Antigen specific expansion of regulatory t cells

Abstract

Disclosed are methods for expanding T regulatory cells (Tregs) that are antigen specific, populations of Tregs, pharmaceutical compositions comprising the populations of Tregs. Also disclosed are methods of using the antigen specific Tregs, e.g., alloantigen specific Tregs for the treatment of tissue rejection, autoimmune, allergic, and other diseases or disorders characterized by a pathological immune response.

Description

ANTIGEN SPECIFIC EXPANSION OF REGULATORY T CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Application No. 63 / 590,317, filed October 13, 2023. The entire content of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.BACKGROUND

[0003] Currently, organ and tissue transplantation rely upon life-long use of nonspecific immunosuppressive agents (IS) to prevent graft rejection. These drugs have significant negative side effects including nephrotoxicity, increased rate of infection, malignancy, diabetes, and hypertension. They are also very costly. Ever since organs were first transplanted in humans, scientists have pursued the elusive goal to establish donor-specific immunologic tolerance, a state where a donated organ is accepted as “self,” eliminating the need for IS agents as well as achieving the goal of “one transplant for life.” Accordingly, novel methods to prevent and treat graft rejection are needed.SUMMARY

[0004] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-p about 14 to about 21 days after beginning step a. to generatea third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF- until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method.In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0005] In an aspect of the current disclosure, methods of expanding T regulator}' cells (Tregs) specific for a target antigen are provided. In some embodiments, the methods comprise: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor P (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subj ect that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1 ,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor isadded to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0006] In an aspect of the current disclosure, methods of expanding alloreactive T regulatory cells (Tregs) are provided. In some embodiments, the methods comprise: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-p about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject inneed of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-0 is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0007] Tn an aspect of the current disclosure, populations of T regulatory cells (Tregs) are provided. In some embodiments, the populations of Tregs are made by a method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1:1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In someembodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0008] In some embodiments, the populations of Tregs are made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor P (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subj ect that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days afterbeginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0009] In some embodiments, the populations of Tregs are made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor (3 (TGF-P) to generate a first population of expanded Tregs; b.contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs atleast about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0010] In an aspect of the current disclosure, ex vivo populations of T regulatory cells (Tregs) are provided. In some embodiments, the populations comprising at least 106Tregs wherein the Tregs in the population are each specific for the same antigen. In some embodiments, the Tregs are CD4+, CD25+, FoxP3+. In some embodiments, the Tregs are CD127'.

[0011] In an aspect of the current disclosure, pharmaceutical compositions are provided.

[0012] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL- 2) and transforming grow th factor (3 (TGF-|3) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-p to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-|3 until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a.In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0013] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the firstpopulation of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-p is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregscomprise at least 106Tregs, at least 107Tregs, or at least I O8Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0014] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1 ,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least IO7Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0015] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition to a subject. In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3agonist and a CD28 agonist in medium comprising IL-2 and TGF- about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-p until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-p is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold.In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0016] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-p about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1 ,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTORinhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0017] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate alloreactive expandedTregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0018] Tn an aspect of the current disclosure, methods of treating transplant rejection in a subject in need thereof are provided. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition to the subject to treat transplant rejection in the subject. In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-p about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-p is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs. or at least 108Tregs. wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0019] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-p to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-p until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneicdonor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0020] Tn some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor 0 (TGF-0) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- 0 to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- 0 about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- 0 until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-0 is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodiesand anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product. In some embodiments, the subject has received an organ transplant, a tissue transplant, or a cellular transplant.

[0021] In an aspect of the current disclosure, methods of inducing tolerance to a target antigen in a subject in need thereof are provided. In some embodiments, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof, wherein the population of Tregs in the pharmaceutical composition is specific for the target antigen. In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulator ■ cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-p until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregsare from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0022] Tn some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-|3 isat a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0023] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In someembodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-0 is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0024] In an aspect of the current disclosure, methods of treating an autoimmune disease in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition, to the subject to treat the autoimmune disease. In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulatorycells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subj ect that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In someembodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0025] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-p about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF- is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0026] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL- 2 is at a concentration of about 1.000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-0 is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least IO6Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, themethod expands the Tregs at least about 1 OO-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product. In some embodiments, the autoimmune disease is selected from scleroderma, Sjogren’s syndrome, vasculitis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and celiac disease. In some embodiments, the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3), 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzymes SAE-1 and SAE-2, topoisomerase- 1, centromere proteins A, B & C (“CENPs”), fibrillarin. nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9. EXOSCIO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B), gamma interferon-inducible protein-16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein. Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMD A) receptor, and tissue transglutaminase.

[0027] In an aspect of the current disclosure, methods of treating an allergy in a subj ect in need thereof are provided. In some embodiments, the method comprise administering a pharmaceutical composition, to the subject to treat the allergy. In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor (3 (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-p to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expandedTregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands theTregs at least about 400-fold 28 days after beginning step a. of the method. Tn some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0028] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor P (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen. In some embodiments, the first and second populations of B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen comprises at least one alloantigen. In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In someembodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

[0029] In some embodiments, the pharmaceutical compositions comprise a population of Tregs made by a method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs. In some embodiments, the Tregs are from a subject in need of allotransplantation and theB cells are from an allogeneic donor. Tn some embodiments, the Tregs are from a subject that has been previously treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the 1L- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-p is at a concentration of at least about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium. In some embodiments, the method is performed under good manufacturing practice (GMP) conditions and. wherein, the method results in a GMP product.

[0030] Tn an aspect of the current disclosure, methods of treating transplant rejection in a subject in need thereof are provided. In some embodiments, the method comprise: a. obtaining T regulatory' cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor P (TGF-P), wherein the target antigen is related to the transplant rejection, to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF- P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen; e. administering an effective amount of the expanded Tregs specific for the target antigen to induce tolerance to the target antigen in the subject. In some embodiments, the B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen is at least one alloantigen. In some embodiments, the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3), 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzymes SAE-1 and SAE-2, topoisomerase- 1, centromere proteins A, B & C (“CENPs”), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSCIO, Ku 70 / 80 components of DNA- dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B), gamma interferon-inducible protein- 16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMDA) receptor, and tissue transglutaminase.In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In someembodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the method produces a population of Tregs, the population comprising at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium.

[0031] In an aspect of the current disclosure, methods of inducing tolerance to a target antigen in a subject in need thereof. In some embodiments, the methods comprise: a. obtaining T regulatory cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforminggrowth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF- about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen; e. administering an effective amount of the expanded Tregs specific for the target antigen to induce tolerance to the target antigen in the subject. In some embodiments, the B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen is at least one alloantigen. In some embodiments, the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3), 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzymes SAE-1 and SAE-2, topoisomerase- 1, centromere proteins A, B & C (“CENPs”), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSC 10, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B). gamma interferon-inducible protein-16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMD A) receptor, and tissue transglutaminase.In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subj ect that has been treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days afterbeginning step a. In some embodiments, the B cells are irradiated. Tn some embodiments, the TL- 2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti- CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the method produces a population of Tregs, the population comprising at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium.

[0032] In an aspect of the current disclosure, methods of treating an autoimmune disease associated with a target antigen in a subject in need thereof are provided. In some embodiments, the methods comprise: a. obtaining T regulatory cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL-2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen; e. administering an effective amount of the expanded Tregs specific for the target antigen to treat the autoimmune disease in the subject. In some embodiments, the B cells are pulsed with the target antigen prior to performing the method. In some embodiments, the target antigen is at least one alloantigen. In some embodiments, the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3), 3- hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene- 5 (MDA5), small ubiquitin-like modifier activating enzy mes SAE-1 and SAE-2, topoisomerase- 1. centromere proteins A, B & C f'CENPs'’). fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSCIO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B), gamma interferon-inducible protein-16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine- deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP. ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMDA) receptor, and tissue transglutaminase.In some embodiments, the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor. In some embodiments, the Tregs are from a subject that has been treated with an allotransplant and the B cells are from the donor of the allotransplant. In some embodiments, step b. is performed about 7 to about 12 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a. In some embodiments, step b. is performed about 12 days after beginning step a. In some embodiments, step c. of the method is performed about 14 to about 21 days after beginning step a. In some embodiments, step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a. In some embodiments, the B cells are irradiated. In some embodiments, the IL-2 is at a concentration of about 1,000 units / ml. In some embodiments, a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is added to the culture medium until about 28 days afterbeginning step a. In some embodiments, the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a. In some embodiments, the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium. In some embodiments, the B cells and the Tregs are present in about a 1: 1 ratio in step a. of the method. In some embodiments, the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method. In some embodiments, the TGF-P is at a concentration of about 5.2 units / ml. In some embodiments, the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively. In some embodiments, the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix. In some embodiments, the method produces a population of Tregs, the population comprising at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the Tregs are each specific for the same antigen. In some embodiments, the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold. In some embodiments, the method expands the Tregs at least about 100-fold 21 days after beginning step a. of the method. In some embodiments, the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method. In some embodiments, the Tregs are cultured in serum free medium.

[0033] In an aspect of the current disclosure, methods of expanding alloreactive T regulatory7cells (Tregs) are provided. In some embodiments, the methods comprise: a. contacting the Tregs with B cells comprising a target antigen, wherein the target antigen is an alloantigen. in serum free medium comprising about 1000 units / ml interleukin 2 (IL-2) and at least about 5.2 units / ml transforming growth factor p (TGF-P); b. contacting the Tregs of step a. with B cells comprising the target antigen in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-P about 7 days after beginning step a.; c. contacting the Tregs of step b. with a CD3 agonist and a CD28 agonist in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-p about 14 days after beginning step a., wherein the CD3 agonist and the CD28 agonist are monoclonal antibodies and are covalently linked to a polymeric nanomatrix: d. culturing the Tregs of step c. in serum free medium comprising about 1000 units / ml IL -2 and at least about 5.2 units / ml TGF-P until about 28 days after beginning step a. togenerate expanded alloreactive Tregs, wherein the alloreactive Tregs are expanded at least about 400-fold and are at least 80% CD4+, CD25+, FoxP3+, CD127’ Tregs.

