Method for enhancing T cell regeneration

Abstract

Described herein are methods for restoring T cell production in a subject in need thereof. TIFF2022519951000002.tif127128

Description

Technical Field

[0001] Description of Rights to Inventions Made Under Federal Government Funding This research was supported by National Institutes of Health grant number DK107784. The government may have certain rights in this invention.

[0002] Cross - References to Related Applications This application is related to U.S. Patent Application No. 62 / 828384, filed on April 2, 2019, and U.S. Patent Application No. 62 / 945290, filed on December 9, 2019, and claims priority from these applications. The entire disclosure content of these applications is incorporated herein by reference.

[0003] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy, created on March 31, 2020, is named 51395 - 002WO3_Sequence_Listing_03.31.20_ST25 and is 87,125 bytes in size. <0000​​​​​​​​​​​​​​In one aspect, the present invention provides a method for increasing T cell production in a T cell-producing tissue or T cell-producing fluid of a subject where such increase is needed, the method comprising the step of administering a composition comprising mesenchymal stromal cells expressing periostin and Pdgfra into the T cell-producing tissue or T cell-producing fluid of the subject, thereby increasing T cell production in the T cell-producing tissue or T cell-producing fluid of the subject.

[0006] In one embodiment, mesenchymal stromal cells do not express Cdh11 and CD248.

[0007] In another embodiment, the T cell-producing tissue is the thymus.

[0008] In another aspect, T cell-producing tissue is lymphocyte-producing tissue.

[0009] In yet another embodiment, the T cell-producing fluid is blood.

[0010] In yet another aspect, the subjects have previously undergone hematopoietic stem cell transplantation.

[0011] In yet another embodiment, the subject has one or more of the following conditions: T lymphopenia-related conditions, impaired T cell production, T cell dysfunction, distortion of the repertoire of cells having T cell receptors, infection, or tumor.

[0012] In yet another embodiment, mesenchymal stromal cells express Flt3 ligand (fms-related receptor tyrosine kinase 3 ligand), Ccl19 (CC motif chemokine ligand 19), BMP2 (osteogenesis protein 2), BMP4 (osteogenesis protein 4), IL-15 (interleukin 15), IL-12a (interleukin-12a), Cxcl14 (CXC motif chemokine ligand 14), Ccl11 (CC motif chemokine ligand 11), (Cxcl10, CXC motif chemokine ligand 10), or IL-34 (interleukin 34), or combinations thereof.

[0013] In one embodiment, mesenchymal stromal cells express Ccl19, Flt3l, and IL-15.

[0014] In yet another embodiment, mesenchymal stromal cells express Flt3 ligand, Ccl19, and IL-15, but do not express Cdh11 and CD248.

[0015] In yet another aspect, the mesenchymal stromal cells are autologous to the subject.

[0016] In yet another embodiment, mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells.

[0017] In yet another embodiment, the mesenchymal stromal cells are derived from embryonic stem cells or their progenitor cells.

[0018] In yet another embodiment, the mesenchymal stromal cells are derived from iPS cells or their precursor cells.

[0019] In another aspect, the present invention provides a method for increasing T cell production in a T cell-producing tissue or T cell-producing fluid of a subject where such production is needed, the method comprising the step of administering a composition comprising Ccl19 (CC motif chemokine ligand 19) into the T cell-producing tissue or T cell-producing fluid of the subject, thereby increasing T cell production in the T cell-producing tissue or T cell-producing fluid of the subject.

[0020] In one embodiment, the T cell-producing tissue is the thymus.

[0021] In another aspect, T cell-producing tissue is lymphocyte-producing tissue.

[0022] In yet another embodiment, the T cell-producing fluid is blood.

[0023] In yet another aspect, the subjects have previously undergone hematopoietic stem cell transplantation.

[0024] In yet another aspect, the subject has one or more of a condition associated with T lymphocytopenia, T cell production disorder, T cell dysfunction, distortion of the repertoire of cells having a T cell receptor, an infectious disease, or a tumor.

[0025] In yet another aspect, the present invention provides isolated mesenchymal stromal cells that express periostin and Pdgfra.

[0026] In one aspect, the mesenchymal stromal cells do not express Cdh11 and CD248.

[0027] In another aspect, the mesenchymal stromal cells express Flt3 ligand (fms-related receptor tyrosine kinase 3 ligand), Ccl19 (C-C motif chemokine ligand 19), BMP2 (bone morphogenetic protein 2), BMP4 (bone morphogenetic protein 4), IL-15 (interleukin 15), IL-12a (interleukin-12a), Cxcl14 (C-X-C motif chemokine ligand 14), Ccl11 (C-C motif chemokine ligand 11), (Cxcl10, C-X-C motif chemokine ligand 10), or IL-34 (interleukin 34), and combinations thereof.

[0028] In yet another aspect, the mesenchymal stromal cells express Ccl19, Flt3l, and IL-15.

[0029] In yet another aspect, the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15 and do not express Cdh11 and CD248.

[0030] In yet another aspect, the mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells.

[0031] In yet another aspect, the mesenchymal stromal cells are derived from embryonic stem cells or their progenitor cells.

[0032] In yet another embodiment, the mesenchymal stromal cells are derived from iPS cells or their precursor cells.

[0033] In another aspect, the present invention provides a population of isolated stem cells that can differentiate into mesenchymal stromal cells expressing periostin and Pdgfra.

[0034] In one embodiment, mesenchymal stromal cells do not express Cdh11 and CD248.

[0035] In yet another aspect, the present invention provides a composition for increasing T cell production in a target T cell-producing tissue or T cell-producing fluid, the composition comprising Ccl19 (CC motif chemokine ligand 19).

[0036] Other features and advantages of the present invention will become apparent from the detailed description and the appended claims. Therefore, other aspects of the present invention are described in the following disclosure and are within the scope of the invention. [Brief explanation of the drawing]

[0037] The following detailed description is illustrative and is not intended to limit the present invention to the specific embodiments described herein, but can be understood in conjunction with the accompanying drawings incorporated herein by reference.

