binds P-selectin to block endothelial adhesion and transmigration (McDonald-Hyman et al. (2018) J Clin Invest. 128(10): 4604-21; Lam et al. (2018) J Immunol. 200(5): 1718-26). Fgl2 (which can exist as both secreted and transmembrane proteins) downregulated, as seen in the data during GVHD, enhances neutrophil migration (Zhou et al. (2019) Sci Adv. 5(11)). scRNAseq analysis of naive (i.e., non-transplanted) WT and IL-17RC+mice did not demonstrate significant differences between the genotypes' representation of monocyte clusters or of differential gene expression within clusters between WT and IL-17RC+genotypes, suggesting that the observed changes emerge specifically in the context of GVHD (FIGS 8A-8F). Similarly, no significant differences in neutrophil cluster representation or differential gene expression were observed in a WT vs. IL-17RCzmanner (FIG. 8G-8I), likewise suggesting that IL-17-mediated influences on neutrophil differentiation arise in the context of transplantation and GVHD. Together, these data suggest that IL-17 acts on a monocyte / macrophage axis in GVHD and identify new cellular and molecular targets for therapeutic targeting. This also identifies I L-17-associated GVHD-related changes in mature neutrophils which presents a candidate for therapeutic targeting and immune signature interrogation.

[0300] CSF-1 R blockade also imprints peripheral blood myeloid chronic GVHD signatures. The effect of CSF-1 R inhibition on peripheral blood gene signatures was examined, a strategy which efficaciously prevents and treats cGVHD in mice and humans (MacDonald et al. (2017) Blood 129(1): 13-21; Alexander et al. (2014) J Clin Invest. 124(10): 4266-80; Kitko et al. (2023) Journal of Clinical Oncology 41(10): 1864-75) (FIG. 9A). Recipient mice were transplanted and CSF-1 R was blocked from day 7 after BMT with an anti-CSF-1R antibody (M279) as previously described (Alexander et al. (2014) J Clin Invest. 124(10): 4266-80). Longitudinal scRNAseq was again undertaken on unseparated peripheral blood at days +14, +21, +28. T-cell and neutrophil cluster designations were applied in theF053-0195PCT / 25-085-WO-PCTsame manner of canonical gene expression as was utilized in the prior analysis of IL-17 signatures. Monocyte cluster designations were newly defined in this experiment Analysis of T-cells identified populations similar to those identified in the IL-17 analyses (FIG. 10A-10C). As expected, given the predominately myeloid-restricted expression of the CSF-1 R, no major changes in proportion of individual clusters' abundance were observed, and there were no significant gene expression differences between the GVHD control and anti-CSF-1 R groups (FIG. 10B). Neutrophil clusters showed relative G5b expansion with CSF-1R inhibition (FIG. 11 A, 11B, 11 E), and differential gene expression was apparent (FIG. 11C). Significantly overexpressed CSF-1R-dependent GVHD gene transcripts included chemotaxis-related (e.g., Cxcl2) and inflammation-related genes (e.g., TNF). Some genes were mutually expressed in an IL-17-, CSF-1R-, and GVHD-dependent fashion, including Cxcl2, in G4, G5b, and G5c clusters, together with Tnf in the G5c cluster. The most dramatic affect of CSF-1R signaling was the expansion of Ly6Cl0clusters within circulating monocytes, in keeping with their very high CSF-1R expression (Alexander et al. (2014) J Clin Invest. 124(10): 4266-80; MacDonald et al. (2005) J Immunol. 175(3): 1399-405) (FIG. 6A-6C). Interestingly, this cluster and a Ly6Chipopulation (Circulating 2; FIGs. 13A, 13B, 13D, 13E, 13F) marked by high expression of activation-related genes was largely ablated with CSF-1R blockade (FIG. 12C). In addition to these profound quantitative effects, qualitative changes also followed CSF-1R signaling blockade, most pronounced in the Circulating 1 and Circulating 3 clusters (Ly6Chi), where GVHD control mice showed higher MHC class II and binding partner gene expression (FIG. 13C).

[0301] Mouse-derived monocyte gene signatures identify IL-17 and CSF-1 signatures in transplant recipients with cGVHD. The preclinical IL-17 and CSF-1 cGHVD signatures were used to interrogate the HCT patients’ peripheral blood, utilizing a DEG-based from mouse Ly6Cl0monocytes (equivalent to human CD14-CD16+ nonclassical monocytes) which bore the greatest degree of differential gene expression in an IL-17 and GVHD-dependent manner Unlike IL-17 inhibition, CSF-1 R inhibition yielded both quantitative and qualitative effects in monocyte subsets in the preclinical studies. It was not feasible to utilize a DEG-based signature approach in Ly6C° cells analogous to that for IL-17 since the CSF-1 R blockade nearly completely ablated the clusters of interest. Instead, it was posited that CSF-1 -dominant immune signatures in patients may be characterized by modulation of CSF-1 -dependnet populations. This caused focus on the marker genes of both the Ly6Cl0and the hitherto unappreciated CSF-1 R-dependent Ly6Chlcluster (FIG. 13G) as the diagnostic clinical gene sets.

