Methods for enhancing the regeneration of T cells

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 - Reference to Related Applications This application is related to and claims priority from 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. 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.

Background Art

[0004] Background of the Invention Deficiency of T cells is an acute and life - threatening complication of hematopoietic stem cell transplantation (HSCT) and a common progressive feature of aging. The generation of new T cells depends on hematopoietic stem cells / hematopoietic progenitor cells entering the thymus and maturing there. Methods to enhance thymic tissue regeneration and long - term T cell reconstitution are highly desirable.

Summary of the Invention

[0005] In one aspect, the present invention provides a method for increasing the production of T cells in the T cell-producing tissue or T cell-producing body fluid of a subject in need thereof, the method comprising administering to the T cell-producing tissue or T cell-producing body fluid of the subject a composition comprising mesenchymal stromal cells expressing periostin and Pdgfra, thereby increasing the production of T cells in the T cell-producing tissue or the T cell-producing body fluid of the subject.

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

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

[0008] In another embodiment, the T cell-producing tissue is lymphopoietic tissue.

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

[0010] In yet another embodiment, the subject has undergone a hematopoietic stem cell transplantation.

[0011] In yet another embodiment, the subject has one or more of a condition associated with T lymphocytopenia, a T cell production disorder, a T cell dysfunction, a distortion of the repertoire of cells having a T cell receptor, an infection, or a tumor.

[0012] In yet another embodiment, 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.

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

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

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

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

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

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

[0019] In another aspect, the present invention provides 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, the method comprising administering a composition comprising Ccl19 (C-C motif chemokine ligand 19) into the 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.

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

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

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

[0023] In yet another aspect, the subject has received a hematopoietic stem cell transplant.

[0024] In yet another aspect, the subject has one or more of a condition associated with T lymphocytopenia, a T cell production disorder, a T cell dysfunction, a distortion of the repertoire of cells having a T cell receptor, an infection, 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 aspect, the mesenchymal stromal cells are derived from iPS cells or their progenitor cells.

[0033] In yet 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 aspect, the mesenchymal stromal cells do not express Cdh11 and CD248.

[0035] In yet another aspect, the present invention provides a composition for increasing the production of T cells in a subject's T cell-producing tissue or T cell-producing body fluid, the composition comprising Ccl19 (C-C motif chemokine ligand 19).

[0036] [The present invention 1001] A method for increasing the production of T cells in a target T cell-producing tissue or T cell-producing body fluid, comprising: administering a composition comprising mesenchymal stromal cells expressing periostin and Pdgfra into the target T cell-producing tissue or T cell-producing body fluid, thereby increasing the production of T cells in the target T cell-producing tissue or T cell-producing body fluid. A method comprising the above. [The present invention 1002] The method of the present invention 1001, wherein the mesenchymal stromal cells do not express Cdh11 and CD248. [The present invention 1003] The method of the present invention 1001, wherein the T cell-producing tissue is the thymus. [The present invention 1004] The method of the present invention 1001, wherein the T cell-producing tissue is lymphopoietic tissue. [The present invention 1005] The method of the present invention 1001, wherein the T cell-producing body fluid is blood. [The present invention 1006] The method of the present invention 1001, wherein the subject has undergone hematopoietic stem cell transplantation. [The present invention 1007] The method of the present invention 1001, wherein 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, infection, or tumor. [The present invention 1008] The method of the present invention 1001, 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. [The present invention 1009] The method of the present invention 1001, wherein the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15 and do not express Cdh11 and CD248. [The present invention 1010] The method of the present invention 1001, wherein the mesenchymal stromal cells are autologous to the subject. [The present invention 1011] The method of the present invention 1001, wherein the mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells. [The present invention 1012] The method of the present invention 1001, wherein the mesenchymal stromal cells are derived from embryonic stem cells or their progenitor cells. [The present invention 1013] The method of the present invention 1001, wherein the mesenchymal stromal cells are derived from iPS cells or their progenitor cells. [The present invention 1014] A method for increasing the production of T cells in the T cell-producing tissue or T cell-producing body fluid of a subject in need thereof, comprising the step of administering a composition comprising Ccl19 (C-C motif chemokine ligand 19) into the 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 the above step. [The present invention 1015] The method of the present invention 1014, wherein the T cell-producing tissue is the thymus. [The present invention 1016] The method of the present invention 1014, wherein the T cell-producing tissue is lymphopoietic tissue. [The present invention 1017] The method of the present invention 1014, wherein the T cell-producing body fluid is blood. [The present invention 1018] The method of the present invention 1014, wherein the subject has undergone hematopoietic stem cell transplantation. [The present invention 1019] The method of the present invention 1014, wherein the subject has one or more of a condition associated with T lymphopenia, T cell production disorder, T cell dysfunction, distortion of the repertoire of cells having a T cell receptor, infection, or tumor. [The present invention 1020] A composition comprising isolated mesenchymal stromal cells expressing periostin and Pdgfra. [The present invention 1021] The composition of the present invention 1020, wherein the mesenchymal stromal cells do not express Cdh11 and CD248. [The present invention 1022] The composition of the present invention 1020, 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. [The present invention 1023] The composition of the present invention 1020, wherein the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15 and do not express Cdh11 and CD248. [The present invention 1024] The composition of the present invention 1020, wherein the mesenchymal stromal cells are derived from mesenchymal stem cells or their progenitor cells. [The present invention 1025] The composition of the present invention 1020, wherein the mesenchymal stromal cells are derived from embryonic stem cells or their progenitor cells. [The present invention 1026] The composition of the present invention 1020, wherein the mesenchymal stromal cells are derived from iPS cells or their progenitor cells. [The present invention 1027] An isolated population of stem cells capable of differentiating into mesenchymal stromal cells expressing periostin and Pdgfra. [The present invention 1028] The isolated population of stem cells of the present invention 1026, wherein the mesenchymal stromal cells do not express Cdh11 and CD248. [The present invention 1029] A composition for increasing the production of T cells in a target T cell-producing tissue or T cell-producing body fluid, the composition comprising Ccl19 (C-C motif chemokine ligand 19). Other features and advantages of the present invention will become apparent from the detailed description and the appended claims. Accordingly, other aspects of the present invention are described in the following disclosure and are within the scope of the present invention.