[0038] [Figure 1A] This study demonstrates that thymic MSCs express important lymphocyte-producing factors. (A) Overview of research using human thymic samples. [Figure 1B] (B) Thymic mesenchymal stem cells (MSCs) express important lymphocyte-producing factors. (A) tSNEs showing annotations of major thymic stromal cell types in humans. [Figure 1C] This shows that thymic MSCs express important lymphocyte-producing factors. (C) Cell count in each population in the human thymus, determined by scRNAseq and flow cytometry. [Figure 1D]This shows that thymic MSCs express important lymphocyte-producing factors. (D) Expression of important lymphocyte-producing regulatory factors in the interstitial compartment of the human thymus, shown as a heatmap. [Figure 1E] This study demonstrates that thymic MSCs express important lymphocyte-producing factors. (E) Summary of research using mouse samples. [Figure 1F] This shows that thymic MSCs express important lymphocyte-producing factors. (F) tSNE showing annotation of major thymic stromal cell types in mice. [Figure 1G] This shows that thymic MSCs express important lymphocyte-generating factors. (G) Cell count in each population in the mouse thymus, determined by scRNAseq and flow cytometry. [Figure 1H] This shows that thymic MSCs express important lymphocyte-producing factors. (H) Expression of important lymphocyte-producing regulatory factors in the interstitial compartment of the human thymus, shown as a heatmap. [Figure 1I] This demonstrates that thymic MSCs express important lymphocyte-producing factors. (I) Quantification of Il15, Ccl19, Flt3l, and Bmp4 expression across all thymic stromal cell types in mouse samples. [Figure 2A] Gating strategies for flow cytometry isolation of human thymic stromal cells. [Figure 2B] Comparison of interstitial yield using two different digestive protocols for human thymus processing. [Figure 2C] tSNE showing all cells sequenced from a human sample, including hematopoietic cells. [Figure 2D] Definition of human hematopoietic cells based on important marker genes. [Figure 2E] tSNE showing annotation of major thymic stromal cell clusters in human samples. [Figure 2F] Gating strategies for flow validation of major thymic stromal cell clusters in humans. [Figure 2G] Gating strategies for flow cytometry isolation of mouse thymic stromal cells. [Figure 2H] UMI and gene count per cell in mouse samples. [Figure 2I] tSNE showing all cells sequenced from a mouse sample, including hematopoietic cells. [Figure 2J] The major stages of T cell development can be tracked through the expression of key marker genes. [Figure 2K] tSNE showing annotation of major thymic stromal cell clusters in mouse samples. [Figure 2L] A heatmap showing the differentially expressed top-level genes among mouse thymic stromal cells. [Figure 2M] Gating strategies for flow validation of major thymic stromal cell clusters in humans. [Figure 3A] tSNEs showing three subsets of thymic MSCs in the human and mouse thymus. [Figure 3B] GO terminology analysis of genes significantly differentially expressed in different mouse MSC subsets. [Figure 3C] Expression of Cl19, Flt3l, and IL15 in human and mouse MSC subsets. [Figure 4A] A heatmap showing the differentially expressed top-level genes among mouse thymic mesenteric cells (MSCs). [Figure 4B] Expression of marker genes that define human and mouse MSC subsets. [Figure 4C] Quantification of thymic MSC subsets in human and mouse samples. [Figure 4D] tSNE showing all stromal cells sequenced by Bornstein et al. [Figure 4E] tSNEs showing three subsets of thymic mesenchymal stem cells (MSCs). [Figure 4F] Expression of MSC subset marker genes in the Bornstein et al. dataset. [Figure 4G] GO terminology analysis of genes significantly differentially expressed in mouse CD248 MSCs. [Figure 5A]This shows the disappearance of periostin + MSC after pre-radiation treatment. (A) Outline of the experiment. [Figure 5B] (B) Disappearance of periostin + MSCs after pre-radiation treatment. Two-photon microscopy images showing GFP-labeled cells that arrived in the tissue 3 days after transplantation and 4 days after radiation exposure. [Figure 5C] (C) tSNE showing thymic stromal cells from untreated control mice (control) and irradiated and transplanted recipient mice (transplant). [Figure 5D] (D) Disappearance of periostin + MSCs after radiotherapy. Changes in composition of the thymic MSC compartment after radiation and transplantation. [Figure 5E] (E) Clearing of periostin + MSCs after radiotherapy. GO terminology analysis of the thymic MSC population after irradiation and transplantation. [Figure 6A] Overview of the experiment. [Figure 6B] Quantification of GFP-labeled cells arriving in tissues by flow cytometry. [Figure 6C] Two-photon microscope images showing the thymus after radiation therapy and transplantation. [Figure 6D] Two-photon microscopy images showing the presence or absence of GFP+ cells in tissue 2, 4, and 5 days after transplantation. [Figure 6E] Compositional changes in the thymic interstitial compartment after radiation therapy and transplantation. [Figure 6F] Changes in the expression of secretory factors Flt3l, Ccl19, and IL15 in MSC subsets after irradiation and transplantation. [Figure 7A] This study demonstrates that the transfer of thymic CD248-MSCs promotes T cell production after radiotherapy pretreatment. (A) Outline of the experiment. [Figure 7B] (B) Thymic CD248-MSC transfer promotes T cell production after radiotherapy. Flow validation of thymic regeneration over 6 days following bone marrow transplantation and intrathymic transfer of CD248-MSCs. [Figure 7C](C) Transplantation of thymic CD248-MSCs promotes T cell production after radiotherapy. Flow validation of the effects of MSC GFP and Ccl19 knockout on thymic regeneration over 6 days following bone marrow transplantation and intrathymic transfer of MSCs. [Figure 7D] (D) Thymic CD248-MSC transfer promotes T cell production after radiotherapy. Flow validation and sjTREC measurements in the thymus for one month after bone marrow transplantation and MSC intrathymic transfer to determine the rate of new T cell generation. [Figure 7E] This shows that thymic CD248-MSC transfer promotes T cell production after radiotherapy. (E) 16-week follow-up of T cell recovery after bone marrow transplantation and intrathymic transfer of MSCs. [Figure 7F] (F) Transfer of thymic CD248-MSCs promotes T cell production after radiotherapy. Estimation of the vaccination response 54 days after bone marrow transplantation and intrathymic transfer of MSCs demonstrates the functionality of newly generated T cells. [Figure 8A] Establishment of CD99l2 and Itgb5 as pan-MSC markers for flow cytometry isolation. [Figure 8B] Colony-forming ability of CD99l2+ Itgb5+ thymic MSCs. [Figure 8C] Validation of Pdgfra and CD248 as flow cytometry markers for distinguishing between MSC subsets. [Figure 8D] Analysis of different T cell development stages during the one-month period following bone marrow transplantation and intrathymic transfer of MSCs. [Figure 8E] A 16-week follow-up study of B-cell and myeloid cell recovery after bone marrow transplantation and intrathymic transfer of MSCs. [Figure 8F] Flow validation of the presence of GFP-labeled MSCs in the thymus of recipient mice 16 weeks after transfer. [Figure 9A](A) Gating strategy for flow cytometry isolation of tdTomato+ (Penk+) MSCs and tdTomato- (Postn+) MSCs from the thymus. [Figure 9B] (B) Summary of the experiment. [Figure 9C] (C) Shows periostin+ MSCs that specifically enhance the recruitment of T cell progenitor cells. Flow validation of thymic regeneration over 6 days following bone marrow transplantation and intrathymic transfer of tdTomato+ (Penk+) MSCs and tdTomato- (Postn+) MSCs. [Modes for carrying out the invention]

[0039] Detailed description of the invention definition All technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains, unless otherwise defined. In case of any conflict, including definitions, the present application shall prevail.

[0040] "Subjects" include humans, domesticated animals and livestock, as well as vertebrates, including any member of the class Mammalia, such as zoo animals, sports animals, or pet animals, including mice, rabbits, pigs, sheep, goats, cattle, and higher primates.

[0041] As used herein, terms such as “to treat,” “to treat,” and “treatment” refer to reducing or relieving the disorder and / or its associated symptoms. It will be recognized, though not excluded, that treatment of a disorder or condition does not require the complete elimination of the disorder, condition, or its associated symptoms.

[0042] "Effective dose" refers to the amount of mesenchymal cells, stem cells, or progenitor cells that produce the desired therapeutic response (i.e., enhanced T cell production in the thymus).

[0043] "Mesenchymal progenitor cells" refer to pluripotent cells that have the potential to be committed to the mesenchymal lineage.

[0044] "Mesenchymal stem cells" refer to pluripotent cells that have the potential to commit to multiple mesenchymal cell types, but do not express genes that define a specific cell type.

[0045] "Isolated" means a material that does not contain, to varying degrees, the components that would normally accompany it when found in its natural state. "To isolate" refers to the degree of separation from the original source or environment.

[0046] As used herein, “increased” means T cell production that is at least about 0.05 times greater than the reference level (e.g., a subject with normal T cell production) (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 25, 50, 100, 1000, 10,000 times or more). Similarly, when referring to T cell production, “increased” means at least about 5% greater than the reference level (e.g., a subject with normal T cell production) (e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% greater). The quantity can be measured according to methods known in the art for determining the quantity of T cells.

[0047] Where used herein, unless otherwise specified or made clear from the context, the term “approximately” is understood to mean within the normal tolerances in the art, for example, within two standard deviations of the mean. “Approximately” is understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “approximately.”

[0048] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1–50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (and their fractions unless otherwise specified in the context).

[0049] In this disclosure, “comprise,” “comprising,” “contain,” and “have,” etc., may have the meanings given to them in U.S. patent law, and may mean “include,” “including,” etc. Similarly, “consisting essentially of” or “consist essentially” may have the meanings given to them in U.S. patent law, and such terms are open-ended, allowing for non-described entities as long as the basic or novel characteristics of the described are not altered by the non-described entities, but excluding aspects of the prior art.

[0050] Other definitions are provided within the context throughout this disclosure.

[0051] Compositions and methods of the present invention Comprehensive analysis of thymic mesenchymal stromal cells has identified a periostin-positive Pdgfra-positive immunophenotype (periostin+Pdgfra+ immunophenotype). This immunophenotype is now known to be essential for T cell production. Adoptive cell transfer of these cell subpopulations into T cell-producing tissues or fluids such as the thymus has been shown to enhance thymic tissue regeneration and long-term T cell rearrangement in hematopoietic stem cell transplantation (HSCT) settings. The creation or isolation of periostin+Pdgfra+ cells and their transfer, as well as / or the specific genes or proteins expressed by these cells, offer therapeutic benefits in the HSCT environment or in other situations, including aging, where T cell depletion / deficiency or dysfunction contributes to adverse effects.

[0052] Periostin is described, for example, by GenBank accession number NM_001135934.2 (SEQ ID NO: 1 and 2). Also known as osteoblast-specific factor 2, periostin is a secreted cell adhesion protein homologous to fasiclin I, an insect cell adhesion molecule. Its N-terminal region contains a signal peptide (SP) for its secretion and a cysteine-rich region (EMI domain) that promotes multimer formation under non-reducing conditions. Adjacent to the SP and EMI domains are four internal homology repeats (FAS domains). These are homologous to fasiclin I, an insect cell adhesion protein, and act as ligands for integrins. The C-terminal region of periostin consists of a hydrophilic domain. While the N-terminal region of periostin is highly conserved, the C-terminal region of the protein differs depending on the isoform. The N-terminal region regulates cellular function by binding to integrins in the cell plasma membrane via its FAS domains. The C-terminal region of this protein regulates cell-matrix organization and interaction by binding to extracellular matrix (ECM) proteins, such as collagen I / V, fibronectin, tenascin C, acidic mucopolysaccharides, such as heparin, and periostin itself.

[0053] Periostin has been shown to be a crucial regulator of bone and tooth formation and maintenance, as well as cardiac development and healing. Periostin also plays a vital role in tumor growth, being upregulated in a wide variety of cancers, including colorectal, pancreatic, ovarian, breast, head and neck, thyroid, and gastric cancers, as well as neuroblastoma. Integrin-bound periostin activates Akt / PKB-mediated and FAK-mediated signaling pathways, which lead to increased cell survival, angiogenesis, invasion, metastasis, and, importantly, epithelial-mesenchymal transition in cancer cells.

[0054] Platelet-derived growth factor receptor alpha, or Pdgfra, is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. These growth factors are mitogens of mesenchymal cells. Pdgfra is known to play a role in organ development, wound healing, and tumor progression. Pdgfra is described, for example, by GenBank accession NM_001347827.2 (SEQ ID NO: 3 and 4). Pdgfra is a typical receptor tyrosine kinase, a transmembrane protein consisting of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. The molecular weight of a mature glycosylated PDGFRα protein is approximately 170 kDA.