[0034] In an aspect of the current disclosure, methods of treating transplant rejection in a subject in need thereof are provided. In some embodiments, the methods comprise: a. obtaining Tregs from the subject and contacting the Tregs with B cells comprising a target antigen, wherein the target antigen is an alloantigen, in serum free medium comprising about 1000 units / ml interleukin 2 (IL-2) and at least about 5.2 units / ml transforming growth factor 0 (TGF-0), wherein the subject is in need of an allotransplant and the B cells are from the donor of the allotransplant; b. contacting the Tregs of step a. with B cells comprising the target antigen in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-0 about 7 days after beginning step a.; c. contacting the Tregs of step b. with a CD3 agonist and a CD28 agonist in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF- 0 about 14 days after beginning step a., wherein the CD3 agonist and the CD28 agonist are monoclonal antibodies and are covalently linked to a polymeric nanomatrix; d. culturing the Tregs of step c. in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-0 until about 28 days after beginning step a. to generate expanded alloreactive Tregs, wherein the alloreactive Tregs are expanded at least about 400-fold and are at least 80% CD4+, CD25+, FoxP3+. CD 127" Tregs; e. administering an effective amount of the Tregs of step d. to the subject to treat transplant rejection in the subject.

[0035] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, or platforms comprise the necessary reagents for performing the methods disclosed herein. In some embodiments, the kits, systems, or platforms further comprise instructions for performing the methods using the kit, system, or platform.BRIEF DESCRIPTION OF THE FIGURES

[0036] FIGs. 1 A and IB show the results of preliminary Treg expansion experiments. Purified Tregs w ere isolated from “recipient’' PBMC using the Treg isolation kit II (Miltenyi Biotec) and were cultured in 24-well culture plates with irradiated (3,000 rad) “donor” B cells that had been expanded and activated in culture as above at a ratio of 1 : 1 at 37°C with 5% CO2. Culture medium consisted of RPMI-1640 supplemented with 15% human AB serum (Sigma), 1,000 lU / mL IL-2(R&D Systems) and Ing / ml TGF- (R&D systems). lOOnM sirolimus (Pfizer) was added to cultures except for the final 7 days. To phenotype Tregs, antibodies against CD4-FITC, CD127- PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter) and FOXP3-PC5 (eBioscience) were used on days 0. 14 and 21, (also on day 28 in longer-term cultures).

[0037] FIG. 2 shows donor specific (Ds) Tregs expanded in cultures with different timelines of sirolimus withdrawal and polyclonal stimulation on Day 14 versus 3 donor specific stimulations. Purified Tregs were isolated from "‘recipient” PBMC using the Treg isolation kit II (Miltenyi Biotec) and were cultured in 24-well culture plates with irradiated (3,000 rad) ‘‘donor” B cells that had been expanded and activated in culture as above at a ratio of 1 : 1 at 37°C with 5% CO2. Culture medium consisted of RPMI-1640 supplemented with 15% human AB serum (Sigma), 1.000 lU / mL IL-2 (R&D Systems) and Ing / ml TGF-(3 (R&D systems). Timing of sirolimus supplementation and Donor specific stimulation strategy was studied according to the table above (FIG. 2, Top panel). 1) Sirolimus supplemented for 21 days with Donor specific stimulations on Day 0, 7 and 14; 2) Sirolimus supplemented for 14 days with donor specific stimulations on Day 0, 7, and 14; 3) Sirolimus supplemented for 14 days with donor specific stimulations on Day 0 and Day 7 and a polyclonal stimulation with MACS GMP ExpAct Treg beads (Miltenyi Biotec) at 1: 1 ratio on day 14. The cultures were harvested on day 21 and compared against the standardized protocol described above (FIG. 1).

[0038] FIGs. 3 A and 3B show Phenotypic and functional assessment of Ds-Tregs expanded in cultures with different timelines of sirolimus withdrawal and polyclonal stimulation on Day 14. Ds-Tregs were cultured as above in FIG. 2. To phenotype Tregs, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter) and FOXP3-PC5 (eBioscience) were used on days 0, 14 and 21, (also on day 28 in longer-term cultures). For functional studies a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMC autologous to the Tregs were stimulated with freshly isolated ‘‘donor”-specific irradiated PBMC from the B cell donor or an irrelevant third part)7at a ratio of 1 : 1 in U-bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3.000 rads. After 7 days,3H-thymidine was added to the cultures during the final 16-20 hours and incorporation of3H-thymidine was used to measure proliferation. The back-response CPM given by the Tregs orcontrol modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds- Treg) and were subtracted from (R + Dx / Ix + Ds-Treg) experimental cultures to obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified according to Equation 1 : % inhibition

[0039] FIG. 4 shows tailoring of Donor Specific Stimulation timeline for Ds-Treg Expansion. Our previous results had demonstrated improved expansion of Ds-Tregs when performing 2 donor specific stimulations with activated B cells followed by a polyclonal stimulation. We next assessed the timing of Donor-specific stimulations as shown in FIG. 4. (Left panel): Donor specific stimulations were performed on Day 0 and Day 7 with a polyclonal stimulation on Day 14. (Right panel): Donor specific stimulations were performed on Day 0 and Day 12 with a polyclonal stimulation on Day 21.

[0040] FIGs. 5A, 5B, 5C, and 5D show a comparison of Donor Specific Stimulation Timelines (Day 0 / 7 versus Day 0 / 12). Isolated, purified CD4+CD25+ cells were cultured in TexMACS GMP medium (Miltenyi Biotec) supplemented with 10% heat inactivated human AB Serum (Valley Biomedical) at 37°C with 5% CO2. Cells were cultured in the presence of 11-2 (lOOOIU / mL media; Clinigen Limited), TGF-beta (Ing / mL; Gibco Inc), and Everolimus (lOOng / mL; Sigma). IL -2, TGF-beta and fresh media were supplemented every' 2-3 days. Everolimus was supplemented through Day 9 of culture, but no additional Everolimus was added after that time. Stimulations were performed on either on a) Day 0 and Day 7 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1: 1 ratio and a final polyclonal stimulation on Day 14 with GMP TransACT (Miltenyi Biotec) OR b) Day 0 and Day 12 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1: 1 ratio and a final polyclonal stimulation on Day 21 with GMP TransACT (Miltenyi Bioetc). Cells were cultured in G-Rex® 6M bioreactors from Wilson Wolf. Cells were phenotypically and functionally characterized on Day 28 of culture. Tregs were phenotyped with the following antibodies: Viakrome Live / Dead IR-808 (Beckmann Coulter), CD3-AP-Cy7 (Biolegend), CD4-AlexaFlour 700 (Biolegend), CD8-PerCp 5.5 (Biolegend), CD25-PE (Biolegend), CD127-BV605 (Biolegend), Foxp3-EFlour450 (Invitrogen), and CD19-PE Cy7(Biolegend). For functional studies a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMCautologous to the Tregs were stimulated with freshly isolated '‘donor”-specific irradiated PBMC from the B cell donor or an irrelevant third part)7at a ratio of 1 : 1 in U-bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3.000 rads. After 6 days, 3H-thymidine was added to the cultures during the final 16-20 hours and incorporation of 3H-thymidine was used to measure proliferation. The back-response CPM given by the Tregs or control modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds- Treg) and were subtracted from (R + Dx / Ix + Ds-Treg) experimental cultures to obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified as above.

[0041] FIG. 6 shows further tailoring of Media Conditions for Ds-Treg Expansion. Although there was significant Ds-Treg expansion with the above conditions, we sought to improve the protocol in order to increase the fold expansion. Serum free media is a good option for large scale cell culture as it provides more consistent results, higher levels of cell growth, and is also better for downstream processing and applications. We compared the optimized conditions from above using human AB serum (left panel) to similar growth conditions where AB serum was substituted with a serum free substitute (Right panel).

[0042] FIG. 7 shows expansion of Ds-Tregs using human AB serum vs Serum Free Media. Isolated, purified CD4+CD25+ cells were cultured in TexMACS GMP medium (Miltenyi Biotec) supplemented with either a) 10% heat inactivated human AB Serum (Valley Biomedical) or b) 5% Cell Vive T Cell CD Serum Substitute, GMP (Biolegend) at 37°C with 5% CO2. Cells were cultured in the presence of 11-2 (lOOOIU / mL media; Clinigen Limited), TGF-beta (Ing / mL; Gibco Inc), and Everolimus (lOOng / mL; Sigma). IL-2, TGF-beta and fresh media were supplemented every 2-3 days. Everolimus was supplemented through Day 9 of culture, but no additional Everolimus was added after that time. Stimulations were performed on Day 0 and Day 7 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1 : 1 ratio, and a final polyclonal stimulation on Day 14 with GMP Trans ACT (Miltenyi Biotec). Cells were cultured in G-Rex® 6M bioreactors (N=2) or G-Rex® lOM / G-Rex® 100M (N=l) from Wilson Wolf. Cells were phenotypically and functionally characterized on Day 21 and Day 28 of culture. Tregs were phenotyped with the following antibodies: Viakrome Live / Dead IR-808 (Beckmann Coulter), CD3-AP-Cy7 (Biolegend), CD4-AlexaFlour 700 (Biolegend), CD8-PerCp 5.5 (Biolegend),CD25-PE (Biolegend), CD127-BV605 (Biolegend), Foxp3-EFlour450 (Invitrogen), and CD19- PE Cy7(Biolegend). For functional studies, a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMC autologous to the Tregs were stimulated with freshly isolatedC“donor”-specific irradiated PBMC from the B cell donor or an irrelevant third party at a ratio of 1 : 1 in U-bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3,000 rads. After 6 days,3H-thymidine was added to the cultures during the final 16-20 hours and incorporation of3H-thymidine was used to measure proliferation. The back- response CPM given by the Tregs or control modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds-Treg) and were subtracted from (R + Dx / Ix + Ds-Treg) experimental cultures to obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified according to Equation 1.