[0302] Fixed RNA single cell profiling on patient and volunteer PBMCs was done. These included 45 patients at approximately day +100 post-HCT without current systemic aGVHD therapy (15 who did not develop cGVHD through at least 18 months post-HCT and 30 who subsequently developed cGVHD). Also included, were ten patients with recently diagnosed cGVHD not yet on systemic immunosuppression and seven patients without cGVHD who were at similar timepoints post-HCT (10-12 months) (FIGs. 14D, 14E).

[0303] Three healthy (i.e, non-transplanted) volunteer samples were included for reference. Analysis demonstrated dominant populations of classical monocytes and smaller subsets of nonclassical monocytes and dendritic cells (FIG.14A, FIG. 15A). After selecting for genes with semi-equivalent top-quartile distributions between the day +100 non-F053-0195PCT / 25-085-WO-PCTcGVHD and future-cGVHD groups and weighting these genes equally into composite scores, generated signature expression thresholds indicative of specific immune pathway dysregulation to classify IL-17 and CSF-1R signals as positive (the lesser of ^2 standard deviations above the day +100 non-cGVHD group's mean or the maximum day +100 non-cGVHD value). The IL-17 signature, derived from mouse Ly6Cl0monocytes, was detectable in seven of 30 patients at day +100 within equivalent human nonclassical monocytes (FIG. 14B, FIG 15C). These signatures were present in half of patients at cGVHD diagnosis and absent in healthy volunteers. These were largely absent in patients without cGVHD ad 10-12 months post-HCT, though incidentally one patient developed sclerotic cGVHD five months later, and another had ongoing active skin aGVHD and expressed an IL-17 signature score above the positive threshold.

[0304] To identify “CSF-1R dysregulated” patients, both the mouse Ly6Cl0Circulating 2 and Ly6Cl0monocyte CSF-1R signature scores were queried in their respective human equivalent CD 14+CD 16- classical and CD14-CD16+ nonclassical monocyte populations. The Ly6Chisignature was positive in 4 of 30 patients at day +100, whereas the Ly6C° signature was positive in 14 patients (including the patients with the Ly6C° signature) (FIG. 14C, FIG. 15C). Conversely in patients at cGVHD diagnosis, three of 10 expressed the Ly6Chisignature and only one separate patient expressed the Ly6Cl0signature. These CSF-1 signatures were negative in non-transplanted volunteers and +10-12 months patients without cGVHD. Altogether, 15 of 30 (50%) day +100 patients who subsequently developed cGVHD expressed at least one signature, of which six expressed both the IL-17 and CSF-1 signatures while eight expressed only the CSF-1 signature and one the IL-17 signature along (FIG. 14C). The overlap of signatures in the nonclassical monocytes is shown in FIG. 15B. Seven of 10 patients at cGVHD onset expressed at least one signature, with two patients expressing both an IL-17 and CSF-1 signature, three the IL-17, and two the CSF-1 in isolation (FIG. 14E).

[0305] Compelling IL-17 and CSF-1 RcGVHD-associated signature expression in monocyte subsets in half of patients analyzed at day +100, prior to their cGVHD development, and 70% of patients at the time of their cGVHD diagnosis, similar to published proportions of patients whose cGVHD sustainedly responds to IL-17 or CSF-1 R-targeting agents belumosudil or axatilimab respectively (Cutler et al. (2021) Blood 138(22): 2278-89; Zeiser et al. (2021) N Engl J Med.385(3): 228-38; Miklos et al. (2017) Blood 130(21): 2243-50; Kitko et al. (2023) Journal of Clinical Oncology 41(10): 1864-75) These signatures were absent in non-transplanted health individuals and patents who did not develop cGVHD through at least 1.5 years post-HCT. Taken together, the preclinical and human findings demonstrate the potential utility of scRNAseq to detect and define causative pathway-specific immune networks in the peripheral blood of HCT recipients.

[0306] Discussion. It has been demonstrated that IL-17- and CSF-1 -related signaling yields gene expression patterns detectable in peripheral blood in a sclerodermatous cGVHD mouse model and that these signatures are present in a compelling subset of patients with newly diagnosed cGVHD and in fact also appear in a cGVHD-specific manner in a significant proportion of patients prior to cGVHD development. These signatures can now be evaluated in patient cohorts receiving targeted agents to delineate their sensitivity / specificity to predict responses. The IL-17 and CSF-1F053-0195PCT / 25-085-WO-PCTeffects in blood include both apparent shifts in cell subset abundance as well as differential gene expression within specific clusters between GVHD and non-GVHD controls and respective GVHD conditions where the IL-17 and CSF-1R pathways are inactive.