[0055] Periostin+Pdgfra+ mesenchymal stromal cells, identified by the periostin+Pdgfra+ immunophenotype, differentially express genes that promote the regenerative phenotype. These genes include, but are not limited to, Flt3 ligand (fms-related receptor tyrosine kinase 3 ligand), Ccl19 (CC-motif chemokine ligand 19), BMP2 (osteogenesis protein 2), BMP4 (osteogenesis protein 4), IL-15 (interleukin 15), IL-12a (interleukin-12a), Cxcl14 (CXC-motif chemokine ligand 14), Ccl11 (CC-motif chemokine ligand 11), Cxcl10 (CXC-motif chemokine ligand 10), and IL-34 (interleukin 34), as well as combinations thereof. Exemplary combinations include Ccl19, Flt31, and IL-15.

[0056] Flt3 ligand is described, for example, by GenBank accession NM_001204502.2 (SEQ ID NO: 7 and 8); Ccl19 is described, for example, by GenBank accession NM_006274.3 (SEQ ID NO: 9 and 10); BMP2 is described, for example, by GenBank accession NM_001200.4 (SEQ ID NO: 11 and 12); BMP4 is described, for example, by GenBank accession NM_001202.6 (SEQ ID NO: 13 and 14); IL-15 is described, for example, by GenBank accession NM_000585.5 (SEQ ID NO: 15 and 16); IL-12a is described, for example, by GenBank accession NM_000882.4 (SEQ ID NO: As described in 17 and 18); Cxcl14 is described, for example, by GenBank accession NM_004887.5 (SEQ ID NO: 19 and 20); Ccl11 is described, for example, by GenBank accession NM_002986.3 (SEQ ID NO: 21 and 22); Cxcl10 is described, for example, by GenBank accession NM_001565.4 (SEQ ID NO: 23 and 24); and IL-34 is described, for example, by GenBank accession NM_001172771.2 (SEQ ID NO: 25 and 26). The mesenchymal stromal cells or their progenitor cells of the present invention may be engineered to express or overexpress these and other regenerative proteins at levels suitable for inducing T cell production.

[0057] In some embodiments, mesenchymal stromal cells identified by the periostin+Pdgfra+ immunophenotype do not express Cdh11 and / or CD248.

[0058] The Cdh11 gene encodes a type II classical cadherin, a membrane-endogenous protein of the cadherin superfamily that mediates calcium-dependent cell-cell adhesion. Cdh11 is described, for example, by GenBank accession number NM_001308392.2 (SEQ ID NO: 27 and 28). Mature cadherin proteins consist of a large N-terminal extracellular domain, a single transmembrane domain, and a highly conserved small C-terminal cytoplasmic domain. Type II (atypical) cadherins are defined based on the absence of the HAV cell adhesion recognition sequence specific to type I cadherins. The expression of this particular cadherin in osteoblast cell lines, and its upregulation during differentiation, suggests a certain function in bone development and maintenance.

[0059] CD248 is also known as tumor endothelial marker 1, tem1, and endosialin. CD248 is described, for example, by GenBank accession number NM_020404.3 (SEQ ID NO: 5 and 6). CD248 is a transmembrane receptor, and its known ligands are fibronectin and type I / IV collagen. CD248 is widely expressed in embryonic mesenchymal cells and is required for the proliferation and migration of pericytes and fibroblasts.

[0060] The mesenchymal stromal cells of the present invention can be obtained from human tissue (e.g., thymus) using methods known in the art, according to their periostin+Pdgfra+ immunophenotype. Methods for purifying and isolating cells are known to those skilled in the art and include, but are not limited to, sorting methods based on the expression of cell surface markers, e.g., fluorescence-activated cell sorting (FACS sorting), positive separation methods, and negative separation, magnetic separation, and combinations thereof. Those skilled in the art can easily determine the proportion of stromal cells, stem cells, or their progenitor cells in a population using various well-known methods such as FACS. In some embodiments, it is desirable to purify the cells first. The stromal cells, stem cells, or their progenitor cells may constitute a population of cells with purities of about 50-55%, 55-60%, 60-65%, and 65-70% (e.g., non-stromal cells, non-stem cells, and / or non-progenitor cells have been removed from the population or are otherwise absent from the population). More preferably, the purity is about 70-75%, 75-80%, and 80-85%; most preferably, the purity is about 85-90%, 90-95%, and 95-100%. The purity of stromal cells, stem cells, or their progenitor cells can be determined according to the genetic marker profile within the population. The therapeutic dose can be easily adjusted by those skilled in the art (for example, a decrease in purity may necessitate an increase in the dose).

[0061] In other embodiments, the mesenchymal stromal cells of the present invention may be derived from appropriate stem cells or progenitor cells. The stem cells of the present invention include mesenchymal stem cells. Mesenchymal stem cells, or "MSCs," are well known in the art. MSCs originate from the embryonic mesoderm and are isolated from the bone marrow of adults, and can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendons. During embryonic development, the mesoderm develops into limb bud mesoderm, which is the tissue that produces bone, cartilage, fat, skeletal muscle, and endothelium. The mesoderm can also differentiate into visceral mesoderm, which can produce cardiac muscle, smooth muscle, or blood islands consisting of endothelium and hematopoietic progenitor cells. Thus, primitive mesoderm, or MSCs, can provide a source of numerous cell and tissue types. Several MSCs have been isolated. (For example, U.S. Patent No. 5,486,359 by Caplan, A., et al.; U.S. Patent No. 5,827,735 by Young, H., et al.; U.S. Patent No. 5,811,094 by Caplan, A., et al.; U.S. Patent No. 5,736,396 by Bruder, S., et al.; U.S. Patent No. 5,837,539 by Caplan, A., et al.; U.S. Patent No. 5,837,670 by Masinovsky, B.; U.S. Patent No. 5,827,740 by Pittenger, M.; Jaiswal, N., et al., (1997). J. Cell Biochem. 64(2):295-312; Cassiede P., et al., (1996). J Bone Miner Res. 9:1264-73; Johnstone, B., et al., (1998) Exp Cell See Res. 1:265-72; Yoo, et al., (1998). J Bon Joint Surg Am. 12:1745-57; Gronthos, S., et al., (1994). Blood 84:4164-73; Pittenger, et al., (1999). Science 284:143-147.

[0062] Mesenchymal stem cells are thought to migrate outside the bone marrow and bind to specific tissues. By enhancing the proliferation and maintenance of mesenchymal stem cells in vitro or ex vivo, an expanded population can be obtained that can be used to generate or regenerate tissues including breast tissue, skin tissue, muscle tissue, endothelial tissue, bone tissue, respiratory tissue, genitourinary tissue, gastrointestinal connective tissue, or fibroblast tissue.

[0063] The stem cells of the present invention also include embryonic stem cells. Embryonic stem (ES) cells have unlimited self-renewal and pluripotent differentiation capabilities (Thomson, J. et al. 1995; Thomson, JA et al. 1998; Shamblott, M. et al. 1998; Williams, RL et al. 1988; Orkin, S. 1998; Reubinoff, BE, et al. 2000). These cells may be derived from the inner cell mass (ICM) of a preimplantation blastocyst (Thomson, J. et al. 1995; Thomson, JA et al. 1998; Martin, GR 1981) or from primordial germ cells from a postimplantation embryo (embryonic germ cells or EG cells). ES cells and / or EG cells have been obtained from numerous species, including mice, rats, rabbits, sheep, goats, and pigs, and more recently from humans and human and non-human primates (U.S. Patents 5,843,780 and 6,200,806).

[0064] Embryonic stem cells are well known in the art. For example, U.S. Patent Nos. 6,200,806 and 5,843,780 refer to embryonic stem cells of primates, including humans. U.S. Patent Applications 20010024825 and 20030008392 describe human embryonic stem cells. U.S. Patent Application 20030073234 describes a cloned human embryonic stem cell line. U.S. Patent Nos. 6,090,625 and 20030166272 describe undifferentiated cells that are described as pluripotent. U.S. Patent Application 20020081724 describes a cell culture that is described as derived from embryonic stem cells.

[0065] The stem cells of the present invention also include iPS cells. iPS cells are mature cells that have been genetically reprogrammed to be in an embryonic stem cell-like state by expressing genes and factors that are important for maintaining the characteristics that define embryonic stem cells.

[0066] Isolated mesenchymal stromal cells, as well as mesenchymal stromal cells derived from suitable stem cells or progenitor cells, may be genetically modified to express a desired nucleic acid by methods known in the art, including all methods known to introduce transient and stable changes in the cell's genetic material. Genetic modification of mesenchymal stromal cells, stem cells, or progenitor cells involves the addition of exogenous genetic material. Exogenous genetic material includes native or synthetic nucleic acids or oligonucleotides introduced into the cell.

[0067] Gene editing systems can be used to perform genetic modification of mesenchymal stromal cells, stem cells, or progenitor cells. For example, the CRISPR / Cas system can be used to inactivate one or more nucleic acids, including CD248 and Cdh11 (Wiedenheft et al. (2012) Nature 482: 331-8). The CRISPR / Cas system has been adapted for use in gene editing (silencing, enhancing, or altering specific genes) in eukaryotes such as mice or primates. This is achieved, for example, by introducing a plasmid containing a specially designed CRISPR and one or more suitable Cass into eukaryotic cells. Generally, a CRISPR / Cas system for gene editing in eukaryotic cells includes (1) a guide RNA molecule (gRNA) containing a target-directed sequence (which can hybridize to a target sequence in genomic DNA) and a sequence that can bind to a Cas, e.g., the Cas9 enzyme, and (2) a Cas protein, e.g., the Cas9 protein. The target-directed sequence and the sequence capable of binding to Cas, such as the Cas9 enzyme, may be located on the same molecule or on different molecules. If located on different molecules, each molecule may include a hybridization domain that allows these molecules to associate, for example, by hybridization.