[0043] FIG. 8 shows representative flow cytometric data from Tregs expanded in AB serum containing medium. On day 0, purified cells highly expressed CD4 and Foxp3 with low expression of CD127. CD25 expression cannot be evaluated on Day 0 given steric hinderance by the microbeads used for Treg isolation. Over 28 days in culture, Tregs continued to highly express CD4, CD25, and Foxp3 with low expression of CD127.

[0044] FIG. 9 shows representative flow cytometric data from Tregs expanded in serum free medium. On day 0, purified cells highly expressed CD4 and Foxp3 with low expression of CD127. CD25 expression cannot be evaluated on Day 0 given steric hinderance by the microbeads used for Treg isolation. Over 28 days in culture, Tregs continued to highly express CD4, CD25, and Foxp3 with low expression of CD 127.

[0045] FIGs. 10A,10B, 10C, and 10D show flow cytometry data of Ds-Treg Expansions using AB serum containing medium versus Serum Free media. (A) Phenotype of Ds-Tregs on Day 21 in AB serum containing media. Cells exhibited high expression of CD4 (97.9% + / - 1.59), CD25 (97.5% + / - 1.19). and Foxp3 (99.4% + / - 0.451). Low expression of CD127 (0.71% + / - 0.20) was observed. (B) Phenotype of Ds-Tregs on Day 21 in serum free media. Cells exhibited high expression of CD4 (98.1% + / - 1.10), CD25 (96.4% + / - 1.42), and Foxp3 (99.2% + / - 0.361). Low expression of CD127 (0.69% + / - 0.36) was observed. (C) Phenotype of Ds-Tregs on Day 28 inAB serum containing media. Cells exhibited high expression of CD4 (98.7% + / - 0.61), CD25 (91.5% + / - 0.15), and Foxp3 (98.1% + / - 1.35). Low expression of CD127 (0.69% + / - 0.27) was observed. (D) Phenotype of Ds-Tregs on Day 28 in serum free media. Cells exhibited high expression of CD4 (98.0% + / - 0.76), CD25 (92.0% + / - 1.57). and Foxp3 (98.1% + / - 1.48). Low expression of CD127 (0.41% + / - 0.10) was observed.

[0046] FIGs. 11A and 11B show flow cy tometry Data of Ds-Treg Expansions using (A) AB serum containing medium versus (B) Serum Free media. Percentage expression of each marker can be seen in FIG. 10 above.

[0047] FIGs. 12A, 12B, 12C, and 12D show a functional assessment of Ds-Tregs expanded in AB serum containing media (A.C) and serum free media (B,D). (A) Day 21 MLR data of Ds- Tregs expanded in AB containing media. (B) Day 21 MLR data of Ds-Tregs expanded in serum free media. (C) Day 28 MLR data of Ds-Tregs expanded in AB serum containing media. (D) Day 28 MLR data of Ds-Tregs expanded in serum free media.

[0048] FIG. 13 shows a tailored protocol for Treg expansion. The protocol utilizes the G- Rex®10M (Days 0-21) and G-Rex® 100 M (Days 21-28) bioreactors which allow for no changes of media with a final volume of lOOmL (G-Rex® 10M) and IL (G-Rex® 100M) after 21 days and 28 days in culture, respectively. Cells are cultured in TexMACS GMP Medium (Miltenyi Biotec) supplemented with 5% Cell Vive T Cell CD Serum Substitute GMP (Biolegend) at 37°C with 5% CO2. Donor specific stimulations are performed on Day 0 and Day 7 with expanded donor B cells. A polyclonal restimulation is performed on Day 14 with Bead-free, TransACT GMP (Miltenyi). IL-2 (lOOOIU / mL; Clinigen Limited), TGF-beta (52U / mL; Miltenyi Biotec) and Everolimus (lOOng / mL; Sigma) are supplemented every 2-3 days of culture. No additional Everolimus is added after day 9 in culture. Seeding concentration of cells is 1 xlO6cells / mL with a starting cell number of 5 xlO6cells per G-Rex® 10M. At Day 21 of culture, all G-Rex® 10M bioreactors are then split (i.e., half of cells removed) into a G-Rex®100M allowing for increased expansion over the final 7 days of culture (right panel above). For a clinical setting, Tregs are isolated utilizing a GMP grade CD8 / CD19 depletion followed by a CD25 selection step using GMP grade microbeads (all from Miltenyi Biotec) and the CliniMACS Plus closed system (Miltenyi Biotec).

[0049] FIG. 14 shows a schematic for an exemplary embodiment of the disclosed technology.

[0050] FIG. 15 shows an exemplary schematic for the isolation of Tregs from subject samples.

[0051] FIG. 16 shows flow cytometry data demonstrating that isolated Tregs that are used as starting material are CD4+, Foxp3+, CD 127".

[0052] FIG. 17 shows an exemplars’ schematic of a mixed lymphocyte reaction (MLR) which is a technique known in the art to test the suppressive function of Tregs.

[0053] FIGs. 18 A. 18B, 18C, and 18D show that the Tregs expanded by the disclosed methods exhibit donor specific suppression.

[0054] FIG. 19 shows exemplary’ flow cytometry’ data demonstrating the assessment of MLR by flow cytometry using cell trace violet (CTV).

[0055] FIG. 20 shows exemplary cumulative data from the flow-based MLR reaction showing that Ds-Tregs are more suppressive than irrelevant targeted (lx) Tregs at all tested ratios of Treg: effector cells.

[0056] FIG. 21 shows that the Tregs expanded by the disclosed methods are phenotypically stable in inflammatory’ conditions.

[0057] FIG. 22 shows that the Tregs expanded by the disclosed methods retain demethylation of FOXP3 gene, which is a hallmark of continued commitment to a Treg lineage. Heat maps display % methylation of the FOXP3 gene at 11 CpG sites. .

[0058] FIG. 23 shows that an exemplary embodiment of the disclosed technology generates large numbers of Tregs that are good manufacturing practices (GMP) grade.DETAILED DESCRIPTION

[0059] The inventors have developed novel methods for expanding regulatory T cells (Tregs) that are specific for a particular antigen of interest ex vivo. Expansion of Tregs to large numbersis necessary for the use of Treg cells in adoptive transfer therapies. Previous methods known in the art induced polyclonal expansion of Tregs using ligands capable of stimulating the proliferation of all T cells. Such polyclonal methods result in Tregs capable of suppressing immune responses to a wide range of targets, but with very limited numbers to any specific antigen.

[0060] The inventors have developed a clinically applicable method for the expansion of antigen specific Tregs using, e g., donor cells as a source of antigen to activate Tregs of interest, e.g., alloreactive Tregs. These antigen specific Tregs are more potent and are more targeted than the polyclonal Tregs produced by other methods. Therefore, fewer of the antigen specific Tregs are required for adoptive cell transfer therapy to be successful and the antigen specific Tregs do not suppress immune responses globally, which may be detrimental to a subject. The disclosed methods of Treg expansion can be used in all clinical situations such as transplantation, autoimmunity', and allergy, where the immune inducing agent or antigen is known.

[0061] In addition, other protocols utilize transfected cells to expand donor B cells as a source of antigen presenting cells (APCs), which has raised regulatory concerns. The disclosed technology does not use transfected cells, improving the overall safety profile of expanded antigen specific Tregs produced by the disclosed methods.Methods of expanding T regulatory cells (Tregs)

[0062] In an aspect of the current disclosure, the inventors disclose herein novel methods to expand Tregs in an antigen-specific manner that is applicable for human adoptive cell transfer therapies.

[0063] The disclosed methods comprise: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF-b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generatea third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a.

[0064] In some embodiments, the methods are methods of expanding T regulatory cells specific for a target antigen comprising: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF-b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen.

[0065] In some embodiments, the methods are methods of expanding alloreactive T regulatory cells (Tregs). the methods comprising: a. contacting a population of Tregs with a first population of B cells comprising a target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF- b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a. to generate expanded alloreactive Tregs.

[0066] As used herein, “contacting” refers to any method suitable to bring the Tregs and B cells into contact in culture, e.g., mixing separate cultures of Tregs and B cells such that the Tregs and B cells occupy the same culture medium and are not separated by a barrier, thereby allowing antigen presentation by the B cells to the Tregs.

[0067] As used herein, '‘culturing” refers to in vitro mammalian cell culture which may be performed according to standard procedures known in the art. Culturing may be performed in GMP grade culture vessels, e.g., G-REX® culture vessels and may utilize GMP grade medium, e.g., TexMACS® GMP Medium.

[0068] As used herein, “alloreactive” refers to cells, e.g., T cells that recognize conspecific non-self antigens, e.g., alloreactive Tregs recognize alloantigens found in an allograft. Similarly, “alloantigens” refers to conspecific non-self antigens and “allografts” refer to con-specific nonself grafts of cells, tissue, or organs.

[0069] The disclosed methods utilize B cells as antigen presenting cells (APCs). Therefore, the B cells may be configured to present any antigen of choice by first pulsing the B cells with the target antigen. Methods of pulsing APCs, and specifically B cells, are known in the art. Suitably, the first and second population of B cells should be similar and should comprise the same antigen. Therefore, in the above method, the first population of B cells and the second population of B cells may, in one example, be from the same population of B cells that is, e.g., from an allograft donor. The first population of B cells for use in step a. of the method may be an aliquot or subpopulation of B cells that is thawed out, e.g., from a frozen stock of allograft donor B cells, and used in step a. Accordingly, the second population of B cells may be a second subpopulation that is thawed from the same frozen stock of allograft donor B cells, as used in step a. Thereby, step a. constitutes a first activation / stimulation with B cells and step b. constitutes a re-stimulation.

[0070] The methods may be performed on isolated Tregs or on samples that have been enriched for Tregs. Methods are known in the art for isolation or enrichment of Tregs and include positive and negative selection with, e.g., magnetic beads coupled to antibodies, fluorescence activated cells sorting (FACS), etc. The Tregs, or B cells, may be isolated using magnetic beads, e.g., with the CD4+CD25+CD127dim / - Regulatory T Cell Isolation Kit II, human available from Miltenyi Biotec.