[0307] While the IL-17 cytokine family is known to behave as monocyte / macrophage chemo-attractants, recruitment and / or differentiation mechanisms are unclear. The data demonstrates that Ly6Cl0monocytes and their precursor Ly6Chicells exhibit IL-17-related expression of activation-related and chemotactic molecules, which may directly influence cell homing to GVHD target tissue. Interestingly, IL-17 also upregulates transcription factors and regulations associated with macrophage differentiation and establishment of tissue resident macrophage status under both homeostatic and infections conditions (Jarjour et al. (2019) Nat Immunol. 20(6): 687-700; Cook et al. (2020) Trends Immunol. 41(11): 1023-36). Conversely, IL-17 deletion upregulates Klf2 and Klf4, transcription factors associated with a “resting" monocyte / macrophage phenotype (Kurotaki et al. (2017) International Immunol. 29(3): 97-107). Taken together, these data suggest that 11-17 may promote monocyte differentiation into tissue macrophages, in addition to is effects on tissue homing. The finding of IL-17- / GVHD-related myeloid Maltl upregulation is intriguing; recent studies have identified Maltl activity in myeloid DCs downstream of pattern recognition receptor Dectin- 1 ligation (Unterreiner et al. (2019) J Vis Exp. 143) during inflammation. Preliminary studies have demonstrated efficacy of an oral Malt 1 inhibitor in a sclerodermatous GVHD mouse model (Dispirito et al. (2021) Blood 138(3810)(S1)); this effect may also act on lymphocytes, including inhibiting Th17 cell development.

[0308] Thus far, neutrophils have not been thought to participate in GVHD pathogenesis. This may, in part, reflect overwhelming use of density-gradient centrifugation sample preparation in which granulocytes are eliminated and not studied. The present G-CSF mobilization study demonstrates terminally differentiated neutrophils' augmented Csf1 expression in response to growth factor. Relevant to transplantation, in a model of cardia allograft tolerance, neutrophil-derived CSF-1 polarized allograft monocytes impacts graft survival (Braza et al. (2018) American Journal of Transplantation 18(5): 1247-55). In peritonitis, which predominately features neutrophil recruitment, inhibiting the ability of neutrophils to secrete CSF-1 and several other factors reduces local macrophage proliferation (Tang et al. (2018) Mol Cell Biol. 38(17)). Likewise, the implication that IL-17 contributes to neutrophil maturation suggests an IL-17-dependent mechanism whereby neutrophils may promote peripheral monocyte differentiation into the tissue-infiltrating macrophages that mediate cGVHD fibrosis. Together, these pathways may contribute to IL-17's ability to promote GVHD. Anti-CSF-1 R treatment depleted Ly6Cl0monocytes in mouse blood. I ntriguingly, this treatment also decreased the abundance of some Ly6Chimonocytes and was associated with gene expression changes in developmentally upstream monocyte clusters. This has not previously been appreciated (and it otherwise unable to be ascertained) by conventional flow cytometry of circulating monocytes. This phenomenon - as well as that of modulated neutrophil gene expression during anti-CSF-1R treatment - are areas for further investigation and potential future therapeutic interventions.

[0309] With respect to the CSF-1 and IL-17 dysregulation signature markers in human HCT patient monocytes, theF053-0195PCT / 25-085-WO-PCTdata demonstrate that these signatures are expressed at varying degrees in patients with or subsequently developing cGVHD. It is posited that different CSF-1 -dependent monocyte subpopulations (i.e., Ly6ChiCirculating 2 vs. Ly6Cl0) and signatures therein may predominate at different timepoints in the evolution of cGVHD and so interrogation of both is relevant. The conservatively proposed patients with signature expression scores 2 standard deviations above a non-cGVHD group's signature scores being most likely to represent those with IL-17- or CSF-1 -dysregulated cGVHD. Critically, these signatures are present in only a subset of patients and might be predicted by cGVHD pathophysiology in both preclinical and clinical studies, and as such they do not represent biomarkers or cGVHD. Instead, the signatures may be well-suited for their ability to predict deep and / or sustained responses to appropriately targeted agents (e.g., belumosudil for IL-17 and axatilimab for CSF-1R). In summary, IL-17- and CSF-1-related gene expression signatures are identifiable in peripheral blood monocytes in preclinical cGVHD mouse models and are present in subsets of patients prior to and at cGVHD onset. These may represent clinically informative tools to identify patients with IL-17 and CSF-1 pathway-predominant cGVHD, facilitating rational therapeutic selection. Importantly, the “reverse engineered” approach may be useful to identify additional pathways of cGVHD (e.g., mediated by alloantibody or defective regulatory T-cell responses) and may be extended to other preclinical models which accurately reflect and predict human disease.

[0310] (viii) Closing Paragraphs. Variants of protein and / or nucleic acid sequences disclosed herein can also be used. Variants include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein and nucleic acid sequences described or disclosed herein wherein the variant exhibits substantially similar or improved biological function.

[0311] “ % sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (WisconsinF053-0195PCT / 25-085-WO-PCTPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0312] In particular embodiments, variant proteins include conservative amino acid substitutions. In particular embodiments, a conservative amino acid substitution may not substantially change the structural characteristics of the reference sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the reference sequence, or disrupt other types of secondary structure that characterizes the reference sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds , Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354:105 (1991).