[0068] CRISPR sequences, sometimes called CRISPR loci, contain alternating repeats and spacers. RNA from CRISPR loci is constitutively expressed and processed into small RNA molecules. These contain spacers adjacent to the repeat sequences. These RNAs induce other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Thus, spacers function as templates for RNA molecules, similar to siRNA. Pennisi (2013) Science 341: 833-836.

[0069] Therefore, the CRISPR / Cas system can be used to make modifications, for example, to delete one or more nucleic acids, such as CD248, or the regulatory elements of CD248, or to introduce immature terminations that result in reduced functional CD248 expression. Alternatively, the CRISPR / Cas system can be used like RNA interference to reversibly turn off CD248. For example, in mammalian cells, the RNA can induce the Cas protein to the promoter of CD248 or Cdh11, thereby sterically interfering with RNA polymerase.

[0070] In another embodiment, one or more nucleic acids can be introduced using the CRISPR / Cas system. Nucleic acids, such as DNA encoding periostin and Pdgfra, can be introduced into cells together with the CRISPR / Cas system. This process can be used to integrate the DNA encoding periostin and Pdgfra, for example, as described herein, into or near a site targeted by the CRISPR / Cas system.

[0071] In other embodiments, the exogenous genetic material may also include native genes that are functionally regulated by a promoter in the expression vector construct. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked plasmids or plasmids contained within liposomes), retrotransposons (e.g., piggyback, sleeping beauty), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), which can be delivered by incorporating recombinant polynucleotides.

[0072] Methods for producing viral expression vectors are known in the art. Generally, the virus of disclosure is produced in a suitable host cell line using conventional methods, which involves culturing transfected or infected host cells under suitable conditions to enable the production of infectious viral particles. The nucleic acid encoding the viral gene, as well as / or sequences encoding, for example, periostin and pdgfra, can be incorporated into a plasmid and introduced into host cells by conventional transfection or transformation methods. Exemplary host cells suitable for the production of the virus of disclosure include human cell lines such as HeLa cells, HeLa-S3 cells, HEK293 cells, 911 cells, A549 cells, HER96 cells, or PER-C6 cells. Specific production and purification conditions vary depending on the virus and the production system employed.

[0073] In some embodiments, the virus may be administered directly to the producing cells, while in other embodiments, infectious virus particles are recovered from the culture after production and optionally purified. Typical purification steps may include plaque purification, centrifugation (e.g., cesium chloride gradient centrifugation), clarification, enzymatic treatment (e.g., benzonase or protease treatment), chromatography (e.g., ion exchange chromatography), or filtration.

[0074] In certain embodiments, the expression vector is a viral vector. In this specification, the term “virus” is used to refer to any obligate intracellular parasite that does not possess either a protein synthesis mechanism or an energy production mechanism. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Epstein-Barr virus (EBV) vectors, polyomavirus vectors (e.g., Simian vacuolated virus 40 (SV40) vectors), poxvirus vectors, and pseudotyped virus vectors.

[0075] Viruses can be RNA viruses (having a genome composed of RNA) or DNA viruses (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asphaviruses, herpesviruses (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papillomaviruses (e.g., HPV), polyomaviruses (e.g., Simian vacuolated virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). In certain embodiments, the viral vector is an RNA viral vector. Exemplary RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, Ebola virus, flaviviruses (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, norovirus (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)), and toroviruses.

[0076] In certain embodiments, an expression vector includes a regulatory sequence or promoter functionally ligated to a nucleotide sequence encoding an exogenous sequence, such as periostin and pdgfra. The term “functionally ligated” refers to the ligation of polynucleotide elements in a functional relationship. A nucleic acid sequence is “functionally ligated” when it is positioned to have a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is functionally ligated to a gene if they affect the transcription of that gene. Typically, functionally ligated nucleotide sequences are contiguous. However, since enhancers generally function when they are several kilobases away from the promoter, and intron sequences can be of varying lengths, some polynucleotide elements may not be directly adjacent even if functionally ligated, and may even function trans from different alleles or chromosomes.

[0077] Further exemplary promoters that may be employed include, but are not limited to, retroviral LTR, SV40 promoter, human cytomegalovirus (CMV) promoter, U6 promoter, or any other promoter (e.g., eukaryotic cell promoters, including, but not limited to, histone promoters, pol III promoter, and β-actin promoter). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, TK promoter, and B19 parvovirus promoter. The selection of an appropriate promoter will be apparent to those skilled in the art from the teachings contained herein.

[0078] In certain embodiments, the expression vector is an adeno-associated virus (AAV) vector. AAV is a small, non-enveloped icosahedral virus belonging to the genus Depend Parvovirus of the family Parvoviridae. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV can infect both dividing and quiescent cells of several tissue types, and different serotypes of AAV exhibit different tissue tropisms. Many cell types, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells, are suitable for AAV vector production (see, for example, U.S. Patents 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, and 8,163,543, U.S. Patent Application Publication 20020081721, and PCT International Publication Nos. 00 / 47757, 00 / 24916, and 96 / 17947). Generally, AAV vectors are produced in these cell types by one plasmid containing an expression cassette adjacent to the ITR, and one or more additional plasmids providing additional AAV genes and helper virus genes.

[0079] Non-exclusive examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EF1α-MCS (System Bio catalog number AAV502A-1), and pAAVK-EF1α-MCS1-CMV-MCS2 (System Bio catalog number AAV503A-1), pAAV-ZsGreen1 (Clontech catalog number 6231), pAAV-MCS2 (Addgene plasmid number 46954), AAV-Stuffer (Addgene plasmid number 106248), pAAVscCBPIGpluc (Addgene plasmid number 35645), AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid number 68375), pAAV-RAM-d2TTA::TRE-MCS-WPRE-pA (Addgene plasmid number 63931), pAAV-UbC (Addgene plasmid number 62806), pAAV These include S1-P-MCS (Addgene plasmid number 80488), pAAV-Gateway (Addgene plasmid number 32671), pAAV-Puro_siKD (Addgene plasmid number 86695), pAAVS1-Nst-MCS (Addgene plasmid number 80487), pAAVS1-Nst-CAG-DEST (Addgene plasmid number 80489), pAAVS1-P-CAG-DEST (Addgene plasmid number 80490), pAAVf-EnhCB-lacZnls (Addgene plasmid number 35642), and pAAVS1-shRNA (Addgene plasmid number 82697). These vectors may be modified to be suitable for therapeutic use. For example, an exogenous nucleic acid sequence of interest can be inserted into the multiplexing site, and a selection marker (e.g., puro, or a gene encoding a fluorescent protein) can be removed or replaced with another (same or different) exogenous gene of interest of interest.Further examples of AAV vectors are disclosed in U.S. Patents Nos. 5,871,982, 6,270,996, 7,238,526, 6,943,019, 6,953,690, 9,150,882, and 8,298,818, U.S. Patent Application Publication No. 2009 / 0087413, and PCT International Publication Nos. 2017075335A1, International Publication Nos. 2017075338A2, and International Publication Nos. 2017201258A1.

[0080] In certain embodiments, the viral vector may be a retroviral vector. Examples of retroviral vectors include Moloney mouse leukemia virus vectors, splenic necrosis virus vectors, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Retroviral vectors are useful as agents that mediate retroviral-mediated gene transfer into eukaryotic cells.

[0081] In certain embodiments, the retroviral vector is a lentiviral vector. In certain embodiments, the recombinant retroviral vector is a lentiviral vector comprising nucleic acid sequences encoding two or more optimal epitopes. Exemplary lentiviral vectors include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and canine arthritis encephalitis virus (CAEV).

[0082] Non-exclusive examples of lentiviral vectors include pLVX-EF1α-AcGFP1-C1 (Clontech catalog number 631984), pLVX-EF1α-IRES-mCherry (Clontech catalog number 631987), pLVX-Puro (Clontech catalog number 632159), pLVX-IRES-Puro (Clontech catalog number 632186), pLenti6 / V5-DEST(trademark) (Thermo Fisher), and pLenti6.2 / V5-DEST(trademark) (Thermo Fisher). Fisher), pLKO.1 (Addgene plasmid number 10878), pLKO.3G (Addgene plasmid number 14748), pSico (Addgene plasmid number 11578), pLJM1-EGFP (Addgene plasmid number 19319), FUGW (Addgene plasmid number 14883), pLVTHM (Addgene plasmid number 12247), pLVUT-tTR-KRAB (Addgene plasmid number 11651), pLL3.7 (Addgene plasmid number 11795), pLB (Addgene plasmid number 11619), pWPXL (Addgene plasmid number 12257), pWPI (Addgene plasmid number 12254), EF.CMV.RFP (Addgene plasmid number 17619), pLenti CMV Puro These include DEST (Addgene plasmid number 17452), pLenti-puro (Addgene plasmid number 39481), pULTRA (Addgene plasmid number 24129), pLX301 (Addgene plasmid number 25895), pHIV-EGFP (Addgene plasmid number 21373), pLV-mCherry (Addgene plasmid number 36084), pLionII (Addgene plasmid number 1730), and pInducer10-mir-RUP-PheS (Addgene plasmid number 44011). These vectors may be modified to be suitable for therapeutic use. For example, a selection marker (e.g., puro, EGFP, or mCherry) may be removed or replaced with an exogenous nucleic acid sequence of a secondary interest.Further examples of lentiviral vectors include U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, and This information is disclosed in Publications No. 6,013,516, No. 6,797,512, No. 6,544,771, No. 5,834,256, No. 6,958,226, No. 6,207,455, No. 6,531,123, and No. 6,352,694, as well as in PCT International Publication No. 2017 / 091786.

