Methods for allogeneic hematopoietic stem cell transplantation

Abstract

To provide methods for improved hematopoietic stem cell transplantations, including methods for enhancing protection from graft versus host disease while maintaining effective immune responses such as graft versus tumor immune responses.SOLUTION: Provided is a pharmaceutical composition for treating cancer in a human subject, the pharmaceutical composition each independently comprising: a population of hematopoietic stem and progenitor cells (HSPC); a population of cells comprising isolated fresh regulatory T cells (Tregs); a population of isolated CD3+ conventional T cells (CD3+Tcons); and a single graft-versus-host disease (GVHD) prophylactic agent.SELECTED DRAWING: Figure 2

Description

[Background technology]

[0001] Patients with high-risk hematological malignancies such as leukemia and lymphoma after initial remission or who have refractory relapses are rarely cured with standard chemotherapy.

[0002] Myeloablative allogeneic hematopoietic cell transplantation (HCT) is associated with improved survival in these patients, with disease-free survival ranging from 10-50% and non-relapse mortality ranging from 30-50%. Early morbidity and mortality associated with acute graft-versus-host disease (aGVHD) are major factors limiting the success of HCT, as are long-term morbidity associated with chronic GVHD.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0004] Disclosed herein in some embodiments is a method of treating a human subject in need thereof, comprising administering to said human subject a) a population of hematopoietic stem and progenitor cells (HSPCs), b) a population of cells comprising regulatory T cells (Tregs), and c) a population of conventional T cells (Tcon), wherein said population of HSPCs and said population of cells comprising Tregs are administered before said population of Tcon, and wherein said population of cells comprising Tregs has not been cryopreserved prior to administration of said cells comprising Tregs.

[0005] Disclosed herein in some embodiments is a method of treating a human subject in need thereof, comprising administering to said human subject a) a population of hematopoietic stem and progenitor cells (HSPCs), b) a population of cells comprising regulatory T cells (Tregs), and c) a population of conventional T cells (Tcon), wherein said population of HSPCs and said population of cells comprising Tregs are administered before said population of conventional T cells, and wherein said human subject does not develop graft-versus-host disease (GVHD) within 30 days after administration of said population of Tcon.

[0006] In some embodiments, a method of treating a human subject in need thereof comprises administering to the human subject a) a population of hematopoietic stem and progenitor cells (HSPCs), b) a population of cells comprising regulatory T cells (Tregs), and c) a population of conventional T cells (Tcon), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to the population of Tcon, and wherein the population of cells comprising Tregs expresses CD45 + cells, + Disclosed herein is a method, wherein greater than 90% of the cells are Tregs. [Brief description of the drawings]

[0007] [Figure 1] Provide patient characteristics for clinical trials involving administration of HSPCs and Tregs prior to Tcon. [Diagram 2] 1 illustrates an original clinical protocol scheme and a modified clinical protocol scheme. [Diagram 3] 1 illustrates the enrichment of Treg cells from a peripheral blood apheresis product for administration to a subject. [Figure 4] 1 illustrates the suppressive capacity of a population of cells, including Tregs, in a mixed lymphocyte reaction (MLR). [Diagram 5] Illustrates a dose escalation scheme for a clinical trial involving administration of HSPCs and Tregs prior to Tcon. [Figure 6] 1 provides a schematic diagram of the generation and administration of fresh Treg grafts. [Figure 7] 1 shows the cell dose yield of donor-derived Tregs in a clinical trial. [Figure 8] Details of cell administration for subjects in clinical trials are provided. [Figure 9] Summarize clinical outcome data from clinical trials. [Figure 10] Illustrates immune cell reconstitution at standardized time points post-HCT. [Figure 11] Illustrates the characteristics of Tregs at 60 days post-HCT and the diversity of the Treg TCR repertoire at 90 days post-HCT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The following description and examples illustrate the embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are many variations and modifications of the present invention, which are encompassed within its scope.

[0009] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0010] As used herein, in connection with a reference numerical value and its grammatical equivalents, the term "about" and its grammatical equivalents can include values ​​in a range of plus or minus 10% from that value. For example, the amount "about 10" includes the amount 9 to 11. In connection with a reference numerical value, the term "about" can also include values ​​in a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0011] overview Provided herein are methods for improving allogeneic hematopoietic stem cell transplantation (HCT). In some embodiments, the methods disclosed herein preserve the graft-versus-tumor (GVT) effect of HCT administered to a subject with cancer while preventing or reducing graft-versus-host disease (GVHD).

[0012] HCT is the transplantation of multipotent hematopoietic stem and progenitor cells (HSPCs), usually derived from the donor's bone marrow, peripheral blood, or umbilical cord blood, into a recipient. The recipient can be subjected to a myeloablative conditioning treatment that can kill hematopoietic cells, including tumor cells and host immune cells. The HSPCs transferred into the recipient can then reconstitute the hematopoietic compartment. HCT can be useful as a treatment for cancer due to the ability of donor T cells to exert an antitumor effect, termed graft versus tumor (GVT). In patients with hematological malignancies that are refractory to chemotherapy, HCT is associated with improved survival.

[0013] However, donor T cells can also attack non-tumor host cells, which can result in graft-versus-host disease (GVHD). GVHD is a major cause of post-HCT complications and can be fatal. Management of GVHD may require immunosuppressive therapy or cytotoxic mediation, which can cause toxicity, increase susceptibility to infection, or blunt anti-tumor immunity. Early morbidity and mortality associated with acute graft-versus-host disease (aGVHD) are major factors limiting the success of HCT, as are long-term morbidity associated with chronic GVHD (cGVHD). GVHD is a risk for both HLA-matched and mismatched transplants. GVHD can occur even when the donor and recipient are HLA-matched because the immune system can still recognize other differences between their tissues. Despite the use of various immunosuppressive agents, the incidence of aGVHD after allogeneic HCT from HLA-matched sibling donors (MSDs) ranges from 20 to 60%.