[0313] In particular embodiments, a "conservative substitution” involves a substitution found in one of the following conservative substitutions groups: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai); and Group 6: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

[0314] Additionally, amino acids can be grouped into conservative substitution groups by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie. Other groups containing amino acids that are considered conservative substitutions for one another include: sulfur-containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information is found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0315] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts The transitional phrase “consisting of' excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of' limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect theF053-0195PCT / 25-085-WO-PCTembodiment. A material effect would cause a statistically significant reduction in detection of an immune signature disclosed herein from a peripheral blood sample.

[0316] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0317] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0318] The terms “a," “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0319] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion orF053-0195PCT / 25-085-WO-PCTdeletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0320] Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0321] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0322] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0323] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0324] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-0195PCT / 25-085-WO-PCTCLAIMSWhat is claimed is:

1. A method of determining that a human transplant recipient has developed or will develop IL-17 chronic graft-versus-host-disease (cGVHD), the method comprisingObtaining monocytes from a peripheral blood sample obtained from the human transplant recipient;Processing the monocytes to detect an expression level of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 wherein differential expression of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop IL-17 related cGVHD.

2. The method of claim 1, wherein the method further comprises treating the human transplant recipient for cGVHD before symptoms emerge.

3. The method of claim 2, wherein the treating comprises administering belumosudil.

4. A method of determining that a human transplant recipient has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the method comprisingObtaining monocytes from a peripheral blood sample obtained from the human transplant recipient;Processing the monocytes to detect an expression level ofat least one or all of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes;at least one or all of APOE, CCL4, CCRL2, CD 14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, GSM, SLC7A11, and TLR2 by classical monocytes; and / orat least one or all of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by non-classical monocytes;wherein differential expression of at least one or all of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop IL-17 related cGVHD;F053-0195PCT / 25-085-WO-PCTwherein differential expression of at least one or all of at least one of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 by classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop CSF-1 related cGVHD; and / orwherein differential expression of at least one or all of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1 , POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by non-classical monocytes in comparison to a reference level determines that the human transplant recipient has developed or will develop CSF-1 related cGVHD.

5. The method of claim 4, comprising processing the monocytes to detect an expression level of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by non-classical monocytes.

6. The method of claim 4, comprising processing the monocytes to detect an expression level of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 by classical monocytes.

7. The method of claim 4, comprising processing the monocytes to detect an expression level of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by non-classical monocytes.

8. The method of claim 4, comprising processing the monocytes to detect an expression level of 1, 2, 3, 4, or 5 of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9 by nonclassical monocytes from a human transplant recipient’s peripheral blood.

9. The method of claim 4, comprising processing the monocytes to detect an expression level of CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1,F053-0195PCT / 25-085-WO-PCTTLR2, and TNFSF9.10 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2 A CSF-1 cGVHD by classical monocytes from a human transplant recipient's peripheral blood.

11. The method of claim 4, comprising processing the monocytes to detect an expression level of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

12. The method of claim 4, comprising processing the monocytes to detect an expression level of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4 by nonclassical monocytes from a human transplant recipient's peripheral blood.

13. The method of claim 4, comprising processing the monocytes to detect an expression level of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.14 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, Ier3, Irf2bp2, Ly6c2, Maff, Maltl , Mapk6, MarcksH, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91 by LyC6lo monocytes from a human transplant recipient’s peripheral blood.

15. The method of claim 4, comprising processing the monocytes to detect an expression level of Acodl, Bcl2l11 , Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, Ier3, Irf2bp2, Ly6c2, Maff, Maltl, Mapk6, MarcksH, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17,F053-0195PCT / 25-085-WO-PCTand Zfp91.16 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3 by LyC6lo monocytes from a transplant recipient’s peripheral blood.

17. The method of claim 4, comprising processing the monocytes to detect an expression level of Cd300e, Clec4a3, Fcgr4, H2-Eb1 , H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.

18. The method of claim 4, comprising processing the monocytes to detect an expression level of Lyz2 by Early monocytes from a transplant recipient’s peripheral blood.

19. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, or 4 of Ier3, Maltl, Mfkbiz, and Tnf by Circulating 1 monocytes from a transplant recipient's peripheral blood.

20. The method of claim 4, comprising processing the monocytes to detect an expression level of Ier3, Maltl , Mfkbiz, and Tnf.

21. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Gm4951, Igtp, ligpl, Klf2, and Rsrpl by Circulating 1 monocytes from a transplant recipient's peripheral blood.

22. The method of claim 4, comprising processing the monocytes to detect an expression level of Gm4951 , Igtp, ligpl , Klf2, and Rsrpl.

23. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Bhlhe40, Btg 1 , Clec4e, Maltl , and Tnf by Circulating 2 monocytes from a transplant recipient’s peripheral blood.

24. The method of claim 4, comprising processing the monocytes to detect an expression level of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.

25. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, or 4 of Fos, Ifitm3, Klf2, and Tsc22d3 by Circulating 2 monocytes from a transplant recipient's peripheral blood.