[0071] In the disclosed methods, the timing of steps b.-d. may be varied by one of skill in the art; In one example, step b. may be performed about 7 days after beginning step a., step c. may be performed about 14 days after beginning step a., and step d. may be performed from about 14days after beginning step a. to about 28 days after beginning step a. In this exemplary protocol, the inventors discovered that antigen-specific Tregs are expanded to about 100-fold by day 21. Alternatively, if step d. is extended to day 28, Tregs are expanded to greater than 400-fold (FIG. 7).

[0072] In another exemplary method, step b. may be performed about 12 days after beginning step a., step c. may be performed about 21 days after beginning step a., and step d. may be performed from about 21 days after beginning step a. to about 28 days after beginning step a.

[0073] It is envisioned that the Tregs expanded by the disclosed methods may be used in the treatment of allograft rejection. Accordingly, the Tregs that are expanded by the methods may be alloreactive and are stimulated by B cells obtained from an allotransplant donor, where the Tregs are derived from a sample from the transplant recipient and the B cells are derived from a sample from the allotransplant donor. In other words, the Tregs that are being expanded by the disclosed methods are reactive toward alloantigens presented by the B cells from the donor.

[0074] It is envisioned that the Tregs expanded by the disclosed methods may be specific for any target antigen.

[0075] As used herein ‘“target antigen” refers to an antigen or set of antigens that are used to expand Tregs using the disclosed methods. The target antigens may be, e.g.. a protein, a fragment of a protein, a small synthetic fragment of a protein, and may be endogenously expressed by B cells or introduced to the B cells, e.g., by “pulsing” the B cells with the target antigen. Methods for pulsing B cells, such that they present a target antigen are known in the art.

[0076] The target antigen may be an antigen that is related to or is the target of an autoimmune disease, i.e., “associated with an autoimmune disease or disorder.” For example, Table 1 below lists several autoantigens, their associated autoimmune diseases and disorders, and particular phenotypes linked to immune responses against the described autoantigens. For further information, see Rosen and Casciola-Rosen “Autoantigens as Partners in Initiation and Propagation of Autoimmune Rheumatic Diseases” Ann. Rev. Immunol. 2016 May 20;34:395- 420, which is incorporated herein by reference.

[0077] Table 1. Partial listing of autoantigens targeted with phenotypic implications in autoimmune rheumatic diseases.

[0078] The target antigen may be selected from any antigen listed in Table 1, or another exemplary autoantigen including, but not limited to, aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1-gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3), 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifieractivating enzymes SAE-1 and SAE-2, topoisomerase- 1, centromere proteins A, B, & C (“CENPs”), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSC IO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B). gamma interferon-inducible protein-16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMD A) receptor, and tissue transglutaminase.

[0079] The target antigen may be related to an autoimmune disease or disorder, or a target of an autoimmune response. The autoimmune disease or disorder may be selected from scleroderma, Sjogren’s syndrome, vasculitis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and celiac disease.

[0080] The disclosed methods use the cytokines interleukin 2 (IL-2) and transforming grow th factor beta (TGF-B) to expand Tregs. The concentration of the IL-2 used in the disclosed methods may be about 10 units / ml to about 100,000 units / ml, about 100 units / ml to about 100,000 units / ml. 1000 units / ml to about 100,000 units / ml, about 10,000 units / ml to about 100,000 units / ml, or about 10,000 units / ml. The concentration of TGF-B used in the disclosed methods may be about 1 unit / ml to about 20 units / ml, about 1 to about 10 units / ml, or about 5.2 units / ml. One of skill in the art will recognize that the cells will deplete the available cytokines in the medium and that fresh cytokines may have to be added at each medium change, e.g., every 2-3 days; however, determining how often to replace medium, or add medium, depending on the culture vessel, is considered routine activity7in the art.

[0081] As used herein, a mammalian target of rapamycin (mTOR) inhibitor may comprise sirolimus, temsirolimus, everolimus, or ridaforolimus. In an exemplary^ embodiment, the mTOR inhibitor is everolimus. The concentration of the mTOR inhibitor may be about 5 ng / ml to about 500 ng / ml or about 50 ng / ml.

[0082] The inventors discovered that removal of the mTOR inhibitor from the culture medium before D14 improves the degree of expansion of Tregs. See, Fig. 2, Ds-Treg (SRL D14) vs. Ds- Treg (SRL-D21). Therefore, the mTOR inhibitor may be added to the culture for the entireduration of the method or added to the medium until about 9 days from the beginning of performing the method, i.e., step a. in the methods disclosed herein. The mTOR inhibitor may be added to the culture until about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days after beginning the method, i.e., step a.

[0083] The ratio of Tregs:B cells during the stimulation steps, i.e., one or more of steps a. or b. may be a 1 : 1 ratio. The ratio of Tregs:B cells may be about 1: 1, about 1:2. about 1 :3, about 1:4, about 2: 1, about 3: 1, or about 4: 1.

[0084] In the disclosed methods, the inventors demonstrated that polyclonal stimulation after antigen specific stimulation, through Treg-B cell interactions, leads to increased expansion of the antigen specific Tregs without compromising their functional / differentiation state (FIGs. 2, 3B). Polyclonal stimulation may be performed with, e.g., a CD3 agonist and a CD28 agonist. The CD3 agonist and / or CD28 agonist may be monoclonal antibodies. The anti-CD3 and anti-CD28 agonistic monoclonal antibodies may be linked to a solid support or a semi-solid support, e.g., beads, gel, a colloidal polymeric nanomatrix, e.g.. MACS® GMP T Cell TransAct™.

[0085] The inventors discovered that the disclosed methods may be performed using culture medium that does not comprise serum, in other words, the medium used in the methods may be serum free. Further, the inventors discovered that, not only could the method be performed in serum-free conditions, the expansion of Tregs in the serum free medium was, surprisingly, significantly greater than in medium comprising serum (FIG. 7). This discovery’ provides several advantages, including, but not limited to, that using serum free defined medium will allow direct translation of the inventors' discoveries to the clinic for use in GMP manufacturing of antigenspecific Tregs.

[0086] The disclosed methods may expand Tregs in a manner that is not polyclonal, i.e., the expansion of the Tregs may not be polyclonal expansion. The disclosed methods may be performed using media that does not comprise serum. The disclosed methods do not require feeder cells. The disclosed methods expand Tregs at least about 100-fold in less than 28 days.Populations of Tregs

[0087] In another aspect of the current disclosure, populations of Tregs are provided. The populations may be an ex vivo population of Tregs comprising at least 106, at least IO7, at least 108, at least 109Tregs, wherein the Tregs are each specific for the same antigen. The antigen specificity of the Tregs may be determined by, e.g., stimulation of the Tregs with the antigen for which the Tregs are specific, which may be accomplished by methods known in the art, e.g., stimulation by APCs pulsed with the antigen and analysis for activation markers / cytokine production by flow cytometry. The Tregs may be specific for the same antigen but may not each recognize the same portion of the antigen.

[0088] In some embodiments, the populations of Tregs may comprise Tregs made by a method comprising: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF- b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF- b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a.

[0089] The populations of Tregs may comprise greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91% greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% CD4+, CD25+, FoxP3+, CD 127" Tregs.Pharmaceutical compositions

[0090] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a population of Tregs of the instant disclosure.

[0091] The pharmaceutical compositions may further comprise at least one pharmaceutically acceptable carrier or excipient.

[0092] The pharmaceutical compositions may be formulated for intravenous administration, subcutaneous administration, intramuscular administration, intrathecal administration, or formulated for any other administration route. Formulation of pharmaceutical compositions is considered to be routine activity for one of skill in the art.Methods of treatment

[0093] The disclosed methods of expanding Tregs may be used to generate pharmaceutical compositions comprising expanded populations of antigen specific Tregs. In an aspect of the current disclosure, methods are provided.

[0094] In some embodiments, the methods comprise administering the pharmaceutical compositions of the instant disclosure to a subject.

[0095] As used herein, a subject may refer to any mammal, e.g., a human subject.

[0096] In another aspect of the current disclosure, methods of treating transplant rejection in a subject in need thereof are provided. In some embodiments, the methods comprise administering a pharmaceutical composition of the instant disclosure to the subject to treat transplant rejection in the subject.

[0097] In some embodiments, the methods of treating transplant rejection comprise: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF-b), wherein the target antigen is related to the transplant rejection, to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a.; e.administering an effective amount of the expanded Tregs specific for the target antigen to induce tolerance to the target antigen in the subject.

[0098] The disclosed methods may treat transplant rejection in a subject by improving one or more signs or symptoms of transplant rejection in the subject, e.g., chills, nausea, vomiting, diarrhea, body aches, headache, fatigue, swelling, or pain in the area of the allograft, increased T cell count in the blood, increased cytokine concentrations in the blood, e.g., increased or detectable interferon gamma (IFN-y) or tumor necrosis factor alpha (TNF-a) in the blood, or graft- or organ-specific complications of rejection, e.g., increased / elevated serum creatinine in the case of a kidney transplant rejection, death or elimination of the graft or organ by the immune system.

[0099] In another aspect of the current disclosure, methods of inducing tolerance to a target antigen in a subject in need thereof are provided. In some embodiments, the methods comprise administering a pharmaceutical composition of the instant disclosure to a subject in need thereof, wherein the population of Tregs in the pharmaceutical composition is specific for the target antigen.

[0100] In some embodiments, the methods of inducing tolerance to a target antigen in a subj ect in need thereof comprise: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF-b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL- 2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF- b until about 21 days to about 28 days after beginning step a. ; e. administering an effective amount of the expanded Tregs specific for the target antigen to induce tolerance to the target antigen in the subject.

[0101] As used herein, "inducing tolerance to a target antigen” refers to a reduction in inflammatory immune responses to a target antigen by cellular or humoral immunity in a subject.For example, the disclosed methods may decrease interferon gamma (IFN-y) or tumor necrosis factor alpha (TNF-a) in the blood and may decrease swelling or inflammation in a subject, especially in the context of inducing tolerance to a target antigen, wherein the target antigen comprises an alloantigen and the subject comprises an allograft comprising the alloantigen.

