Analysis of CD25 positive switched memory b cells in osteoarthritis

Abstract

Compositions and methods are provided for determining the presence of early-stage osteoarthritis (OA) in an individual by single cell profiling of B cell population(s) in a blood sample. Expansion of CD25hi switched memory B cells is detectable in the early stages of OA and can be utilized for early detection of the disease.

Description

ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217ANALYSIS OF CD25 POSITIVE SWITCHED MEMORY B CELLS IN OSTEOARTHRITISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 708,170, filed October 16, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Osteoarthritis (OA) is an age-associated, chronic disease that affects 1 in every 5 adults above the age of 60, leading to joint dysfunction and persistent adverse effects on the quality of life. No disease-modifying drugs are available for OA, with treatment options being limited to pain management until the eventual surgery for total joint replacement. Part of the reason why OA disease modification has been challenging is the late identification of the disease making the reversal difficult, along with a lack of biomarkers that can accurately reflect the molecular causation and manifestation of the disease.

[0003] Assaying joint tissues like cartilage and synovium from patients are difficult because procurement procedures are invasive and can even be detrimental to patients for tissues like cartilage. Therefore, the joint tissues that are generally available to study the disease are the ones harvested from patients undergoing total joint replacement surgeries, often at advanced stages of OA. Efforts have been made for biomarker identification in longitudinal cohorts of OA patients using imaging modalities and readily available tissues such as blood or urine specimens. However, such studies have hitherto been limited to serum proteins and bulk transcriptomics or epigenomic feature identification.

[0004] OA pathophysiology is distinct from the autoimmune disease rheumatoid arthritis (RA), however significant trafficking of immune cells is evident in OA joints. Details regarding the immune cell types involved, the tissues that are infiltrated in the joint, the timing of infiltration, and the cellular cross-talk that results in the breakdown of joint homeostasis are still unclear. Identification of relevant cell subsets is of great interest for early diagnosis and treatment of this disease.SUMM RY

[0005] Compositions and methods are provided for determining the presence of osteoarthritis (OA) in an individual by single cell analysis of a blood sample. It is shown herein that individuals with OA, including early OA, can be identified by an expansion of a switched memory B cell subpopulation (CD25h'CD24h'CD27+lgD ) in peripheral blood, relative to aATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 healthy control. It is also shown that there is a depletion of the naive B cell population in peripheral blood, where such cells may be identified as CD27 lgD+.

[0006] Analysis of B cell populations in a subject, including determination of an expansion of switched memory B CD25hiCD27+lgD CD24tligtlcells, is used to determine the presence of OA, including early stage OA. Analysis of the B cell population may further comprise determination of a decrease in naive B cells, e.g. CD27 lgD+CXCR5+CD38+cells. The presence of altered levels of B cell populations is determined in a patient sample comprising circulating immune cells.

[0007] The analysis of B cell populations in an individual is useful in classifying the individual for early detection of OA, where the individual may be asymptomatic for OA, or may have joint abnormalities detectable only by imaging modalities. In some embodiments the individual is a human. In some embodiments the individual has a condition or injury that can pre-dispose to OA, e.g. a joint injury such as anterior cruciate ligament (ACL) tears, degenerative meniscal tears (DMT), etc.; genetic history; sports injuries, bone deformity; repetitive stress; metabolic disease, e.g. diabetes, hemochromatosis; and the like. Treatment at a pre-clinical, sometimes asymptomatic, point in disease progression is exceptionally valuable, in that degeneration and loss of function can prevented. In addition to classification and treatment of patients, the switched memory B cell population provides a therapeutic target for screening and design of novel therapeutic agents for treatment of OA.

[0008] In some embodiments of the invention a device or kit is provided for the analysis of patient samples, e.g. from an individual suspected of having osteoarthritis. Such devices or kits comprise reagents that specifically identify one or more cell populations, including without limitation CD25hiCD24hiCD27+lgD_B cells, from a patient sample. The reagents can be provided in isolated form, or pre-mixed as a cocktail suitable for the methods of the invention. A kit can include instructions for using the plurality of reagents to determine data from the sample; and instuctions for statistically analyzing the data. The kits may be provided in combination with a system for analysis, e.g. a system implemented on a computer. Such a system may include a software component configured for analysis of data obtained by the methods of the invention.

[0009] In an embodiment, a device or kit comprises reagents for the enrichment of B cells from a patient blood sample, e.g. using flow cytometry, magnetic bead selection, etc. For example, a bulk selection for expression of B cell marker(s) may be performed on a population of peripheral blood monocytic cells (PBMC). The B cell enriched population is then analyzed at a single cell level for the presence of switched B cells that are CD27+CD25hi, e.g. with a device or kit comprising comprising or consisting of affinity reagents specific for CD27 and for CD25, and the representation of such cells in the B cell population is determined. In someATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 embodiments a device or kit comprises or consists of reagents specific for each of CD25, CD27 and IgM. In some embodiment flow cytometry or mass cytometry is used to perform single cell profiling of the sample. Affinity reagents are optionally labeled for use in flow or mass cytometry. Alternatively microscopy can be used for single cell profiling. In other embodiments the enriched B cell population is analyzed for expression of genetic markers characteristic of the switched memory B cell population, for example as shown in FIG 12 and 14. In an embodiment the B cell population is assessed for the upregulation of one or more genes, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18 or 20 genes, which may be selected from LINC01619, CMIP, BCL2, HIVEP2, VOPP1 , TNRC6C, IQCN, ABCG1 , RASGRP1 , ETV6, EEF2K, PALM2AKAP, SMAD3, GALNT2, ECE1 , KDM6B, CD96, FMNL1 , TET3 and EXT1. Optionally, the B cell population is further analyzed for the antigenic specificity of the switched memory B cells. It will be understood by one of skill in the art that cells express multiple proteins on their surface that can form the basis for phenotyping, and that these proteins provide a useful subset of possible markers for phenotyping.

[0010] In an embodiment, a device or kit comprises reagents for a one-step analysis of a patient sample to determine whether there is an expansion of CD25hiswitched memory B cells. Such a device or kit may comprise affinity reagents that identify B cells, e.g. reagents specific for a pan-B cell marker such as CD20, CD19, etc; or reagents specific for switched B cells, e.g. anti-IgG. Alternative the kit comprises reagents for negative selection of B cells, e.g. with reagents specific for immune cells other than B cells. The devide or kit further comprises reagents specific for CD27 and for CD25. The device or kit may further comprise reagents specific for IgM. In some embodiment flow cytometry is used to perform single cell profiling of the sample.

[0011] In an embodiment of the invention, the methods of determining the presence of OA in an individual include obtaining a patient sample comprising circulating immune cells for analysis. Blood samples are a convenient source of circulating immune cells, particularly whole blood, although PBMC fractions also find use. The sample may be enriched for B cells.

[0012] In some embodiments, a patient sample is contacted with a combination of reagents, e.g. affinity reagents, which reagents are specific for markers that identify CD25hiswitched memory B cells. Reagents may be provided in a kit or device as discussed above. A combination of reagents may comprise, for example, at least reagents specific for CD25 and CD27. A combination of reagents may further comprise a reagent specific for IgM. The combination of reagents may further comprise reagents sufficient to identify immune cell subsets as disclosed in WO 2023 / 224985, herein specifically incorporated by reference.

[0013] The detectably labeled population is analyzed at a single cell level, e.g. by flow cytometry, microscopy, etc. In some embodiments the flow cytometry is time-of-flight (TOF)ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 mass cytometry. In some embodiments the flow cytometry is fluorescence activated flow cytometry. In some embodiments at least 103cells; at least 104cells; at least 105cells are analyzed. The resulting dataset is input into a predictive classification algorithm for a determination of whether the individual has early stage OA. The single cell data may be clustered into cell populations, e.g. by FlowSOM clustering. The algorithm may utilize a previously determined model for the prediction, e.g. a random forest model that has been trained on: samples from healthy individuals; individuals with ACL tear; individuals with DMT ; etc.

[0014] In some embodiments data is analyzed with an algorithm including a predetermined a cut-off for determining a diagnosis of OA, e.g. where the total B cell population comprises greater than about 1 .5% CD25hiswitched memory B cells, greater than about 2%, greater than about 2.5%, greater than about 3%, greater than about 3.5%, greater than about 4%, greater than about 4.5%, greater than about 5%. The analysis optionally includes a decrease in naive B cells in the total B cell population. The analysis optionally includes analysis of additional immune cell subsets as disclosed in WO 2023 / 224985.

[0015] In other embodiments a population of B cells is analyzed for expression of genetic markers characteristic of the switched memory B cell population, e.g. by determined upregulated expression of more or more of genes selected from LINC01619, CMIP, BCL2, HIVEP2, VOPP1 , TNRC6C, IQCN, ABCG1 , RASGRP1 , ETV6, EEF2K, PALM2AKAP, SMAD3, GALNT2, ECE1 , KDM6B, CD96, FMNL1 , TET3 and EXT1 .

[0016] The predictive analysis may further comprise analysis other than cell profiling, e.g. arthroscopy, radiographic imaging, ultrasound imaging, magnetic resonance imaging (MRI), computed tomography (CT), etc. The predictive analysis may further comprise determination of the presence of a molecule, e.g. C-reactive protein (CRP), a cytokine, antibody, cartilage component, protease, etc. or other clinical laboratory marker of inflammation, e.g. erythrocyte sedimentation rate (ESR), and compared to a control or reference value, wherein altered level of the molecular marker, in combination with the predictive cell classification, is indicative of early OA.

[0017] The predictive analysis may further comprise determining the antigenic specificity of the CD25+switched memory B cells, for example where there is specific reaction with cartilage proteins, including osteopontin, cartilage intermediate layer protein (CILP), YKL-39, fibulin-4, collagen, aggrecan core protein, etc.

[0018] In some embodiments, following a determination that an individual has OA, including early OA, the individual is treated to ameliorate, diminish, actively treat, reverse or prevent injury, damage, or loss of articular cartilage or subchondral bone subsequent to the early stage of disease. Treatment may prevent progression or reduce severity of OA. In someATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 embodiments treatment is pharmacologic. In some embodiments treatment comprises physical and / or occupational therapy. In some embodiments treatment is surgical. In some embodiments, treatment comprises clinical trial enrollment, where individuals can be stratified by likelihood of OA developing prior to the clinical trial.

[0019] Also provided herein is a method for prediction of the presence of OA, including early OA, comprising: obtaining a dataset associated with an immune sample obtained from the subject, wherein the dataset comprises quantitative data from the markers disclosed herein; and analyzing the dataset classification with a predictive model, wherein a statistically significant match with a model disclosed herein is indicative of early OA. The data may be analyzed by a computer processor. The processor may be communicatively coupled to a storage memory for analyzing the data. The processor may be coupled to a flow cytometer, and may include agorithms for clustering cell populations, and predictive classification. Also described herein is a computer-readable storage medium storing computer-executable program code, the program code comprising code for storing and analyzing data obtained by the methods of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0021] FIGS. 1A-1 E. Traditional immunophenotyping identifies significant differences between healthy and OA PBMC. (A) Schematic representation of experimental design. Briefly, whole blood PBMCs of healthy donors (n=21 ) and OA patients (n=17) were isolated and labeled with metal isotope-conjugated antibodies, followed by mass cytometry and analysis by traditional phenotyping. (B) Spearman’s correlation matrix demonstrates significant correlations (p<0.05) between the immune population frequencies and social and clinical demographics of healthy and OA cohorts. (C) Major immune populations identified by traditional hierarchical gating from parent CD45+CD66b- population are visualized by UMAP plots in healthy and OA samples. (D) UMAP highlighting significantly different immune subpopulations between healthy and OA cohorts. (E) Half boxplots of significantly different immune cell subtypes of major populations. Dots in the half boxplots represent the distribution of individual samples in a cohort. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under the 75thand 25thpercentiles, respectively.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 Statistical significance was calculated by a non-parametric Quade test to control for age and gender, represented by p values.

[0022] FIGS. 2A-2I. FlowSOM identifies perturbations in the naive and memory B cell repertoire in OA (A) Schematic of experimental design and mass cytometry analysis by FlowSOM. (B) UMAR representation of significantly different clusters of B cells in healthy (n=21 ) and OA cohorts (n=17), identified by unsupervised hierarchical clustering by FlowSOM. (C) Heatmap represents a median expression of markers (z-scored column-wise) in differential clusters across all samples. (D, E) UMAP and half boxplots of significantly different frequencies of switched memory B cell (IgDj clusters (D, E) and naive B cell clusters (F, G). Dots in the half boxplots represent the cluster abundance of individual samples. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under the 75thand 25thpercentiles, respectively. Statistical significance between healthy and OA clusters was calculated by non-parametric Quade test to control for age and represented by p values after comparisons by the Wilcoxon test and adjusting for multiple hypotheses by the Dunnett T3 test. (H) Comparative analysis of median expression of CXCR5 in differentially abundant switched B cell memory clusters. Statistical significance was calculated by pairwise Wilcoxon rank sum test. (I) Principal component analysis (PCA) performed on the frequencies of all FlowSOM-defined B cell clusters in all healthy and OA samples. Plots depict the projection of samples on the first two principal components.