[0014] Both GVT and GVHD are primarily mediated by conventional T cells (Tcon), which initiate immune responses upon recognition of cognate antigens by T cell receptors. Depleting T cells from HCT grafts can alleviate GVHD, but can also cause alleviation of GVT and increased likelihood of cancer recurrence. In addition to Tcon, Tregs are an additional subset of T cells that negatively regulate inflammation and promote immune tolerance. Tregs can prevent or alleviate GVHD, for example, by negative regulation of inflammation, including inflammation induced by donor Tcon recognizing recipient antigens.

[0015] Provided herein are methods for improving HCT, including administering to a subject specific populations of cells, including a population comprising HSPCs, a population comprising Tregs, and a population comprising Tcon. Without wishing to be bound by theory, administration of Tregs can reduce GVHD, and administration of Tcon enhances GVT, and thus the present disclosure provides methods for administering both to enhance GVT while minimizing GVHD. Without wishing to be bound by theory, these beneficial aspects of the present invention may be associated with the order of administration of the cells described herein (e.g., administering Tregs before Tcon) or not cryopreserving Tregs prior to administration. Thus, the methods disclosed herein can preserve the graft-versus-tumor (GVT) effect of HCT provided to a subject with cancer, while preventing or reducing graft-versus-host disease (GVHD). In some embodiments, two or more populations of cells are administered at different times, for example, HSPCs and Tregs can be administered before Tcon.

[0016] The one or more cell populations can be fresh, and the one or more cell populations can be cryopreserved before thawing and administration to a subject.Without wishing to be bound by theory, administering fresh Treg can reduce GVHD in a subject, and / or administering cryopreserved Tcon can reduce GVHD in a subject.For example, administering fresh Treg followed by cryopreserved Tcon can reduce GVHD in a subject, promote GVT in a subject, or promote GVT in a subject in the absence of GVHD.

[0017] The present disclosure also provides a population of cells containing Tregs, which is selected to generate a population of cells containing a high percentage of Tregs. In some embodiments, a population of cells containing a high percentage of fresh Tregs is administered before administering Tcon to reduce GVHD in a subject.

[0018] Hematopoietic Cell Transplantation and Cancer Provided herein is a method for a recipient subject with cancer to undergo hematopoietic stem cell transplantation (HCT).HCT can be useful for treating or alleviating cancer in the subject.In some embodiments, a population of conventional T cells (Tcon) is administered to the subject to induce graft-versus-tumor (GVT) immune response.

[0019] Patients with high-risk hematologic malignancies are rarely cured with standard chemotherapy. High-risk malignancies include, for example, leukemia or lymphoma that has progressed after first remission or that has refractory relapse. Myeloablative allogeneic hematopoietic stem cell transplantation (HCT) is associated with improved survival in these patients, with disease-free survival ranging from 10-50% and non-relapse mortality ranging from 30-50%.

[0020] Hematopoietic stem cell transplantation (HCT) is the transplantation of multipotent hematopoietic stem and progenitor cells (HSPCs), usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSPCs can have extensive self-renewal capacity and the ability to differentiate into specialized cell types, e.g., to reconstitute all hematopoietic cell lineages. HSPCs can undergo asynchronous replication, in which two daughter cells are produced with different phenotypes. Hematopoietic stem cells can exist in a mitotically quiescent form.

[0021] HSPCs can be obtained by harvesting from bone marrow or peripheral blood. Bone marrow can be aspirated from the posterior or anterior iliac crest while the donor is under local or general anesthesia. HSPCs can be obtained by harvesting from peripheral blood, for example, by peripheral blood apheresis. The number of harvested stem cells can be increased by treating the donor with a mobilization agent, i.e., an agent that mobilizes stem cells from bone marrow to peripheral blood. Non-limiting examples of mobilization agents include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, and combinations thereof. Techniques for mobilizing stem cells into peripheral blood can include, for example, administering 10-40 μg / kg / day of a mobilization agent to the donor. The mobilization agent can be administered to the donor in, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. The apheresis product can be isolated from the donor, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, or 30 hours after administration of the mobilizing agent.

[0022] The method of the present disclosure can be used to treat a human subject with cancer. In some embodiments, the subject is being treated for cancer, for example, by treatment with chemotherapy or radiation. The method of the present disclosure can be useful for treating hematological malignancies, for example, leukemia or lymphoma. Examples of hematological malignancies that can be treated by the method of the present disclosure include, but are not limited to, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, and lymphomas, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma. The cancer can be a solid tumor. In some embodiments, the cancer is a primary or metastatic tumor.

[0023] The types of cancer that can be treated using the methods of the present invention include leukemia, lymphoma, adrenocortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastases, brain cancer, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, cervical cancer, childhood non-Hodgkin's lymphoma, colon and rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and pharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, These include, but are not limited to, leukemia, liver cancer, lung cancer, pulmonary carcinoid tumor, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndromes, myeloproliferative disorders, paranasal sinus and nasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, melanoma skin cancer, non-melanoma skin cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarcinoma, head and neck cancer, teratoma, and Waldenstrom's macroglobulinemia.

[0024] Hematopoietic Cell Transplantation and Graft-Versus-Host Disease The present disclosure provides a method for improving hematopoietic stem cell transplantation (HCT), which reduces or prevents GVHD. In some embodiments, a population of cells described herein is administered to an HCT recipient subject, and the recipient subject does not develop GVHD within a certain period of time (e.g., within 30, 100, or 200 days) after administration of the cells. For example, a population of cells described herein can be administered to an HCT recipient subject, and the likelihood of the recipient subject developing GVHD can be reduced within a certain period of time (e.g., within 30, 100, or 200 days) after administration of the cells compared to a subject who has undergone HCT according to a different protocol. A population of cells comprising Tregs can be administered to an HCT recipient subject, and the population of Tregs can play a role in reducing or preventing GVHD in the HCT recipient subject. A population of cells comprising fresh Tregs can be administered to an HCT recipient subject, and can play a role in reducing or preventing GVHD in the HCT recipient subject. In some embodiments, a population of cells comprising a high percentage of Tregs (eg, CD4+FOXP3+ or CD4+CD25+CD127dim cells) is administered to an HCT recipient subject and serves to reduce or prevent GVHD in the HCT recipient subject.