[0102] In another aspect of the current disclosure, method of treating an autoimmune disease in a subject in need thereof are provided. In some embodiments, the methods comprise administering a pharmaceutical composition of the instant disclosure to the subject to treat the autoimmune disease.

[0103] In some embodiments, methods of treating an autoimmune disease associated with a target antigen in a subject in need thereof are provided and the methods comprise: a. contacting a population of Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor b (TGF-b) to generate a first population of expanded Tregs; b. contacting the expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-b to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-b about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; and d. culturing the third population of Tregs in the presence of IL-2 and TGF-b until about 21 days to about 28 days after beginning step a.; e. administering an effective amount of the expanded Tregs specific for the target antigen to the autoimmune disease in the subject.

[0104] Many autoimmune diseases and disorders are characterized by immune responses against one or more known autoantigen or autoantigen that is commonly detected in subjects suffering from the autoimmune disease or disorder, e.g., anti-nuclear antibodies (ANA) in systemic lupus erythematosus (SLE). Therefore, as used herein, “associated with a target antigen” refers to autoimmune diseases or disorders for which there are known autoantigens and the immune response to the autoantigen is either causative or characteristic of the autoimmune disease or disorder. See, e.g., Table 1, which describes several such autoimmune diseases that are associated with a target antigen.

[0105] The disclosed methods may improve one or more sign or symptom associated with the autoimmune disease or disorder including, but not limited to, prevention of progression of the autoimmune disease or disorder or remission of the autoimmune disease or disorder.

[0106] Administration may comprise any suitable route including, but not limited to intravenous administration, subcutaneous administration, intramuscular administration, intrathecal administration, intracranial administration. Suitably, administration comprises intravenous administration.

[0107] A “therapeutically effective amount” or an “effective amount” may comprise administering about IxlO2, about 2xl02, about 3xl02, about 4xl02, about 5xl02, about 6xl02, about 7xl02, about 8xl02, about 9xl02, IxlO3, about 2x103, about 3x103, about 4x103, about 5xl03, about 6xl03, about 7xl04, about 8xl04, about 9xl04, IxlO5, about 2xl05, about 3xl05, about 4xl05, about 5x105, about 6xl05, about 7xl05, about 8xlO5, about 9x105, IxlO6, about 2x106, about 3x106, about 4xl06, about 5xl06, about 6xl06, about 7xl06, about 8xl06, about 9xl06, IxlO7, about 2xl07, about 3xl07, about 4xl07, about 5xl07, about 6xl07, about 7xl07, about 8xl07, about 9xl07, IxlO8, about 2xl08, about 3x108, about 4xl08. about 5xl08, about 6xl08, about 7xl08. about 8x108, about 9xl08, Ixl O9, about 2x109, about 3xl09, about 4x109, about 5x109, about 6xl 09, about 7x l09, about 8xl09, about 9xl09, IxlO10, about 2xlO10, about 3xl010, about 4xlO10, about 5xl010, about 6xlO10, about 7xlO10, about 8xl010, about 9xlO10, or more Tregs in the disclosed pharmaceutical compositions.Kits, systems, and platforms

[0108] In another aspect of the current disclosure, kit, systems, and platforms comprising the reagents necessary to perform the disclosed methods are provided. The kits, systems, and platforms may further comprise instructions for using the kits, systems, or platforms.

[0109] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0110] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “asubstituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0111] As used herein, "‘about”, "approximately.” "substantially,” and "significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0112] As used herein, the terms “include” and “including” have the same meaning as the terms "comprise” and “comprising.” The terms “comprise” and "comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0113] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to "such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0114] Furthermore, in those instances where a convention analogous to "at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0115] All language such as “up to." “at least;' “greater than,’7“less than." and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1. 2, 3, 4, or 6 members, and so forth.

[0116] The modal verb “may’’ refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”EXAMPLES

[0117] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Antigen-specific expansion of T regulatory (Treg) cells

[0118] FIG. 1: Ds-Treg Expansions.

[0119] Methods: Purified Tregs were isolated from “recipient” PBMC using the Treg isolation kit II (Miltenyi Biotec) and were cultured in 24-well culture plates with irradiated (3,000 rad) “donor” B cells that had been expanded and activated in culture as above at a ratio of 1 : 1 at 37°C with 5% CO2. Culture medium consisted of RPMI-1640 supplemented with 15% human AB serum (Sigma), 1,000 lU / mL IL-2 (R&D Systems) and Ing / ml TGF-(3 (R&D systems). lOOnM sirolimus (Pfizer) was added to cultures except for the final 7 days. To phenotype Tregs, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter)and FOXP3-PC5 (eBioscience) were used on days 0, 14 and 21, (also on day 28 in longer-term cultures).

[0120] Results:

[0121] (A) Tregs proliferated ~20-fold during 28 days in culture, with robust proliferation occurring after SRL was removed from culture on day 21.

[0122] (B) Phenotypically, the purified Tregs on day 0 prior to culture, were >95% CD4+ and>85% FOXP3+. A minor population of conventional T cells staining CD127dim was present in the purified product. However, it gradually disappeared with the final product becoming virtually devoid of CD127+ cells. Because of the flow cytometric configuration available in the laboratory at the time, the CD25+ population could not be assessed on day 0 for the starting product, as the selection antibody sterically hindered the detection antibody. However, there was a progressive enrichment with >90% being CD25+FOXP3+ cells at the end of the culture period. Thus, >80% of the cells were CD4+CD127-CD25+FOXP3+ Tregs in the final product.

[0123] Conclusion: Preliminary Ds-Treg expansions resulted in approximately 20-fold expansion with high expression of CD4, CD25 and Foxp3 with low expression of CD127 in the final product.

[0124] FIG. 2: Ds-Tregs expanded in cultures with different timelines of sirolimus withdrawal and polyclonal stimulation on Day 14 versus 3 donor specific stimulations. Although initial expansions were successful, clinical applicability requires greater numbers of expanded Ds-Tregs. We addressed this by testing if amplified expansion without the loss of antigen specificity could be obtained by polyclonal restimulation after two rounds of donor specific stimulations rather than three donor specific stimulations.

[0125] Methods: Purified Tregs were isolated from “recipient” PBMC using the Treg isolation kit II (Miltenyi Biotec) and were cultured in 24-well culture plates with irradiated (3,000 rad) “donor” B cells that had been expanded and activated in culture as above at a ratio of 1 : 1 at 37°C with 5% CO2. Culture medium consisted of RPMI-1640 supplemented with 15% human AB serum (Sigma). 1,000 lU / mL IL-2 (R&D Systems) and Ing / ml TGF-(3 (R&D systems). Timing of sirolimus supplementation and Donor specific stimulation strategy was studied according tothe table above (FIG. 2, Top panel). 1 ) Sirolimus supplemented for 21 days with Donor specific stimulations on Day 0, 7 and 14; 2) Sirolimus supplemented for 14 days with donor specific stimulations on Day 0, 7 and 14; 3) Sirolimus supplemented for 14 days with donor specific stimulations on Day 0 and Day 7 and a polyclonal stimulation with MACS GMP ExpAct Treg beads (Miltenyi Biotec) at 1: 1 ratio on day 14. The cultures were harvested on day 21 and compared against the standardized protocol described above (FIG. 1).

[0126] Results: There was a two-fold increase in the absolute number of Tregs when polyclonal restimulation was used as compared to donor-specific stimulation throughout, both in the absence of SRL during the final 7-day culture period (FIG. 2, bottom panel).

[0127] Conclusion: Utilizing two donor specific stimulations followed by a polyclonal stimulation with MACS GMP ExpAct Treg Beads improved expansion of Ds-Tregs. Withdrawal of sirolimus prior to Day 14 of culture additionally provided increased expansion when compared to sirolimus supplementation for 21 days of culture.

[0128] FIG. 3: Phenotypic and functional assessment of Ds-Tregs expanded in cultures with different timelines of sirolimus withdrawal and polyclonal stimulation on Day 14.

[0129] Methods: Ds-Tregs were cultured as above in FIG. 2. To phenotype Tregs, antibodies against CD4-FITC. CD127-PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter) and FOXP3- PC5 (eBioscience) were used on days 0, 14 and 21, (also on day 28 in longer-term cultures).

[0130] For functional studies a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMC autologous to the Tregs were stimulated with freshly isolated “donor’- specific irradiated PBMC from the B cell donor or an irrelevant third party at a ratio of 1 : 1 in U- bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3,000 rads. After 7 days,3H-thymidine was added to the cultures during the final 16-20 hours and incorporation of3H-thymidine was used to measure proliferation. The back-response CPM given by the Tregs or control modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds-Treg) and were subtracted from (R + Dx / Ix + Ds-Treg) experimental culturesto obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified according to Equation 1.

[0131] Results:

[0132] (A) On day 21, all the three cultures showed equivalent proportion of cells that wereCD4+and CD 127" as well as CD25+and FOXP3+, proving polyclonal restimulation does not negatively affect Treg phenotype.

[0133] (B) When assayed functionally in MLR inhibition assays on day 21, all three cultures demonstrated equivalently potent inhibition against the specific “donor” stimulator used in expanding the Tregs. However, there were subtle differences in the inhibition against the third party irrelevant stimulated MLRs; the cultures from which SRL was withdrawn on day 14 (middle and right) had higher inhibition at 1 : 10 and 1 :50 Treg: responder ratios, when compared to the culture with SRL present throughout the 21 day culture (left). Importantly, the inhibition was “donof’-specific in all cultures at higher and more clinically relevant Treg: responder ratios (>1 :250).

[0134] Conclusion: These results demonstrate that amplified expansion without the loss of antigen specificity can be obtained by polyclonal restimulation on day 14 subsequent to antigen specific stimulations on days 0 and 7. Further, early sirolimus withdrawal (prior to day 14) does not substantially impact phenotype or function of Ds-Tregs.