[0023] FIGS. 3A-3N. Expansion of a CD25hiswitched memory B cell population in OA (A) UMAP visualization of cell clusters from the scRNA-seq analysis depicting the different immune cell types after B cell enrichment. (B) Distribution of the major types of B cells within each group. (C) Proportion testing plot using ‘scProportiontest’ depicting the Iog2 fold change of proportions of different B cell subtypes (dashed line indicates a cut-off of 5% change in proportion, red dots represent statistically significant changes). (D) Cell cluster UMAP plots representing switched memory B cell types with distinct gene expression profiles and (E) distribution across control and OA samples. (F) Barplot representing the proportions of (SMB) cell clusters within each sample. (G) Density plot of CD25 (IL2RA) gene expression within the switched memory B cell population and (H) violin plot of CD25 across control and OA samples. (I) Enriched pathways for each switched memory B cell subtype. (J) Cell cluster UMAP plots representing naive B cell types with distinct gene expression profiles and (K) distribution across control and OA samples. (L) Barplot representing the proportions of naive B cell clusters within each sample. Violin plots representing (M) the top 10 downregulated and top 10 upregulated genes, respectively, in OA within Naive B cells. (N) Pathway analysis representing enriched pathways for each naive B cell subtype.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0024] FIGS. 4A-4H. The immune landscape of Degenerative Meniscal Tear (DMT) patients resembles OA. (A) UMAR of major immune cell types identified by traditional phenotyping in healthy (n=21), OA (n=17) and DMT (n=10) PBMCs. (b) Half boxplot of statistically significant switched memory B cell frequency among healthy, OA and DMT samples derived by gating. (C) UMAP of significantly different FlowSOM-identified clusters of B cells and (D) the overall median expression of markers in the differential clusters across samples. (E) Half boxplots of the abundance of significantly different switched memory and naive B cell clusters between healthy, OA, and DMT samples. (F) UMAP of significantly different CD4T cell clusters defined by FlowSOM and (G) identified by overall marker expression across all samples. (H) Half boxplots of significantly different frequencies of CD4T cell clusters in healthy, OA, and DMT cohorts. Dots in the half boxplots represent the cluster abundance of individual samples. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under the 75thand 25thpercentiles respectively. Statistical significance between healthy, OA, and DMT clusters was calculated by a non-parametric Quade test to control for age and represented by p values after pairwise comparisons by Dunnett’s test for unequal variances.

[0025] FIGS. 5A-5D. Specific immune correlates of OA are already evident in the early stages of the disease. (A) Spearman correlation matrix of significantly different FlowSOM-identified immune clusters between healthy and OA cohorts, and donor demographic information such as age and gender along with Kellgren / Lawrence (KL) scores for three different regions of the medial, lateral and patellofemoral (PF) areas of the knee in OA individuals. Only statistically significant correlations (p<0.01) are depicted as dots. (B) Heatmap of the cluster frequencies in individual OA and DMT samples wherein each cluster was differential in the OA cohort compared to healthy. Hierarchical clustering was performed on the dataset by using cosine similarity as a distance metric and Ward’s minimum variance method (Ward.D2). (C) Dotplot of CXCR5 expression across individual OA and DMT samples. Each dot represents the frequency of each cluster in every sample while the color gradient represents the median expression of CXCR5 per sample per cluster. (D) Dotplot of CD25 expression across individual OA and DMT samples. Each dot represents the frequency of each cluster in every sample, and the color gradient represents the median expression of CD25 per sample per cluster.

[0026] FIG. 6. Manual gating strategy for the identification of immune cells. Cells were first gated on the expression of DNA (labeled with Ir191 ) and cisplatin to identify live cells as positive for DNA and negative for cisplatin. Cells were gated as CD45+CD66b_on live cells were the parent population to identify major immune cells and their subtypes, namely monocytes (classical and non-classical), B cells (plasmoblasts, naive, transitional, memoryATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 resting and switched memory), NK cells (CD16+and CD16 ), dendritic cells (mDCs and pDCs), CD8 T cells (activated, naive, central memory, effector memory and terminal effector) and CD4 T cells (activated, naive, central memory, effector memory and terminal effector, Th1 , Th2, Th17, activated CCR4+ Tregs, CCR4+ memory Treg and CCR4+ naive Tregs).

[0027] FIG. 7: Half boxplots of major immune populations by manual phenotyping of healthy (n=21 ) and OA groups (n=17). Dots in the half boxplots represent the distribution of individual samples in a cohort. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under the 75thand 25thpercentiles respectively.

[0028] FIGS. 8A-8F. Perturbations in monocyte and CD8 T cell repertoire in OA as defined by FlowSOM. (A) UMAP of significant differentially abundant monocyte clusters in healthy (n=21) and OA (n=17) cohorts. (B) Heatmap represents the medians of markers used for identifying the monocyte clusters. (C) The frequency of significantly different classical monocyte clusters in healthy and OA cohorts is represented as a percentage of total PBMCs in half boxplots. (D) UMAP of significantly abundant CD8T clusters in healthy (n=21) and OA (n=17). (E) Heatmap represents the medians of markers used for identifying the CD8T cell clusters. (F) Half boxplots of significantly different CD8 T cell clusters in OA when compared to healthy. Dots in the half boxplots represent the distribution of individual samples in a cohort. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under the 75thand 25thpercentiles respectively. Statistical significance between healthy and OA clusters was calculated by non-parametric quade test to control for age and gender, p values were calculated by the Wilcoxon test after adjusting for multiple hypotheses by the Bonferroni method.

[0029] FIGS. 9A-9I. CD4 T cell repertoire in OA as defined by FlowSOM. (A) UMAP of significantly expanded CD4T cell clusters in healthy (n=21) and OA (n=17) cohorts. (B) Heatmap represents the medians of markers used for the identification of the significantly different FlowSOM defined CD4T cell clusters. The frequency of significantly expanded (C) naive (D) terminal effector CD4T cell clusters in OA is represented as a percentage of total CD4T cells in half boxplots. (E) UMAP of significantly depleted CD4T clusters in healthy (n=21 ) and OA (n=17). Half boxplots of significantly different (F) naive, (G) Th2 and effector memory CD4 T cell clusters in OA when compared to healthy. (H) Principal component analysis (PCA) performed on the frequency of FlowSOM defined CD4T cell clusters in all healthy and OA samples. Plots depict the projection of samples on the first 2 principal components, and the contribution of the cluster frequencies to the PC plot (I) . Dots in the half boxplots represent the distribution of individual samples in a cohort. Line inside the boxplot is the median at 50thATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 percentile, and upper and lower whiskers denote data within 1 .5 times the interquartile range under 75thand 25thpercentiles respectively. Statistical significance between healthy and OA clusters was calculated by non-parametric quade test to control for age and gender, p values were calculated by Wilcoxon test after adjusting for multiple hypotheses by Bonferroni method.

[0030] FIGS. 10A-10D. B cell identification from single cell analysis (A) Uniform Manifold Approximation and Projection (UMAP) visualization of cell clusters from scRNA-seq analysis. (B) Feature plots of known B cell gene markers - CD19, CD20 (MS4A1 ), PAX5 and CD79A. (C) Violin plots of known immune cell markers enriched in specific clusters. (D) Automated cluster labeling using the Monacolmmune database of immune cell gene expression profiles.

[0031] FIGS. 1 1A-1 1 E. CXCR5 and IL2RA enrichment in OA-specific clusters of Switched Memory B cells (A) UMAP visualization of cell clusters of switched memory B cells from scRNA-seq analysis. Density plots of (B) CD25 (IL2RA) and (C) CXCR5 gene expression within the switched memory B cell population. (D) Barplot representing the proportions of switched memory B cell clusters within each sample. (E) Dot plots of the average expression of IL2RA and CXCR5 within each switched memory B cell cluster.

[0032] FIGS. 12A-12F. Validation of previous CyTOF findings using an independent cohort. (A) viSNE plots showing clustering of major immune cell populations and (B) the expression of markers used to characterize these populations. (C) FlowSOM clustering of B cell subset of PBMCs identifies 25 clusters. (D) Heatmap of scaled median expression of select markers within the significantly altered clusters of B cells in OA patients versus PBMCs of healthy controls. Significance was determined by the diffcyt-DA-GLMM significance testing function in the ‘diffcyt’ package in R (E) Box and whisker plots of significantly expanded Switched Memory B cell clusters with the CD25hicluster 22 highlighted and (F) significantly depleted Naive B cell clusters in OA versus healthy PBMCs. p-values : * < 0.05, ** < 0.01.

[0033] FIG. 13. Table of upregulated genes expressed in normal and OA B cells.

[0034] FIGS. 14A-14F. A. Clustering of cell types in a PBMC sample. B. Markers of immune cells. C. Clustering of B cells. D. Expression of markers in a B cell population. E. Representation of switched memory B cell Cluster 16 and 22 B cells in an OA and control sample. F. Representation of naive B cell clusters 3, 9, 20 and 24 in OA and control samples.DETAILED DESCRIPTION

[0035] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purposeATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0038] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0039] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0040] Naive B cells. Human naive B cells are immature B cells that have not yet encountered an antigen. These cells express specific surface markers that distinguish them from other B cell subsets, such as memory or activated B cells. Common markers for human naive B cells include CD19; and CD20. Naive B cells lack the expression of CD27. Naive B cells express surface immunoglobulin M (IgM) and immunoglobulin D (IgD) as their B cell receptors (BCRs).ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 This dual expression is a hallmark of naive B cells. Naive B cells typically express low to intermediate levels of CD38.

[0041] Switched B cells. Switched B cells, also known as class-switched B cells or isotype- switched B cells, are B lymphocytes that have undergone class switch recombination (CSR). This process changes the isotype (class) of the antibody they produce without altering the antigen specificity. Instead of producing immunoglobulin M (IgM), which is the default antibody produced by naive B cells, switched B cells produce other antibody isotypes such as IgG, IgA, or IgE. Switched B cells can be identified by the loss of IgM and IgD expression and the presence of other immunoglobulin isotypes (IgG, IgA, IgE). They express CD19 and CD20, and low levels of CD38.

[0042] Memory B cells. Many switched B cells are part of the memory B cell pool, which is responsible for long-term immune memory and rapid response upon re-exposure to an antigen. However, some activated B cells may also switch and differentiate into plasma cells that secrete antibodies immediately. Memory B cells persist in the body for extended periods, often for many years, even in the absence of antigen. Memory B cells are specific to the antigen that originally stimulated their formation, allowing for a targeted immune response during re-infection. Memory B cells typically express immunoglobulins other than IgM and IgD. CD27+is a key marker of memory B cells, distinguishing them from naive B cells (CD27j. Memory B cells typically express high levels of CD21 , compared to naive or activated B cells.

[0043] Breg cells, or regulatory B cells, are a subset of B lymphocytes that play a critical role in suppressing immune responses and maintaining immune tolerance. They act as modulators of immune activity, preventing excessive inflammation and autoimmunity. Breg cells exert their regulatory functions mainly through the production of anti-inflammatory cytokines and interactions with other immune cells. Breg cells suppress immune responses primarily through the secretion of anti-inflammatory cytokines, including IL-10, IL-35, TGF-p. Expression of CD25 can be characteristic of certain Breg cells.

[0044] Osteoarthritis (OA). OA affects nearly 27 million people in the United States, accounting for 25% of visits to primary care physicians, and half of all prescriptions for nonsteroidal anti-inflammatory drugs (NSAIDs). It is a chronic arthropathy characterized by disruption and potential loss of joint cartilage along with other joint changes, including bone remodeling such as bone hypertrophy (osteophyte formation), subchondral sclerosis, and formation of subchondral cysts. OA results in the degradation of joints, including degradation of articular cartilage and subchondral bone, resulting in mechanical abnormalities and joint dysfunction. Symptoms may include joint pain, tenderness, stiffness, sometimes an effusion,ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217 and impaired joint function. A variety of causes can initiate processes leading to loss of cartilage in OA.

[0045] OA may begin with joint damage caused by trauma to the joint; mechanical injury to the meniscus, articular cartilage, a joint ligament, or other joint structure; defects in cartilage matrix components; and the like. Mechanical stress on joints may underlie the development of OA in many individuals, with the sources of such mechanical stress being many and varied, including misalignment of bones as a result of congenital or pathogenic causes; mechanical injury; overweight; loss of strength in muscles supporting joints; and impairment of peripheral nerves, leading to sudden or dys-coordinated movements that overstress joints.