26. The method of claim 4, comprising processing the monocytes to detect an expression level of Fos, Ifitm3, Klf2, and Tsc22d3.

27. The method of claim 4, comprising processing the monocytes to detect an expression level of Cd6 by G4 neutrophils from a transplant recipient’s peripheral blood.

28. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip by G5a neutrophils from a transplant recipient’s peripheral blood.

29. The method of claim 4, comprising processing the monocytes to detect an expression level of Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.30 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, or 4 of Csf1, Fgl2, Vim, and Xpc by G5a neutrophils from a transplant recipient's peripheral blood.

31. The method of claim 4, comprising processing the monocytes to detect an expression level of Csf1, Fgl2, Vim,F053-0195PCT / 25-085-WO-PCTand Xpc.32 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36 by G5b neutrophils from a transplant recipient's peripheral blood.

33. The method of claim 4, comprising processing the monocytes to detect an expression level of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.

34. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1 or 2 of Ifitm2 and Vim by G5b neutrophils from a transplant recipient's peripheral blood35. The method of claim 4, comprising processing the monocytes to detect an expression level of Ifitm2 and Vim.

36. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, and Traf by G5c neutrophils from a transplant recipient’s peripheral blood.

37. The method of claim 4, comprising processing the monocytes to detect an expression level of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.

38. The method of claim 4, comprising processing the monocytes to detect an expression level of Fgl2 by G5c neutrophils from a transplant recipient’s peripheral blood.

39. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1 or 2 of Ccl3 and F13a1 by Circulating 1 monocytes from a transplant recipient’s peripheral blood.

40. The method of claim 4, comprising processing the monocytes to detect an expression level of Ccl3 and F13a1.41 The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21 by Circulating 1 monocytes from a transplant recipient’s peripheral blood.

42. The method of claim 4, comprising processing the monocytes to detect an expression level of Wfdc17, Gm 19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.

43. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of H2-Ab1, H2-Eb1, H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1 by Circulating 3 monocytes from a transplant recipient's peripheral blood.

44. The method of claim 4, comprising processing the monocytes to detect an expression level of H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.

45. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of TTmem71, PI16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21 by Circulating 3 monocytes from a transplant recipient’s peripheral blood.

46. The method of claim 4, comprising processing the monocytes to detect an expression level of TTmem71, Pi 16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.F053-0195PCT / 25-085-WO-PCT47. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Cstdc5, Cxcl2, II 1b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21 by G4 neutrophils from a transplant recipient's peripheral blood.

48. The method of claim 4, comprising processing the monocytes to detect an expression level of Cstdc5, Cxcl2, 111 b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.

49. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1, 2, 3, 4, or 5 of Acodl, Ccrl2, Cd 14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17 by G5a neutrophils from a transplant recipient's peripheral blood50. The method of claim 4, comprising processing the monocytes to detect an expression level of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.

51. The method of claim 4, comprising processing the monocytes to detect an expression level of Tsc22d3 by G5a neutrophils from a transplant recipient’s peripheral blood.

52. The method of claim 4, comprising processing the monocytes to detect an expression level of at least 1 or 2 of Cxcl2 and Nfkbia by G5b neutrophils from a transplant recipient’s peripheral blood.

53. The method of claim 4, comprising processing the monocytes to detect an expression level of Cxcl2 and Nfkbia.

54. The method of claim 4, wherein processing the monocytes to detect an expression level comprises bulk monocyte processing.

55. The method of claim 4, wherein processing the monocytes to detect an expression level comprises single cell monocyte processing.56 The method of claim 4, wherein processing the monocytes to detect an expression level comprises RNA sequencing.

57. The method of claim 56, wherein the RNA sequencing comprises bulk RNA sequencing.

58. The method of claim 56, wherein the RNA sequencing comprises single cell RNA sequencing.

59. A kit for determining that a human transplant recipient has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the kit comprisingat least one RNA annealing primer that binds CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, or TNFSF9;at least one RNA annealing primer that binds APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, or TLR2;at least one RNA annealing primer that binds ABCG1 , ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A,F053-0195PCT / 25-085-WO-PCTPRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, S0CS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, or TREML4;at least one RNA annealing primer that binds AcocH, Bcl2l11 , Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcl1, Ehd1, Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl , Mapk6, MarcksH, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl , Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdcl 7, or Zfp91;at least one RNA annealing primer that binds Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, or Tsc22d3;at least one RNA annealing primer that binds Lyz2;at least one RNA annealing primer that binds Ier3, Maltl, Mfkbiz, or Tnf;at least one RNA annealing primer that binds Gm4951, Igtp, ligpl, Klf2, or Rsrpl;at least one RNA annealing primer that binds Bhlhe40, Btg1, Clec4e, Maltl, or Tnf;at least one RNA annealing primer that binds Fos, Ifitm3, Klf2, or Tsc22d3;at least one RNA annealing primer that binds Cd6;at least one RNA annealing primer that binds Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, orTxnip; at least one RNA annealing primer that binds Csf 1 , Fgl2, Vim, or Xpc;at least one RNA annealing primer that binds Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, or Zfp36;at least one RNA annealing primer that binds Ifitm2 or Vim;at least one RNA annealing primer that binds Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, or Traf;at least one RNA annealing primer that binds Fgl2;at least one RNA annealing primer that binds Ccl3 or F13a1;at least one RNA annealing primer that binds Wfdcl 7, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, or Wfdc21;at least one RNA annealing primer that binds Klf2, Rsrpl, Tsc22d3, Ifitml, or Wfdc21;at least one RNA annealing primer that binds H2-Ab1, H2-Eb1, H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, or Fth1 ;at least one RNA annealing primer that binds TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, or Wfdc21;at least one RNA annealing primer that binds Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, or Wfdc21;at least one RNA annealing primer that binds Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1 , Steap4, Tnf, Tnfaip3, or Wfdc17;F053-0195PCT / 25-085-WO-PCTat least one RNA annealing primer that binds Tsc22d3; and / orat least one RNA annealing primer that binds Cxcl2 or Nfkbia.