[0025] Graft-versus-host disease (GVHD) is an inflammatory disease that can occur in an allogeneic transplant environment. GVHD involves donor cells (graft) attacking recipient cells (host). GVHD can be classified as acute GVHD (aGVHD) and chronic GVHD (cGVHD). aGVHD typically occurs in the first 3 months after transplantation. aGVHD can be life-threatening, for example, infiltrating the skin, intestine, and / or liver. cGVHD typically occurs after the first 3 months after transplantation. cGVHD is the leading cause of late treatment-related complications and can be life-threatening. In addition to inflammation, cGVHD can cause the development of fibrosis, which can lead to functional impairment.

[0026] Early morbidity and mortality associated with acute graft-versus-host disease (aGVHD) are major factors limiting the success of HCT, as are long-term morbidity associated with chronic GVHD (cGVHD). Despite the use of various immunosuppressive agents, such as tacrolimus, cyclosporine, methotrexate, mycophenolate, antithymocyte globulin, and corticosteroids, the incidence of aGVHD after allogeneic HCT from HLA-matched sibling donors (MSDs) ranges from 20 to 60%. Approximately one-third of patients who undergo allogeneic HCT with MSDs and T cell-rich (T cell replete) grafts develop chronic GVHD.

[0027] GVHD severity can be graded, for example, using the Glucksberg grade (I-IV) or the International Bone Marrow Transplant Registry (IBMTR) grading system (A-D). The severity of acute GVHD is determined by assessment of the extent of skin, liver, and gastrointestinal involvement. The stage of individual organ involvement is combined (Glucksberg) or not (IBMTR) with the patient's performance status to generate an overall grade that may be significant in terms of prognosis. Grading is important in terms of assessing response to prophylaxis or treatment, impact on survival, and association with the graft-versus-leukemia effect.

[0028] Grade I (A) GVHD is characterized as mild disease, Grade II (B) GVHD as moderate, Grade III (C) as severe, and Grade IV (D) as life-threatening. According to the IBMTR grading system, the severity of acute GVHD is defined as follows: Grade A: Stage 1 skin involvement only (maculopapular rash over <25% of the body) with no liver or gastrointestinal involvement, Grade B: Stage 2 skin involvement, Stage 1-2 intestinal or liver involvement, Grade C: Stage 3 involvement of any organ system (generalized erythroderma, bilirubin 6.1-15.0 mg / dL, diarrhea 1500-2000 mL / day), and Grade D: Stage 4 involvement of any organ system (generalized erythroderma with blistering, bilirubin >15 mg / dL, diarrhea >2000 mL / day, or pain or ileus). Patients with moderate to severe GVHD have a significantly higher mortality rate compared to patients with mild disease; for example, patients with grade III (C) aGVHD have an estimated 5-year survival rate of 25%, while patients with grade IV (D) have an estimated 5-year survival rate of 5%.

[0029] Management of GVHD may require immunosuppressive therapy (e.g., high-dose corticosteroids, long-term administration of immunosuppressants) or cytotoxic mediators, all of which are associated with toxicity. In many cases, immunosuppressive therapy may not effectively treat GVHD or may result in increased susceptibility to infection or blunted antitumor immunity.

[0030] In some embodiments, the methods disclosed herein prevent or reduce GVHD in HCT recipient subjects. For example, the methods disclosed herein can prevent any manifestation of GVHD in subjects who have undergone HCT.

[0031] The methods disclosed herein can, for example, prevent any stage 1 or higher GVHD, any stage 2 or higher GVHD, any stage 3 or higher GVHD, or any stage 4 GVHD in a subject who has undergone HCT.

[0032] In some embodiments, the methods disclosed herein provide a method for determining whether or not a population of cells disclosed herein is effective to reduce or eliminate the risk of developing a disease that is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, Prevent any stage 1, 2, 3, or 4 GVHD in subjects who have undergone HCT for more than 0, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more.

[0033] The present disclosure provides a method for treating a patient having a disease in which the patient is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, Methods are provided that can prevent any stage 1, 2, 3, or 4 GVHD in a subject that has undergone HCT for 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less.

[0034] The disclosure provides methods that can prevent, for example, any GVHD grade A or higher, any GVHD grade B or higher, any GVHD grade C or higher, or any GVHD grade D in a subject who has undergone HCT.

[0035] In some embodiments, the methods described herein provide a method for treating a patient with a disease that is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1 Any grade A, B, C, or D GVHD can be prevented in subjects who have undergone HCT for more than 5, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more.

[0036] The present disclosure provides a method for treating a patient having a disease in which the patient is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, Methods are provided that can prevent any grade A, B, C, or D GVHD in a subject who has undergone HCT for 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less.

[0037] The methods disclosed herein can, for example, reduce the proportion of HCT recipient subjects exhibiting stage 1 or higher, stage 2 or higher, stage 3 or higher, or stage 4 GVHD compared to other HCT methods.

[0038] The methods disclosed herein can provide, for example, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, The proportion of HCT recipient subjects exhibiting Stage 1 or greater, Stage 2 or greater, Stage 3 or greater, or Stage 4 GVHD for 80, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more may be reduced.

[0039] In some embodiments, the methods disclosed herein provide for up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 445, 446, 447, 448, 449, 450, 4 , 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less, reduce the proportion of HCT recipient subjects exhibiting stage 1 or greater, stage 2 or greater, stage 3 or greater, or stage 4 GVHD.

[0040] Disclosed herein are methods that can reduce the proportion of HCT recipient subjects exhibiting grade A or higher, grade B or higher, grade C or higher, or grade D GVHD, for example, compared to other HCT methods.

[0041] The methods disclosed herein may, for example, provide a method for treating a patient with a disease that is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 685, 690, 700, 710, 720, 730, 740, 750, 765, 770, 785, 790, 800, 810, 820, 830, 840, 850, 860, 870, 885, 890, 900, 910, 920, 930, 940, 950, 960, 970, 985, , 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more, or the proportion of HCT recipient subjects exhibiting grade A or greater, grade B or greater, grade C or greater, or grade D GVHD can be reduced.