Brief Description of the Drawings

[0037] The following detailed description is given by way of example only and is not intended to limit the present invention to the specific embodiments described. It can be understood in conjunction with the accompanying drawings incorporated herein by reference.

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Mode for Carrying Out the Invention

[0039] Detailed Description of the Invention Definitions All technical and scientific terms used in this specification, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including the definitions, will prevail.

[0040] "Subject" refers to a vertebrate including any member of the class Mammalia, including humans, domestic and farm animals, and zoo, sports, or pet animals such as mice, rabbits, pigs, sheep, goats, cows, and higher primates.

[0041] As used herein, the terms "treat", "treating", "treatment", etc. refer to reducing or alleviating a disorder and / or its associated symptoms. It will be recognized that treatment of a disorder or condition, although not excluding it, does not necessarily require the complete elimination of the disorder, condition, or their associated symptoms.

[0042] "Effective amount" means the amount of mesenchymal cells, stem cells, or progenitor cells that results in a desired therapeutic response (i.e., enhanced production of T cells in the thymus).

[0043] "Mesenchymal progenitor cell" means a pluripotent cell that has the potential to commit to the mesenchymal lineage.

[0044] "Mesenchymal stem cell" means a multipotent cell that has the potential to commit to multiple mesenchymal cell types but does not express genes that define a specific cell type.

[0045] "Isolated" means a material that contains, to varying degrees, fewer of the components that are normally associated with it when found in its natural state. "Isolating" refers to the degree of separation from the original source or original environment.

[0046] As used herein, "increased", when referring to T cell production, means at least about 0.05-fold more (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 25, 50, 100, 1000, 10,000-fold or more) T cell production compared to a reference level (e.g., a subject having normal T cell production). Also, "increased" when referring to T cell production means at least about 5% more (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% more) compared to the T cell production level compared to a reference level (e.g., a subject having normal T cell production). The amount can be measured according to methods known in the art for determining the amount of T cells.

[0047] As used herein, unless otherwise specifically described or apparent from the context, the term "about" is understood to be within the normal tolerance range in the art, e.g., within 2 standard deviations of the mean. "About" is understood to be 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 specified from the context, all numerical values provided herein are modified by the term "about".

[0048] The ranges provided herein are understood to be shorthand for all values within that range. For example, the range of 1-50 is understood to include any number, combination of numbers, or sub-range 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, unless otherwise clearly specified in the context, fractions thereof).

[0049] In the present disclosure, terms such as "comprise", "comprising", "contain", and "have" can have the meanings given to them in the United States Patent Law and can mean "include", "including", etc. Similarly, "consisting essentially of" or "consist essentially" have the meanings given in the United States Patent Law, and the term is open-ended and allows for the presence of elements other than those recited, provided that the basic or novel characteristics of the recited elements are not changed by the presence of elements other than those recited, except for aspects of the prior art.

[0050] Other definitions are set forth contextually throughout the present disclosure.

[0051] Compositions and methods of the present invention By comprehensive analysis of thymus-derived mesenchymal stromal cells, a periostin-positive Pdgfra-positive immunophenotype (periostin+Pdgfra+ immunophenotype) was identified. It has now been found that this immunophenotype is essential for T cell production. By adoptive transfer of these cell subpopulations into T cell-producing tissues or fluids such as the thymus, these cells have been shown to enhance thymic tissue regeneration and long-term T cell reconstitution in the context of hematopoietic stem cell transplantation (HSCT). Generation or isolation of periostin+Pdgfra+ cells and their transfer, and / or specific genes or proteins expressed by these cells, provide therapeutic benefits in the context of HSCT or in other situations where T cell depletion / deficiency or dysfunction, including aging, contributes to adverse effects.

[0052] Periostin is described, for example, by GenBank accession number NM_001135934.2 (SEQ ID NO: 1 and 2). Periostin, also known as osteoblast-specific factor 2, is a secreted cell adhesion protein and is homologous to fasciclin 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 fasciclin I, an insect cell adhesion protein, and act as ligands for integrins. The C-terminal region of periostin consists of a hydrophilic domain. The N-terminal region of periostin is highly conserved, whereas the C-terminal region of the protein varies among isoforms. The N-terminal region controls cell function by binding to integrins at the plasma membrane of cells via its FAS domains. The C-terminal region of the protein controls 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 an important regulatory factor in bone and tooth formation and maintenance, as well as in heart development and healing. Periostin also plays an important role in tumor growth and is upregulated in a variety of cancers such as colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, head and neck cancer, thyroid cancer, and gastric cancer, as well as in neuroblastoma. Periostin bound to integrins activates Akt / PKB-mediated and FAK-mediated signaling pathways, and these signaling pathways lead to increased cell survival, angiogenesis, invasion, metastasis, and importantly, epithelial-mesenchymal transition of cancer cells.

[0054] Platelet-derived growth factor receptor alpha, i.e., Pdgfra, is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. These growth factors are mitogens for mesenchymal-derived cells. Pdgfra is known to play roles in organ development, wound healing, and tumor progression. Pdgfra is described, for example, by GenBank accession NM_001347827.2 (SEQ ID NOs: 3 and 4). Pdgfra is a typical receptor-type tyrosine kinase and is a transmembrane protein consisting of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. The molecular weight of the 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 a regenerative phenotype, including but not limited to 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), and IL-34 (interleukin 34), and 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: 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); IL-34 is described, for example, by GenBank accession NM_001172771.2 (SEQ ID NO: 25 and 26). The mesenchymal stromal cells of the present invention, or their progenitor cells, may be engineered to express or overexpress these and other regenerative proteins at levels suitable to induce the production of T cells.