[0135] FIG. 4: Optimization of Donor Specific Stimulation timeline for Ds-Treg Expansion.

[0136] Our previous results had demonstrated improved expansion of Ds-Tregs when performing 2 donor specific stimulations with activated B cells followed by a polyclonal stimulation. We next assessed the timing of Donor-specific stimulations as shown in FIG. 4.

[0137] FIG. 5: Comparison of Donor Specific Stimulation Timelines (Day 0 / 7 versus Day 0 / 12).

[0138] Methods: Isolated, purified CD4+CD25+ cells were cultured in TexMACS GMP medium (Miltenyi Biotec) supplemented with 10% heat inactivated human AB Serum (ValleyBiomedical) at 37°C with 5% CO2. Cells were cultured in the presence of 11-2 ( 1 OOOIU / mL media; Clinigen Limited), TGF-beta (Ing / mL; Gibco Inc), and Everolimus (lOOng / mL; Sigma). IL-2, TGF-beta and fresh media were supplemented every 2-3 days. Everolimus was supplemented through Day 9 of culture, but no additional Everolimus was added after that time. Stimulations were performed on either on a) Day 0 and Day 7 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1 : 1 ratio and a final polyclonal stimulation on Day 14 with GMP TransACT (Miltenyi Biotec) OR b) Day 0 and Day 12 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1: 1 ratio and a final polyclonal stimulation on Day 21 with GMP TransACT (Miltenyi Bioetc). Cells were cultured in G-Rex® 6M bioreactors from Wilson Wolf.

[0139] Cells were phenotypically and functionally characterized on Day 28 of culture. Tregs were phenotyped with the following antibodies: Viakrome Live / Dead IR-808 (Beckmann Coulter), CD3-AP-Cy7 (Biolegend), CD4-AlexaFlour 700 (Biolegend), CD8-PerCp 5.5 (Biolegend), CD25-PE (Biolegend), CD127-BV605 (Biolegend), Foxp3-Eflour450 (Invitrogen), and CD19-PE Cy7(Biolegend).

[0140] For functional studies a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMC autologous to the Tregs were stimulated with freshly isolated “donor”- specific irradiated PBMC from the B cell donor or an irrelevant third party at a ratio of 1 : 1 in U- bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3,000 rads. After 6 days,3H-thymidine was added to the cultures during the final 16-20 hours and incorporation of3H-thymidine was used to measure proliferation. The back-response CPM given by the Tregs or control modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds-Treg) and were subtracted from (R + Dx / Ix + Ds-Treg) experimental cultures to obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified according to Equation 1.

[0141] Results:

[0142] (A) Ds-Tregs expanded in both conditions exhibited similar fold expansion over 28 days in culture (Day 0 / 7 vs Day 0 / 12; 91.3 fold vs 93.1 fold, respectively).

[0143] (B) Phenotypically, Tregs in the Day 0 / 7 stimulation strategy had high expression ofCD4 (98.0%), CD25 (91.6%), and Foxp3 (96.7%) with low expression of CD127 (0.8%). Tregs in the Day 0 / 12 stimulation strategy had high expression of CD4 (98.2%), CD25 (90.0%), and Foxp3 (99.3%) with low expression of CD127 (0.51%).

[0144] I Tregs in the Day 0 / 7 stimulation strategy exhibited high inhibitory effects in a donor specific fashion even at a 1:250 Treg:effector cell ratio (63.7% vs 16.4%).

[0145] (D) Tregs in the Day 0 / 12stimulation strategy exhibited high inhibitory effects in a donor specific fashion even at a 1:250 Treg:effector cell ratio (70.9% vs 6.7%).

[0146] Conclusion: There was no observed difference in the fold expansion, phenotype, or function of Ds-Tregs expanded when Donor specific stimulations were performed on Day 0 / 7 versus Day 0 / 12. However, the protocol utilizing the Day 0 / 7 donor specific stimulation with polyclonal stimulation on Day 14 allows for a truncated culture of 21 days if desired, and also allows for additional “in-process” phenotypic (i.e., Flow cytometry’) and functional assessment (i.e., MLR) of Tregs prior to use in a clinical setting. Moving forward, the Day 0 / 7 donor specific stimulation protocol will be used.

[0147] FIG. 6: Optimization of Media Conditions for Ds-Treg Expansion.

[0148] Although there was significant Ds-Treg expansion with the above conditions, we sought to improve the protocol in order to increase the fold expansion. Serum free media is a good option for large scale cell culture as it provides more consistent results, higher levels of cell growth, and is also better for downstream processing and applications. We compared the optimized conditions from above using human AB serum (left panel) to similar growth conditions where AB serum was substituted w ith a serum free substitute (Right panel).

[0149] FIG. 7: Expansion of Ds-Tregs using human AB serum vs Serum Free Media

[0150] Methods: Isolated, purified CD4+CD25+ cells were cultured in TexMACS GMP medium (Miltenyi Biotec) supplemented with either a) 10% heat inactivated human AB Serum (Valley Biomedical) or b) 5% Cell Vive T Cell CD Serum Substitute. GMP (Biolegend) at 37°C with 5% CO2. Cells were cultured in the presence of 11-2 (lOOOIU / mL media; Clinigen Limited),TGF-beta (Ing / mL; Gibco Inc), and Everolimus (l OOng / mL; Sigma). IL-2, TGF-beta and fresh media were supplemented every 2-3 days. Everolimus was supplemented through Day 9 of culture, but no additional Everolimus was added after that time. Stimulations were performed on Day 0 and Day 7 of culture with irradiated (3000 rad) expanded Donor B cells (from above) in a 1 : 1 ratio, and a final polyclonal stimulation on Day 14 with GMP TransACT (Miltenyi Biotec). Cells were cultured in G-Rex® 6M bioreactors (N=2) or G-Rex® lOM / G-Rex® 100M (N=l) from Wilson Wolf.

[0151] Cells were phenotypically and functionally characterized on Day 21 and Day 28 of culture. Tregs were phenotyped with the following antibodies: Viakrome Live / Dead IR-808 (Beckmann Coulter), CD3-AP-Cy7 (Biolegend), CD4-AlexaFlour 700 (Biolegend), CD8-PerCp 5.5 (Biolegend), CD25-PE (Biolegend). CD127-BV605 (Biolegend), Foxp3-Eflour450 (Invitrogen), and CD19-PE Cy7(Biolegend).

[0152] For functional studies, a standard mixed lymphocyte reaction (MLR) was performed. Isolated responder PBMC autologous to the Tregs were stimulated with freshly isolated “donor”- specific irradiated PBMC from the B cell donor or an irrelevant third party at a ratio of 1 : 1 in U- bottom 96-well plates in triplicate. Tregs or irradiated R-PBMC (control) were added at indicated modulator: responder ratios at the initiation of the suppression assays. The cells were irradiated at 3,000 rads. After 6 days,?H-thymidine was added to the cultures during the final 16-20 hours and incorporation of3H-thymidine was used to measure proliferation. The back-response CPM given by the Tregs or control modulators at the tested doses were also assessed in separate cultures (Rx + Dx / Ix + Ds-Treg) and w ere subtracted from (R + Dx / Ix + Ds-Treg) experimental cultures to obtain the delta CPM (A CPM). The mixed lymphocyte reaction (MLR) inhibition was quantified according to Equation 1.

[0153] Results: Ds-Tregs expanded in serum free conditions exhibited greater fold expansion over 28 days in culture as compared to AB serum supplemented media (445 fold vs 189 fold, p=0.01). Cell counts at various time points in culture are shown below in Table 2.

[0154] Conclusion: Serum Free supplement from Biolegend provides the optimal expansion of Ds-Tregs as compared to human AB serum containing medium.

[0155] FIG. 10: Flow Cytometry Data of Ds-Treg Expansions using AB serum containing medium versus Serum Free media.

[0156] (A) Phenotype of Ds-Tregs on Day 21 in AB serum containing media. Cells exhibited high expression of CD4 (97.9% + / - 1.59), CD25 (97.5% + / - 1.19), and Foxp3 (99.4% + / - 0.451). Low expression of CD127 (0.71% + / - 0.20) was observed.

[0157] (B) Phenotype of Ds-Tregs on Day 21 in serum free media. Cells exhibited high expression of CD4 (98.1% + / - 1.10), CD25 (96.4% + / - 1.42), and Foxp3 (99.2% + / - 0.361). Low expression of CD127 (0.69% + / - 0.36) was observed.

[0158] I Phenotype of Ds-Tregs on Day 28 in AB serum containing media. Cells exhibited high expression of CD4 (98.7% + / - 0.61), CD25 (91.5% + / - 0.15), and Foxp3 (98.1% + / - 1.35). Low expression of CD127 (0.69% + / - 0.27) was observed.

[0159] (D) Phenotype of Ds-Tregs on Day 28 in serum free media. Cells exhibited high expression of CD4 (98.0% + / - 0.76), CD25 (92.0% + / - 1.57), and Foxp3 (98.1% + / - 1.48). Low expression of CD127 (0.41% + / - 0.10) was observed.

[0160] Conclusions: Ds-Tregs cultured in AB serum containing medium and serum free medium have comparable phenotype with no significant differences.

[0161] FIG. 12: Functional assessment of Ds-Tregs expanded in AB serum containing media (A,C) and serum free media (B,D).

[0162] (A) Day 21 MLR data of Ds-Tregs expanded in AB serum containing media.

[0163] (B) Day 21 MLR data of Ds-Tregs expanded in serum free media.

[0164] I Day 28 MLR data of Ds-Tregs expanded in AB serum containing media.

[0165] (D) Day 28 MLR data of Ds-Tregs expanded in serum free media.

[0166] In both AB containing serum and serum free conditions Ds-Tregs exhibited a strong inhibitory effect in a donor specific fashion at Day 21 (N=3) and Day 28 (N=2). There was noobserved difference in level of inhibition between Ds-Tregs grown in AB serum containing medium vs serum free medium.

[0167] Conclusions: Ds-Tregs cultured in AB serum containing medium and serum free medium have similar levels of inhibitory function in a donor-specific fashion.