[0046] In synovial joints there are at least two movable bony surfaces that are surrounded by the synovial membrane, which secretes synovial fluid, a transparent alkaline viscid fluid that fills the joint cavity, and articular cartilage, which is interposed between the articulating bony surfaces. The earliest gross pathologic finding in OA is softening of the articular cartilage in habitually loaded areas of the joint surface. This softening or swelling of the articular cartilage is frequently accompanied by loss of proteoglycans from the cartilage matrix. As OA progresses, the integrity of the cartilage surface is lost and the articular cartilage thins, with vertical clefts extending into the depth of the cartilage in a process called fibrillation. Joint motion may cause fibrillated cartilage to shed segments and thereby expose the bone underneath (subchondral bone). In OA, the subchondral bone is remodeled, featuring subchondral sclerosis, subchondral cycts, and ectopic bone comprising osteophytes. The osteophytes (bone spurs) form at the joint margins, and the subchondral cysts may be filled with synovial fluid. The remodeling of subchondral bone increases the mechanical strain and stresses on both the overlying articular cartilage and the subchondral bone, leading to further damage of both the cartilage and subchondral bone.

[0047] The tissue damage stimulates chondrocytes to attempt repair by increasing their production of proteoglycans and collagen. However, efforts at repair also stimulate the enzymesthat degrade cartilage, as well as inflammatory cytokines, which are normally present in only small amounts. Inflammatory mediators trigger an inflammatory cycle that further stimulates the chondrocytes and synovial lining cells, eventually breaking down the cartilage. Chondrocytes undergo programmed cell death (apoptosis) in OA joints.

[0048] OA should be suspected in patients with gradual onset of joint symptoms and signs, particularly in older adults, usually beginning with one or a few joints. Pain can be the earliest symptom, sometimes described as a deep ache. Pain is usually worsened by weight bearing and relieved by rest but can eventually become constant. Joint stiffness in OA is associated with awakening or inactivity. If OA is suspected, plain x-rays should be taken of the most symptomatic joints. X-rays generally reveal marginal osteophytes, narrowing of the jointATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 space, increased density of the subchondral bone, subchondral cyst formation, bony remodeling, and joint effusions. Standing x-rays of knees are more sensitive in detecting jointspace narrowing. Magnetic resonance imaging (MRI) can be used to detect cartilage degeneration, and several MRI-based based scoring systems exist for characterizing the severity of OA (Hunter et al, PM R. 2012 May;4(5 Suppl) :S68-74).

[0049] OA commonly affects the hands, feet, spine, and the large weight-bearing joints, such as the hips and knees, although in theory any joint in the body can be affected. As OA progresses, the affected joints appear larger, are stiff and painful, and usually feel better with gentle use but worse with excessive or prolonged use. Treatment generally involves a combination of exercise, lifestyle modification, and analgesics. If pain becomes debilitating, joint-replacement surgery may be used to improve quality of life.

[0050] In addition to affecting humans, OA and joint degeneration also frequently impacts animals, including dogs, cats, horses, and other animals in which it can causes significant joint pain and dysfunction. Osteoarthritis (OA) is the most common form of arthritis in dogs, affecting approximately a quarter of the population. It is a chronic joint disease characterized by loss of joint cartilage, thickening of the joint capsule and new bone formation around the joint (osteophytosis) and ultimately leading to pain and limb dysfunction. The majority of OA in dogs occur secondarily to developmental orthopedic disease, such as cranial cruciate ligament disease, hip dysplasia, elbow dysplasia, OCD, patella (knee cap) dislocation. In a small subset of dogs, OA occurs with no obvious primary causes and can be related to genetic and age. Other contributing factors to OA in dogs include body weight, obesity, gender, exercise, and diet.

[0051] T reatment of OA includes, for example, pharmacologic treatment such as doxycycline, bisphosphonates, and licofelone. Other OA drugs and targets include, for example, inhibition of cartilage matrix degradation with MMP-inhibitor PG-1 16800; Cartilage matrix regeneration with Sprifermin, BMP-7, or OP-1 ; bisphosphonates; bone turnover with Zoledronic acid, Risedronate; AXS-02 (disodium zoledronate tetrahydrate); inhibition of bone degradation with Cathepsin K inhibitor MIV-711 ; inhibition of IL-1 with Anakinra (IL-1 receptor antagonist), AMG 108 (fully human monoclonal antibody to IL-1 R1 ), Lutikizumab (anti IL-1 a / p antibody); antitumor necrosis factor-alpha, e.g. Adalimumab, Etanercept, Infliximab; Hydroxychloroquine; inhibition of I-KB kinase with SAR1 13945 (l-kB kinase inhibitor); inhibition of p38 MAP kinase with FX-005; agents that act on Cox-2, e.g. metformin; agents that act on HMG-CoA reductase, e.g. simvastatin, atorvastatin, Fluvastatin, lovastatin, nystatin, pravastatin, rosuvastatin, and the like. Anti-inflammatories such as NSAIDs, opiates, intra-articular corticosteroids, and hyaluronic acid derivatives injected into the joint are also used.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0052] Early osteoarthritis. Assessment of OA may use the Kellgren Lawrence (KL) grading system (Kellgren and Lawrence, Ann. Rheum. Dis., 16:494-502, 1957, herein specifically incorporated by reference). The KL grading system relies on an anterior-posterior (AP) radiograph and is as follows: grade 0 = no features of OA; grade 1 = presence of OA is doubtful, presence of minute osteophyte(s), unchanged joint space; grade 2 = minimal OA, definite osteophyte(s), unchanged joint space; grade 3 = moderate OA, moderate diminution of joint space; grade 4 = severe OA, joint space greatly reduced with sclerosis of subchondral bone. For the purposes of the present invention, the KL score is less than 3, desirably less than 2, and in some embodiments is less than one. In some embodiments, the presence of early stages of arthritis is indicated by lack of definite joint space narrowing, lack of osteophytes (Kellgren-Lawrence Grade <2) but with positive results in at least one imaging marker, e.g. from an examination of one or more joints using noninvasive procedures including radiographic imaging and MRI for features including, for example, cartilage breakdown, decreased synovial space, and the like.

[0053] Individuals with pre-clinical or early-OA are those at increased risk of developing OA, as evidenced by biochemical, imaging, or clinical markers. Conditions or events that predispose to the development of OA include, without limitation, a history of injury to a joint; clinically or radiographically diagnosed meniscal injury with or without surgical intervention; a ligamentous sprain with clinically or radiographically diagnosed anterior or posterior cruciate or medial or lateral collateral ligament injury (Chu et al, Arthritis Res Ther. 2012 14(3):212. PMID: 22682469); clinically measured limb-length discrepancy; obesity with a current, or prolonged historical period of, BMI >27; or biomechanical features of abnormal gait or joint movement. In general, a determination of pre-clinical OA is associated with one or more, two or more, three or more parameters of joint pathology including, without limitation and relative to a healthy control sample, cartilage proteoglycan loss; cartilage damage; or elevated levels of degradative enzymes, the presence of products of cartilage or extracellular matrix degradation or bone remodeling. Humans at risk for OA, who have pre-OA, and who have early-stage OA are often asymptomatic, but a subset of patients experience joint pain due to cartilage injury (e.g. meniscal injury), ligamentous injury (e.g. tearing of the anterior cruciate ligament), or another joint abnormality.

[0054] MRI-detected imaging markers indicative of the presence of early or pre-clinical OA include cartilage edema, cartilage proteoglycan loss, cartilage matrix loss, bone marrow edema, articular cartilage fissures, articular cartilage degeneration, a meniscal tear, an anterior cruciate ligament tear, a posterior cruciate ligament tear, and other abnormalities of the cartilage or ligaments in the joint. Ultrasound will show evidence of cartilage edema or damage. Arthroscopy can allow direct detection or visualization of cartilage edema, cartilageATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 softening, cartilage thinning, cartilage fissures, cartilage erosion, or other cartilage abnormalities. Cartilage damage is frequently defined by the Outerbridge classification criteria or similar directly observed changes within the joint. For example, one such scoring system defines the presence of damage is as follows: grade 0= normal cartilage; grade I: softening and swelling of cartilage; grade II: a partial-thickness defect in the cartilage with fissures on the surface that do not reach subchondral bone or exceed 1 .5 cm in diameter; grade III: fissures in the cartilage that extend to the level of subchondral bone in an area with a diameter of more than 1.5 cm. Humans at risk for OA or with “pre-clinical OA” may be asymptomatic but may have signs of cartilage damage, meniscal damage, ligament damage, or other abnormalities of the joint.

[0055] The term "flow cytometry" as used herein refers to a method and a process whereby cells within a sample can be detected and identified when transversing past a detector within an apparatus containing a detecting source and a flowing apparatus, e.g. FACS and mass cytometry. Flow cytometry can provide an alternative analysis means, rather than mass cytometry.

[0056] Cells are characterized by their expression of cell surface markers. For several of these markers, the expression is “high” in level. While it is commonplace to refer to cells as “positive” or “negative” for a particular marker, actual expression levels are a quantitative trait. The number of molecules on the cell surface can vary by several logs, yet still be characterized as “positive”. Characterization of the level of staining permits subtle distinctions between cell populations.

[0057] The staining intensity of cells can be monitored by flow cytometry. Although the absolute level of staining may differ with a particular fluorochrome and antibody preparation, the data can be normalized to a control. In order to normalize the distribution to a control, each cell is recorded as a data point having a particular intensity of staining. These data points may be displayed according to a log scale, where the unit of measure is arbitrary staining intensity. In one example, the brightest cells in a bone marrow sample are designated as 4 logs more intense than the cells having the lowest level of staining. When displayed in this manner, it is clear that the cells falling in the highest log of staining intensity are bright, while those in the lowest intensity are negative. The “high” staining cells, which fall in the highest log of staining intensity, can have properties that are unique from the negative and positive cells.

[0058] Fluorescence-activated flow cytometry (FACs) combines flow cytometry, the measurement of cells as they flow in a liquid stream through a laser, with fluorescence detection, allowing for the identification and quantification of different cell populations. Cells orATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 particles are suspended in a fluid and passed through a narrow channel, creating a single-file stream of cells that can be analyzed one by one. As the cells pass through the flow cytometer, they are illuminated by lasers. These lasers provide the energy to excite fluorescent molecules (fluorophores) attached to the cells or particles. Cells are typically labeled with fluorophore- conjugated affinity reagents, e.g. antibodies, that bind to specific cell surface or intracellular markers.

[0059] Mass cytometry. Elemental mass spectrometry-based flow cytometry (mass cytometry) is a method to characterize single cells or particles with elemental metal isotopelabeled binding reagents. Because there are many stable metal isotopes available, and little overlap between measurement channels, dozens of molecules (parameters) can be readily measured. An example of a mass cytometer used to read the metal tags is an inductively- coupled plasma mass spectrometer (ICP-MS). In a typical workflow (similar to fluorescence based cytometry), cells are first incubated with antibodies / affinity binders conjugated to pure isotopes and subsequently the cell suspension is injected as a single cell stream into the mass cytometer. Single cell droplets are generated via nebulization and are carried by an argon gas stream into about 7500 degrees Kelvin plasma where each single cell is completely atomized and ionized. Thereby generated metal ions are then directed into a time-of-flight (TOF) mass spectrometer and the mass over charge ratio and number of metal ions is measured per cell and thereby the abundance of the target epitope / molecules.

[0060] As used herein, the term "elemental analysis" refers to a method by which the presence and / or abundance of elements of a sample are evaluated. "Capacitively coupled plasma" (CCP) means a source of ionization in which a plasma is established by capacitive coupling of radiofrequency energy at atmospheric pressure or at a reduced pressure (typically between 1 and 500 Torr) in a graphite or quartz tube.

[0061] "Mass spectrometer" means an instrument for producing ions in a gas and analyzing them according to their mass / charge ratio. "Microwave induced plasma" (MIP) means a source of atomization and ionization in which a plasma is established in an inert gas (typically nitrogen, argon or helium) by the coupling of microwave energy. The frequency of excitation force is in the GHz range. "Glow discharge" (GD) means a source of ionization in which a discharge is established in a low pressure gas (typically between 0.01 and 10 Torr), typically argon, nitrogen or air, by a direct current (or less commonly radiofrequency) potential between electrodes. "Graphite furnace" means a spectrometer system that includes a vaporization and atomization source comprised of a heated graphite tube. Spectroscopic detection of elements within the furnace may be performed by optical absorption or emission, or the sample may be transported from the furnace to a plasma source (e.g. inductively coupled plasma) for excitation and determination by optical or mass spectrometry.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0062] In some embodiments the methods utilize ICP-MS. In some embodiments the ICP-MS is performed with solution analysis, for example ELAN DRC II, Perkin-Elmer. In other embodiments the analysis is performed with a mass cytometer (e.g. CyTOF, DVS Sciences), which uses a nebulizer to administer a suspension of cells, beads, or other particles in a singleparticle stream to an ICP-MS chamber, thereby yielding single particle / cell data similar to a flow cytometer. Alternatively the analysis is performed by an elemental analysis-driven imaging system (e.g. laser ablation ICP-MS). Devices for such analytic methods are known in the art.

[0063] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0064] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome.

[0065] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, may include treatment of arthritis in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms.