60. The kit of claim 59, comprising RNA annealing primers that bind CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

61. The kit of claim 59, comprising RNA annealing primers that bind APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, PGS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

62. The kit of claim 59, comprising RNA annealing primers that bind ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MYO18A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.

63. The kit of claim 59, comprising RNA annealing primers that bind Acodl, Bcl2l11 , Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcll, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, Marcksll, Mbnl2, Ndufb1-ps, Nfkbid, Nfkbiz, Ninjl , Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1COa6, S1COa8, S1COa9, Sdc4, Socs3, Sqstml, Tagap, Tlr2, Tnf, TnfaipS, Tnfsfp, Wfdc17, and Zfp91.

64. The kit of claim 59, comprising RNA annealing primers that bind Cd300e, Clec4a3, Fcgr4, H2-Eb1, H2-K1, Ier5, Klf2, Pfn1 , Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.

65. The kit of claim 59, comprising RNA annealing primers that bind Lyz2.

66. The kit of claim 59, comprising RNA annealing primers that bind Ier3, Maltl, Mfkbiz, and Tnf.

67. The kit of claim 59, comprising RNA annealing primers that bind Gm4951, Igtp, ligpl, Klf2, and Rsrpl.

68. The kit of claim 59, comprising RNA annealing primers that bind Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.

69. The kit of claim 59, comprising RNA annealing primers that bind Fos, Ifitm3, Klf2, and Tsc22d3.70 The kit of claim 59, comprising RNA annealing primers that bind Cd6.

71. The kit of claim 59, comprising RNA annealing primers that bind Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.

72. The kit of claim 59, comprising RNA annealing primers that bind Csf 1 , Fgl2, Vim, and Xpc.

73. The kit of cl aim 59, comprising RNA annealing primers that bind Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.

74. The kit of claim 59, comprising RNA annealing primers that bind Ifitm2 and Vim.75 The kit of claim 59, comprising RNA annealing primers that bind Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, Marcksll, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spatai 3, Tgifl, Tnf, Tnfaip3, and Traf.

76. The kit of claim 59, comprising RNA annealing primers that bind Fgl2.F053-0195PCT / 25-085-WO-PCT77. The kit of claim 59, comprising RNA annealing primers that bind Ccl3 and F13a1.78 The kit of claim 59, comprising RNA annealing primers that bind Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.

79. The kit of claim 59, comprising RNA annealing primers that bind H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.

80. The kit of claim 59, comprising RNA annealing primers that bind TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.81 The kit of claim 59, comprising RNA annealing primers that bind Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.

82. The kit of claim 59, comprising RNA annealing primers that bind Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1 , Steap4, Tnf, Tnfaip3, and Wfdc17.

83. The kit of claim 59, comprising RNA annealing primers that bind Tsc22d3.

84. The kit of claim 59, comprising RNA annealing primers that bind Cxcl2 and Nfkbia.85 The kit of claim 59, wherein the RNA-annealing primer is a polyT primer or a gene-specific primer.

86. The kit of claim 59, wherein the kit further comprises a DNA polymerase, a reverse transcriptase, a transcription buffer, a reverse transcription buffer, a ligase, an adapter, and / or a detection molecule.

87. A method of treating a human transplant recipient that has developed or will develop IL-17 and / or CSF-1 related chronic graft-versus-host-disease (cGVHD), the method comprising:obtaining a report indicating that the human transplant recipient has an immune signature associated with IL-17 and / or CSF-1 related (cGVHD); andadministering an anti-IL-17 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with IL-17 related cGVHD;administering an anti-CSF-1 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with CSF-1 related cGVHD; or administering an anti-IL-17 cGVHD therapeutic and an anti-CSF-1 cGVHD therapeutic to the human transplant recipient when the report indicates that the human transplant recipient has an immune signature associated with IL-17 related cGVHD and an immune signature associated with CSF-1 related cGVHD.