[0168] FIG. 13: Protocol for Treg Expansion. The protocol utilizes the G-Rex®10M (Days 0-21) and G-Rex® 100 M (Days 21-28) bioreactors which allow for no changes of media with a final volume of lOOmL (G-Rex® 10M) and IL (G-Rex® 100M) after 21 days and 28 days in culture, respectively. Cells are cultured in TexMACS GMP Medium (Miltenyi Biotec) supplemented with 5% Cell Vive T Cell CD Serum Substitute GMP (Biolegend) at 37°C with 5% CO2. Donor specific stimulations are performed on Day 0 and Day 7 with expanded donor B cells. A polyclonal restimulation is performed on Day 14 with Bead-free, TransACT GMP (Miltenyi). IL-2 (lOOOIU / mL; Clinigen Limited), TGF-beta (52U / mL; Miltenyi Biotec) and Everolimus (lOOng / mL; Sigma) are supplemented every72-3 days of culture. No additional Everolimus is added after day 9 in culture. Seeding concentration of cells is 1 xl06cells / mL with a starting cell number of 5 xlO6cells per G-Rex® 10M. At Day 21 of culture, all G-Rex® 10M bioreactors are then split (i .e., half of cells removed) into a G-Rex® 1 OOM allowing for increased expansion over the final 7 days of culture (right panel). For a clinical setting, Tregs are isolated utilizing a GMP grade CD8 / CD19 depletion followed by a CD25 selection step using GMP grade microbeads (all from Miltenyi Biotec) and the CliniMACS Plus closed system (Miltenyi Biotec).A. Reagents a. Source of Recipient Cells (Leukapheresis product) b. Source of donor irradiated B cells (B Cell optimization described above) c. CD8 GMP microbeads (Miltenyi Biotec) d. CD 19 GMP microbeads (Miltenyi Biotec) e. CD25 GMP microbeads (Miltenyi Biotec) f. CiiniMacs PBS / EDTA buffer (Miltenyi Biotec) g. Human Serum Albumin (Takeda) h. LS Tubing set (Miltenyi Biotec) i. TS Tubing set (Miltenyi Biotec) j. G-Rex® 10M (Wilson Wolf) k. G-Rex® 100M (Wilson Wolf) l. TexMACS GMP medium (Miltenyi Biotec)m. Proleukin / IL-2 (Clinigen Limited) n. Everolimus (Sigma) o. GMP grade TGF-beta (Miltenyi Biotec) p. Cell Vive T Cell CD Serum Substitute GMP (Biolegend)

[0169] Table 2. Comparison of Ds-Treg fold expansion in AB serum (Mean AB) versus serum free (Mean SF) conditions. Expansion under serum free conditions provides greater expansion than grow th in AB serum containing media.

[0170] Table 3. Flow cytometric Data of Treg Isolation products on Day 0 of Ds-Treg culture (N=3). Cells highly expressed CD4 (95.7% + / - 0.961) and Foxp3 (82.2% + / - 2.33), with low expression of CD127 (8.24% + / - 8.74). CD25 expression cannot be evaluated due to steric hinderance from selection beads.

[0171]

[0172] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0173] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.Example 2 - Methods of treating transplant rejection in a subject in need thereof

[0174] In one example, a subject suffering from transplant rejection is administered a therapeutically effective amount of a pharmaceutical composition comprising a population of Tregs made by the methods of the instant disclosure. The pharmaceutical compositions may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal,intrathecal, intracerebral, or intrarectal routes. The pharmaceutical compositions may be administered intravenously. Signs and symptoms of the transplant rejection may be reduced by the administration of the pharmaceutical compositions. Treatment may be administered daily, every other day, every third day, once per week, once per month, once per year, once per lifetime, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject will experience an increase in body mass, appetite, or amelioration of other metrics associated with transplant rejection, e.g., chills, nausea, vomiting, diarrhea, body aches, headache, fatigue, swelling, or pain in the area of the allograft, as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of transplant rejection are known in the art and may include, but are not limited to, measuring increased T cell count in the blood, measuring increased cytokine concentrations in the blood, e.g., increased or detectable interferon gamma (IFN-y) or tumor necrosis factor alpha (TNF-a) in the blood.Example 3 - Methods of generating alloreactive T regulatory cells and use thereof in the treatment of transplant rejection

[0175] In one example, a subject in need of an allotransplant receives the allotransplant from a suitable donor. The donor further provides, in addition to the allotransplant material, B cells for use in the disclosed methods. Thus, the allotransplant and the donor B cells have the same HLA composition and share the potential minor mismatches, relative to the subject in need of the allotransplant. The donor B cells are used in the disclosed methods to generate Tregs that are alloreactive, i.e., reactive to the allograft. The subject may receive a pharmaceutical composition comprising the alloreactive B cells in advance of any signs or symptoms of rejection or the subject may be suffering from transplant rejection. The subject is administered a therapeutically effective amount of a pharmaceutical composition comprising a population of Tregs made by the methods of the instant disclosure. The pharmaceutical compositions may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The pharmaceutical compositions may be administered intravenously. Signs and symptoms of the transplant rejection may be reduced by the administration of the pharmaceutical compositions. Treatment may be administered daily, every other day, every third day, once per week, once per month, once per year, once per lifetime, or on a schedule asdetermined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject will experience an increase in body mass, appetite, or amelioration of other metrics associated with transplant rejection, e.g., chills, nausea, vomiting, diarrhea, body aches, headache, fatigue, swelling, or pain in the area of the allograft, as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of transplant rejection are known in the art and may include, but are not limited to, measuring increased T cell count in the blood, measuring increased cytokine concentrations in the blood, e.g., increased or detectable interferon gamma (IFN-y) or tumor necrosis factor alpha (TNF-a) in the blood.Example 4 - Methods of treating an autoimmune disease in a subject in need thereof

[0176] In one example, a subject suffering from an autoimmune disease, e.g., scleroderma. Sjogren’s syndrome, vasculitis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or celiac disease, is administered a therapeutically effective amount of a pharmaceutical composition comprising a population of Tregs made by the methods of the instant disclosure. The Tregs are made using B cells from the subject that are pulsed with a known autoantigen, or a cocktail of known autoantigens. The pharmaceutical compositions may suitably be administered by any route that is indicated by the treatment needs of the subject, e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The pharmaceutical compositions may be administered intravenously. Signs and symptoms of the transplant rejection may be reduced by the administration of the pharmaceutical compositions. Treatment may be administered daily, every other day, every third day, once per week, once per month, once per year, once per lifetime, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject will experience amelioration of metrics associated with their particular autoimmune disease. In a subject with scleroderma, the methods may improve tightening, hardening, and thickening of the skin on the hands, feet, face, and extremities associated with scleroderma. In a subject with Sjogren’s syndrome, the methods may improve joint pain, swelling, stiffness, swollen salivary glands, skin rashes, cough, or fatigue. In a subject with vasculitis, the methods may improve fever, tiredness, w eight loss, loss of appetite, aches or pains, headaches, cough, shortness of breath, lung infiltrates, cavities in the lungs, unusual rashes or skin discoloration, skin sores, or blisters. In a subject with rheumatoid arthritis, the methods may improve aching, orstiffness in more than one joint, especially in the hands, wrists, and feet, tenderness, redness, and warmth in the joints, fever, weight loss, poor appetite, or exhaustion. In a subject with systemic lupus erythematosus (SLE), the methods may improve fatigue, skin rash, e.g., a butterfly-shaped rash that appears on the cheeks and bridge of the nose, painful and swollen joints, especially in the fingers, hands, wrists, and knees, fever, hair loss, mouth sores, sensitivity to sunlight, swollen lymph nodes, or chest pain. In a subject with celiac disease, the methods may improve Diarrhea, constipation, bloating, gas, abdominal pain, nausea, and vomiting, itchy skin and blisters on the buttocks, elbows, knees, scalp, or torso, headaches, anemia, fatigue, delayed puberty, infertility, weak bones, and teeth changing color or losing their enamel. Methods of measuring reductions in signs and symptoms of the autoimmune diseases are known in the art.

Claims

CLAIMS1. A method of expanding T regulatory cells (Tregs) specific for a target antigen, the method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL- 2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen.

2. The method of claim 1, wherein the first and second populations of B cells are pulsed with the target antigen prior to performing the method.

3. The method of claim 1, wherein the target antigen comprises at least one alloantigen.

4. The method of claim 1, wherein the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor.

5. The method of claim 1, wherein the Tregs are from a subject that has been previously- treated with an allotransplant and the B cells are from the donor of the allotransplant.

6. The method of claim L wherein step b. is performed about 7 to about 12 days after beginning step a.

7. The method of claim 1, wherein step b. is performed about 7 days after beginning step a.

8. The method of claim 1. wherein step b. is performed about 12 days after beginning step a.

9. The method of claim 1 , wherein step c. of the method is performed about 14 to about 21 days after beginning step a.

10. The method of claim 1 , wherein step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a.

11. The method of claim 1. wherein the B cells are irradiated.

12. The method of claim 1, wherein the IL-2 is at a concentration of about 1,000 units / ml.

13. The method of claim 1 , wherein a mammalian target of rapamy cin (mTOR) inhibitor is added to the culture medium.

14. The method of claim 13, wherein the mTOR inhibitor is everolimus.

15. The method of claim 13, wherein the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a.

16. The method of claim 13, wherein the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a.

17. The method of claim 14, wherein the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium.

18. The method of claim 1 , wherein the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method.

19. The method of claim 1. wherein the B cells and the Tregs are present in about a 1: 1 ratio in step a. and b. of the method.

20. The method of claim 1, wherein the TGF-P is at a concentration of at least about 5.2 units / ml.

21. The method of claim 1 , wherein the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively.

22. The method of claim 21, wherein the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix.

23. The method of claim 1. wherein the expanded Tregs comprise at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the expanded Tregs are each specific for the same antigen.

24. The method of claim 1, wherein the method expands the Tregs at least about 100- fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445-fold.