[0066] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0067] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of osteoarthritis. AATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0068] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0069] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0070] A “sample” in the context of the present teachings refers to any biological sample that is isolated from a subject, generally a blood sample, which may comprise circulating immune cells. "Blood sample" can refer to whole blood or a fraction thereof, including blood cells, plasma, white blood cells or leucocytes. Samples can be obtained from a subject by means including but not limited to venipuncture, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0071] The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations.

[0072] Cells for use in the methods, as described above, may be collected from a sample from a subject or a donor, and may optionally may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0073] The collected and optionally enriched cell population may be used immediately or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0074] Enrichment for B cells. A patient sample may be enriched for B cells prior to analysis. B-cells are typically enriched by negative selection — in which B-cells remain “untouched” in their native state; or by positive selection, in which B-cells are labelled and actively removed from the sample. Commercially available kits can be used for this purpose, e.g. Negative B- cell isolation kits or Positive B-cell isolation kit from Miltenyi, StemCell, etc. Methods may include column chromatography, e.g. with magnetic labeled beads, FACS, etc. For example, memory B cells can be isolated by depletion of unwanted cells and subsequent positive selection with CD27 MicroBeads. Unwanted cells, for example, T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells are depleted using a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A), performing a first magnetic separation (depletion): LD or autoMACS® Columns and a magnetic separation (positive selection): MS or autoMACS Columns.

[0075] In another protocol, non-target cells are labeled with a cocktail of biotinylated CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin A), Anti-IgM, and Anti-lgD antibodies. These cells are subsequently magnetically labeled with Anti-Biotin MicroBeads. Highly pure switched memory B cells are obtained by depletion of the magnetically labeled cells.

[0076] A “dataset” 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 suchATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217 as a laboratory, or from a database or a server on which the dataset has been stored. Similarly, the term “obtaining a dataset associated with a sample” encompasses obtaining a set of data determined from at least one sample. Obtaining a dataset encompasses obtaining a sample, and processing the sample to experimentally determine the data, e.g., via measuring antibody binding, or other methods of quantitating a signaling response. The phrase also encompasses receiving a set of data, e.g., from a third party that has processed the sample to experimentally determine the dataset.

[0077] “Measuring” or “measurement” in the context of the present teachings refers to determining the presence, absence, quantity, amount, or effective amount of a cell population in a clinical or subject-derived sample, including the presence, absence, or concentration levels of such cells, and / or evaluating the values or categorization of a subject's clinical parameters based on a control, e.g. baseline levels of the cell population.

[0078] Classification can be made according to predictive modeling methods that set a threshold for determining the probability that a sample belongs to a given class. The probability is preferably at least 50%, or at least 60% or at least 70% or at least 80% or higher. Classifications also can be made by determining whether a comparison between an obtained dataset and a reference dataset yields a statistically significant difference, where the reference dataset may correspond to the results for a healthy control cell population. If so, then the sample from which the dataset was obtained is classified as not belonging to the reference dataset class. Conversely, if such a comparison is not statistically significantly different from the reference dataset, then the sample from which the dataset was obtained is classified as belonging to the reference dataset class.

[0079] Classification is the process of recognizing, understanding, and grouping ideas and objects into preset categories or “sub-populations.” Using pre-categorized training datasets, machine learning programs use a variety of algorithms to classify future datasets into categories. Classification algorithms in machine learning use input training data to predict the likelihood that subsequent data will fall into one of the predetermined categories. An analytic classification process may use any one of a variety of statistical analytic methods to manipulate the quantitative data and provide for classification of the sample. Examples of useful methods include linear discriminant analysis, recursive feature elimination, a prediction analysis of microarray, a logistic regression, a CART algorithm, a FlexTree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, machine learning algorithms; etc. Using any one of these methods, a protein distribution pattern may be used to generate a predictive model. In the generation of such a model, a dataset comprising control, and OA are used as a training set. A training set will contain data for one or more different distributions of interest. In some embodiments a decision tree is used to order classes on a precise level, forATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217 example with a random forest algorithm.

[0080] The predictive ability of a model can be evaluated according to its ability to provide a quality metric, e.g. AUG or accuracy, of a particular value, or range of values. In some embodiments, a desired quality threshold is a predictive model that will classify a sample with an accuracy of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or higher. As an alternative measure, a desired quality threshold can refer to a predictive model that will classify a sample with an AUG (area under the curve) of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[0081] As is known in the art, the relative sensitivity and specificity of a predictive model can be “tuned” to favor either the selectivity metric or the sensitivity metric, where the two metrics have an inverse relationship. The limits in a model as described above can be adjusted to provide a selected sensitivity or specificity level, depending on the particular requirements of the test being performed. One or both of sensitivity and specificity can be at least about at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[0082] The raw data may be initially analyzed by measuring the values for each marker, usually in triplicate or in multiple triplicates; and the cells may be clustered into populations, e.g. with flowSOM. The data may be manipulated, for example, raw data may be transformed using standard curves, and the average of triplicate measurements used to calculate the average and standard deviation for each patient. These values may be transformed before being used in the models, e.g. log-transformed, Box-Cox transformed (see Box and Cox (1964) J. Royal Stat. Soc., Series B, 26:211 —246), etc. The data are then input into a predictive model, which will classify the sample according to the state. The resulting information may be transmitted to a patient or health professional.

[0083] The term "specific binding member" as used herein refers to a member of a specific binding pair, i.e. two molecules, usually two different molecules, where one of the molecules through chemical or physical means specifically binds to the other molecule. For the purposes of the present invention, one of the molecules is an analyte as defined above, and generally the specific binding member is labeled for detection of fluorescence or elemental analysis, as known in the art.

[0084] The complementary members of a specific binding pair are sometimes referred to as a ligand and receptor; or receptor and counter-receptor. Specific binding indicates that the agent can distinguish a target antigen, or epitope within it, from other non-target antigens. It is specific in the sense that it can be used to detect a target antigen above background noiseATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 ("non-specific binding"). For example, a specific binding partner can detect a specific sequence or a topological conformation. A specific sequence can be a defined order of amino acids or a defined chemical moiety (e.g., where an antibody recognizes a phosphotyrosine or a particular carbohydrate configuration, etc.) which occurs in the target antigen. The term "antigen" is issued broadly, to indicate any agent which elicits an immune response in the body. An antigen can have one or more epitopes.

[0085] Binding pairs of interest include antigen and antibody specific binding pairs, complementary nucleic acids, peptide-MHC-antigen complexes and T cell receptor pairs, biotin and avidin or streptavidin; carbohydrates and lectins; complementary nucleotide sequences; peptide ligands and receptor; effector and receptor molecules; hormones and hormone binding protein; enzyme cofactors and enzymes; enzyme inhibitors and enzymes; and the like. The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc. so long as an epitope is present.

[0086] Immunological specific binding pairs include antigens and antigen specific antibodies; and T cell antigen receptors, and their cognate MHC-peptide conjugates. Suitable antigens may be haptens, proteins, peptides, carbohydrates, etc. Recombinant DNA methods or peptide synthesis may be used to produce chimeric, truncated, or single chain analogs of either member of the binding pair, where chimeric proteins may provide mixture(s) or fragment(s) thereof, or a mixture of an antibody and other specific binding members. Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies and their suitability for use as specific binding members are well- known to those skilled in the art.

[0087] A nucleic acid based binding partner such as an oligonucleotide can be used to recognize and bind DNA or RNA based analytes. The term "polynucleotide" as used herein may refer to peptide nucleic acids, locked nucleic acids, modified nucleic acids, and the like as known in the art. The polynucleotide can be DNA, RNA, LNA or PNA, although it is not so limited. It can also be a combination of one or more of these elements and / or can comprise other nucleic acid mimics.

[0088] Binding partners can be primary or secondary. Primary binding partners are those bound to the analyte of interest. Secondary binding partners are those that bind to the primary binding partner.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0089] In one embodiment analysis is performed on a mass cytometer, in which cells are introduced into a fluidic system and introduced into the mass cytometer one cell at a time. In one embodiment, cells are carried in a liquid suspension and sprayed into a plasma source by means of a nebulizer. In another embodiment, the cells may be hydrodynamically focused one cell at a time through a flow cell using a sheath fluid. In particular embodiments, the cells may be compartmentalized in the flow cell by introduction of an immiscible barrier, e.g., using a gas (e.g., air or nitrogen) or oil, such that the cell is physically separated from other cells that are passing through the flow cell. The cells may be compartmentalized prior to or during introduction of the cell into the flow cell by introducing an immiscible material (e.g., air or oil) into the flow path.

[0090] The general principles of mass cytometry, including methods by which single cell suspensions can be made, methods by which cells can be labeled, methods for atomizing particles and methods for performing elemental analysis on particles, as well as hardware that can be employed in mass cytometry, including flow cells, ionization chambers, reagents, mass spectrometers and computer control systems are known and are reviewed in a variety of publications including, but not limited to Bandura et al Analytical Chemistry 2009 81 6813- 6822), Tanner et al (Pure Appl. Chem 2008 80: 2627-2641), U.S. Pat. No. 7,479,630 (Method and apparatus for flow cytometry linked with elemental analysis) and U.S. Pat. No. 7,135,296 (Elemental analysis of tagged biologically active materials); and published U.S. patent application 20080046194, for example, which publications are incorporated by reference herein for disclosure of those methods and hardware.

[0091] The results of such analysis may be compared to results obtained from reference compounds, concentration curves, controls, etc. The comparison of results is accomplished by the use of suitable deduction protocols, Al systems, statistical comparisons, etc.

[0092] In particular embodiments, the method described above may be employed in a multiplex assay in which a heterogeneous population of cells is labeled with a plurality of distinguishably labeled binding agents (e.g., a number of different antibodies). After the population of cells is labeled, the cells are introduced into the flow cell, and individually analyzed using the method described above, where the viable cells are distinguished from non-viable cells by the presence of platinum derived from the viability reagent.

[0093] The analyte distribution pattern may be generated from a cell sample using any convenient protocol. The readout may be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement. The readout information may be further refined by direct comparison with the corresponding reference or control pattern. A pattern may be evaluated on a number of points: to determine if there is a statistically significant change at any point in the data matrix; whether the change is anATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 increase or decrease in prevalence of an isoform; and the like. The absolute values will display a variability that is inherent in live biological systems.Methods

[0094] Analysis of biological samples, e.g. blood-based samples, obtained from an individual is used to obtain a determination of changes in immune cell subsets, which changes are shown herein to be predictive of the presence of early OA.

[0095] The sample can be any suitable type that allows for the analysis of one or more cells, proteins and metabolites, preferably a blood sample. Samples can be obtained once or multiple times from an individual, e.g. a time course may be used to monitor high risk patients for changes in status. The cells can be separated from body samples by red cell lysis, centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, solid supports (magnetic beads, beads in columns, or other surfaces) with attached antibodies, etc.

[0096] A phenotypic profile of a population of cells is determined by measuring at a single cell level the presence of specific markers. It is understood that marker levels can exist as a distribution and that a marker used to classify a cell can be a particular point on the distribution but more typically can be a portion of the distribution. In some embodiments of the invention, different gating strategies can be used in order to analyze a specific cell population (e.g., only CD4+T cells) in a sample of mixed cell population. These gating strategies can be based on the presence of one or more specific surface markers. The following gate can differentiate between dead cells and live cells and the subsequent gating of live cells classifies them into, e.g. myeloid blasts, monocytes and lymphocytes. A clear comparison can be carried out by using two-dimensional contour plot representations, two-dimensional dot plot representations, and / or histograms.

[0097] The profiling measures the relative abundance in the B cell population of CD25hiswitched memory B cells, which may be characterized as CD27+lgD CD24highcells. The abundance of naive B cells may also be determined, naive B cells may be characterized as CD27 lgD+CXCR5+CD38+cells.

[0098] A population that is “expanded” relative to a healthy control population may be increased at least about 1 .5-fold, at least about 1 .75-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold or more. A population that is depleted relative to a healthy control population may be decreased at least about 1 .25-fold, at least about 1 .5-fold, at least about 1 .75-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold or more.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0099] Samples may be obtained at one or more time points. Where a sample at a single time point is used, comparison is made to a reference “base line” level for the feature, which may be obtained from a training set data as disclosed herein.

[0100] In some embodiments a B cell population is assessed for upregulation more or more of genes selected from LINC01619, CMIP, BCL2, HIVEP2, VOPP1 , TNRC6C, IQCN, ABCG1 , RASGRP1 , ETV6, EEF2K, PALM2AKAP, SMAD3, GALNT2, ECE1 , KDM6B, CD96, FMNL1 , TET3 and EXT1 , which genes are upregulated in OA B cell populations. Changes in gene expression may be increased at least about 1 .5-fold, at least about 1 .75-fold, at least about 2- fold, at least about 2.5-fold, at least about 3-fold or more.