[0057] In some embodiments, the 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 member of the cadherin superfamily of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. Cdh11 is described, for example, by GenBank accession number NM_001308392.2 (SEQ ID NOs: 27 and 28). The mature cadherin protein consists 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 characteristic of type I cadherins. Expression of this specific cadherin in osteoblast cell lines and its upregulation during differentiation suggest a role 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 NOs: 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 tissues (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 techniques based on the expression of cell surface markers, such as fluorescence-activated cell sorting (FACS sorting), positive separation techniques, and negative separation, magnetic separation, and combinations thereof. A person skilled in the art can easily determine the ratio of stromal cells, stem cells, or their progenitor cells within a population using various well-known methods such as FACS. In some embodiments, it is desirable to first purify the cells. The stromal cells, stem cells, or their progenitor cells may constitute a population of cells having a purity 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%, 80-85%; most preferably, the purity is about 85-90%, 90-95%, and 95-100%. The purity of the stromal cells, stem cells, or their progenitor cells can be determined according to the gene marker profile within the population. The therapeutic dosage can be easily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in the dosage).

[0061] In other aspects, the mesenchymal stromal cells of the present invention may be derived from suitable stem cells or progenitor cells. The stem cells of the present invention include mesenchymal stem cells. Mesenchymal stem cells, namely "MSCs", are well known in the art. MSCs are originally derived from embryonic mesoderm and are isolated from adult bone marrow, and can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendon. During embryogenesis, mesoderm grows into limb bud mesoderm, which is a tissue that generates bone, cartilage, fat, skeletal muscle, and endothelium. Mesoderm also differentiates into visceral mesoderm, which can give rise to blood islands consisting of cardiomyocytes, smooth muscle, or endothelium and hematopoietic progenitor cells. Thus, primitive mesoderm or MSCs can provide a source of numerous cell types and tissue types. Several MSCs have been isolated. (See, for example, U.S. Patent No. 5,486,359 to Caplan, A., et al.; U.S. Patent No. 5,827,735 to Young, H., et al.; U.S. Patent No. 5,811,094 to Caplan, A., et al.; U.S. Patent No. 5,736,396 to Bruder, S., et al.; U.S. Patent No. 5,837,539 to Caplan, A., et al.; U.S. Patent No. 5,837,670 to Masinovsky, B.; U.S. Patent No. 5,827,740 to 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 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 out of 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 is provided that can be used to generate or regenerate tissues including breast tissue, skin tissue, muscle tissue, endothelial tissue, bone tissue, respiratory tissue, urogenital 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 ability and pluripotent differentiation ability (Thomson, J. et al. 1995; Thomson, J.A. et al. 1998; Shamblott, M. et al. 1998; Williams, R.L. et al. 1988; Orkin, S. 1998; Reubinoff, B.E., et al. 2000). These cells may be derived from the inner cell mass (ICM) of a pre-implantation blastocyst (Thomson, J. et al. 1995; Thomson, J.A. et al. 1998; Martin, G.R. 1981), or may be derived from primordial germ cells from a post-implantation embryo (embryonic germ cells or EG cells). ES cells and / or EG cells have been obtained from a number of species including mouse, rat, rabbit, sheep, goat, pig, and more recently from humans and human and non-human primates (U.S. Patent Nos. 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 primate embryonic stem cells including humans. U.S. Patent Application Nos. 20010024825 and 20030008392 describe human embryonic stem cells. U.S. Patent Application No. 20030073234 describes a cloned human embryonic stem cell line. U.S. Patent No. 6,090,625 and U.S. Patent Application No. 20030166272 describe undifferentiated cells stated to be pluripotent. U.S. Patent Application No. 20020081724 describes what is stated to be a cell culture 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 an embryonic stem cell-like state by expressing genes and factors important for maintaining the characteristics that characterize 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 genetic material of the cells. Genetic modification of mesenchymal stromal cells, stem cells, or progenitor cells includes the addition of exogenous genetic material. Exogenous genetic material includes natural or synthetic nucleic acids or oligonucleotides introduced into the cell.

[0067] To achieve genetic modification of mesenchymal stromal cells, stem cells, or progenitor cells, a gene editing system can be used. 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 modified for use in gene editing (silencing, enhancing, or changing specific genes) in eukaryotes such as mice or primates. This is achieved, for example, by introducing into eukaryotic cells a plasmid containing a specially designed CRISPR and one or more appropriate Cas. Generally, the CRISPR / Cas system for gene editing in eukaryotic cells comprises (1) a guide RNA molecule (gRNA) comprising a target-directed sequence (capable of hybridizing to a target sequence of genomic DNA) and a sequence capable of binding to a Cas, such as a Cas9 enzyme, and (2) a Cas protein, such as a Cas9 protein. The target-directed sequence and the sequence capable of binding to a Cas, such as a Cas9 enzyme, may be located on the same molecule or on different molecules. When located on different molecules, each molecule comprises a hybridization domain, for example, enabling these molecules to associate by hybridization.

[0068] The CRISPR sequence, sometimes called the CRISPR locus, contains alternating repeats and spacers. RNA from the CRISPR locus is constitutively expressed and processed into small RNAs. These contain the spacer adjacent to the repeat sequence. These RNAs induce other Cas proteins to silence foreign 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, the spacer functions as a template for an RNA molecule, similar to siRNA. Pennisi (2013) Science 341: 833-836.

[0069] Thus, using the CRISPR / Cas system, modifications can be made, for example, deleting one or more nucleic acids, such as CD248, or gene regulatory elements of CD248, or introducing premature termination that results in decreased expression of functional CD248. Alternatively, the CRISPR / Cas system can be used like RNA interference to reversibly turn off CD248. For example, in mammalian cells, the RNA can direct the Cas protein to the promoter of CD248 or Cdh11 and sterically interfere with RNA polymerase.

[0070] In another aspect, 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. Using this process, DNA encoding periostin and Pdgfra, as described herein for example, can be integrated at or near the site targeted by the CRISPR / Cas system.

[0071] In other aspects, the exogenous genetic material may also include a native gene placed under the functional regulation of a promoter in an 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., lentivirus, retrovirus, adenovirus, and adeno-associated virus), which can incorporate and deliver recombinant polynucleotides.

[0072] Methods for producing viral expression vectors are known in the art. Generally, the disclosed viruses are produced in suitable host cell lines using conventional techniques, which include culturing transfected or infected host cells under appropriate conditions to allow for the production of infectious viral particles. Nucleic acids encoding the genes of the virus, and / or sequences encoding, for example, periostin and pdgfra, can be incorporated into plasmids and introduced into host cells by conventional transfection or transformation techniques. Exemplary host cells suitable for the production of the disclosed viruses 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 production cells may be administered directly to the subject, while in other embodiments, after production, the infectious viral particles are recovered from the culture and optionally purified. Typical purification steps can include plaque purification, centrifugation, such as cesium chloride gradient centrifugation, clarification, enzymatic treatment, such as benzonase or protease treatment, chromatography steps, such as ion exchange chromatography, or filtration steps.