25. The method of claim 1 , wherein the method expands the Tregs at least about 100- fold 21 days after beginning step a. of the method.

26. The method of claim 1, wherein the method expands the Tregs at least about 400- fold 28 days after beginning step a. of the method.

27. The method of claim 1, wherein the Tregs are cultured in serum free medium.

28. The method of claim 1 , wherein the method is performed under good manufacturing practice (GMP) conditions and, wherein, the method results in a GMP product.

29. A population of T regulatory cells (Tregs) made by the method of claim 1.

30. An ex vivo population of T regulatory cells (Tregs), the population comprising at least 106Tregs wherein the Tregs in the population are each specific for the same antigen.

31. The population of Tregs of any one of claims 29 or 30, wherein the Tregs are CD4+, CD25+, FoxP3+.

32. The population of Tregs of claim 31. wherein the Tregs are CD 127’.

33. A pharmaceutical composition comprising the population of Tregs of claim 29.

34. A method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 33 to a subject.

35. A method of treating transplant rejection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 33 to the subject to treat transplant rejection in the subject.

36. The method of claim 35, wherein the subject has received an organ transplant, a tissue transplant, or a cellular transplant.

37. A method of inducing tolerance to a target antigen in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 33 to a subject in need thereof, wherein the population of Tregs in the pharmaceutical composition is specific for the target antigen.

38. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 33, to the subject to treat the autoimmune disease.

39. The method of claim 38, wherein the autoimmune disease is selected from scleroderma, Sjogren’s syndrome, vasculitis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and celiac disease.

40. The method of claim 38, wherein the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIF I -y; TRIM 33), nuclear matric protein-2 (NXP2; MORC3). 3-hydroxy-3-methylglutaryl-coA reductase (HMGCR), melanoma- associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzymes SAE-1 and SAE-2, topoisomerase-1, centromere proteins A, B & C (“CENPs”), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSCIO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60. La (SS-B), gamma interferon-inducible protein-16 (1F116), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMDA) receptor, and tissue transglutaminase.

41. A method of treating an allergy in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 33, to the subject to treat the allergy.

42. A method of treating transplant rejection in a subject in need thereof, the method comprising: a. obtaining T regulatory cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF-P), wherein the target antigen is related to the transplant rejection, to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL- 2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen;e. administering an effective amount of the expanded Tregs specific for the target antigen to the subject to induce tolerance to the target antigen in the subject.

43. A method of inducing tolerance to a target antigen in a subject in need thereof, the method comprising: a. obtaining T regulatory cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF- ) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL- 2 and TGF-P until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen; e. administering an effective amount of the expanded Tregs specific for the target antigen to induce tolerance to the target antigen in the subject.

44. A method of treating an autoimmune disease associated with a target antigen in a subject in need thereof, the method comprising: a. obtaining T regulatory cells (Tregs) from the subject and contacting the Tregs with a first population of B cells comprising the target antigen in medium comprising interleukin 2 (IL-2) and transforming grow th factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-p to generate a second population of expanded Tregs;c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL- 2 and TGF- until about 21 days to about 28 days after beginning step a. to generate expanded Tregs specific for the target antigen; e. administering an effective amount of the expanded Tregs specific for the target antigen to treat the autoimmune disease in the subject.

45. The method of any one of claims 42-43, wherein the B cells are pulsed with the target antigen prior to performing the method.

46. The method of any one of claims 42-44, wherein the target antigen is at least one alloantigen.

47. The method of any one of claims 42-44, wherein the target antigen is selected from the group consisting of: aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matric protein-2 (NXP2; M0RC3). 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzy mes SAE-1 and SAE-2, topoisomerase-1, centromere proteins A, B & C (“CENPs'’), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSCIO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B), gamma interferon-inducible protein- 16 (IF116), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul- RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl- D-aspartate (NMD A) receptor, and tissue transglutaminase.

48. The method of claim 38, wherein the Tregs are from a subject in need of allotransplantation and the B cells are from an allogeneic donor.

49. The method of any one of claims 38 or 44, wherein the Tregs are from a subject that has been treated with an allotransplant and the B cells are from the donor of the allotransplant.

50. The method of any one of claims 38-45, wherein step b. is performed about 7 to about 12 days after beginning step a.

51. The method of claim 50, wherein step b. is performed about 7 days after beginning step a.

52. The method of claim 50, wherein step b. is performed about 12 days after beginning step a.

53. The method of any one of claims 42-52, wherein step c. of the method is performed about 14 to about 21 days after beginning step a.

54. The method of any one of claims 42-53, wherein step b. is performed about 7 days after beginning step a., step c. is performed about 14 days after beginning step a., and step d. is performed until about 28 days after beginning step a.

55. The method of any one of claim 42-54. wherein the B cells are irradiated.

56. The method of any one of claims 42-55, wherein the IL-2 is at a concentration of about 1,000 units / ml.

57. The method of any one of claims 42-56, wherein a mammalian target of rapamycin (mTOR) inhibitor is added to the culture medium.

58. The method of claim 57, wherein the mTOR inhibitor is everolimus.

59. The method of claim 57 or 58, wherein the mTOR inhibitor is added to the culture medium until about 28 days after beginning step a.

60. The method of claim 59, wherein the mTOR inhibitor is added to the culture medium until about 9 days after beginning step a.

61. The method of any one of claims 57-60, wherein the mTOR inhibitor is at a concentration of about 50 ng / ml in the culture medium.

62. The method of any one of claims 42-61 , wherein the B cells and the Tregs are present in about a 1 : 1 ratio in step a. of the method.

63. The method of any one of claims 42-62. wherein the B cells and the Tregs are present in about a 1 : 1 ratio in step a. and b. of the method.

64. The method of any one of claims 42-63, wherein the TGF-P is at a concentration of about 5.2 units / ml.

65. The method of any one of claims 42-64, wherein the CD3 agonist and the CD28 agonist are anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies, respectively.

66. The method of claim 65, wherein the anti-CD3 and anti-CD28 monoclonal antibodies are covalently linked to a colloidal polymeric nanomatrix.

67. The method of any one of claims 42-66, wherein the method produces a population of Tregs, the population comprising at least 106Tregs, at least 107Tregs, or at least 108Tregs, wherein the Tregs are each specific for the same antigen.

68. The method of any one of claims 42-67, wherein the method expands the Tregs at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 445 -fold.

69. The method of claim 68, wherein the method expands the Tregs at least about 100- fold 21 days after beginning step a. of the method.

70. The method of any one of claims 42-69, wherein the method expands the Tregs at least about 400-fold 28 days after beginning step a. of the method.

71. The method of any one of claims 42-70, wherein the Tregs are cultured in serum free medium.

72. A method of expanding alloreactive T regulatory cells (Tregs), the method comprising: a. contacting the Tregs with B cells comprising a target antigen, wherein the target antigen is an alloantigen, in serum free medium comprising about 1000 units / ml interleukin 2 (IL-2) and at least about 5.2 units / ml transforming growth factor p (TGF- ); b. contacting the Tregs of step a. with B cells comprising the target antigen in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-p about 7 days after beginning step a.; c. contacting the Tregs of step b. with a CD3 agonist and a CD28 agonist in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-P about 14 days after beginning step a, wherein the CD3 agonist and the CD28 agonist are monoclonal antibodies and are covalently linked to a polymeric nanomatrix;d. culturing the Tregs of step c. in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-P until about 28 days after beginning step a. to generate expanded alloreactive Tregs, wherein the alloreactive Tregs are expanded at least about 400-fold and are at least 80% CD4+, CD25+, FoxP3+, CD 127’ Tregs.

73. A method of treating transplant rejection in a subject in need thereof, the method comprising: a. obtaining Tregs from the subject and contacting the Tregs with B cells comprising a target antigen, wherein the target antigen is an alloantigen, in serum free medium comprising about 1000 units / ml interleukin 2 (IL-2) and at least about 5.2 units / ml transforming growth factor p (TGF-P), wherein the subject is in need of an allotransplant and the B cells are from the donor of the allotransplant; b. contacting the Tregs of step a. with B cells comprising the target antigen in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-P about 7 days after beginning step a.; c. contacting the Tregs of step b. with a CD3 agonist and a CD28 agonist in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-p about 14 days after beginning step a, wherein the CD3 agonist and the CD28 agonist are monoclonal antibodies and are covalently linked to a polymeric nanomatrix; d. culturing the Tregs of step c. in serum free medium comprising about 1000 units / ml IL-2 and at least about 5.2 units / ml TGF-p until about 28 days after beginning step a. to generate expanded alloreactive Tregs, wherein the alloreactive Tregs are expanded at least about 400-fold and are at least 80% CD4+, CD25+, FoxP3+, CD 127’ Tregs; e. administering an effective amount of the Tregs of step d. to the subject to treat transplant rejection in the subject.

74. A kit, system, or platform comprising the necessary reagents for performing the methods of any one of claims 1-32 or 42-73.

75. The kit, system, or platform of claim 74, further comprising instructions for performing the methods using the kit, system, or platform.

76. A method comprising: a. contacting a population of T regulatory cells (Tregs) with a first population of B cells comprising a target antigen in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of Tregs in the presence of IL-2 and TGF-P until about 21 days to about 28 days after beginning step a.

77. A method of expanding alloreactive T regulatory cells (Tregs), the method comprising: a. contacting the Tregs with a first population of B cells comprising the target antigen, wherein the target antigen comprises at least one alloantigen, in medium comprising interleukin 2 (IL-2) and transforming growth factor p (TGF-P) to generate a first population of expanded Tregs; b. contacting the first population of expanded Tregs with a second population of B cells comprising the target antigen in medium comprising IL-2 and TGF-P to generate a second population of expanded Tregs; c. contacting the second population of expanded Tregs with a CD3 agonist and a CD28 agonist in medium comprising IL-2 and TGF-P about 14 to about 21 days after beginning step a. to generate a third population of expanded Tregs; d. culturing the third population of expanded Tregs in medium comprising IL- 2 and TGF-p until about 21 days to about 28 days after beginning step a. to generate alloreactive expanded Tregs.

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