[0101] In some embodiment, the methods of the invention include the use of liquid handling components. The liquid handling systems can include robotic systems comprising any number of components. In addition, any or all of the steps outlined herein can be automated; thus, for example, the systems can be completely or partially automated. See USSN 61 / 048,657. As will be appreciated by those in the art, there are a wide variety of components which can be used, including, but not limited to, one or more robotic arms; plate handlers for the positioning of microplates; automated lid or cap handlers to remove and replace lids for wells on noncross contamination plates; tip assemblies for sample distribution with disposable tips; washable tip assemblies for sample distribution; 96 well loading blocks; cooled reagent racks; microtiter plate pipette positions (optionally cooled); stacking towers for plates and tips; and computer systems.

[0102] Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications. This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration. These manipulations are cross-contamination- free liquid, particle, cell, and organism transfers. This instrument performs automated replication of microplate samples to filters, membranes, and / or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.

[0103] In some embodiments, platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity. This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipetteATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 tips, and an active wash station. In some embodiments, the methods of the invention include the use of a plate reader.

[0104] In some embodiments, interchangeable pipet heads (single or multi-channel) with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms. Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.

[0105] In some embodiments, the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay. In some embodiments, useful detectors include a mass cyometer; and a computer workstation.

[0106] In some embodiments, the robotic apparatus includes a central processing unit which communicates with a memory and a set of input / output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this can be in addition to or in place of the CPU for the multiplexing devices of the invention. The general interaction between a central processing unit, a memory, input / output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.

[0107] Individuals may be treated appropriately for a diagnosis of early stage osteoarthritis. Osteoarthritis treatment goals are relieving pain, maintaining joint flexibility, and optimizing joint and overall function. Primary treatments include physical measures that involve rehabilitation; support devices; exercise for strength, flexibility, and endurance; patient education; and modifications in activities of daily living. Adjunctive therapies include drug treatment and surgery.

[0108] Moderate weight loss in patients with overweight often reduces pain and may even reduce progression of knee osteoarthritis. Rehabilitation techniques are best begun before disability develops. Exercises (range of motion, isometric, isotonic, isokinetic, postural, strengthening) maintain range of motion and increase the capacity for tendons and muscles to absorb stress during joint motion. Exercise can sometimes arrest or even reverse hip and knee osteoarthritis. Aquatic exercises are recommended because they spare the joints from stress. Stretching exercises should be done daily. In osteoarthritis of the spine, knee, or thumb carpometacarpal joint, various supports can relieve pain and increase function, but to preserve flexibility, they should be accompanied by specific exercise programs. For medial knee osteoarthritis, orthoses designed to reduce knee load are preferred to lateral wedge insoles, which have yielded equivocal outcomes. In erosive osteoarthritis, range-of-motion exercises done in warm water can help prevent contractures.

[0109] Drug therapy is an adjunct to the physical program. Acetaminophen in dosages of up to 1 g orally 4 times a day may relieve pain and is generally safe in the absence of hepaticATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 disease or considerable alcohol intake. More potent analgesics, such as tramadol or rarely opioids, may be required; however, these medications can cause confusion in older patients and are generally avoided. Duloxetine, a serotonin norepinephrine reuptake inhibitor, may modestly reduce pain caused by osteoarthritis. Topical capsaicin has been helpful in relieving pain in superficial joints by disrupting pain transmission.[001 10] Nonsteroidal anti-inflammatory drugs (NSAIDs), including selective cyclooxygenase-2 (COX-2) inhibitors or coxibs, may be considered if patients have refractory pain or signs of inflammation (eg, redness, warmth). NSAIDs may be used simultaneously with other analgesics (eg, tramadol, rarely opioids) to provide better relief of symptoms. Topical NSAIDs may be of value for superficial joints, such as the hands and knees. Topical NSAIDs may be of particular value in older patients, because systemic NSAID exposure is reduced, minimizing risk of drug adverse effects. Gastric protection should be considered when using NSAIDs on a regular basis in older patients.[001 11] Muscle relaxants such as cyclobenzaprine, metaxalone, and methocarbamol (usually in low doses) occasionally relieve pain that arises from muscles strained by attempting to support osteoarthritis joints, yet strong evidence is lacking unless there is coexistent central sensitization. In older patients, however, they may cause more adverse effects than relief.[001 12] Hyaluronic acid formulations can be injected into the knee and provide some pain relief in some patients for prolonged periods of time. They should not be used more often than every 6 months. The treatment is a series of 1 to 5 weekly injections.[001 13] Glucosamine sulfate 1500 mg orally once / day has been suggested to relieve pain and slow joint deterioration; chondroitin sulfate 1200 mg once / day has also been suggested for pain relief.[001 14] Other adjunctive measures may reduce pain, including massage, heating pads, weight loss, acupuncture, and transcutaneous electrical nerve stimulation (TENS). Laminectomy, osteotomy, and total joint replacement should be considered if nonsurgical approaches fail.[001 15] Disease-modifying anti-rheumatic drugs (DMARDs) may improve articular pain and functional disability in OA patients. The most common conventional DMARDs are methotrexate, sulfasalazine, hydroxychloroquine, and leflunomide. Biological therapies include adalimumab, the IL-6R inhibitor tocilizumab, interleukin 1 inhibitors, etanercept, etc. Targeted synthetic DMARDs, include, for example, the JAK inhibitors baricitinib and tofacitinibData Analysis[001 16] The abundance of altered B cell populations can be generated from a biological sample using any convenient protocol, for example as described below, and may use a device or kit designed for that purpose. The readout can be a mean, average, median or the variance orATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 other statistically or mathematically-derived value associated with the measurement. The marker readout information can be further refined by direct comparison with the corresponding reference or control pattern. A population distribution pattern can be evaluated on a number of points: to determine if there is a statistically significant change at any point in the data matrix relative to a reference value; whether the change is an increase or decrease in the population frequency; and the like. The absolute values obtained for each marker under identical conditions will display a variability that is inherent in live biological systems and also reflects the variability inherent between individuals.[001 17] Following obtainment of the signature pattern from the sample being assayed, the signature pattern can be compared with a reference or base line profile to make a prognosis regarding the phenotype of the patient from which the sample was obtained / derived.[001 18] In certain embodiments, the obtained signature pattern is compared to a single reference / control profile to obtain information regarding the phenotype of the patient being assayed. In yet other embodiments, the obtained signature pattern is compared to two or more different reference / control profiles to obtain more in depth information regarding the phenotype of the patient. For example, the obtained signature pattern can be compared to a positive and negative reference profile to obtain confirmed information regarding whether the patient has the phenotype of interest.[001 19] The data can be subjected to non-supervised hierarchical clustering to reveal relationships among profiles. For example, hierarchical clustering can be performed, where the Pearson correlation is employed as the clustering metric. One approach is to consider a patient disease dataset as a “learning sample” in a problem of “supervised learning”. CART is a standard in applications to medicine (Singer (1999) Recursive Partitioning in the Health Sciences, Springer), which can be modified by transforming any qualitative features to quantitative features; sorting them by attained significance levels, evaluated by sample reuse methods for Hotelling's T2statistic; and suitable application of the lasso method. Problems in prediction are turned into problems in regression without losing sight of prediction, indeed by making suitable use of the Gini criterion for classification in evaluating the quality of regressions.

[0120] Other methods of analysis that can be used include logistic regression. One method of logic regression Ruczinski (2003) Journal of Computational and Graphical Statistics 12:475- 512. Logic regression resembles CART in that its classifier can be displayed as a binary tree. It is different in that each node has Boolean statements about features that are more general than the simple “and” statements produced by CART.

[0121] Another approach is that of nearest shrunken centroids (Tibshirani (2002) PNAS 99:6567-72). The technology is k-means-like, but has the advantage that by shrinking clusterATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 centers, one automatically selects features (as in the lasso) so as to focus attention on small numbers of those that are informative. The approach is available as Prediction Analysis of Microarrays (PAM) software, a software “plug-in” for Microsoft Excel, and is widely used. Two further sets of algorithms are random forests (Breiman (2001 ) Machine Learning 45:5-32 and MART (Hastie (2001 ) The Elements of Statistical Learning, Springer). These two methods are already “committee methods.” Thus, they involve predictors that “vote” on outcome. Several of these methods are based on the “R” software, developed at Stanford University, which provides a statistical framework that is continuously being improved and updated in an ongoing basis.

[0122] Other statistical analysis approaches including principle components analysis, recursive partitioning, predictive algorithms, Bayesian networks, random forest, and neural networks.

[0123] The analysis and database storage can be implemented in hardware or software, or a combination of both. In one embodiment of the invention, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention. Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, clinical trial analysis, and the like. Preferably, the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.

[0124] Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language. Each such computer program is preferably stored on a storage media or device readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0125] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern.

[0126] The signature patterns and databases thereof can be provided in a variety of media to facilitate their use. “Media” refers to a manufacture that contains the signature pattern information of the present invention. The databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic / optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present database information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.Kits

[0127] In some embodiments of the invention a device or kit is provided for the analysis of patient samples, e.g. from an individual suspected of having osteoarthritis. Such devices or kits comprise reagents that specifically identify one or more cell populations, including without limitation CD25hiCD24hiCD27+lgD_B cells, from a patient sample. The reagents can be provided in isolated form, or pre-mixed as a cocktail suitable for the methods of the invention. A kit can include instructions for using the plurality of reagents to determine data from the sample; and instuctions for statistically analyzing the data. The kits may be provided in combination with a system for analysis, e.g. a system implemented on a computer. Such a system may include a software component configured for analysis of data obtained by the methods of the invention.

[0128] In an embodiment, a device or kit comprises reagents for the enrichment of B cells from a patient blood sample, e.g. using flow cytometry, magnetic bead selection, etc. For example, a bulk selection for expression of B cell marker(s) may be performed on a population of peripheral blood monocytic cells (PBMC). The B cell enriched population is then analyzed at a single cell level for the presence of switched B cells that are CD27+CD25hi, e.g. with aATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 device or kit comprising affinity reagents specific for CD27 and for CD25, and the representation of such cells in the B cell population is determined. In some embodiment flow cytometry or mass cytometry is used to perform single cell profiling of the sample. Affinity reagents are optionally labeled for use in flow or mass cytometry. Alternatively microscopy can be used for single cell profiling. In other embodiments the enriched B cell population is analyzed for expression of genetic markers characteristic of the switched memory B cell population. Optionally, the B cell population is further analyzed for the antigenic specificity of the switched memory B cells. It will be understood by one of skill in the art that cells express multiple proteins on their surface that can form the basis for phenotyping, and that these proteins provide a useful subset of possible markers for phenotyping.

[0129] In an embodiment, a device or kit comprises reagents for a one-step analysis of a patient sample to determine whether there is an expansion of CD25hiswitched memory B cells. Such a device or kit may comprise affinity reagents that identify B cells, e.g. reagents specific for a pan-B cell marker such as CD20, CD19, etc; or reagents specific for switched B cells, e g. anti-IgG. Alternative the kit comprises reagents for negative selection of B cells, e.g. with reagents specific for immune cells other than B cells. The devide or kit further comprises reagents specific for CD27 and for CD25. In some embodiment flow cytometry or mass cytometry is used to perform single cell profiling of the sample.

[0130] In some embodiments reagents are provided for assessing the expression of more or more of genes selected from LINC01619, CMIP, BCL2, HIVEP2, VOPP1 , TNRC6C, IQCN, ABCG1 , RASGRP1 , ETV6, EEF2K, PALM2AKAP, SMAD3, GALNT2, ECE1 , KDM6B, CD96, FMNL1. TET3 and EXT1.

[0131] Kits provided by the invention can comprise one or more labeling elements. Nonlimiting examples of labeling elements include small molecule fluorophores, proteinaceous fluorophores, radioisotopes, enzymes, antibodies, chemiluminescent molecules, biotin, streptavidin, digoxigenin, chromogenic dyes, luminescent dyes, phosphorous dyes, luciferase, magnetic particles, beta-galactosidase, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, quantum dots , chelated or caged lanthanides, isotope tags, radiodense tags, electron- dense tags, radioactive isotopes, paramagnetic particles, agarose particles, mass tags, e-tags, nanoparticles, and vesicle tags.

[0132] Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and / or summaries of these and the like, which indicate or establish the activities and / or advantages of the composition, and / or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on humanATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 clinical trials. Kits described herein can be provided, marketed and / or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.Reports

[0133] In some embodiments, providing an evaluation of a subject for a classification, diagnosis, prognosis, theranosis, and / or prediction of early OA includes generating a written report that includes the artisan’s assessment of the subject’s state of health, including, for example, a “diagnosis assessment”, of the subject’s prognosis, i.e. a “prognosis assessment”, and / or of possible treatment regimens, i.e. a “treatment assessment”. Thus, a subject method may further include a step of generating or outputting a report providing the results of an assessment, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).

[0134] A “report,” as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to a diagnosis assessment, a prognosis assessment, and / or a treatment assessment and its results. A subject report can be completely or partially electronically generated. A subject report includes at least a diagnosis assessment, and / or a suggested course of treatment to be followed. A subject report can further include one or more of: 1 ) information regarding the testing facility; 2) service provider information; 3) subject data; 4) sample data; 5) an assessment report, which can include various information including: a) test data, where test data can include an analysis of cellular signaling responses to activation, b) reference values employed, if any.