[0042] In some embodiments, the methods disclosed herein provide for up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 445, 446, 447, 448, 449, 450, 4 , reduce the proportion of HCT recipient subjects exhibiting grade A or greater, grade B or greater, grade C or greater, or grade D GVHD for 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less.

[0043] Immune cells in HCT and GVHD Provided herein is a method for improving hematopoietic stem cell transplantation (HCT), where GVHD is reduced or prevented, for example, compared to comparable treatment methods in which Tcon is not administered after HSPC and / or Treg, or Treg is cryopreserved, or Tcon is not cryopreserved, or any combination thereof. Subsets of immune cells, such as conventional T cells (Tcon), regulatory T cells (Treg), invariant natural killer T cells (iNKT), and memory T cells (Tmem), can contribute to aspects of GVHD after HCT, for example, GVT immune response, immune reconstitution, infection susceptibility, and survival rate.

[0044] Disclosed herein is a method comprising administering a population of Tcon and Treg to an HCT recipient subject. In some embodiments, iNKT is also administered. In some embodiments, Tmem is also administered. This disclosure provides parameters for administering these populations of cells that contribute to successful clinical outcomes in HCT recipient subjects. Without wishing to be bound by theory, parameters that can contribute to successful clinical outcomes in HCT recipient subjects include, for example, the population administered, the order and timing of administration of different populations, the purity criteria of the population, the method for obtaining the population, the method for handling or storing the population (e.g., using a population of fresh cells versus a population of frozen cells), the dosage of the population administered, the method for obtaining the population, and combinations thereof.

[0045] GVHD is primarily mediated by donor T cells, which can trigger inflammatory responses upon recognition of recipient antigens. T cell depletion (TCD) of donor grafts can be performed to reduce the likelihood of acute and chronic GVHD. T cells can be depleted using methods including, but not limited to, physical adsorption of T cells to protein ligands such as lectins, immunodepletion with T cell-specific antibodies, and immunoaffinity techniques (e.g., use of T cell or lymphocyte-specific antibodies in immunoadsorption columns, magnetic activated cell sorting (MACS), or fluorescence activated cell sorting (FACS)). Application of TCD techniques to donor grafts can result in a 10-fold increase in T cells to 10 5 TCD may reduce the incidence of GVHD by depleting T cells. However, TCD may also increase the incidence of cancer recurrence because the lack of T cells may reduce graft-versus-tumor (GVT) immune responses. Furthermore, TCD may impair immune recovery and increase susceptibility to infection.

[0046] Both GVT and GVHD are primarily mediated by conventional T cells (Tcon), which initiate immune responses upon recognition of cognate antigens (tumor antigens in GVT, non-tumor recipient antigens in GVHD) by T cell receptors. Tcon may contribute, for example, to GVT, GVHD, or a combination thereof. Without wishing to be bound by theory, Tcon administration following Treg administration can enhance GVT immunity and / or reduce susceptibility to infection.

[0047] As used herein, Tcon can broadly refer to all CD3+ T cells, cells expressing moderate to high levels of CD127, cells expressing CD3 and moderate to high levels of CD127, or cells expressing CD3, moderate to high levels of CD127, and CD4 or CD8. In some embodiments, Tcon does not express Vα24Jα18 TCR. Tcon and Treg can be populations of cells that are not mutually exclusive. In some embodiments, Tcon and Treg are populations of cells that are mutually exclusive.

[0048] Regulatory T cells ("Tregs") are a specialized subpopulation of T cells that can suppress immune system activation and thereby maintain immune tolerance. Without wishing to be bound by theory, the population of Tregs administered by the methods of the present disclosure may contribute to better clinical outcomes, for example, by reducing or preventing GVHD in transplant recipients and / or improving immune reconstitution in transplant recipients. Administering Tregs with HSPCs before administering conventional T cells (Tcon) can, for example, promote graft-versus-tumor (GVT) retention and prevent GVHD. Administering Tregs with HSPCs before administering conventional T cells (Tcon) can, for example, promote graft-versus-tumor (GVT) retention and reduce GVHD, compared to alternative HCT methods. Administering Tregs with HSPCs before administering conventional T cells (Tcon) can, for example, promote graft-versus-tumor (GVT) retention and reduce GVHD, compared to alternative HCT methods. Administering Tregs with HSPCs before administering conventional T cells (Tcon) can, for example, promote graft-versus-tumor (GVT) retention and reduce the risk of GVHD, compared to alternative HCT methods. Without wishing to be bound by theory, for example, administration of Tregs can prevent GVHD and administration of Tcon can promote the GVT effect, in some embodiments, administration of Tregs reduces the risk of developing GVHD and administration of Tcon promotes the GVT effect, in comparison to alternative HCT methods.

[0049] There are various kinds of Tregs, including natural regulatory T cells (nTregs) and inducible regulatory T cells (iTregs), for example, TCRαβ+CD4+ regulatory T cells. nTregs are T cells that are produced in the thymus and delivered to the periphery as long-lived autoantigen-specific lymphoid cells. iTregs are mobilized from circulating lymphocytes and acquire regulatory properties under certain stimulation conditions in the periphery. nTregs and iTregs are CD4+CD25+, both can inhibit the proliferation of CD4+CD25- T cells in a dose-dependent manner, and both are anergic and do not proliferate upon TCR stimulation. In addition to being positive for CD4 and CD25, Tregs are positive for the transcription factor FOXP3, an intracellular marker. Tregs can be identified or selected based on various marker expression profiles. Non-limiting examples of marker expression profiles that can be used to select Tregs include: (1) CD4+CD25+CD127dim, (2) CD4+FOXP3+, (3) CD3+CD4+CD25+, (5) CD3+CD4+CD25+CD127dim, (6) CD3+CD4+CD25+CD127dimFOXP3+, (7) CD3+FOXP3+, (8) CD3+CD4+FOXP3+, (9) These include CD3+CD4+CD25+FOXP3+, (10)CD3+CD25+FOXP3+, (11)CD3+CD25+CD127dim, (12)CD4+CD25+, (13)CD4+CD25+CD127dimFOXP3+, (14)FOXP3+, CD4+FOXP3+, (15)CD4+CD25+FOXP3+, (16)CD25+FOXP3+, or (17)CD25+CD127dim.