[0074] In certain embodiments, the expression vector is a viral vector. As used herein, the term "virus" is used to refer to any obligate intracellular parasite that has neither a protein synthesis mechanism nor an energy generation mechanism. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Epstein-Barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotype viral vectors.

[0075] The virus can be an RNA virus (having a genome composed of RNA) or a DNA virus (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvovirus (e.g., adeno-associated virus), adenovirus, asfarvirus, herpesvirus (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papillomavirus (e.g., HPV), polyomavirus (e.g., simian vacuolating virus 40 (SV40)), and poxvirus (e.g., vaccinia virus, cowpox virus, variola virus, fowlpox virus, sheep pox virus, myxoma virus). In certain embodiments, the viral vector is an RNA viral vector. Exemplary RNA viruses include bunyavirus (e.g., hantavirus), coronavirus, Ebola virus, flavivirus (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis virus (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza virus (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), retrovirus (e.g., human immunodeficiency virus-1 (HIV-1)), and torovirus.

[0076] In certain embodiments, the expression vector comprises a control sequence or promoter operably linked to a nucleotide sequence encoding an exogenous sequence, such as one encoding periostin and pdgfra. The term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a gene if they affect the transcription of the gene. Usually, operably linked nucleotide sequences are contiguous. However, since enhancers generally function when located several kilobases away from a promoter and intron sequences can be of variable length, some polynucleotide elements may be operably linked but not directly adjacent and may even function in trans from different alleles or chromosomes.

[0077] Additional 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 such as histone promoters, pol III promoters, and β-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoter, 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 of the genus Dependoparvovirus in the Parvoviridae family. 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 the production of AAV vectors (see, for example, U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, and 8,163,543, U.S. Patent Application Publication No. 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 that provide additional AAV genes and helper virus genes.

[0079] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EF1α-MCS (System Bio catalog number AAV502A-1), 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), pAAVS1-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 multiple cloning site, and a selectable 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.Further examples of AAV vectors are disclosed in U.S. Patent 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. 2017075335 A1, 2017075338 A2, and 2017201258 A1.

[0080] In certain embodiments, the viral vector may be a retroviral vector. Examples of retroviral vectors include Moloney murine leukemia virus vectors, spleen necrosis virus vectors, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Retroviral vectors are useful as agents that mediate retrovirus-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 a nucleic acid sequence 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 caprine arthritis encephalitis virus (CAEV).

[0082] Non-limiting 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), pLenti6.2 / V5-DEST (trademark) (Thermo 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 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), pInducer10-mir-RUP-PheS (Addgene plasmid number 44011). These vectors may be modified to be suitable for therapeutic use. For example, a selectable marker (e.g., puro, EGFP, or mCherry) can be removed or replaced with an exogenous nucleic acid sequence of a second interest.Further examples of lentiviral vectors are disclosed in 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, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, and PCT International Publication No. 2017 / 091786.

[0083] In some embodiments, the viral vector may be an adenoviral vector. An adenovirus is a non-enveloped (naked) icosahedral virus of medium size (90 - 100 nm) composed 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 adenovirus subgroups of human, bovine, ovine, equine, canine, porcine, murine, and simian. Generally, an adenoviral vector is produced by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus to allow insertion of a non-native nucleic acid sequence, for example, for gene transfer.

[0084] Adenoviral vectors may be replication-competent, conditionally replication-competent, or replication-deficient. Replication-competent adenoviral vectors can replicate in typical host cells, i.e., cells that can be generally infected by adenoviruses. Conditionally replicating adenoviral vectors are adenoviral vectors engineered to replicate under predetermined conditions. For example, gene functions essential for replication, such as those encoded by adenoviral early regions, may be operably linked to inducible, repressible, or tissue-specific transcriptional regulatory sequences, such as promoters. Conditionally replicating adenoviral vectors are further described in U.S. Patent No. 5,998,205. Replication-deficient adenoviral vectors are adenoviral vectors that require complementation of one or more gene functions or gene regions of the adenoviral genome necessary for replication as a result of the deletion of one or more gene functions or gene regions essential for replication, and thus, this adenoviral vector does not replicate in typical host cells, particularly in human cells infected with this adenoviral vector.

[0085] The replication-deficient adenovirus vector of the present invention may be produced at an appropriate level in a complementing cell line that provides gene functions required for virus growth but is not present in the replication-deficient adenovirus vector in order to produce a high-titer virus vector stock. Such complementing 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. WO 1997 / 000326 and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (e.g., described in PCT International Publication No. WO 1995 / 034671 and Brough et al. (1997) J. Virol. 71: 9206-9213). Other complementing cell lines suitable for producing the replication-deficient adenovirus vector of the present invention include complementing cells engineered to grow an adenovirus vector encoding a transgene whose expression inhibits virus growth in the host cell (see, e.g., U.S. Patent Application Publication No. 2008 / 0233650). Further suitable complementing cells are described, for example, in U.S. Pat. Nos. 6,677,156 and 6,682,929, and PCT International Publication No. WO 2003 / 020879. Furthermore, formulations for compositions containing adenovirus vectors are described, for example, in U.S. Pat. Nos. 6,225,289 and 6,514,943, and PCT International Publication No. WO 2000 / 034444.

[0086] Additional exemplary adenoviral vectors, and / or methods for making or propagating adenoviral vectors, are described in U.S. Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, 6,083,716, 6,113,913, 6,303,362, 7,067,310, and 9,073,980.

[0087] Commercially available adenoviral vector systems include the ViraPower™ Adenoviral Expression System available from Thermo Fisher Scientific, the AdEasy™ Adenoviral Vector System available from Agilent Technologies, and the Adeno-X™ Expression System 3 available 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 that may be useful in gene therapy. HSV-1 vectors have been used to transfer genes into muscle and to treat brain tumors in mice. Helper virus-dependent mini-virus vectors have been developed for easier manipulation and greater insert capacity (up to 140 kb). In the art, replication-deficient HSV amplicons have been constructed. These HSV amplicons contain large deletions of the HSV genome to provide a site for insertion of exogenous DNA. Generally, these include the HSV-1 packaging site, the HSV-1 "ori S" replication site, and the IE 4 / 5 promoter sequence. These virions are dependent on helper virus for propagation.