[0083] In some embodiments, the viral vector may be an adenovirus vector. Adenoviruses are medium-sized (90–100 nm) non-enveloped (naked) icosahedral viruses consisting of a nucleocapsid and a double-stranded linear DNA genome. The term “adenovirus” refers to any virus of the genus Adenoviridiae, including but not limited to the adenovirus subgenuses of humans, cattle, sheep, horses, dogs, pigs, mice, and monkeys. Generally, adenovirus vectors are constructed by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome of an adenovirus to allow insertion of non-natural nucleic acid sequences into the adenovirus, for example, for gene transfer.

[0084] Adenovirus vectors may be replication-capable, conditionally replication-capable, or replication-deficient. Replication-capable adenovirus vectors can replicate in typical host cells, i.e., cells that adenoviruses can commonly infect. Conditionally replication-capable adenovirus vectors are adenovirus vectors engineered to replicate under specific conditions. For example, gene functions essential for replication, such as those encoded by the adenovirus initial region, may be functionally linked to inducible, repressive, or tissue-specific transcriptional regulatory sequences, such as promoters. Conditionally replication-capable adenovirus vectors are further described in U.S. Patent No. 5,998,205. Replication-deficient adenovirus vectors are adenovirus vectors that require the completion of one or more gene functions or gene regions of the adenovirus genome required for replication, for example, as a result of the loss of one or more gene functions or gene regions essential for replication, and therefore, these adenovirus vectors will not replicate in typical host cells, in particular, in human cells infected with these adenovirus vectors.

[0085] The replication-deficient adenovirus vector of the present invention may be produced at appropriate levels in complementary cell lines that provide gene functions necessary for viral replication but are not present in the replication-deficient adenovirus vector, in order to produce high-titer viral vector stocks. Such complementary cell lines are known and include, but are not limited to, 293 cells (e.g., described in Graham et al. (1977) J. Gen. Virol. 36: 59-72), PER.C6 cells (e.g., described in PCT International Publication No. 1997 / 000326, and U.S. Patents No. 5,994,128 and 6,033,908), and 293-ORF6 cells (e.g., described in PCT International Publication No. 1995 / 034671 and Brough et al. (1997) J. Virol. 71: 9206-9213). Other complementary cell lines suitable for producing the replication-deficient adenovirus vector of the present invention include complementary cells prepared to grow an adenovirus vector whose expression encodes a transgene that inhibits viral growth in host cells (see, for example, U.S. Patent Application Publication 2008 / 0233650). Further suitable complementary cells are described, for example, in U.S. Patents 6,677,156 and 6,682,929, and PCT International Publication 2003 / 020879. Furthermore, formulations for compositions containing the adenovirus vector are described, for example, in U.S. Patents 6,225,289 and 6,514,943, and PCT International Publication 2000 / 034444.

[0086] Further exemplary adenovirus vectors and / or methods for producing or propagating adenovirus vectors are described in U.S. Patents No. 5,559,099, No. 5,837,511, No. 5,846,782, No. 5,851,806, No. 5,994,106, No. 5,994,128, No. 5,965,541, No. 5,981,225, No. 6,040,174, No. 6,020,191, No. 6,083,716, No. 6,113,913, No. 6,303,362, No. 7,067,310, and No. 9,073,980.

[0087] Commercially available adenovirus vector systems include the ViraPower® adenovirus expression system from Thermo Fisher Scientific, the AdEasy® adenovirus vector system from Agilent Technologies, and the Adeno-X® expression system 3 from Takara Bio USA, Inc.

[0088] In certain embodiments, the viral vector may be a herpes simplex virus plasmid vector. Herpes simplex virus type 1 (HSV-1) has been demonstrated as a gene delivery vector system with potential usefulness in gene therapy. HSV-1 vectors have been used to transfer genes into muscle and have been used to treat brain tumors in mice. For simpler handling and a larger insertion capacity (up to 140 kb), helper virus-dependent miniviral vectors have been developed. In the art, non-replicating HSV amplicons have been constructed. These HSV amplicons contain a large deletion in the HSV genome to provide a site for inserting exogenous DNA. Generally, these contain the HSV-1 packaging site, the HSV-1 "ori S" replication site, and the IE 4 / 5 promoter sequence. These virions rely on helper viruses for replication.

[0089] The method of the present invention can be used to treat any disease or disorder in which increasing the amount of T cells is desirable. Often, subjects who require the treatment method according to the present invention are subjects who are undergoing or are scheduled to undergo immune cell depletion treatments such as chemotherapy. Most chemotherapeutic agents work by killing all cells that are undergoing cell division. Therefore, for example, the method of the present invention can be used to treat patients who require bone marrow transplantation or hematopoietic stem cell transplantation, for example, cancer patients undergoing chemotherapy and / or radiotherapy. In particular, the method of the present invention is useful in treating patients undergoing chemotherapy or radiotherapy for cancer, including patients with myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, or leukemia.

[0090] The disorders treated by the method of the present invention may be the result of undesirable side effects or complications of other primary treatments, such as radiotherapy, chemotherapy, or treatment with immunosuppressants such as zidovadine, chloramphenicol, or gangciclovir. Such disorders include neutropenia, anemia, thrombocytopenia, and immune dysfunction.

[0091] Reduced levels of immune function compared to normal individuals can result from a variety of disorders, disease infections, or conditions, including but not limited to leukemia, immunosuppressive states resulting from renal failure; autoimmune disorders including but not limited to systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroiditis, scleroderma, and inflammatory bowel disease; various cancers and tumors; viral infections including but not limited to human immunodeficiency virus (HIV); bacterial infections; and parasitic infections, and may also occur as a result of aging.

[0092] Accordingly, the present invention provides a method for treating a disease and / or disorder or its symptoms, the method comprising administering a therapeutically effective amount of a composition comprising periostin + Pdgfra + mesenchymal stromal cells as described herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method for treating a subject having a disease characterized by a deficiency of T cells or alteration of the complexity of T cell receptors within a T cell population. The method comprises administering to the subject, under conditions such as the treatment of the disease or disorder, a therapeutic amount sufficient to treat the disease or disorder or its symptoms, of periostin + Pdgfra + mesenchymal stromal cells or mesenchymal stem cells expressing CCL19, or a mixture comprising such cell types. The identification of a subject requiring such treatment may be at the discretion of the subject or a medical professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods).

[0093] Periostin + Pdgfra + mesenchymal stromal cells are administered by methods known in the art. Such compositions may be administered by any conventional route, including injection or timed, stepwise infusion. Administration may be, for example, intrathymic, transpulmonary, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, or percutaneous, depending on the composition being administered. Periostin + Pdgfra + mesenchymal stromal cells are administered in an "effective dose," or alone or in combination with further administration to produce the desired therapeutic response. The cells administered in this invention may be autologous ("self") cells or non-autologous ("non-self," e.g., allogeneic, syngeneic, or heterogeneic) cells. Generally, cell administration can be performed within a short period after treatment (e.g., within 1, 2, 5, 10, 24, or 48 hours after treatment) and according to the requirements of each desired treatment regimen. For example, if radiotherapy or chemotherapy is performed prior to administration, the treatment and cell transplantation of this invention should be optimally provided within about one month of the interruption of treatment. However, transplantation at a later stage after discontinuation of treatment can be performed with induced clinical outcomes.

[0094] Periostin + Pdgfra + mesenchymal stromal cells can be combined with pharmaceutically active ingredients known in the art to improve the preservation and maintenance of cells before administration. In some embodiments, the cell compositions of the present invention can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0095] Sterile injectable solutions can be prepared, as desired, by incorporating the cells to be used in the implementation of the present invention into the required amount of a suitable solvent, along with various amounts of other components. Such compositions may be miscibles with suitable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, or dextrose. Compositions can also be lyophilized. Depending on the desired administration route and preparation, compositions may contain auxiliary substances such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity-enhancing additives, preservatives, flavoring agents, and dyes. To prepare suitable preparations without excessive experimentation, one may refer to standard textbooks such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985, which is incorporated herein by reference.

[0096] Various additives can be added to improve the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be reliably achieved with various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid.

[0097] The composition may be isotonic; that is, it may have the same osmotic pressure as blood and tears. The desired isotonicity of the composition of the present invention may be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffer solutions containing sodium ions.

[0098] A method that potentially increases cell viability when introducing cells to a target where this is necessary is to incorporate the cells of interest into a biopolymer or synthetic polymer. Depending on the condition of the target, the injection site may turn out to be unsuitable for cell seeding and growth due to scarring or other obstacles. Examples of biopolymers include, but are not limited to, cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. These can be constructed with or without growth factors or differentiation factors. Furthermore, these may be in suspension, but their residence time at the site of flow is minimal. Another alternative is a three-dimensional gel in which cells are encapsulated in the interstitial spaces of a cell biopolymer mixture. In this case as well, growth factors or differentiation factors may be included with the cells. These can be delivered by injection via the various routes described herein.

[0099] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and will not affect the viability or efficacy of stem cells or their progenitor cells as described herein. This is not a problem for those familiar with chemical and pharmaceutical principles, or can be easily avoided by referring to standard textbooks or by simple experiments (without excessive experimentation) from the literature cited herein and herein.

[0100] One consideration regarding the therapeutic use of cells is the amount of cells required to achieve the optimal effect. In different situations, it may be necessary to optimize the amount of cells injected into the tissue of interest. Therefore, the amount of cells administered varies depending on the subject being treated. The precise determination of what is considered an effective dose can be based on individual factors for each patient, including the patient's size, age, sex, weight, and condition. As few as 100 to 1000 cells may be administered to a selected patient for a specific desired use. Therefore, those skilled in the art can easily determine the dosage from this disclosure and knowledge in the art.