[0050] Selection based on a particular expression profile can be achieved based on extracellular markers, without the need for cell permeabilization, for example by selection based on CD4+CD25+CD127dim.

[0051] In the methods of the disclosure, administering Tregs to a subject who has undergone HCT can, for example, serve to prevent graft rejection, serve to prevent GVHD, serve to alleviate GVHD, promote hematopoietic reconstitution, promote immune reconstitution, promote mixed chimerism, or a combination thereof.

[0052] In the methods of the present disclosure, invariant natural killer T cells (iNKT) can be administered to a subject. iNKT cells express the Vα24Jα18 TCR α chain in humans (referred to herein as "Vα24Jα18 + iNKT cells are a subclass of CD1d-restricted natural killer T (NKT) cells that express a highly conserved αβ-T cell receptor containing the αβ-T cell receptor (referred to as "α-galactosylceramide (GalCer)", PBS-57, PBS-44, or other natural or synthetic glycolipids. They can be found as tetramers, dendrimers, and other structures, Fc fusions, or any combination of these. Another method of identification is an antibody or combination of antibodies that specifically recognize the Vα24Jα18 region. Examples include Vα24 antibodies, Jα18 antibodies, or monoclonal antibody clone 6B11 that specifically binds to a unique region of the Vα24Jα18 TCR and can be used to identify iNKT cells. iNKT cells are characterized by their binding to CD3 + Vα24Jα18 + It could be.

[0053] In the method of the present disclosure, iNKT can be administered to a subject who has undergone HCT, for example, to promote engraftment, promote GVT, alleviate GVHD, reduce susceptibility to cancer recurrence, reduce susceptibility to infection, or combinations thereof.In some embodiments, iNKT promotes the activity of Treg.In some embodiments, iNKT promotes the activity of HSPC.

[0054] In the methods of the present disclosure, memory T cells (Tmem) may be administered to a subject. Tmem may refer to antigen experienced T cells, for example, expressing phenotypic markers CD45RO, TCRα, TCRβ, CD3, CD4 or CD8, CD95, and IL-2Rβ. Memory T cells can provide immunity and persist for long periods in a non-activated state. Memory T cells can rapidly acquire effector function upon re-challenge with antigen. A population of memory T cells may include any combination of the subclasses central memory T cells and effector memory T cells. In some embodiments, the Tmem is CD3+CD45RA-CD45RO+. In the methods of the present disclosure, Tmem administered to a subject who has undergone HCT may, for example, promote GVT, reduce GVHD, reduce susceptibility to cancer recurrence, reduce susceptibility to infection, or a combination thereof.

[0055] HLA typing and donor / host matching The present disclosure provides a method comprising administering a population of cells to a subject.The population of cells administered to the recipient can be derived from an allogeneic donor.Candidate and recipient can be HLA typed to determine whether they are compatible in any HLA allele.

[0056] Human leukocyte antigens (HLA), also commonly referred to as major histocompatibility complex (MHC) antigens, are protein molecules expressed on the surface of cells that can confer antigenic identity to cells. HLA / MHC antigens are target molecules that can be recognized by T cells and natural killer (NK) cells as originating from the same hematopoietic stem cell source as the immune effector cells ("self") or from a different hematopoietic cell source ("non-self"). HLA class I antigens (A, B, and C in humans) are expressed by the majority of cells, whereas HLA class II antigens (DR, DP, and DQ in humans) are expressed primarily on professional antigen-presenting cells. Both HLA classes have been implicated in GVHD.

[0057] HLA antigens are encoded by highly polymorphic genes, and various alleles exist for each HLA class I and II gene. The allele products differ in one or more amino acids in the α and / or β domain(s). Panels of specific antibodies or nucleic acid reagents are used to type an individual's HLA haplotype using white blood cells expressing class I and class II molecules. HLA alleles can be described at various levels of detail. Most nomenclature begins with the HLA and locus name, then a * and several (even) digits that identify the allele. The first two digits identify the group of alleles. Older typing methodologies often could not completely distinguish alleles and stopped at this level. The third to fourth digits identify synonymous alleles. The fifth to sixth digits indicate any synonymous mutations within the coding frame of the gene. The seventh and eighth digits distinguish mutations outside the coding region. A letter such as L, N, Q, or S may follow the allele designation to specify the expression level or other known non-genomic data for this. Thus, a fully described allele may be up to 9 digits long, not including the HLA-prefix and locus designation.

[0058] Three HLA genes are considered to be most important for HLA typing of potential transplant donors and recipients: HLA-A, HLA-B, and HLA-DR. Human subjects carry two copies of each, giving a total of six genes to be HLA typed.

[0059] HLA matched refers to a donor-recipient pair in which all six of the six HLA-A, HLA-B, and HLA-DR alleles are matched between the donor and the recipient. HLA incompatibility refers to a donor-recipient pair in which at least one HLA antigen is incompatible between the donor and the recipient. GVHD can occur even if the donor and the recipient are HLA matched, because the immune system can still recognize other differences between their tissues.

[0060] A set of HLA alleles inherited from one parent forms a haplotype. Some alleles and haplotypes are more common than others, and they are distributed at different frequencies in different racial and ethnic groups, so identifying the patient's haplotype can help predict the probability of finding a compatible donor and aid in the development of search strategies. HLA haploidentical refers to a donor-recipient pair in which one chromosome is matched between the donor and the recipient at least in HLA-A, HLA-B, and HLA-DR. A haploidentical pair may or may not match in other alleles, such as other HLA genes on other chromosomes, or additional histocompatibility loci on either chromosome. Such donors often occur in families, for example, parents may be haploidentical with their children, and siblings may be haploidentical.

[0061] The present disclosure provides methods that include administering to a subject a population of cells. The population of cells administered to the recipient can be derived from an allogeneic donor.