[0089] The method of the present invention can be used to treat any disease or disorder for which it is desirable to increase the amount of T cells. Often, the subject in need of the treatment method according to the present invention is a subject who has received or is scheduled to receive a treatment that depletes immune cells, such as chemotherapy. Most chemotherapeutic agents act by killing all cells that are undergoing cell division. Thus, for example, the method of the present invention can be used to treat patients in need of bone marrow transplantation or hematopoietic stem cell transplantation, such as cancer patients who are undergoing chemotherapy and / or radiotherapy. In particular, the method of the present invention is useful in the treatment of patients undergoing chemotherapy or radiotherapy for cancer, including patients suffering from multiple 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 another primary treatment, such as radiotherapy, chemotherapy, or treatment with an immunosuppressive drug such as zidovadine, chloramphenical, or gangciclovir. Such disorders include neutropenia, anemia, thrombocytopenia, and immune dysfunction.

[0091] Reduced levels of immune function compared to normal subjects can occur due to various disorders, diseases, infections, or conditions, including, but not limited to, leukemia, immunosuppression due to renal failure; autoimmune disorders including systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroiditis, scleroderma, 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 a symptom thereof, the method comprising administering to a subject (e.g., a mammal such as a human) a therapeutically effective amount of a composition comprising the perlecan+Pdgfra+ mesenchymal stromal cells described herein. Accordingly, one aspect is a method for treating a subject having a disease characterized by a lack of T cells or a change in the complexity of the T cell receptor within the T cell population. The method comprises administering to the subject, under conditions such that the disease or disorder is treated, a therapeutically sufficient amount of perlecan+Pdgfra+ mesenchymal stromal cells or mesenchymal stem cells expressing CCL19, or a mixture comprising such cell types, or CCL19 itself. Identification of a subject in need of such treatment can be made by the judgment of the subject or a medical professional and may be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0093] The perlecan+Pdgfra+ mesenchymal stromal cells are administered by methods known in the art. Such compositions may be administered by any conventional route, including injection or slow, stepwise infusion over time. Administration may be, for example, intrathymic, transthoracic, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, or transdermal, depending on the composition being administered. The perlecan+Pdgfra+ mesenchymal stromal cells are administered in an "effective amount," or in an amount that produces the desired therapeutic response, either alone or in combination with further dosages. The cells administered in the present invention may be autologous ("self") cells or allogeneic ("non-self", e.g., syngeneic, isogeneic, or xenogeneic) cells. Generally, administration of the cells can be carried out within a short period after treatment (e.g., within 1, 2, 5, 10, 24, or 48 hours after treatment) and in accordance with the requirements of each desired treatment regimen. For example, if radiotherapy or chemotherapy is performed prior to administration, the treatment and transplantation of the cells of the present invention should optimally be provided within about one month from the interruption of the treatment. However, transplantation at a later time point after discontinuation of treatment can be carried out with an inducible clinical outcome.

[0094] The periostin+Pdgfra+mesenchymal stromal cells can be combined with pharmaceutically known excipients in the art to improve the preservation and maintenance of the 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 usually easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient for administration, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid composition or the viscous composition can contain a carrier, which may be a solvent or a dispersion medium containing, for example, water, physiological saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0095] Sterile injection solutions can be prepared by incorporating the cells utilized in the practice of the present invention, in the required amounts, into a suitable solvent together with various amounts of other ingredients, as desired. Such compositions may be mixtures with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (such as methylcellulose), pH buffering agents, additives that enhance gelling or viscosity, preservatives, flavoring agents, dyes, etc., depending on the desired route of administration and the preparation. Standard textbooks such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference, may be referred to for preparing suitable preparations without undue experimentation.

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

[0097] The composition may be isotonic. That is, the composition can 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 buffers containing sodium ions.

[0098] A method for potentially increasing cell survival when introducing cells into a subject in need thereof is to incorporate the cells of interest into a biopolymer or synthetic polymer. Depending on the condition of the subject, the injection site may prove unsuitable for seeding and growth of the cells due to scarring or other obstacles. Examples of biopolymers include, but are not limited to, cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycan. This can be constructed with or without growth factors or differentiation factors. Further, these may be in suspension, but the residence time at the site subjected to the flow is short. Another alternative is a three-dimensional gel in which cells are encapsulated in the interstitial spaces of a cell-biopolymer mixture. In this case too, growth factors or differentiation factors may be included with the cells. These can be placed by injection via the various routes described herein.

[0099] One of ordinary skill in the art will recognize that the components of the composition should be selected to be chemically inert and not to affect the viability or effectiveness of the stem cells or their progenitor cells as described in the present invention. This will not pose a problem for those skilled in chemical and pharmaceutical principles, or the problem can be easily avoided by referring to standard textbooks or by simple experiments (without undue experimentation) from the present disclosure and the literature cited herein.

[0100] One consideration in the therapeutic use of cells is the amount of cells necessary to achieve an optimal effect. In different situations, it may be necessary to optimize the amount of cells injected into the tissue of interest. Thus, the amount of cells administered will vary depending on the subject being treated. The exact determination of what is considered an effective dose can be based on individual factors for each patient, including the size, age, gender, weight, and condition of the particular patient. As few as 100 - 1000 cells can be administered to a selected patient for a particular desired use. Thus, one of ordinary skill in the art can readily ascertain dosages from the present disclosure and knowledge in the art.

[0101] One of ordinary skill in the art can readily determine the amounts of the cells and any optional additives, vehicles, and / or carriers that are in the compositions of the present invention and that are administered in the methods of the present invention. Thus, of course, for any composition administered to an animal or human and for any particular method of administration, it is preferred to determine toxicity (e.g., by determining the lethal dose (LD) and LD 50 in a suitable animal model, e.g., a rodent such as a mouse); and to determine the dosage of the composition, the concentration of the components in the composition, and the timing of administration of the composition that elicits an appropriate response. Such determinations do not require undue experimentation, given the knowledge of one of ordinary skill in the art, the present disclosure, and the literature cited herein. Also, the time for continuous administration can be ascertained without undue experimentation.