[0101] Those skilled in the art can easily determine the amounts of cells and optional additives, vehicles, and / or carriers present in the compositions of the present invention and administered in the methods of the present invention. Therefore, naturally, for any composition administered to animals or humans, and for any particular method of administration, the toxicity (e.g., in a suitable animal model, such as a rodent like a mouse, the lethal dose (LD) and LD500) can be determined. 50 It is preferable to determine the appropriate dosage of the composition, the concentration of its components, and the timing of administration of the composition, which will induce an appropriate response. Such determination does not require excessive experimentation, as can be determined by the knowledge of those skilled in the art, this disclosure, and the literature cited herein. Furthermore, the timing for continuous administration can be determined without performing excessive experimentation.

[0102] The present invention also provides a method for treating a disease and / or disorder or its symptoms, the method comprising the step of administering a therapeutically effective amount of a composition comprising Ccl19 (CC motif chemokine ligand 19) into a T cell-producing tissue or T cell-producing fluid of interest, for example, into the thymus. Ccl19 is a cytokine that plays a role in normal lymphocyte recirculation and homing. Ccl19 also plays an important role in T cell transport in the thymus and in the migration of T and B cells to secondary lymphoid organs. Ccl19 is expressed in periostin + Pdgfra + mesenchymal stromal cells according to the present invention.

[0103] Ccl19 can be administered in an effective dose by any suitable method of administration known in the art (e.g., injection or infusion). The effective dose depends on the method of administration, the specific condition being treated, and the desired outcome. The effective dose may also depend on the stage of the condition, the age and physical condition of the subject, the nature of any concomitant therapy, and similar factors known to the physician. In the case of therapeutic application, the effective dose is the amount sufficient to achieve the medically desired outcome (increased T cell production). Generally, the dose of the active Ccl19 polypeptide compound of the present invention will be about 0.01 mg / kg / day to about 1000 mg / kg / day. A dose in the range of about 50 to about 2000 mg / kg is expected to be appropriate. Lower doses are provided by specific forms of administration, such as intravenous administration. If the response in the subject is insufficient with the initial dose, a higher dose (or a higher effective dose via a more localized different delivery route) may be employed as far as the patient's tolerance allows. Multiple daily doses are intended to achieve an appropriate systemic level of the Ccl19 composition of the present invention.

[0104] The present invention will be further illustrated through the following non-limiting examples for illustrative purposes. These examples will provide a better understanding of the present invention and its many advantages. [Examples]

[0105] The following embodiments illustrate several aspects and aspects of the present invention. Various modifications, additions, substitutions, etc., can be made without altering the spirit or scope of the invention, and it will be apparent to those skilled in the art that such modifications and variations fall within the scope of the invention as defined in the following claims. The following embodiments are not intended to limit the invention in any way.

[0106] The materials and methods used to perform the assays in the following examples are described in detail below in this specification.

[0107] Animals: Eight-week-old male and female C57Bl / 6 mice were used for all transplantation and sequencing experiments. B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) and C57BL / 6-Tg(UBC-GFP)30Scha / J mice were used as bone marrow donors. B6;129S-Penktm2(cre)Hze / J mice were crossed with B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J to produce donors for mesenchymal stromal cell (MSC) transfer. All mice were obtained from Jackson Laboratories, and all animal experiments were conducted in accordance with national and institutional guidelines.

[0108] Tissue Collection and Processing: All human tissue samples were collected with the approval of the Institutional Review Board (IRB). Tissues were processed immediately after isolation to ensure the highest possible cell quality. Mouse samples were cut into fine fragments and digested in Medium 199 (M199, Gibco) containing 2% (v / v) fetal bovine serum (FBS, Gibco), liberase (0.5 WU / ml, Roche), and DNAse I (0.1 KU, Invitrogen) at 37°C for 3 × 15 minutes with constant agitation. Human samples were processed by digesting in M199 containing 2% FBS, DNAse I (0.1 KU), and 2 mg / ml Stemxyme 1 (Worthington) at 37°C for 2 × 30 minutes with constant agitation. For the last 30 minutes, the samples were digested with a combination of Stemxyme / DNAse I cocktail and 0.125% trypsin (Gibco). All samples were digested in the presence of RNase inhibitors (RNasin (Promega) and RNase OUT (Invitrogen)).

[0109] FACS sorting for single-cell RNA sequencing: Human single-cell suspensions were blocked with anti-human CD16 / 32 Fc-block (BD Biosciences) at 4°C for 10 minutes, then stained with cell lineage cocktails -FITC, CD66b-FITC, CD45-BV711, CD235a-BV711, CD8a-APC / Cy7, and CD4-BV605 (all from BD Biosciences). Mouse samples were also blocked with anti-mouse CD16 / 32 Fc-block (BD Biosciences) at 4°C for 10 minutes, then stained with CD45-PE / Cy7 and Ter119-PE (both from BioLegend). Samples were stained at 4°C for 45 minutes under constant agitation. 7-AAD (ThermoFisher) was added to samples immediately before analysis to detect dead cells. Flow sorting of viable, non-hematopoietic cells (7-AAD, CD45-CD235a / Ter119- cell lineage) was performed using a BD FACS Aria III (BD Biosciences) equipped with a 70 μm nozzle.

[0110] FACS sorting and analysis of thymic stromal cell populations: For the analysis of various thymic stromal cell populations, human samples were stained with cell lineage cocktails—FITC, CD66b-FITC, CD45-BV711, CD235a-BV711, CD8a-APC / Cy7, and CD4-BV605—in combination with CD326-BV421 (BD Bioscience) and CD31-PE / Dazzle594 (BioLegend). Mouse stromal cell types were characterized and sorted by surface staining with CD45-APC / Cy7 and Ter119-APC / Cy7 (both from BD Biosciences), as well as CD31-BUV737, CD326-BV77, and CD140a-BV785 (all from BD Biosciences). Itgb5, CD99l2, and CD248 (R&D Systems) were conjugated in-house with PE / Cy7 and APC (Abcam), respectively, and similarly used as part of stromal cell sorting.

[0111] Single-cell RNA sequencing: Selected thymic stromal cells were encapsulated in emulsion droplets using Chromium Controller (10X Genomics). Subsequently, an scRNA sequencing library was prepared using the Chromium single-cell 3' v2 reagent kit (10X Genomics). The library was diluted to 4 nM, pooled, and then sequenced using the NextSeq 500 sequencing system (Illumina).

[0112] Bone marrow, lymphoid progenitor cell, and MSC transplantation: 8-week-old C57Bl / 6 mice were given a single dose of 9.5 Grey 12–24 hours prior to transplantation. For lymphoid progenitor cell transplantation, bone marrow from a C57BL / 6-Tg(UBC-GFP)30Scha / J donor was depleted according to the manufacturer's instructions (Miltenyi). Subsequently, cells were stained with biotinylated cell lineage antibodies (CD3e, B220, CD4, CD8a, Gr-1, Cd11b), ckit-APC, and CD135-BV421 at 4°C for 30 minutes. Then, they were incubated with streptavidin-PE / Cy7 for 15 minutes. Cell lineage - CD135+ cKit+GFP+ lymphoid progenitor cells were sorted using BD FACS Aria III, and 40,000 cells were selected from 10 nucleated whole bone marrow cells from a B6.SJL-Ptprca Pepcb / BoyJ donor. 6 Along with the cells, each recipient was injected into a lethally irradiated cell. In the case of MSC adoption, the recipient was irradiated 12 hours prior to the transfer to ensure that the thymus was large enough to allow for intrathymic injection. 2,000 to 10,000 MSCs (CD45-Ter119-CD31-CD326-CD248+CD9912+Itgb5+CD140+) were injected into the thymus along with 10 nucleated whole bone marrow cells from B6.SJL-Ptprca Pepcb / BoyJ mice. 6 The individual was injected into the posterior orbital region.

[0113] Tissue clearing and two-photon imaging: For imaging of spontaneous fluorescence, tissue was fixed in vivo by injecting 4% paraformaldehyde (PFA, Electron Microscopy Sciences) and then incubating with 4% PFA for a further 6 hours. Tissue was dehydrated by successive incubation steps in progressively increasing concentrations of tert-butanol solution (Sigma, v / v, 50%, 70%, 80%, 90%, and 100%). Lipids were removed by exposure to dichloromethane (Sigma) for 45 minutes. Finally, refractive index matching was achieved by incubation in benzyl alcohol, benzyl benzoate, and diphenyl ether (BABB-D4, Sigma, 26%:53%:20%). Prior to imaging, the sample was mounted between two coverslips and immersed in BABB-D4. Images were acquired using the Olympus FVMPE-RS multiphoton imaging platform (Olympus).

[0114] Example 1. Single-cell sequencing of human and mouse thymus cells identifies mesenchymal cell subsets with different T cell support characteristics. Inefficient T-cell rearrangement after bone marrow transplantation is a major cause of morbidity and mortality. The success of T-cell-mediated immunity rearrangement then depends entirely on the regenerative capacity of the thymus. However, the mechanisms underlying thymic recovery failure are not well understood. In particular, stromal cell regeneration supporting T-cell development is still not well understood. To characterize the thymic microenvironment, CD45-CD235-CD45-Lin-thymic stromal cells were isolated and single-cell RNA sequencing was performed on a single human thymic sample (Figure 1A, Figure 2A). Initial attempts demonstrated that digestion conditions are crucial for successful isolation of thymic stromal cells from human tissue. Shorter digestion times resulted in insufficient enrichment of stromal cells and lower cell type diversity compared to longer protocols (Figure 2B). Flow sorting of non-hematopoietic cells always resulted in contamination with hematopoietic cells (Figure 2C and D), so all cells expressing PTPRC and CD3E were removed from further analysis (Figure 2D).