[0062] In some embodiments, the population of cells disclosed herein is administered to a recipient subject after HLA typing of the donor and the recipient, for example, by typing the HLA alleles HLA-A, HLA-B and HLA-DR in the donor and the recipient to determine whether the donor and the recipient are HLA-compatible or HLA-mismatched. The donor-recipient pair of the present disclosure can be HLA-compatible, HLA-mismatched, or haplotype-matched. The donor and the recipient can be relatives (e.g., parents, children, siblings, grandparents, grandchildren, aunts, uncles, or cousins). The donor and the recipient can be relatives and can be HLA-compatible, HLA-mismatched, or haplotype-matched. The donor and the recipient can be non-relatives.

[0063] Chimerism After administration of transplanted cells from donor to recipient, chimerism can be monitored in the recipient. Chimerism can refer to the mixing of donor cells and host cells in an individual who has undergone HCT. The risk of GVHD is significantly reduced in patients with mixed chimerism, but not complete chimerism, and for this reason it is desirable to achieve mixed chimerism. In addition, immune deficiency and infection are observed more frequently in complete chimerism versus mixed chimerism. In some embodiments, the methods provided herein allow human subjects to achieve mixed chimerism.

[0064] Subjects that show more than 95% donor cells in a given cell lineage at any time after transplantation can be referred to as having complete donor chimerism.Subjects that show more than 1% but less than 95% donor DNA in such analysis can be referred to as having mixed chimerism.Subjects that show mixed chimerism can be further classified according to the progression of chimerism, and improvement of mixed chimerism can include a continuous increase in the proportion of donor cells over at least 6 months.Stable mixed chimerism can include the fluctuation of the proportion of recipient cells over time without a complete loss of donor cells.

[0065] The determination of whether a subject is fully chimeric, mixed chimeric, or non-chimeric can be performed by analysis of hematopoietic cell samples from the graft recipient, for example, peripheral blood or bone marrow. The analysis can be performed by any convenient typing method. In some embodiments, the degree of chimerism between total mononuclear cells, T cells, B cells, CD56+ NK cells, and CD15+ neutrophils is monitored periodically using PCR with probes for microsatellite analysis. For example, commercially available kits can be used to quantify donor and host genetic material extracted from cells based on polymorphisms in short terminal repeat length. Automated readers provide the percentage of donor seed cells based on standard curves from artificial donor and host cell mixtures.

[0066] method Cell acquisition One or more populations of cells of the present disclosure can be obtained from a single donor, for example, from mobilized peripheral blood apheresis of a single donor. HSPCs, Tregs, Tcon, iNKT, Tmem, or any combination thereof can be obtained from a single donor.

[0067] One or more populations of cells of the present disclosure can be obtained from one donor, and one or more additional populations of cells of the present disclosure can be obtained from a second donor. One population of cells of the present disclosure can be obtained from a single donor, and a second population of cells of the present disclosure can be obtained from multiple donors.

[0068] The populations of the present disclosure can be obtained from multiple donors, for example, from mobilized peripheral blood apheresis of multiple donors. HSPCs can be obtained from multiple donors. Tregs can be obtained from multiple donors. Tcon can be obtained from multiple donors. iNKT can be obtained from multiple donors. Tmem can be obtained from multiple donors.

[0069] The donor and the recipient of the present disclosure can be allogeneic. The donor and the recipient of the present disclosure can be HLA-matched. The donor and the recipient of the present disclosure can be HLA-mismatched, for example, mismatched at 1, 2, 3, 4, 5, or 6 of the major HLA alleles. The donor and the recipient of the present disclosure can be haploidentical.

[0070] In some embodiments, the population of cells of the present disclosure can be obtained from whole blood. The population of cells of the present disclosure can be obtained from peripheral blood apheresis products, such as mobilized peripheral blood apheresis products. The population of cells of the present disclosure can be obtained from at least one apheresis product, two apheresis products, three apheresis products, four apheresis products, five apheresis products, six apheresis products, or more products.

[0071] In some embodiments, the population of cells of the present disclosure can be obtained from bone marrow, hi some embodiments, the population of cells of the present disclosure can be obtained from umbilical cord blood.

[0072] Cell treatment The population of cells of the present disclosure can be purified by selection from another population of cells, for example, peripheral blood or peripheral blood apheresis products. The selection method of the population of cells can include a method involving positive or negative selection of the population of cells of interest. The selection method of the population of cells can include affinity reagents, including but not limited to antibodies, full length antibodies, fragments of antibodies, naturally occurring antibodies, synthetic antibodies, engineered antibodies, full length affibodies, fragments of affibodies, full length affilins, fragments of affilins, full length anticalins, fragments of anticalins, full length avimers, fragments of avimers, full length DARPins, fragments of DARPins, full length finomers, fragments of finomers, full length Kunitz domain peptides, fragments of Kunitz domain peptides, full length monobodies, fragments of monobodies, peptides, or polyamino acids. In some embodiments, the affinity reagent is directly bound to the detection reagent and / or purification reagent. In some cases, the detection reagent and purification reagent are the same. In other cases, the detection reagent and purification reagent are different. For example, the detection reagent and / or purification reagent are fluorescent, magnetic, etc. In some cases, the detection and / or purification reagents are magnetic particles for column purification. For example, magnetic column purification may be performed using the Miltenyi system of columns, antibodies, buffers, preparation materials and reagents (CliniMACs).

[0073] The affinity reagents can include immunoaffinity reagents that utilize the binding specificity of an antibody or a fragment or derivative thereof to positively or negatively select for a population of cells of interest. The selection method for a population of cells can include affinity agents and columns, such as magnetic activated cell sorting (MACS) using specific antibodies and microbeads. The selection method for a population of cells can include fluorescence activated cell sorting (FACS), where a population of cells is sorted based on a staining profile using one or more fluorescently conjugated antibodies. The selection method for a population of cells can include physical adsorption, e.g., physical adsorption of T cells to a protein ligand such as a lectin.

[0074] A population of HSPCs of the present disclosure can be selected based on expression of CD34. For example, a population of HSPCs of the present disclosure can be selected using an anti-CD34 antibody as part of a magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS) system.