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

[0103] Ccl19 can be administered in an effective amount by any suitable method of administration known in the art (e.g., injection or infusion). The effective amount depends on the method of administration, the particular condition being treated, and the desired outcome. The effective amount may also depend on the stage of the condition, the age and physical condition of the subject, in some cases the nature of the combination therapy, and similar factors well known to the physician. In the case of therapeutic application, the effective amount is an amount sufficient to achieve a medically desirable result (an increase in T cell production). Generally, the dosage of the active Ccl19 polypeptide compound of the present invention will be from about 0.01 mg / kg / day to about 1000 mg / kg / day. Dosages in the range of about 50 to about 2000 mg / kg are expected to be appropriate. Lower dosages may be brought about by certain forms of administration such as intravenous administration. If the response in the subject is insufficient with the initial application dosage, higher dosages (or higher effective dosages by a more local and different delivery route) may be employed to the extent tolerated by the patient. Multiple dosings per day are contemplated 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 purposes of illustration. These examples provide a better understanding of the present invention and many of its advantages.

Examples

[0105] The following examples illustrate some aspects and aspects of the present invention. Various modifications, additions, substitutions, etc. can be made without changing the spirit or scope of the present invention, and it will be apparent to those skilled in the relevant technical field that such modifications and variations are included within the scope of the present invention as defined in the following claims. The following examples are in no way intended to limit the present invention.

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

[0107] Animals: 8-week-old male and female C57Bl / 6 mice were used in all transplantation and sequencing experiments. B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) and C57BL / 6-Tg(UBC-GFP)30Scha / J mice were used as donors for bone marrow transplantation. B6;129S-Penktm2(cre)Hze / J mice were mated with B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J to generate 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 specimens were collected with approval from the Institutional Review Board (IRB). Tissues were processed immediately after isolation to ensure the highest possible cell quality. Mouse samples were cut into small pieces 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 sample was digested by combining the Stemxyme / DNAse I cocktail with 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: After blocking with anti-human CD16 / 32 Fc-block (BD Biosciences) for 10 minutes at 4°C, human single-cell suspensions were stained with a cocktail of lineage markers - 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) for 10 minutes at 4°C and then stained with CD45-PE / Cy7 and Ter119-PE (both from BioLegend). Samples were stained for 45 minutes at 4°C with constant agitation. To detect dead cells, 7-AAD (ThermoFisher) was added to the samples immediately before analysis. Flow sorting of viable and non-hematopoietic cells (7-AAD, CD45-CD235a / Ter119-lineage negative) was performed on a BD FACS Aria III (BD Biosciences) equipped with a 70um 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 cocktail-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 were similarly used as part of the stromal cell sorting.

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

[0112] Transplantation of bone marrow, lymphoid progenitor cells, and MSCs: 8-week-old C57Bl / 6 mice were given a single dose of 9.5 Gy 12 - 24 hours before transplantation. For lymphoid progenitor cell transplantation, bone marrow from C57BL / 6-Tg(UBC-GFP)30Scha / J donors was lineage-depleted according to the manufacturer's instructions (Miltenyi). Subsequently, cells were stained with biotinylated 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. Lineage-CD135+ cKit+GFP+ lymphoid progenitor cells were sorted using a BD FACS Aria III, and 40,000 cells were injected into each lethally irradiated recipient together with 10 6 nucleated total bone marrow cells from B6.SJL-Ptprca Pepcb / BoyJ donors. In the case of MSC adoptive transfer, recipients were irradiated 12 hours before transfer to ensure that the thymus was of a size that allowed intrathymic injection. 2000 - 10,000 MSCs (CD45-Ter119-CD31-CD326-CD248+CD99l2+Itgb5+CD140+) were injected intrathymically, together with 10 6 nucleated total bone marrow cells from B6.SJL-Ptprca Pepcb / BoyJ mice, injected retroorbitally.

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

[0114] Example 1. Single-cell sequencing of human and mouse thymus identifies subsets of mesenchymal cells with different T cell support properties Inefficient T cell reconstitution after bone marrow transplantation is a major cause of morbidity and mortality. Success of the reconstitution of T cell-mediated immunity then depends entirely on the regenerative capacity of the thymus. However, the mechanisms underlying thymic recovery impairment are not fully understood. In particular, the regeneration of stromal cells that support T cell development has not yet been fully understood. To clarify the characteristics of the thymic microenvironment, CD45-CD235-CD45-Lin- thymic stromal cells were isolated and single-cell RNA sequencing was performed on one human thymic sample (Figure 1A, Figure 2A). A first attempt demonstrated that digestion conditions are crucial for successfully isolating thymic stromal cells from human tissues. 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 blood 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 pericytes (RGS5). (Figs. 1B and 2E). The proportions of the different populations were similar between samples, and this observation was largely confirmed by flow cytometry (Figs. 1C and 2F). Interestingly, the largest proportion of stromal cells was composed of mesenchymal stromal cells (MSCs) expressing PRRX1, a cell population that has received little attention from the perspective of T cell development in the thymus, despite being abundant (Fig. 1C).

[0116] The main function of thymic stromal cells is to provide factors that mobilize, maintain, and commit hematopoietic progenitor cells to the T cell lineage. Many of the molecules involved in this process have been defined. By evaluating which cell types express these lymphopoietic factors, several predicted combinations became apparent. Thymic epithelium (TEC) was found to be particularly rich in the chemokines CCL21 and CCL25, which mobilize T cell progenitor cells (Fig. 1D). However, notably, human thymic MSCs were thought to express at high levels several well-established regulators of lymphoid cell development, including FLT3LG, CCL19, and IL15 (Fig. 1D). This suggests that a substantial pool of thymic mesenchymal cells may be important for contributing to T cell development.