[0135] The report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and / or data generation was conducted. This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted the assay and / or who entered the input data, the date and time the assay was conducted and / or analyzed, the location where the sample and / or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like. Report fields with this information can generally be populated using information provided by the user.

[0136] The report may include information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and / or data generation. Report fields with this information canATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu). Other service provider information in the report can include contact information for technical information about the result and / or about the interpretive report.

[0137] The report may include a subject data section, including subject medical history as well as administrative subject data (that is, data that are not essential to the diagnosis, prognosis, or treatment assessment) such as information to identify the subject (e.g., name, subject date of birth (DOB), gender, mailing and / or residence address, medical record number (MRN), room and / or bed number in a healthcare facility), insurance information, and the like), the name of the subject's physician or other health professional who ordered the susceptibility prediction and, if different from the ordering physician, the name of a staff physician who is responsible for the subject's care (e.g., primary care physician).

[0138] The report may include a sample data section, which may provide information about the biological sample analyzed, such as the source of biological sample obtained from the subject (e.g. blood, type of tissue, etc.), how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as pre-scripted selections (e.g., using a drop-down menu).

[0139] The report may include an assessment report section, which may include information generated after processing of the data as described herein. The interpretive report can include a prognosis of the likelihood that the patient will develop preeclampsia. The interpretive report can include, for example, results of the analysis, methods used to calculate the analysis, and interpretation, i.e. prognosis. The assessment portion of the report can optionally also include a Recommendation(s).

[0140] It will also be readily appreciated that the reports can include additional elements or modified elements. For example, where electronic, the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report. For example, the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database. This latter embodiment may be of interest in an in-hospital system or in-clinic setting. When in electronic format, the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.

[0141] It will be readily appreciated that the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient toATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 provide the analysis requested by the user (e.g., a diagnosis, a prognosis, or a prediction of responsiveness to a therapy).Screening Assays

[0142] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0143] Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable forthis invention are those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).

[0144] Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water,ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.

[0145] The term samples also includes the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 ml to 1 ml of a biological sample is sufficient.

[0146] Candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0147] Agents are screened for biological activity by adding the agent to at least one and usually a plurality of cells of interest to form a panel, usually in conjunction with cells lacking the agent. The change in parameter readout in response to the agent is measured, desirably normalized, and the resulting analysis may then be evaluated by comparison to reference readouts. The reference readouts may include basal readouts in the presence and absence of the factors, readouts obtained with other agents, which may or may not include known inhibitors of known pathways, etc. Agents of interest for analysis include any biologically active molecule with the capability of modulating, directly or indirectly, the phenotype of interest of a cell of interest.

[0148] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compoundATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0149] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0150] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0151] Various methods can be utilized for quantifying the presence of the selected markers. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity Fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to autofluoresce, e.g. by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12):477-81).

[0152] Multiple labels, e.g. mass labels and / or fluorescent labels, can be used on the same sample and individually detected quantitatively, permitting measurement of multiple cellular responses simultaneously. Many quantitative techniques have been developed to harness the unique properties of fluorescence including: direct fluorescence measurements, fluorescence resonance energy transfer (FRET), fluorescence polarization or anisotropy (FP), time resolved fluorescence (TRF), fluorescence lifetime measurements (FLM), fluorescence correlation spectroscopy (FCS), and fluorescence photobleaching recovery (FPR) (Handbook of Fluorescent Probes and Research Chemicals, Seventh Edition, Molecular Probes, Eugene Oreg.).ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0153] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0154] Flow or mass cytometry may be used to quantitate parameters such as the presence of cell surface proteins or conformational or posttranslational modification thereof; intracellular or secreted protein, where permeabilization allows antibody (or probe) access, and the like. Brefeldin A is commonly utilized to prevent secretion of intracellular substances. Flow cytometry methods are known in the art, and described in the following: Flow Cytometry and Cell Storing (Springer Lab Manual), Radbruch, Ed., Springer Verlag, 2000; Ormerod, Flow Cytometry, Springer Verlag, 1999; Flow Cytometry Protocols (Methods in Molecular Biology, No 91 ), Jaroszeski and Heller, Eds., Humana Press, 1998; Current Protocols in Cytometry, Robinson et al., eds, John Wiley & Sons, New York, N.Y., 2000. The readouts of selected parameters are capable of being read simultaneously, or in sequence during a single analysis, as for example through the use of fluorescent antibodies to cell surface molecules. As an example, these can be tagged with different fluorochromes, fluorescent bead, tags, e.g. quantum dots, etc., allowing analysis of up to 4 or more fluorescent colors simultaneously by flow cytometry. Plug-flow flow cytometry that has the potential to automate the delivery of small samples from unpressurized sources at rates compatible with many screening and assay applications, may allow higher throughput, compatible with high throughput screening, Edwards et al. (1999) Cytometry 37:156-9.ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217

[0155] Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Pat. No. 5,989,833 issued Nov. 23, 1999.

[0156] The quantitation of nucleic acids, especially messenger RNAs, is also of interest as a parameter. These can be measured by hybridization techniques that depend on the sequence of nucleic acid nucleotides. Techniques include polymerase chain reaction methods as well as gene array techniques. See Current Protocols in Molecular Biology, Ausubel et al., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999) Biotechniques 26(1 ):112- 225; Kawamoto et al. (1999) Genome Res 9(12):1305-12; and Chen et al. (1998) Genomics 51 (3) :313-24, for examples.EXPERIMENTAL

[0157] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of howto make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.EXAMPLE 1Multi-parametric profiling of circulating immune cells identifies an expansion of CD25hiswitched memory B cells in Osteoarthritis

[0158] Osteoarthritis (OA) is a chronic, debilitating disease with no available diseasemodifying drugs. Biomarker identification in OA patients has hitherto been limited to serum proteins and bulk epigenomic feature identification. Peripheral blood mononuclear cells (PBMC) from 21 healthy donors, 17 OA patients, and 10 patients with degenerative meniscal tears (DMT) were immunophenotyped at single-cell resolution by mass cytometry by time-of- flight (cyTOF) using a 29-marker panel to identify OA-associated features in the circulating immune cells. Single-cell RNA sequencing was utilized to discern mechanistic attributes of perturbed immune populations in OA. A comparison of healthy donors and OA patients’ PBMC revealed distinct perturbations in OA. While subsets of naive B cells were depleted, switchedATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 memory B cells were significantly expanded in OA including a CD25hiCXCR5hiCD27+lgD_subpopulation. Single cell-RNA sequencing revealed IL2 / Stat3 and TNF signaling dysfunction in the CD25hi-switched memory B cells in OA. A similar expansion of CD25hiswitched memory B cells was observed in patients with DMT, a population at enhanced risk of OA. CD25hiswitched memory B cells were identified as a cellular biomarker for early and late OA in readily accessible blood.Materials and Methods

[0159] Study design. The primary objective of the study was to profile immune cells in the peripheral blood of OA patients to highlight differences in their immune populations compared to healthy individuals. The next aim was to investigate the alignment of the immune landscape observed in OA to the immune profile of patients with degenerative meniscal tears (DMT), considered at-risk of developing OA post-injury. To this end, peripheral blood mononuclear cells (PBMC) were isolated from clinically diagnosed OA patients with radiographic evidence of OA (n=17), healthy individuals (n=21 ) with no known history of OA, and DMT patients before corrective surgery (Tables 1 and 2). An additional cohort of PBMCs from healthy individuals (n = 6) and OA patients (n = 5) was collected to validate our findings from the initial datasets (Tables 3 and 4). Radiographic severity of OA in the OA patient cohort ranged from 1 -4 on the modified Kellgren / Lawrence score (KL) score recommended by OARSI in at least one region i.e medial, lateral and patellofemoral regions of the joint (0 being normal). No treatment required blinding, and the unblinded grades were prospectively assigned as the patient was enrolled. Subjects in the OA cohort were symptomatic as evidenced by KOOS and WOMAC scores on pain and function (Table 2). PBMCs were profiled by mass cytometry using a 29- marker mass cytometry panel (Human Immune Monitoring Panel Kit, catalog number 201234, Standard Bio Tools). Immune profiles of healthy (n=21), OA (n=17), and DMT (n=10) were analyzed together, as the original cohort, to identify differentially abundant populations. The same 29-marker mass cytometry panel was used to confirm the findings from the original cohort in the validation cohort comprising of healthy individuals (n = 6) and OA patients (n = 5).

[0160] Peripheral blood collection and PBMC isolation. The whole blood of patients diagnosed with either OA or DMT was collected by clinicians. The protocols, policies, and human sample collection methods were approved by the Stanford University Institutional Review Board (IRB- 31943) and informed consent was obtained from all participants. The whole blood of healthy donors was procured from Stanford Blood Center. All blood samples were collected in EDTA vacutainer tubes and processed to isolate PBMCs within 6 hours of blood draw. PBMCs wereATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 isolated by Ficoll-based density gradient centrifugation. Briefly, whole blood was centrifuged at 200g for 10minutes at room temperature to remove plasma in the supernatant. Plasmadeficient blood was diluted with an equal volume of phosphate-buffered saline (PBS) free of Ca++and Mg++ions and layered over Ficoll-Paque Plus (density 1 .077 g / mL, GE Healthcare). Density gradient centrifugation of blood layered over Ficoll was conducted at 1000g for 15 minutes with minimum acceleration and zero deceleration settings at room temperature. The straw-colored buffy coat was carefully collected and treated with ACK lysing buffer (Gibco) to remove red blood cells. PBMCs were washed thrice with PBS, centrifuged, the PBMC pellet was frozen in FBS+20% DMSO and stored in liquid Nitrogen until staining for mass cytometry.

[0161] PBMC staining and mass cytometry. All staining and barcoding steps were performed using buffers, reagents, and protocols provided by Fluidigm. Briefly, frozen PBMCs were thawed in a 37 °C water bath, and approximately 3 x106cells per sample were stained with 10 LIM cisplatin to stain dead cells for 5 minutes at room temperature. Cisplatin was quenched using a serum-containing RPMI medium and washed in Maxpar cell staining buffer. PBMCs were fixed in Fix I buffer and permeabilized in 1 X barcode perm buffer before barcoding using cell-ld 20-plex barcoding kit (Fluidigm) per manufacturer’s instructions. Once barcoded, the PBMCs from all samples were combined into one tube and stained for 29 surface markers using metal-conjugated antibodies provided in the Maxpar human immune cell monitoring panel kit (catalog no. 210324, Fluidigm) following manufacturer’s guidelines. Finally, stained PBMCs were labeled with cell-ID intercalator (dilution 1 :1000, Fluidigm) to stain DNA. To normalize signal over runtime, the PBMCs were diluted in EQ four-element calibration beads (Fluidigm) diluted with water (1 :10) before injection into cyTOF2 mass cytometer using the supersampler, housed at Shared FACS facility at Beckman Center, Stanford University. As only 20 samples could be barcoded and run at a time, 4 batches of barcoding were performed and run at different times in the same mass cytometer. One common sample was included in all four batches to normalize batch effects.

[0162] Data cleaning, normalization, and de-barcoding. Data were acquired in fcs files. Signal normalization over runtime for each run was performed using Normalizer vO.3. All fcs files were concatenated and de-barcoded using ‘Premessa’ package in R software. Channel data was arcsine transformed and batch normalized across three runs concerning the common sample using BatchAdjust() command-line application in R. Files were then uploaded into the cloudbased platform Cytobank for gating, clustering, and visualization.

[0163] Traditional phenotyping, hierarchical clustering by FlowSOM and UMAP projections. CD45+CD66b_cell population gated upon cisplatin-negative viable cells were classified into major immune populations and their subtypes using manual gating strategy provided byATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 Fluidigm (FIG. 6). Unsupervised hierarchical clustering by FlowSOM (Cytobank) was used to identify subpopulations of major immune cell types from gated parent populations with optimized metacluster and cluster inputs namely: (a) CD45+CD66b CD14 CD3+CD8 CD4+population CD4T cell subpopulations (20 metaclusters and 64 clusters); (b) CD45+CD66b_CD14- CD3+CD4 CD8+ population CD8T cell subpopulations (10 metaclusters and 25 clusters); (c) CD45+CD66b CD14 CD16 CD161 CD3 CD19+population B cell subpopulations (10 metaclusters and 25 clusters); and (d) CD45+CD66b CD20 CD19 CD3 CD14+ population monocyte subpopulations (5 metaclusters and 9 clusters). All events (cells) per sample were utilized for FlowSOM analyses and abundances were represented as percent of total cells analyzed per sample. UMAP of manually gated populations and FlowSOM clusters were created using equal downsampled files in the Cytobank platform with perplexity = 60 and theta = 0.5.