88. The method of claim 87, wherein the anti-IL-17 cGVHD therapeutic comprises belumosudil.

89. The method of claim 87, wherein the anti-CSF-1 cGVHD therapeutic comprises Axatilimab.

90. An interleukin (IL)-17 chronic graft versus host disease (cGVHD) immune signature expressed by nonclassical monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of CCL3, CLEC4E, EHD1, EIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, ODC1, SDC4. SOCS3, SQSTM1, TLR2, and TNFSF9.

91. The IL-17 cGVHD immune signature of claim 90, comprising differential expression of CCL3, CLEC4E, EHD1,F053-0195PCT / 25-085-WO-PCTEIF3J, FTH1, GADD45B, GPX4, IER3, MAFF, MBNL2, NDUFB1, NFKBID, NFKBIZ, 0DC1, SDC4. S0CS3, SQSTM1, TLR2, and TNFSF9.

92. A colony-stimulating factor-1 (CSF-1) cGVHD immune signature expressed by classical monocytes and detected from a transplant recipients peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11 , and TLR2.

93. The CSF-1 cGVHD immune signature of claim 92, comprising differential expression of APOE, CCL4, CCRL2, CD14, CSRNP1, CXCL2, CXCL10, DUSP1, EGR1, POS, GADD45B, NFKBIZ, NLRP3, OSM, SLC7A11, and TLR2.

94. A CSF-1 cGVHD immune signature expressed by nonclassical monocytes and detected from a transplant recipient's peripheral blood sample comprising differential expression of at least 1, 2, 3, 4, or 5 of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.

95. The CSF-1 cGVHD immune signature of claim 94, comprising differential expression of ABCG1, ABHD12, ABI3, ACE, ACP2, ADCY7, AGPAT4, BATF3, BBC3, BTG2, CBLB, CCL3, CCL4, CCRL2, CD274, CD300C, CD300E, CD74, CD82, CD9, CDC42EP2, CEACAM1, CFB, CHKA, CIITA, CX3CR1, CYFIP2, CYTH3, DOCK5, DOK2, DUSP2, EGR1, ENO3, ENTPD1, FAM43A, FGD2, FILIP1L, FYN, GNGT2, HAVCR2, HES1, ITGA2, ITGAL, ITGAV, JUN, LACC1, LDLRAP1, LPP, MAN1A1, MY018A, NABP1, NDST1, NFKBID, NFKBIE, NFKBIZ, NR4A1, PDE4B, PILRB, PLA2G7, PLTP, PMAIP1, POT1, PP1R15A, PRR5L, PTPN12, PTPRJ, RAP1GAP2, RAP2A, RASGRP1, RUNX2, SDC3, SEMA4D, SH2D1B, SKIL, SLAMF8, SLC11A1, SLC8A1, SMPDL3B, SOCS3, SPN, SRGN, STAP1, STK10, TBC1D2B, TCF7L2, TGFBR1, TGFBR2, TGIF1, TGM2, THEMIS2, TMEM51, TRAF1, and TREML4.96 An IL-17 cGVHD immune signature expressed by LyCSIo monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcl1, Ehd1, Eif3j1, Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, Marcksll, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4, S1C0a6, S1COa8, S1COa9, Sdc4, Socs3, Sqstnd, Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.97 The IL-17 cGVHD immune signature of claim 96, comprising differential expression of Acodl, Bcl2l11, Bhlhe40, Ccl2, Cdkmla, Clec4e, Crrnpl, Cxcl1, Ehd1, Eif3j1 , Fn1, Fth1, Gadd45b, Gm10076, Fna13, Gpx4, ier3, irf2bp2, Ly6c2, Maff, Maltl, Mapk6, Marcksll, Mbnl2, Ndufbl -ps, Nfkbid, Nfkbiz, Ninjl, Npc2, Odd, Pde4b, Ptafr, Rasgeflb, S1COa4,F053-0195PCT / 25-085-WO-PCTS1C0a6, S1C0a8, S1C0a9, Sdc4, Socs3, Sqstml , Tagap, Tlr2, Tnf, Tnfaip3, Tnfsfp, Wfdc17, and Zfp91.98 An IL-17 cGVHD immune signature expressed by LyC6lo monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1 , 2, 3, 4, or 5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1 , H2-K1, Ier5, Klf2, Pfn1 , Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.

99. The IL-17 cGVHD immune signature claim 98, comprising differential expression of at least 1, 2, 3, 4, or 5 of Cd300e, Clec4a3, Fcgr4, H2-Eb1 , H2-K1 , Ier5, Klf2, Pfn1, Pou2f2, Psmb9, Rgs2, Rsrpl, Themis2, and Tsc22d3.

100. An IL-17 cGVHD immune signature expressed by Early monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of Lyz2.

101. An IL-17 cGVHD immune signature expressed by Circulating 1 monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, or 4 of Ier3, Maltl , Mfkbiz, and Tnf.

102. The IL-17 cGVHD immune signature of claim 101, comprising differential expression of Ier3, Maltl, Mfkbiz, and Tnf.

103. An IL-17 cGVHD immune signature expressed by Circulating 1 monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Gm4951, Igtp, ligpl, Klf2, and Rsrpl.104.The IL-17 cGVHD immune signature of claim 103, comprising differential expression of Gm4951, Igtp, ligpl, Klf2, and Rsrpl.