[0117] To further understand the populations identified in humans and clarify their characteristics, scRNA-seq was performed on the resting thymus of 8-week-old mice (Figures 1E and 2G). A total of four samples were sequenced, and after quality control and filtering of hematopoietic cells, a total of 6,491 mouse stromal cells were obtained (Figures 2H, 1I, and 1J). All thymic stromal cell populations found in humans were also present in mice: endothelial cells (Pecam1), mesenchymal stromal cells (Prrx1), two types of perivascular cells (Rgs5), and thymic epithelial cells (Epcam), respectively (Figures 1F, 2K, and 2L). In addition, the mouse thymus contains two other stromal subsets. Recently described thymic tuft cells were defined by the expression of Trpm5 and IL25 (Figures 1F, 2K, and 2L). A small cell population was also found to express Lrrn4, a marker previously associated with mesothelial stem and progenitor cells (Figures 1F, F2K, and 2L). These differences in the content of thymic stromal cells may reflect actual interspecies differences, but may also be due to inherent differences in sample preparation and sample source. Most human samples, for example, were derived from infants, whereas mouse tissues were isolated from adults. Nevertheless, studies using adult mice were continued because this is a more appropriate population for studying thymic regeneration.

[0118] Similar to what was seen in human samples, it was determined by scRNA sequencing and flow cytometry analysis that the largest proportion of mouse stromal cells are MSCs (Figures 1G, 2M). It was also found that important thymocyte support factors are 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-supportive MSCs are thought to be present in human and mouse thymic tissues.

[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 (Figs. 3A, 4A). Both species were found to have CD248+ and Postn+ MSC populations, albeit at varying frequencies (Figs. 3A, 4B, and 4C). The third MSC subset was found to be characterized by CDH11 expression in humans, whereas in mouse samples, it was characterized by cells defined by Cdh11 and Penk (Figs. 3A, 4B, and 4C). Comparison with previously published mouse thymic stromal datasets further confirmed the presence of the three MSC subpopulations (Figs. 4D and 4E). The relative abundance of total MSC and the three subtypes were found to be different (Figs. 4D and 4E). However, since thymic epithelial cells were the main focus of this study, a different isolation protocol was used, which likely accounts for this difference. Notably, MSCs expressing Cd248, Penk, and Postn were also found in this dataset (Fig. 4F).

[0120] GO term analysis of mouse samples further revealed potentially different functions among MC subtypes. CD248+ MSCs were found to be enriched mainly in terms related to protein translation and secretion (Figure 4G). This, combined with the upregulation of multiple extracellular matrix components (Fn1 and Ogn) expressed by these cells (Figure 4A), suggests fibroblast function of these cells. On the other hand, Penk+ Cdh11+ MCs were found to be characterized by terms related to adipogenesis or stress response (Figure 3B). This is particularly interesting given that the epithelial compartment of the aging thymus is gradually replaced by adipocytes through an unknown process. The expression of the epithelial control program in Postn+ MSCs (Figure 3B) is consistent with what is previously known about the function of thymic MSCs, that mesenchymal cells during embryogenesis are involved in the recruitment of epithelial progenitor cells. Postn+ cells also showed significant activation of the angiogenesis pathway (Figure 3B), suggesting that these cells may play an important role in controlling other thymic stromal cell types. However, most importantly, Postn+ MSCs were found to be a subtype significantly enriched 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 express the lymphopoietic cytokines Ccl19, Flt3l, and IL15 at significantly higher levels than other MSC subpopulations (Figure 3C). It is shown that Postn+ MSCs are responsible for most of the interactions with T cells that occur in the thymus.

[0121] Example 3. Disappearance of periostin+ MSCs after radiation pretreatment Thymic regeneration is of particular interest from the perspective of bone marrow transplantation. Therefore, the inventors considered comparing steady-state scRNA sequencing with samples that had received cytotoxic pretreatment and transplantation. A major obstacle in early thymic regeneration is the inefficient mobilization of T cell progenitor cells from the bone marrow. To better understand what is lacking in the microenvironment at this stage, the inventors aimed to collect thymic stroma at the time when T cell progenitor cells first seed the tissue after transplantation. To this end, 40,000 GFP-labeled lymphoid progenitor cells (LPCs, cell lineage - cKit+CD135+) were transplanted into lethally irradiated recipient mice together with 1 million helper bone marrow cells, and an attempt was made to track thymic seeding using flow cytometry (Figures 6A and B). This was found to be an unreliable approach. GFP+ cells were easily found in the bone marrow, but in the thymus, only very few could be detected, if any, at early time points after transplantation (Figure 6B). In addition, many of the cells were positive for cell lineage-defining markers (Figure 6B), suggesting that they were not early thymic progenitor cells (ETPs). As a result, the tracking was switched to recent thymic colonizers by tissue clearing, which allows imaging from top to bottom while minimizing the loss of material (Figure 5A, Figure 6C). This led to the first detection of rare GFP+ cells in the thymus 3 days after transplantation (Figure 5B, Figure 6D), and at subsequent stages, the tissue was found to be rich in migrated cells (Figure 6D). Therefore, thymic seeding is thought to start on day 3 after transplantation, and this was selected as the time point for scRNA sequencing analysis of thymic stromal cells.

[0122] As performed in the steady-state analysis, CD45−Ter119− cells were sorted from 8-week-old mice that had received a single lethal dose of radiation 4 days prior and bone marrow transplantation of 40,000 GFP+ LPC and unlabeled helper marrow 3 days prior to isolation (Figure 5A). A total of three samples were sequenced, yielding 8,873 cells that passed quality control and were found to be negative for Ptprc and CD3e (Figure 5C). No complete disappearance of a cell type or emergence of a new subset occurred due to the radiation pretreatment (Figure 5C, 5D, and Figure 6E). Multiple cell populations, such as TEC B and endothelial cells, showed a large decrease in relative abundance, but these did not reach statistical significance (Figure 6E). However, the MSC compartment showed a large change (Figure 5C). The stress-responsive Penk+ Cdh11+ MSC were found to be significantly expanded, whereas the frequency of the T cell-supportive Postn+ MSC decreased dramatically (Figure 5D). It is suggested that this imbalance observed in the thymic MSC subset is partly due to the cytotoxic pretreatment and inefficient T cell production after bone marrow transplantation.