[0164] B cell isolation. PBMC samples from OA patients and healthy donors (n = 6 per group) that were previously profiled using CyTOF were enriched for B-cells using the magneticactivated cell sorting (MACS) bead isolation kit from (Human Pan B cell isolation kit 130-101 - 638, Miltenyi Biotec) according to manufacturer’s instructions.

[0165] Single cell (sc) RNA library preparation, sequencing and analyses. Cells were fixed and cDNA libraries were generated using the Evercode WT Mini v1 kit (Parse Biosciences, Seattle) according to manufacturer’s instructions. 3 samples each were pooled to obtain 2 single cell cDNA libraries per group. Sequencing files were processed using the Parse Biosciences Pipeline (v1 .0.3) and aligned to the human GRCh38 genome. The count matrix was loaded into R (v4.3.0) using the Seurat package (v4.3.0.1) to cluster, visualize and analyze the dataset. 4,018 cells with a median of 7,861 transcripts (and 2,706 genes) per cell were successfully recovered from sequencing after filtering out low quality reads. While median mitochondrial read percentage was 3.2%, we filtered out cells with above 15% mitochondrial reads. Further cells with less than 5000 features and 20,000 transcripts were retained for downstream analysis. Detailed analysis procedures are made available in the code uploaded. Sc RNA sequencing data was deposited in GEO data repository and can be accessed using GSE253539 accession number.

[0166] Statistics. Spearman’s correlation was performed across demographic and patient- reported outcome measures and immune cell populations to identify significant correlations (p<0.05). Statistical differences between means of immune cell populations and cluster frequencies were first controlled for age and sex-associated effects by non-parametric Quade test using SPSS software. Wilcoxon rank-sum test was used to measure significance when comparing two groups. When more than two groups were compared, pairwise comparisonsATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 were calculated by Dunnet’s test for groups with unequal variances after correcting for multiple hypotheses by the Dunnett T3 test. Graphical data and heatmaps were created using RStudio software. Half boxplots were used for the graphical representation of cyTOF data using gghalves and ggplot2 libraries in RStudio. Dots in the half boxplots represent the distribution of individual samples in a cohort. The line inside the boxplot is the median at the 50thpercentile, and upper and lower whiskers denote data within 1.5 times the interquartile range under the 75thand 25thpercentiles, respectively. Correlation plots were generated using corrplot, ’ PGA using ‘factoextra,’ and heatmap using the ‘pheatmap’ package in R.Results

[0167] Peripheral blood mononuclear cells (PBMC) were isolated from OA patients with evident radiographic OA (n=17; 33-70 years) and healthy individuals with no known history of OA (n=21 ; 42-76 years). The OA patients had radiographic and symptomatic OA as shown by modified KL scores (OARSI) majorly between 2-4 in one of the three knee joint compartments, i.e. , medial, lateral, or patellofemoral compartment (Table 1). The mean age of the OA cohort, 55.59 ± 10.81 years, was comparable to that of the healthy cohort at 57.29 ± 10.31 years. Patient-reported outcomes measured by Knee Injury and Osteoarthritis Outcome Score (KOOS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), as listed in symptoms.

[0168] Traditional immunophenotyping identifies significant differences between healthy and OA PBMC. To identify whether any immune cell populations were differentially populated in the circulating blood of OA patients, we utilized cytometry by time-of-flight (cyTOF), a massspectrometry-based high-dimensional method for single-cell detection of isotope-labeled antibodies. Peripheral blood mononuclear cells (PBMC) were isolated and stained with a 29- marker antibody panel (Fluidigm) to identify major immune cell populations by cyTOF (Fig. 1 A, Table 1 and 2). Before using algorithmic analyses, we employed a traditional manual gating strategy (Supplementary FIG. 6) for immunophenotyping and identifying known immune populations in healthy and OA individuals (Fig 1A, Fig S1).

[0169] Correlations between the individual subtypes of major immune populations and demographic data (sex, age, Kellgren / Lawrence scores, also known as KL scores for radiographic severity of OA, WOMAC subscales, and KOOS scores) were explored to identify any evident trends (Fig 1 B, Table 1 ). Age and sex information was available for all individuals in the healthy and OA cohorts; clinical joint pathology and pain information was included for the OA and DMT cohorts. While no associations were observed between immune cell frequencies and sex, radiographic severity of OA in the medial region of the joint, asATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 determined by the modified KL scores, was found to be negatively correlated with Th2 Th17 cells but positively correlated with switched memory B cells, resting memory B cells, plasmoblasts, transitional B cells, memory Treg cells and central memory (CM) CD4T cells. KL score of the lateral region of the joint was negatively correlated with Th2 cells but positively correlated with switched memory and memory resting B cells (Fig 1 B).

[0170] Naive subsets of CD8T and CD4T, along with Th1 cells, myeloid dendritic cells (mDCs), and memory-resting B cells, were shown to be negatively correlated with age. Among the patient-reported outcomes, WOMAC stiffness was positively correlated with naive CD4T cells, while WOMAC pain was negatively correlated with activated Treg cells. No significant correlation of immune cells with BMI, except for CD16- NK cells, was observed. Given the observed correlations between multiple immune cells and either age or sex, we controlled for age and sex-associated immune populations and their subtypes resolved into distinct populations by Uniform manifold approximation and projection (UMAP) (Fig 1 C). No significant differences were noted between healthy and OA PBMC in the proportion and frequencies of the major immune cell populations, namely, CD14+ monocytes, NK cells, dendritic cells, CD4T cells, CD8T cells, and B cells (FIG. 7). However, the mean frequencies of switched memory B cells (CD20+CD27+lgD-), resting memory B cells, memory Treg cells (CD25+CD127- CCR4+CD45RA-CD45RO+), activated Treg and central memory (CM) CD4T cells (CD4+CCR7+CD45RO+CD45RA-) were found to be significantly expanded in the OA cohort compared to healthy (Fig 1 E).

[0171] Differential abundance of T cell repertoire in OA. A comprehensive analysis via unsupervised hierarchical clustering by the FlowSOM algorithm identified distinct immune cell subpopulations that could not be distinguished by traditional markers used in manual gating (Fig 8A). With FlowSOM analyses, fluctuations in the classical monocyte clusters were observed in OA, where CD127+ classical monocyte cluster 3 was expanded however, CD14|OWand CD25+classical monocyte clusters 2 and 6, respectively, were depleted (Fig 8B-C). Cluster 1 belonging to central memory (CM) CD8T cell was expanded while cluster 7 of the naive CD8T cell population was depleted (Fig 8D-F) in OA. FlowSOM analysis of the CD4T cell population identified differentially abundant clusters of naive (CD4+CCR7+CD45RO- CD45RA+), effector memory (EM) phenotypes (CD4+CCR7- CD45RO+CD45RA ), and Th subsets in OA patients when compared to healthy cohort. Naive CD4T cell clusters 4 and 14 and EM CD4T cell clusters 37, 41 , 42, 51 , and 58 were expanded in OA (Fig 9A-D). On the other hand, Th2 cluster 26, naive CD4T clusters 15 and 21 , and EM CD4T clusters 43 and 8 were depleted in OA (Fig 9E-G). Interestingly, we note that CD56 was expressed by all the naive and EM CD4T cell clusters that were depleted in the OA cohort compared to those thatATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 were expanded (Fig 9C). While these differences were interesting, in principal component analysis (PCA) analysis, the healthy and OA cohorts overlapped in the respective CD4T repertoires. Thus, the CD4T subsets could not explicitly distinguish OA from healthy cohorts (Fig 9H, I).

[0172] FlowSOM identifies perturbations in the naive and memory B cell repertoire in OA. FlowSOM revealed distinct B cell clusters with perturbed frequencies in OA compared to the healthy cohort across all B cells (Fig 2A-B). The differential clusters were identified as naive and switched memory B cell phenotypes (Fig 2C). CD27+lgD_switched memory B cell clusters 6, 7, and 11 were significantly expanded in OA after controlling for age and gender by the Quade test (Fig 2D-E). This observation is consistent with our results from immunophenotyping by manual gating, where the switched memory B cells subtype was significantly expanded in OA versus healthy (Fig 1 G). Within the switched memory B cells, cluster 6 was marked by the higher expression of CD25 and CD24 hence, we termed it as the CD25hiswitched memory B cell cluster. Among the naive B cells, clusters 15 and 21 , marked by high CD38 expression, were significantly depleted in OA (Fig 2F-G). Higher expression of CD56 differentiated naive B cell cluster 21 from cluster 15 (Fig 20). Interestingly, CXCR5 was significantly enriched in all the switched memory B cell clusters 6, 7, and 11 that were expanded in OA compared to the healthy donors (Fig 2H). Contrary to the PCA of CD4T cell clusters in OA and healthy donors (Fig 9H, I), we noted a greater separation of OA from healthy individuals in the PCA analyses in the B cell repertoire (Fig 2I).

[0173] Expansion of a CD25hiswitched memory B cell population in OA. Since the CyTOF data identified multiple B cell clusters that could differentiate OA and healthy PBMC, we performed scRNA-seq on a subset of healthy (n=6) and OA (n=6) samples to validate the results. Isolated PBMC was enriched for B cells using a pan B cell magnetic-activated cell sorting (MACS) kit, and scRNA libraries were constructed and sequenced. Seurat package (v4.3.0.1 ) was used to cluster, visualize, and analyze the dataset (see methods for details). The enriched B cells clustered into 14 unique clusters (Figure 10A), 7 of which had a high expression for B cell markers, including CD19, MSA1 , PAX5, and CD79A (Fig 10B-C). Automated clustering algorithms also identified these as B cells (and their subsets) when immune cells (Fig 3A and Fig 4D). The proportion of naive B cells was reduced (Iog2 fold change = - 0.16), and that of switched memory B cells was expanded (Iog2 fold change = 0.60) in OA samples (Figure 3B-C), consistent with the CyTOF data. Single-cell clusters of switched memory (total 625 cells) and naive B cells (total 2461 cells) were extracted and re-examined independently. The switched memory B cells grouped into four sub- clusters, wherein clusters 1 and 2 were disproportionately higher in OA (Fig 3 D-F). In agreement with the findings of theATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 cyTOF analyses, a CD25hicluster (cluster 1 ) was expanded in OA samples (Fig 3 G-H). CD25 denotes the IL2 receptor, IL2RA, and genes in cluster 1 were accordingly enriched for the IL2 STAT5 signaling pathway (Fig 3I). Other pathways associated with the CD25hicluster were the TNFa signaling via the NFKB and the TGF|3 signaling pathway, also associated with OA pathology in the joint cells i.e. cartilage, bone, and synovium. The IL2-stat and TNFa pathways were also overrepresented in the genes expressed in cluster 2, along with the IL6-JAK-STAT pathway. Consistent with the cyTOF data, the CD25hiswitched memory cell cluster also showed enrichment for the CXCR5 gene (Fig 1 1 C).

[0174] Naive B cells were grouped into seven sub-clusters with surprisingly little overlap of clusters between healthy control and OA samples (Fig 3J-L). While the transcript for CD56 (NCAM1 ) was not widely expressed in any of these clusters, CD38 gene expression was enriched in cluster 1 but was not significantly different between groups, unlike the cyTOF analyses where CD38- expressing clusters were depleted in OA. However, CD38 was found to be a negative marker for OA-enriched clusters 6 and 7, implying that it was significantly underrepresented in these subsets of cells. The bulk of the differentially expressed genes belonged to the heat shock protein family, and almost all of these were downregulated in Naive B cells of OA patients (Fig 3M). Interestingly, IL2 STAT5 signaling emerged as a significantly enriched pathway in clusters 4 and 7, each disproportionately higher in OA (Fig 3N).

[0175] Validation of CD25hiswitched memory B cell expansion in an independent cohort. To confirm our findings from the discovery cohort of patients, we conducted a comprehensive 29- immune cell marker cyTOF experiment in an independent, validation cohort of five OA patients and six healthy controls, distinct from our original dataset (Fig. 12A-B, Table 3 and 4). FlowSOM clustering of B cells identified 25 distinct clusters (Fig. 12C), which were distributed between Naive (lgD+CD27-CD38+ / lo), Switched Memory (lgD-CD27+), Unswitched Memory (lgD+CD27-) and lgD-CD27- B cell subpopulations. Similar to our initial findings, we found significantly altered B cell subpopulations in our validation cohort. Nine of the 25 FlowSOM clusters, with distinct marker expression patterns, had significantly altered abundances in OA versus healthy PBMCs (Fig. 12D). Notably, the CD25hi Switched Memory B cell cluster 22 was expanded along with another cluster of the same B cell subtype (Fig. 12E). In concordance with our original findings, all of the four significantly different Naive cell clusters were depleted in OA compared to healthy PBMCs (Fig 12F).