[0075] The number of HPSCs in a population of cells can be determined, for example, by quantifying CD34+ cells by flow cytometry. In some embodiments, dose calculations are adjusted based on a measure of cell viability, for example, viability determined by flow cytometry with propidium iodide or 7-AAD or trypan blue dye exclusion.

[0076] The population of cells comprising Tregs of the present disclosure can be selected based on the expression of markers including CD3, CD4, CD25, CD127, FOXP3, and combinations thereof.

[0077] The population of cells containing Tregs can be selected using magnetic activated cell sorting (MACS). The population of cells containing Tregs can be selected using fluorescence activated cell sorting (FACS). The population of cells containing Tregs can be selected using multiple procedures, such as multiple MACS selections, multiple FACS selections, or a combination of MACS and FACS selections. For example, a first selection can be performed for expression of CD25, for example, using MACS to isolate CD25+ cells from a hematopoietic cell sample. A second selection can be performed by contacting CD25+ cells with antibodies specific for CD4 and CD127, and isolating cells that are CD4+CD127dim using FACS.

[0078] The population of cells that comprises Tregs can be isolated from whole blood. The population of cells that comprises Tregs can be isolated from peripheral blood apheresis products. The population of cells that comprises Tregs can be isolated from a population of cells that have been previously enriched and / or depleted for one or more other cell types, for example, from a population of cells that have been depleted of CD34+ cells. In some embodiments, Tregs are isolated from the flow-through fraction of CD34+ MACS selection.

[0079] The number of Tregs in a population of cells can be determined, for example, by flow cytometry, and Tregs can be identified, for example, as CD4+CD25+CD127dim or CD4+FOXP3+. Dose calculations can be adjusted based on a measure of cell viability, for example, viability determined by flow cytometry using propidium iodide or 7-AAD or trypan blue dye exclusion.

[0080] The Tcon population can be sourced from peripheral blood. The Treg population can be sourced from a peripheral blood apheresis product.

[0081] In some embodiments, no selection step is performed and the Tcon population is sourced directly from an aliquot of peripheral blood or apheresis product. In some embodiments, the cell population can be enriched for Tcon by sorting based on the expression of various markers using, for example, MACS, FACS, or a combination thereof. The Tcon population can be enriched by sorting for CD3+ cells. The Tcon population can be enriched by sorting for CD4+ and CD8+ cells. The Tcon population can be enriched by negative selection, and non-Tcon cells are removed, for example, by MACS depletion of cells expressing CD34, CD19, CD25, or a combination thereof.

[0082] The number of Tcon present in the population can be quantified, for example, by quantifying CD3+ cells by flow cytometry. The number of CD3+ cells in an aliquot can be determined, and a volume containing an appropriate dose of CD3 cells can be administered to the recipient. Dose calculations can be adjusted based on a measure of cell viability, for example, viability determined by flow cytometry using propidium iodide or 7-AAD or trypan blue dye exclusion.

[0083] The apheresis product of the present disclosure can be divided into two parts, one part is used to provide Tcon cells and the other part is used to isolate and purify HSPCs and Tregs. In some embodiments, CD34+ cells are isolated and purified from the apheresis product, which creates a CD34-negative cell fraction, and then Tregs are isolated.

[0084] The iNKT population can be sourced from peripheral blood. The iNKT population can be sourced from a peripheral blood apheresis product.

[0085] The population of cells can be enriched for iNKT by sorting based on the expression of various markers, for example, using MACS, FACS, or a combination thereof. The population of iNKT can be enriched for iNKT by sorting based on the expression of various markers, for example, CD3 + Vα24Jα18 + Enrichment can be achieved by cell sorting.

[0086] The number of iNKTs present in a population can be determined, for example, by flow cytometry using CD3 + Vα24Jα18 + The CD3+ / CD4+ cells in the aliquots can be quantified by quantifying + Vα24Jα18 + The number of cells can be determined and a volume containing the appropriate dose of iNKT can be administered to the recipient. In some embodiments, the dose calculation is adjusted based on a measure of cell viability, such as viability determined by flow cytometry using propidium iodide or 7-AAD or trypan blue dye exclusion.

[0087] The population of Tmem can be sourced from peripheral blood.The population of Tmem can be sourced from a peripheral blood apheresis product.

[0088] A population of cells can be enriched for Tmem by sorting based on expression of various markers, for example, using MACS, FACS, or a combination thereof. A population of Tmem can be enriched, for example, by sorting for CD3+CD45RA-CD45RO+ cells.

[0089] The number of Tmem present in a population can be quantified, for example, by quantifying CD3+CD45RA-CD45RO+ cells by flow cytometry. The number of CD3+CD45RA-CD45RO+ cells in an aliquot can be determined, and a volume containing an appropriate dose of Tmem can be administered to the recipient. Dose calculations can be adjusted based on a measure of cell viability, for example, viability determined by flow cytometry using propidium iodide or 7-AAD or trypan blue dye exclusion.

[0090] Use of fresh or cryopreserved cells The populations of cells of the present disclosure can be administered fresh after isolation, or after cryopreservation and subsequent thawing.

[0091] Freshly isolated cells from a donor ("fresh cells") can be administered to a recipient subject. Fresh cells can be stored in a buffer, for example, CliniMACS PBS-EDTA buffer with 0.5% human serum albumin, or Plasma-Lyte-A at pH 7.4 supplemented with 2% human serum albumin. Fresh cells can be stored at low temperatures (e.g., 2-8 °C) but cannot be cryopreserved / frozen.

[0092] After obtaining the population of fresh cells from the donor, the fresh cells may be diluted with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 1 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 120, 150, 200, or 300 hours or more.

[0093] After obtaining a population of fresh cells from a donor, the fresh cells may be incubated for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 1 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 120, 150, 200, 300 hours or less.

[0094] The cells of the present disclosure can be cryopreserved.In some embodiments, cryopreservation can be beneficial for the methods disclosed herein.For example, cryopreservation of Tcon before subsequent thawing and administration to subject can alleviate GVHD.