[0123] To further explore the functional characteristics of MSCs after transplantation, another GO term analysis of significantly differentially expressed genes was performed. Notably, Penk+ Cdh11+ MSCs were still characterized by terms related to adipogenesis and responses to various stress factors but were also significantly enriched for pathways that inhibit leukocyte proliferation (Figure 5E).

[0124] Therefore, expansion of these cells after radiation pretreatment may further inhibit T cell production. On the other hand, Postn+ MSCs were found to still support T cells and endothelial cells (Figs. 5E and 6F), but they also showed increased adipogenic activity. In the bone marrow, it is well established that MSCs differentiate into adipocytes in response to radiation exposure. Whether bone marrow adipocytes enhance or impede hematopoiesis remains a matter of debate. However, thymic adipocytes cannot support T cell development, suggesting a further adverse effect of the changes in MSCs observed after radiation exposure and bone marrow transplantation.

[0125] Example 4. Transplantation of CD248-thymic MSCs promotes T cell production after radiation pretreatment To investigate the functional significance of thymic MSCs, scRNA sequencing data were queried for potential cell surface markers that could be used to facilitate flow cytometric sorting of individual MSC subsets. Unfortunately, no suitable markers were found that would allow discrimination between Penk+ Cdh11+ MSCs and the Postn+ population. Two markers, CD99l2 and Itgb5, were identified that label all MSCs and show little overlap with perivascular cells (Fig. 8A). The specificity of these markers in the MSC compartment was further confirmed by flow cytometry analysis and sorting and plating of CD99l2+Itgb5+ thymocytes (Figs. 8A and 8B). These cells were found to adhere to plastic and have colony-forming ability equivalent to that of bone marrow MSCs (Fig. 8B). In addition, Penk+ Cdh11+ MSCs and Postn+ MSCs were found to express Pdgfra and, as described above, these cells were negative for Cd248 (Fig. 8C). As a result, sorting CD45-Ter119-CD31-CD326-CD248-CD99l2+Itgb5+Pdgfra+ cells enriched the MSCs that most support T cells (CD248- MSCs) while excluding CD248+ MSCs, which are thought to be of less importance.

[0126] Ubiquitin-GFP mice were used as donors to isolate CD248-MSCs, which were then injected into the thymus of irradiated recipients that also received bone marrow transplantation (Figure 7A). In parallel with the MSC-treated mice, sham recipients received bone marrow injection but were injected with PBS into the thymus (Figure 7B). To verify the introduction of cells into the tissue, a cohort of mice that received an 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 engrafted MSCs was further associated with an improvement in the numbers of both ETPs and endothelial cells (Figure 7B), although the numbers of MSCs and epithelial cells (data not shown) did not change compared with sham-treated and CD8+ T cell-treated mice. This indicates that thymic regeneration can be improved by injecting fresh thymic CD248-MSCs after irradiation pretreatment.

[0127] Ccl19, one of the factors significantly enriched in thymic MSCs, has previously been associated with the mobilization of ETPs. To determine whether Ccl19 expression in MSCs is required for the improved ETP seeding observed after transplantation, CD248-MSCs were isolated from Cas9-GFP-expressing mice. Subsequently, these cells were infected with lentiviral vectors expressing guide RNAs directed against Ccl19 or the control locus GFP. Transplantation of these modified MSCs demonstrated that knockout of Ccl19 abrogated the improvement in ETP mobilization after CD248 MSC treatment (Figure 7C).

[0128] To determine whether the increased influx of progenitor cells on day 6 leads to an increase in de novo T cell generation, transplantation experiments were repeated. This time, the thymus was analyzed 4 weeks later. GFP+ CD248− MSCs were still found to be present in the tissue (Figure 7C), and the thymus weight and cellularity were significantly higher in MSC-treated mice (Figure 8D). sjTREC analysis further demonstrated that the production of newly rearranged T cells was significantly improved in mice injected with CD248− MSCs (Figure 7C). This was further supported by a greater number of cells at all stages of T cell development (Figure 8D). In addition, 16 weeks of follow-up observation of the transplanted mice revealed that in CD248− MSC recipients, the number of CD4+ T H cells and CD8+ T CTL cells was dramatically improved (Figure 7D), and there was no effect 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 viable in the tissue (Figure 8F).

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

[0130] Example 5. Periostin+ MSCs specifically enhance the mobilization of T cell progenitors Penk-Cre mice were crossed with Rosa26-LSL-tdTomato reporters to generate mice in which Penk+ Cdh11+ MSCs and Postn MSCs could be separated. 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 was faithful to the scRNA sequencing data. Indeed, when tdTomato+ or tdTomato- cells were transferred under conditions of bone marrow transplantation, recipients of putative Postn+ MSCs were shown to have improved numbers of ETPs and endothelial cells 6 days later (Figure 9B). Thus, the effects mediated by thymic MSCs are thought to be contained within the Postn+ MSC population.

[0131] References All patents, patent applications, and publications referred to herein are hereby incorporated by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

Claims

**Claim 1**: A composition for increasing the production of T cells in a target T cell-producing tissue or T cell-producing body fluid, comprising mesenchymal stromal cells isolated from CD45−, CD235−, Lin− thymic stromal cells, expressing PRRX1, periostin, and Pdgfra, and not expressing CD248. **Claim 2** The composition according to claim 1, wherein the mesenchymal stromal cells do not further express Cdh11. **Claim 3** The composition according to claim 1, wherein the T cell-producing tissue is the thymus. **Claim 4** The composition according to claim 1, wherein the T cell-producing tissue is a lymphopoietic tissue. **Claim 5** The composition according to claim 1, wherein the T cell-producing body fluid is blood. **Claim 6** The composition according to claim 1, wherein the subject has undergone a hematopoietic stem cell transplantation. **Claim 7** The composition according to claim 1, wherein the subject has one or more of a condition associated with T lymphopenia, T cell production disorder, T cell dysfunction, distortion of the repertoire of cells having a T cell receptor, an infectious disease, or a tumor. **Claim 8** The composition according to 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. **Claim 9** The composition according to claim 1, wherein the mesenchymal stromal cells express Ccl19, Flt3 ligand, and IL-15 and do not express Cdh11. **Claim 10** The composition according to claim 1, wherein the mesenchymal stromal cells are autologous to the subject.

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