[0176] The immune landscape of Degenerative Meniscal Tear (DMT) patients resembles OA.Joint injuries have been implicated in increasing the risk of developing OA, with post-traumatic OA accounting for 12% of the overall burden of disease. Degenerative meniscal tears (DMT) are identified as one such injury-related risk factor of OA. To investigate whether correlationsATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 exist between the DMT and OA immune landscapes, we explored the immune landscape in PBMCs of DMT patients’ whole blood obtained before any surgical interventions were made (n=10; 25-69 years). The DMT cohort was relatively younger, with a mean age of 48.50 ± 13.91 years compared to the mean age of the OA cohort, 55.59 ± 10.81 years, and of the healthy cohort, 57.29 ± 10.31 years. Interestingly, the BMI of the DMT cohort was higher at 33.40 ± 7.60 kg / m2compared to that of the OA cohort at 27.41 ± 4.87 kg / m2. The blood PBMC isolated from the blood of the DMT patients was stained using the same 29-marker cyTOF panel. All statistical measurements were performed by controlling for age by way of a nonparametric Quade test as previously. Immunophenotyping by manual gating for defined immune populations revealed a significant expansion of the switched memory B cells in DMT, similar to OA patients compared to healthy donors (Fig 4 A-B).

[0177] To further discern OA-like perturbations in the DMT cohort, we selected the FlowSOM clusters significantly different between healthy and OA as a framework to compare the frequencies of the same clusters in the DMT cohort. FlowSOM analyses revealed significant differences in the frequencies of immune clusters belonging to B cells and CD4T cell populations. Switched memory B cell cluster 6, identified previously as CD25hiswitched memory B cell phenotype, was significantly expanded in the DMT cohort while naive B cell cluster 21 was significantly depleted (Fig 4 C-E). Meanwhile, among the CD4T cell clusters significantly different between healthy and OA, naive CD4T cluster 14 and EM CD4T cell cluster 51 were significantly elevated in the DMT cohort compared to healthy (Fig 4 F-H). We note that the trend observed in the frequencies of the clusters differentially abundant in OA versus healthy was maintained in DMT versus healthy comparisons. Also, the frequencies of these clusters were not significantly different between the OA and DMT cohorts, suggesting that the specific perturbations in the B and CD4 T cell compartments in the OA cohorts were similar and present in the DMT cohort.

[0178] Specific immune correlates of OA are already evident in the early stages of the disease. We next evaluated the correlations among the clinical features, demographics, and abundances of FlowSOM-identified differential immune clusters in all our datasets, including the healthy, OA, and DMT cohorts (Fig 5A). No correlation between age or sex and immune cluster abundances was noted. BMI, however, was anti-correlated with switched memory B cell cluster 7. Interestingly, significant associations were observed between CD4T cell clusters. Expanded EM CD4T cell cluster 42 in OA was negatively correlated with WOMAC pain and function. Expanded naive CD4T cluster 14 in OA was positively correlated with WOMAC stiffness (Figure 5A). OA severity, as represented by KL scores in the medial region of the joint, was negatively correlated (p<0.05) with the CD56+naive CD4T cell cluster that wasATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 differentially depleted in OA versus healthy (Fig 5A). Utilizing all the FlowSOM-identified immune subpopulations that were significantly differential in abundance in OA compared to healthy, hierarchical clustering showed that switched memory B cell clusters 6, 7, and 1 1 , in addition to classical monocyte cluster 3 and central memory CD8T cell cluster 1 clustered separately from other differential cell populations among the OA and DMT patients (Fig 5B), suggesting agreement between OA and DMT profiles for this set of immune subpopulations. Switched memory B cell types expanded in OA (clusters 6, 7, and 1 1 ) were similarly enriched for CXCR5 expression in a majority of DMT patients (Fig 5C), aligning with the trend observed in OA versus healthy (Fig 2H). CD25 expression was uniformly high in all the DMT patients (Fig 5D), consistent with the expansion of the CD25hiswitched memory B cells in the blood of early and late OA patients.

[0179] Recent studies have highlighted the role of memory B cells in inflammation, pathogenesis, and relapse of various diseases, including RA, prompting the exploration of B cell depletion approaches for therapeutic intervention in their pathogenesis. The role of B cells in OA pathogenesis is, however, relatively unexplored, although a flow cytometry-based analysis reported a skewing of immune cells in the B cell compartment of OA blood. A remarkable feature that we have observed in the circulating immune cells in the OA cohort is a significant expansion of a switched memory B cell subpopulation (CD25hiCD24hiCD27+lgD ) in peripheral blood. This CD25hiswitched memory B cell population was found to be enriched in OA by two independent approaches, cyTOF and scRNA-seq, validating the observation. These data highlight the power and significance of utilizing higher resolution techniques like mass cytometry since the precise differences in B cell repertoire could not be detected using flow cytometry with a limited number of cell-surface markers.

[0180] Expansion of the CD25hiCD24hiswitched memory B cell population in OA blood warrants further studies in the role of the ILR2A / CD25 gene and this memory B cell population in OA pathogenesis. Recent studies have reported perturbations in pre-switched and switched memory B cells in the blood, synovium, and synovial fluid of patients diagnosed with rheumatoid arthritis (RA). Interestingly, the ILR2A / CD25 gene has been identified as a susceptibility locus for juvenile idiopathic arthritis (JIA) and implicated in other autoimmune diseases, however, its function in regulatory T cells rather than B cells has been in focus. RNA- seq data also validated that the cluster of switched memory B cells with enriched CD25 (IL2RA receptor) expressed genes associated with the IL2 STAT5 signaling pathway. Further, TNFa signaling via NFKB and the TGFp signaling pathway were enriched in the CD25hi-ATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 switched memory B cells. These pathways play important roles in OA pathogenesis in the multiple cells constituting the joints, namely cartilage, bone, and synovium.

[0181] Contrary to the expansion of memory B cells, we noted fewer frequencies of naive B cell populations in the peripheral blood of OA patients. RNA-seq data demonstrated stark differences in the naive B cell populations between the normal and OA patient samples (Fig 3K). While naive B cell clusters 2, 3, and 5 were largely depleted in OA, clusters 4 and 6 were expanded. Interestingly, the IL2-STAT pathway was operational in the naive B cell cluster 4 similar to CD25hiswitched memory B cell, both differentially abundant in OA. An imbalance in the T cell repertoire was also prominent in the OA landscape of immune cells, even though the T cell signature did not completely distinguish between normal and OA patients when assessed by cyTOF. In the CD4T cell repertoire, select naive CD4T cell clusters and EM CD4T cell clusters were expanded, whereas two CD56+-expressing clusters were depleted. A circulating Th2 cell cluster was depleted in the OA peripheral blood, consistent with a previous study that showed a loss of IL-4-producing Th2 subset in OA peripheral blood.

[0182] OA patients are clinically defined by joint space narrowing detected by X-ray along with patient- reported outcomes on pain. Since these clinical attributes of OA pathogenesis can be reached through multiple molecular routes, including joint trauma, mechanical instabilities in gait, inflammation, metabolic disorders, and more, it is becoming increasingly clear that the identification of OA biomarkers and potential stratification of patients based on their molecular characteristics is required. Such a stratification is useful in risk identification and clinical treatments in patients, for a precision medicine approach. The quest for OA biomarkers has not yet utilized single-cell approaches, focusing on bulk analyses of blood cells or proteomic analyses of cell-free blood and urine samples instead. Our recent CyTOF studies of OA cartilage also identified distinct patient subtypes based on abundance of cell populations and as drug response, however such an approach for prospective patient stratification before a clinical trial, for example, is not feasible. Immune cell profiling of easily accessible peripheral blood, on the other hand, can be greatly informative for patient stratification independently or in conjunction with clinical features like pain, BMI, and radiological imaging in a larger cohort of OA patients. In this study, we demonstrate that identifying an OA-specific signature of immune cell features through single-cell data is an approach to define OA biomarkers.

[0183] Another major bottleneck in OA treatment is the relatively late detection of the disease, which makes it difficult to reverse the pathogenesis and is the main reason for the lack of a disease- modifying OA drug in the clinic. Early detection of OA, before the irreversible clinical symptoms are apparent, is a significant clinical breakthrough to accelerate drug discovery efforts and devise effective clinical trials for candidate drugs in early or moderate OA ratherATTORNEY DOCKET NO.: STAN-2228WO CLIENT REFERENCE NO.: S24-217 than late-stage OA. Although it takes many years for OA to manifest, it is known that patients with degenerative meniscal tears (DMT) are at a much higher risk for OA development.

[0184] Novel methods in magnetic resonance imaging (MRI) have been applied for visualizing and quantifying changes in the cartilage before the advent of irreversible damages. However, high inter-patient variability in cartilage extracellular matrix composition may hinder the accuracy of predicting early OA based on MRI alone, especially in patients where initial matrix changes are not apparent. Our study, therefore, highlights the potential of utilizing patientspecific immune cell features either as a stand-alone or in combination with advanced MRI methods to improve the current clinical methods for OA prognosis. On profiling the immune cell landscape in these populations at risk for OA, we observed significant similarities in the immune landscape of the OA cohort with the DMT cohort. These analyses show that the immune cell features associated with OA are present and detectable in the early stages of OA, and can be utilized for early detection of the disease.References

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[0221] Chu CR, Williams AA, Erhart-Hledik JC, et al. Visualizing pre-osteoarthritis: Integrating MRI UTE-T2* with mechanics and biology to combat osteoarthritis-The 2019 Elizabeth Winston Lanier Kappa Delta Award. J Orthop Res 2021 ;39:1585-1595.Table 1Social and clinical demographic data of patient and donor samples used in the study.ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217Age (Mean ± SD) Percent Male Percent Female BMI (Mean ± SD)Healthy 57.29 ± 10.31 38.10% 47.62% N / AOA 55.59 ± 10.81 52.94% 47.06% 27.41 ± 4.87DMT 48.50 ± 13.91 90.00% 10.00% 33.40 ± 7.60Table 2Patient -reported outcomes as measured by the KOOS and WOMAC scales. Higher KOOS indicates less pain and better function, while high WOMAC scores reflect more pain and worse function. N / A denotes data not available.ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217Table 3Social and clinical demographic data of OA patient and healthy donor PBMC validation cohort.ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217Table 4Patient -reported outcomes as measured by the KOOS and WOMAC scales

[0222] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-217T HAT WHICH IS CLAIMED IS:1 . A method of determining the presence of osteoarthritis (OA) in an individual, the method comprising: quantitating CD25hiswitched memory B cells in a circulating cell sample from the individual; wherein an expansion of CD25hiswitched memory B cells as a percentage of total B cells is indicative of osteoarthritis in the individual.

2. The method of claim 1 , wherein a determination of osteoarthritis is made when the CD25hiswitched memory B cells comprise greater than 2% of the total B cell population.

3. The method of claim 1 or claim 2, wherein the individual is asymptomatic for OA.

4. The method of any of claims 1 -3, wherein the individual is a human individual with a condition or injury that can pre-dispose to OA.

5. The method of any of claims 1 -4, wherein the circulating cell sample is a blood sample.

6. The method of claim 5, comprising a step of enriching the blood sample for B cells prior to the quantitating step.

7. The method of any of claims 1 -6, wherein the quantitating step comprises contacting the cell sample with reagents specific for at least CD25 and CD27, and determining the presence of cells that are CD27+CD25hi.

8. The method of claim 7, wherein the quantitating step further comprises contacting the cell sample with a reagent specific for IgM, and determining the presence of cells that are IgM CD27+CD25tli.

9. The method of any of claims 1 -6, wherein the quantitating step comprises determining the expression of a gene set indicative of CD25hiswitched memory B cells, which may include more or more of genes selected from LINC01619, CMIP, BCL2, HIVEP2, VOPP1 , TNRC6C, IQCN, ABCG1 , RASGRP1 , ETV6, EEF2K, PALM2AKAP, SMAD3, GALNT2, ECE1 , KDM6B, CD96, FMNL1 , TET3 and EXT1 .ATTORNEY DOCKET NO.: STAN-2228WOCLIENT REFERENCE NO.: S24-21710. The method of any of claims 1 -8, further comprising: quantitating naive B cells in the circulating cell sample; wherein a depletion of naive B cells as a percentage of total B cells is indicative of osteoarthritis in the individual.1 1 . The method of any of claims 1 -9, wherein quantitation is performed by single cell flow cytometry.

12. The method of claim 1 1 , wherein the flow cytometry is mass cytometry or fluorescence activated flow cytometry.

13. The method of any of claims 1 -12, wherein the individual is treated in accordance with the classification.

14. The method of any of claims 1-13, wherein the individual is stratified for a clinical trial in accordance with the classification.

15. The method of any of claims 1 -14, wherein a report of the classification is provided to the individual.

16. A device or kit for diagnosis of osteoarthritis, comprising: affinity reagents to quantitate CD25hiswitched memory B cells in a circulating cell sample.

17. The device or kit of claim 16, comprising or consisting of affinity reagents specific for CD27 and for CD25.

18. The device or kit of claim 16 or 17, further comprising reagents to enrich for B cells.

19. The device or kit of any of claims 16-18, further comprising an affinity reagent specific for IgM.

20. The device or kit of claim 16, further comprising reagents to identify gene expression indicate of CD25hiswitched memory B cells.

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