105. An IL-17 cGVHD immune signature expressed by Circulating 2 monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.

106. The IL-17 cGVHD immune signature of claim 105, comprising differential expression of Bhlhe40, Btg1, Clec4e, Maltl, and Tnf.

107. An IL-17 cGVHD immune signature expressed by Circulating 2 monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, or 4 of Fos, Ifitm3, Klf2, and Tsc22d3.108.The IL-17 cGVHD immune signature of claim 107, comprising differential expression of Fos, Ifitm3, Klf2, and Tsc22d3.

109. An IL-17 cGVHD immune signature expressed by G4 and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of Cd6.

110. An IL-17 cGVHD immune signature expressed by G5a neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Cxcl6, Egr1, Fermt3,G0s2, Junb, Mafk, Nampt, Osginl, and Txnip.

111. The IL-17 cGVHD immune signature of claim 110, comprising differential expression ofCxcl6, Egr1, Fermt3,G0s2,F053-0195PCT / 25-085-WO-PCTJunb, Mafk, Nampt, Osginl , and Txnip.

112. An IL-17 cGVHD immune signature expressed by G5a neutrophils and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, or 4 of Csf1, Fgl2, Vim, and Xpc.

113. The IL-17 cGVHD immune signature of claim 112, comprising differential expression of Csf1, Fgl2, Vim, and Xpc.114.An IL-17 cGVHD immune signature expressed by G5b neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.115.The IL-17 cGVHD immune signature of claim 114, comprising differential expression of Fos, G0s2, H2-Q6, Ier2, Ifi27l2a, Osm, and Zfp36.

116. An IL-17 cGVHD immune signature expressed by G5b neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1 or 2 of Ifitm2 and Vim.

117. The IL-17 cGVHD immune signature of claim 116, comprising differential expression of Ifitm2 and Vim.118.An IL-17 cGVHD immune signature expressed by G5c neutrophils and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, and Traf.

119. The IL-17 cGVHD immune signature of claim 118, comprising differential expression of Ccrl2, Cstdc4, Cxcl2, Dusp2, Fth1, G0s2, Junb, MarcksH, Nfkbia, Nfkbiz, Osginl, Osm, Bde4b, Spata13, Tgifl, Tnf, Tnfaip3, and Traf.

120. An IL-17 cGVHD immune signature expressed by G5c neutrophils and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of Fgl2.

121. A CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1 or 2 of Ccl3 and F13a1.

122. The CSF-1 cGVHD immune signature claim 121, comprising differential expression of Ccl3 and F13a1.

123. A CSF-1 cGVHD immune signature expressed by Circulating 1 monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.124.The CSF-1 cGVHD immune signature of claim 123, comprising differential expression of Wfdc17, Gm19951, Lmna, Uba52, S100a8, Klf2, Rsrpl, Tsc22d3, Ifitml, and Wfdc21.

125. A CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of H2-Ab1, H2-Eb1 , H2-Aa, Mafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.

126. The CSF-1 cGVHD immune signature of claim 125, comprising differential expression of H2-Ab1, H2-Eb1, H2-Aa,F053-0195PCT / 25-085-WO-PCTMafb, Cd74, Mareks, H2-DMb1, Hsp90ab1, and Fth1.127.A CSF-1 cGVHD immune signature expressed by Circulating 3 monocytes and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.

128. The CSF-1 cGVHD immune signature of claim 127, comprising differential expression of TTmem71, Pi16, Bachl, Rsrpl, Gpr141, Stxbp3, Gm15987, Stk38, Tsc22d3, Klf2, and Wfdc21.

129. A CSF-1 cGVHD immune signature expressed by G4 neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.

130. The CSF-1 cGVHD immune signature of claim 129, comprising differential expression of Cstdc5, Cxcl2, 111b, Junb, Nfkbia, Prok2 Sfa2, Wfdc17, and Wfdc21.

131. A CSF-1 cGVHD immune signature expressed by G5a neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1, 2, 3, 4, or 5 of Acodl, Ccrl2, Cd14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.

132. The CSF-1 cGVHD immune signature of claim 131, comprising differential expression of Acodl, Ccrl2, Cd 14, Cxcl2, Igfbp6, Nfkbia, Pim1, Steap4, Tnf, Tnfaip3, and Wfdc17.

133. A CSF-1 cGVHD immune signature expressed by G5a neutrophils and detected from a transplant recipient's peripheral blood sample, the immune signature comprising differential expression of Tsc22d3.

134. A CSF-1 cGVHD immune signature expressed by G5b neutrophils and detected from a transplant recipient’s peripheral blood sample, the immune signature comprising differential expression of at least 1 or 2 of Cxcl2 and Nfkbia.135.The CSF-1 cGVHD immune signature of claim 134, comprising differential expression of at least 1 or 2 of Cxcl2 and Nfkbia.

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