[0115] In the stromal cell compartment, six cell populations with distinct expression patterns were subsequently identified: endothelial cells (CDH5), mesenchymal stromal cells (PRRX1), two types of thymic epithelial cells (EPCAM), and two types of perivascular cells (RGS5) (Figure 1B, Figure 2E). The proportions of these different populations were similar across samples, and this observation was largely confirmed by flow cytometry (Figure 1C and Figure 2F). Interestingly, the largest proportion of stromal cells was found to consist of PRRX1-expressing mesenchymal stromal cells (MSCs), a cell population that, despite its abundance, has received little attention from the perspective of T cell development in the thymus (Figure 1C).

[0116] The primary function of thymic stromal cells is to recruit, maintain, and commit hematopoietic progenitor cells to the T cell lineage. Many of the molecules involved in this process are defined. By evaluating which cell types express these lymphocyte-generating factors, several expected combinations were revealed. Thymic epithelium (TEC) was found to be particularly rich in CCL21 and CCL25, chemokines that recruit T cell progenitor cells (Figure 1D). Notably, however, human thymic MSCs were thought to express high levels of several well-established regulators of lymphoid cell development, including FLT3LG, CCL19, and IL15 (Figure 1D). This suggests that a substantial pool of thymic mesenchymal cells may play a crucial role in T cell development.

[0117] To further understand and characterize the populations identified in humans, scRNA-seq was performed on the quiescent thymus of 8-week-old mice (Figure 1E and Figure 2G). A total of four samples were sequenced, and after quality control and hematopoietic cell filtering, a total of 6491 mouse stromal cells were obtained (Figure 2H, 1I, and 1J). All thymic stromal cell populations found in humans were similarly present in mice: endothelial cells (Pecam1), mesenchymal stromal cells (Prrx1), two types of perivascular cells (Rgs5), and thymic epithelial cells (Epcam), respectively (Figure 1F, Figure 2K, and 2L). In addition, the mouse thymus contains two other stromal subsets. Recently described thymic tuft (Tuft) cells were defined by the expression of Trpm5 and IL25 (Figure 1F, Figure 2K, and 2L). It was also found that small cell populations expressed Lrrn4, a marker previously associated with mesothelial stem cells and progenitor cells (Figure 1F, F2K, and 2L). While these differences in the contents of thymic stromal cells may reflect actual interspecies differences, they could also be attributable to inherent differences in sample preparation and source. Most human samples were derived from infants, for example, while mouse tissues were isolated from adults. Nevertheless, studies using adult mice continued because they represent a more suitable population for studying thymic regeneration.

[0118] Similar to what was observed in human samples, scRNA sequencing and flow cytometry analysis revealed that the largest proportion of mouse stromal cells were MSCs (Figure 1G, Figure 2M). Important thymocyte-supporting factors were also found to be abundant in mouse thymic MSCs (F1H). Indeed, IL-15, Flt3l, Ccl19, and Bmp4 were expressed at significantly higher levels in the MSC subset compared to all other stromal cell types (Figure 1I). Therefore, T cell-supporting MSCs are thought to be present in the thymic tissue of both humans and mice.

[0119] Example 2. Periostin + thymic MSCs preferentially express T cell regulatory factors. Further exploration of the MSC compartment identified three distinct subpopulations in both human and mouse thymus (Figures 3A and 4A). Both species were found to possess CD248+ and Postn+ MSC populations, albeit at varying frequencies (Figures 3A, 4B, and 4C). The third MSC subset was found to be characterized by cells defined by Cdh11 and Penk in mouse samples, while in human samples it was characterized by CDH11 expression (Figures 3A, 4B, and 4C). Comparison with previously published mouse thymic stroma datasets further confirmed the presence of the three MSC subpopulations (Figures 4D and 4E). The relative abundances of MSCs as a whole and the three subtypes were found to differ (Figures 4D and 4E). However, since thymic epithelial cells were the primary focus of this study, a different isolation protocol was used, which likely explains this difference. Notably, MSCs expressing Cd248, Penk, and Postn were also found in this dataset (Figure 4F).

[0120] GO terminology analysis of mouse samples further revealed potentially different functions among MC subtypes. CD248+ MSCs were found to be rich in terminology primarily related to protein translation and secretion (Figure 4G). This, combined with the upregulation of several extracellular matrix components (Fn1 and Ogn) exhibited by these cells (Figure 4A), suggests fibroblastic function in these cells. On the other hand, Penk+ Cdh11+ MCs were found to be characterized by terminology related to adipogenesis or stress response (Figure 3B). This is particularly interesting given that the epithelial compartments of the aging thymus are gradually replaced by adipocytes through an unknown process. The expression of epithelial regulatory programs in Postn+ MSCs (Figure 3B) is consistent with what is previously known about the function of thymic MSCs, that mesenchymal cells are involved in the recruitment of epithelial progenitor cells during embryonic development. Postn+ cells also showed significant activation of the angiogenic pathway (Figure 3B), suggesting that these cells may play a crucial role in regulating other thymic stromal cell types. However, most importantly, Postn+ MSCs were found to be a remarkably rich subtype in terms of T cell development and differentiation (Figure 3B). This observation was further confirmed by the fact that both human and mouse Postn+ MSCs expressed lymphocyte-generating cytokines Ccl19, Flt3l, and IL15 at significantly higher levels than other MSC subpopulations (Figure 3C). This suggests that Postn+ MSCs are responsible for the majority of interactions with T cells developing in the thymus.

[0121] Example 3. Disappearance of periostin + MSC after pre-radiation treatment. Because thymic regeneration is of particular interest from the perspective of bone marrow transplantation, we wanted to compare steady-state scRNA sequencing with samples that had undergone cytotoxic pretreatment and transplantation. A major obstacle in early thymic regeneration is the inefficient recruitment of T cell progenitor cells from the bone marrow. To better understand what is missing in the microenvironment at this stage, we aimed to collect thymic stroma at the point when T cell progenitor cells are first seeded in the tissue after transplantation. To this end, we attempted to track thymic seeding using flow cytometry by transplanting 40,000 GFP-labeled lymphoid progenitor cells (LPCs, cell lineage-cKit+CD135+) along with 1 million helper bone marrow cells into lethally irradiated recipient mice (Figures 6A and B). This proved to be an unreliable approach. GFP+ cells were readily found in the bone marrow, but could only be detected in very small numbers, if any, in the thymus early after transplantation (Figure 6B). In addition, many of the cells were positive for cell lineage-defining markers (Figure 6B), suggesting they were not early thymic progenitor cells (ETPs). As a result, follow-up was switched to recent thymic settlements by tissue clearing, which allowed for top-to-bottom imaging while minimizing material loss (Figures 5A, 6C). This allowed for the first detection of rare GFP+ cells in the thymus three days after transplantation (Figures 5B, 6D), and at subsequent stages, the tissue was found to be rich in migrated cells (Figure 6D). Therefore, thymic dissemination was thought to begin on day 3 post-transplantation, and this was chosen as the point in time for scRNA sequencing analysis of thymic stromal cells.

[0122] As performed in the steady-state analysis, CD45-Ter119- cells were selected from 8-week-old mice that had received a single lethal dose of radiation 4 days prior and a bone marrow transplant of 40,000 GFP+ LPCs and unlabeled helper bone marrow 3 days prior to isolation (Figure 5A). A total of three samples were sequenced, yielding 8873 cells that passed quality control, which were found to be negative for Ptprc and CD3e (Figure 5C). Pre-radiation treatment did not result in the complete disappearance of any cell type or the emergence of new subsets (Figures 5C, 5D, and 6E). Several cell populations, such as TEC B and endothelial cells, showed significant relative decreases in abundance, but these were not statistically significant (Figure 6E). However, the MSC compartment showed significant changes (Figure 5C). Stress-responsive Penk+ Cdh11+ MSCs were found to be significantly enlarged, while the frequency of T-cell-supporting Postn+ MSCs was dramatically reduced (Figure 5D). It is suggested that cytotoxic pretreatment and inefficient T cell production after bone marrow transplantation may be partly responsible for this imbalance observed in the thymic MSC subset.

[0123] To further explore the functional characteristics of MSCs after transplantation, we performed another GO terminology analysis of genes that were significantly differentially expressed. Notably, Penk+ Cdh11+ MSCs were still characterized by terms related to adipogenesis and responses to various stressors, but were also significantly rich in pathways that inhibit leukocytosis (Figure 5E).

[0124] Therefore, the expansion of these cells after radiotherapy may further inhibit T cell production. On the other hand, Postn+ MSCs still supported T cells and endothelial cells (Figures 5E and 6F), but they also showed increased adipogenic activity. In the bone marrow, it is well confirmed that MSCs differentiate into adipocytes in response to irradiation. Whether bone marrow adipocytes enhance or hinder hematopoiesis is still debated. However, thymic adipocytes were unable to support T cell development, suggesting further adverse effects of the changes in MSCs observed after irradiation and bone marrow transplantation.

[0125] Example 4. Transfer of CD248-thymic MSCs promotes T cell production after radiotherapy. To investigate the functional significance of thymic MSCs, scRNA sequencing data were queryed for potential cell surface markers that could be used to facilitate flow cytometry sorting of individual (induvial) MSC subsets. Unfortunately, no suitable markers existed that would allow for differentiation between Penk+ Cdh11+ MSCs and Postn+ populations. Two markers, CD99l2 and Itgb5, were identified that labeled all MSCs while showing little overlap with perivascular cells (Figure 8A). The specificity of these markers in the MSC compartment was further confirmed by flow cytometry analysis, as well as sorting and plating of CD99l2+Itgb5+ thymocytes (Figures 8A and 8B). These cells were found to adhere to plastic and to have colony-forming ability comparable to bone marrow MSCs (Figure 8B). In addition, Penk+ Cdh11+ MSCs and Postn+ MSCs were found to express Pdgfra, and as previously mentioned, these cells were negative for Cd248 (Figure 8C). As a result, by selecting CD45-Ter119-CD31-CD326-CD248-CD9912+Itgb5+Pdgfra+ cells, the most T cell-supporting MSCs (CD248- MSCs) were enriched, while CD248+ MSCs, which were considered to be of lower importance, were excluded.

[0126] CD248-MSCs were isolated from ubiquitin-GFP mice used as donors and injected into the thymus of recipients who had undergone radiation therapy and bone marrow transplantation (Figure 7A). In parallel with the MSC-treated mice, sham-treated recipients were injected with bone marrow but PBS into their thymus (Figure 7B). To match the introduction of cells into the tissue, a cohort of mice that received intrathymic injection of single-positive CD8 thymocytes, a cell population not previously associated with thymic regeneration, was included (Figure 7B). Six days after transplantation, flow cytometry analysis demonstrated the persistence of GFP-labeled CD248-MSCs in the tissue (Figure 7B). The presence of the transferred MSCs was further associated with improvements in both ETP and endothelial cell counts (Figure 7B), while the number of MSCs and epithelial cells (data not shown) was unchanged compared to sham-treated mice and CD8+ T cell-treated mice. This suggests that injecting fresh thymic CD248-MSCs after radiation pre-treatment can improve thymic regeneration.

[0127] Ccl19, one of the factors significantly abundant in thymic MSCs, has previously been associated with ETP recruitment. To determine whether Ccl19 expression in MSCs is necessary for the improved ETP dissemination observed after transplantation, CD248- MSCs were isolated from Cas9-GFP expressing mice. These cells were then infected with lentiviral vectors expressing guide RNA directed to either Ccl19 or the control locus GFP. When these modified MSCs were transplanted, it was demonstrated that knockout of Ccl19 rendered the improved ETP recruitment after CD248 MSC treatment ineffective (Figure 7C).

[0128] To determine whether the increased influx of progenitor cells on day 6 led to increased T cell generation, the transplantation experiment was repeated. This time, the thymus was analyzed after 4 weeks. GFP+ CD248- MSCs were still found to be present in the tissue (Figure 7C), and thymic weight and cell density were significantly higher in MSC-treated mice (Figure 8D). sjTREC analysis further demonstrated that CD248- MSC-injected mice showed significantly improved production of newly rearranged T cells (Figure 7C). This was further supported by higher cell counts at all stages of T cell development (Figure 8D). In addition, 16-week follow-up of transplanted mice showed that CD248- MSC recipients had increased CD4+ T H Cells and CD8+ T CTL The number of cells was dramatically improved (Figure 7D), and there was no impact on B cells or the myeloid population (Figure 8E). Surprisingly, analysis of the thymic stromal compartment 16 weeks after transplantation revealed that GFP+ MSCs were still surviving in the tissue (Figure 8F).

[0129] The definitive goal of improving T cell counts after bone marrow transplantation is to enhance functional immunity. Therefore, transplant recipients were vaccinated against ovalbumin 44 days later (Figure 7F). After reloading, CD248-MSC treated mice showed ovalbumin-specific CD8+ T CTL The immune response was found to be significantly improved, as evidenced by an increase in cell number and IFNγ production (Figure 7F). Thus, the initial improvement in thymic regeneration observed after CD248-MSC transfer ultimately leads to robust production of functional T cells.

[0130] Example 5. Periostin + MSC specifically enhances the recruitment of T cell progenitor cells. Penk-Cre mice were crossed with the Rosa26-LSL-tdTomato reporter to create mice capable of separating Penk+ Cdh11+ MSCs from Postn MSCs. Initial flow cytometry analysis of these mice showed that the CD45-Ter119-CD31-CD326-CD248-CD99l2+Itgb5+Pdgfra+ subset separated into distinct tdTomato+ (Penk+) and tdTomato- (Postn+) populations (Figure 9A), suggesting that this reporter is faithful to scRNA sequencing data. Indeed, when tdTomato+ or tdTomato- cells were transferred under bone marrow transplantation conditions, the putative Postn+ MSC recipients demonstrated improved ETP and endothelial cell counts after 6 days (Figure 9B). Therefore, the effect mediated by thymic MSCs is thought to be included in the Postn+ MSC population.

[0131] References All patents, patent applications, and publications referenced herein are incorporated by reference to the same extent as each individual patent and publication is incorporated by reference specifically and individually.

Claims

1. 1. A method for increasing the production of T cells in a T cell-producing tissue or T cell-producing body fluid of a subject in need thereof, comprising: administering a composition comprising mesenchymal stromal cells expressing periostin and Pdgfra into a T cell-producing tissue or T cell-producing body fluid of the subject, thereby increasing T cell production in the T cell-producing tissue or T cell-producing body fluid of the subject. A method comprising:

2. The method of claim 1, wherein the mesenchymal stromal cells do not express Cdh11 and CD248.

3. The method of claim 1, wherein the T cell-producing tissue is the thymus.

4. The method of claim 1, wherein the T cell-producing tissue is a lymphocyte-producing tissue.

5. The method of claim 1, wherein the T cell-producing body fluid is blood.

6. The method of claim 1, wherein the subject has undergone a hematopoietic stem cell transplant.

7. 10. The method of claim 1, wherein the subject has one or more of a condition associated with T lymphopenia, impaired T cell production, impaired T cell function, a skewed repertoire of cells bearing T cell receptors, an infection, or a tumor.

8. 2. The method of claim 1, wherein the mesenchymal stromal cells express Flt3 ligand (fms-related receptor tyrosine kinase 3 ligand), Ccl19 (C-C motif chemokine ligand 19), BMP2 (bone morphogenetic protein 2), BMP4 (bone morphogenetic protein 4), IL-15 (interleukin 15), IL-12a (interleukin-12a), Cxcl14 (C-X-C motif chemokine ligand 14), Ccl11 (C-C motif chemokine ligand 11), (Cxcl10, C-X-C motif chemokine ligand 10), or IL-34 (interleukin 34), and combinations thereof.

9. 2. The method of claim 1, wherein the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15, but do not express Cdh11 and CD248.

10. The method of claim 1, wherein the mesenchymal stromal cells are autologous to the subject.

11. The method of claim 1, wherein the mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells.

12. The method of claim 1, wherein the mesenchymal stromal cells are derived from embryonic stem cells or progenitor cells thereof.

13. The method of claim 1, wherein the mesenchymal stromal cells are derived from iPS cells or their precursor cells.

14. 1. A method for increasing the production of T cells in a T cell-producing tissue or T cell-producing body fluid of a subject in need thereof, comprising: administering a composition comprising Ccl19 (CC motif chemokine ligand 19) into a T cell-producing tissue or T cell-producing body fluid of the subject, thereby increasing the production of T cells in the T cell-producing tissue or T cell-producing body fluid of the subject. A method comprising:

15. 15. The method of claim 14, wherein the T cell-producing tissue is the thymus.

16. 15. The method of claim 14, wherein the T cell-producing tissue is a lymphopoietic tissue.

17. 15. The method of claim 14, wherein the T cell-producing body fluid is blood.

18. 15. The method of claim 14, wherein the subject has undergone a hematopoietic stem cell transplant.

19. 15. The method of claim 14, wherein the subject has one or more of a condition associated with T lymphopenia, impaired T cell production, impaired T cell function, a skewed repertoire of cells bearing T cell receptors, an infection, or a tumor.

20. A composition comprising isolated mesenchymal stromal cells that express periostin and Pdgfra.

21. 21. The composition of claim 20, wherein the mesenchymal stromal cells do not express Cdh11 and CD248.

22. 21. The composition of claim 20, wherein the mesenchymal stromal cells express Flt3 ligand (fms-related receptor tyrosine kinase 3 ligand), Ccl19 (C-C motif chemokine ligand 19), BMP2 (bone morphogenetic protein 2), BMP4 (bone morphogenetic protein 4), IL-15 (interleukin 15), IL-12a (interleukin-12a), Cxcl14 (C-X-C motif chemokine ligand 14), Ccl11 (C-C motif chemokine ligand 11), (Cxcl10, C-X-C motif chemokine ligand 10), or IL-34 (interleukin 34), and combinations thereof.

23. 21. The composition of claim 20, wherein the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15, but do not express Cdh11 and CD248.

24. 21. The composition of claim 20, wherein the mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells.

25. 21. The composition of claim 20, wherein the mesenchymal stromal cells are derived from embryonic stem cells or progenitor cells thereof.

26. 21. The composition of claim 20, wherein the mesenchymal stromal cells are derived from iPS cells or precursor cells thereof.

27. A population of isolated stem cells capable of differentiating into mesenchymal stromal cells that express periostin and Pdgfra.

28. 27. The isolated stem cell population of claim 26, wherein said mesenchymal stromal cells do not express Cdh11 and CD248.

29. A composition for increasing T cell production within a subject's T cell-producing tissue or T cell-producing body fluid, the composition comprising Ccl19 (CC motif chemokine ligand 19).

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