[0095] Cryopreservation can include the addition of a preservative (e.g., DMSO) and slowly cooling the cells in a controlled-rate freezer to prevent osmotic cytotoxicity during ice crystal formation. Cryopreservation can include commercially available cryopreservation agents and materials, such as Cryobags and CryoStor® CS10.

[0096] Cryopreserved cells can be stored at low temperatures for periods ranging from a few hours to a few years. Cryopreserved cells can be stored at ultra-low temperatures, for example, -50°C, -60°C, -70°C, -80°C, -90°C, -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, -196°C or lower. Cryopreserved cells can be stored in a storage device containing liquid nitrogen.

[0097] The cells can be cryopreserved before or after other methods of the disclosure, e.g., before or after a sorting method, or before or after a characterization method such as determining cell viability or the concentration of a particular type of cell.

[0098] In some embodiments, whole blood can be cryopreserved. Whole blood can be cryopreserved without sorting or characterization. Whole blood can be cryopreserved after sorting but without characterization. Whole blood can be cryopreserved after characterization but without sorting. Whole blood can be cryopreserved after characterization and sorting. Whole blood can be cryopreserved after quantifying the cell types of the present disclosure. Whole blood can be cryopreserved after quantifying conventional T cells (Tcon, e.g., CD3+ cells). Whole blood can be cryopreserved after quantifying the viability of all cells or populations of cells of the present disclosure (e.g., conventional T cells).

[0099] The peripheral blood apheresis product of the present disclosure can be cryopreserved. The peripheral blood apheresis product can be cryopreserved without sorting or characterization. The peripheral blood apheresis product can be cryopreserved after sorting but without characterization. The peripheral blood apheresis product can be cryopreserved after characterization but without sorting. The peripheral blood apheresis product can be cryopreserved after characterization and sorting. The peripheral blood apheresis product can be cryopreserved after quantifying the cell types of the present disclosure. The peripheral blood apheresis product can be cryopreserved after quantifying conventional T cells (Tcon, e.g., CD3+ cells). The peripheral blood apheresis product can be cryopreserved after quantifying the viability of all cells or populations of cells of the present disclosure (e.g., conventional T cells).

[0100] A population of cells that has been sorted or selected from another population of cells can be cryopreserved, for example, a population of HSPCs, Tregs, Tcon, iNKT, or Tmem can be cryopreserved.

[0101] The cells of the present disclosure can be cryopreserved for any amount of time. The cells of the present disclosure can be cryopreserved for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 11 The cells may be frozen for more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours or longer.

[0102] The cells of the present disclosure may be thawed and administered to a subject for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 12 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours or less.

[0103] The cells of the present disclosure may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 12 The cells may be frozen for greater than 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 days or more.

[0104] The cells of the present disclosure may be thawed and administered to a subject for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 12 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 days or less.

[0105] Cell administration Sequence and timing of administration Disclosed herein are methods for enhancing allogeneic hematopoietic stem cell transplantation comprising administering to a subject a population of cells.

[0106] In some embodiments, a population of hematopoietic stem and progenitor cells (HSPCs), a population of cells comprising regulatory T cells (Treg), and a population of conventional T cells (Tcon) are administered to a subject.

[0107] The population of HSPCs and the population of cells comprising Tregs can be administered at the same or similar time or at different times.

[0108] The population of HSPCs and the population of cells comprising Tregs can be administered up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours apart.

[0109] The population of Tcon may be administered to the subject after the population of HSPCs.

[0110] The Tcon population is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, The compound may be administered to the subject more than 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0111] In some embodiments, the population of Tcon is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0112] The Tcon population was approximately 6–96, 12–84, 12–72, 12–66, 12–60, 12–54, 12–48, 12–42, 12–36, 12–30, 12–24, 12–18, 18–72, 18–66, 18–60, 18–54, 18–48, 18–42, 18–36, 18–30, 18–24, 24–72, 24–66, 24–60, 24–54, 24–48, 24–42, 24–36, 24–30, 24–24, 24–26, 24–28, 24–36, 24–30 ... The subject may be administered within 30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0113] The population of Tcon may be administered to the subject after the population of cells containing Treg.

[0114] The population of Tcon is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 ...9, The compound may be administered to the subject more than 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0115] In some embodiments, the population of Tcon is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0116] The Tcon population is, for example, approximately 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, The subject may be administered 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0117] The population of Tcon may be administered to the subject after the population of HSPCs and the population of cells comprising Tregs.

[0118] The Tcon population may be, for example, at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 110, 111, 112, 113, 123, 124, 125, 126, 127, 128, 130, The compound may be administered to the subject more than 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0119] In some embodiments, the population of Tcon is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0120] The Tcon population, for example, is approximately 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42 , 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later, the subject may be administered the

[0121] In some embodiments, a population of hematopoietic stem and progenitor cells (HSPCs), a population of cells including regulatory T cells (Tregs), a population of conventional T cells (Tcon), and a population of invariant natural killer T cells (iNKTs) are administered to a subject.

[0122] The population of iNKTs can be administered to the subject at the same time or at a similar time as the population of HSPCs. In some embodiments, the population of iNKTs is administered to the subject after the population of HSPCs.

[0123] The iNKT population is at least approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, The compound may be administered to the subject more than 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0124] In some embodiments, the population of iNKT is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0125] The iNKT population, for example, is approximately 6–96, 12–84, 12–72, 12–66, 12–60, 12–54, 12–48, 12–42, 12–36, 12–30, 12–24, 12–18, 18–72, 18–66, 18–60, 18–54, 18–48, 18–42, 18–36, 18–30, 18–24, 24–72, 24–66, 24–60, 24–54, 24–48, 24–42, 24–36, The subject may be administered 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0126] The population of iNKT cells can be administered to the subject at the same time or at the same time as the population of cells comprising Tregs. In some embodiments, the population of iNKT cells is administered to the subject after the population of cells comprising Tregs.

[0127] The iNKT population is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, The compound may be administered to the subject 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0128] In some embodiments, the population of iNKT cells is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 104, 105, 106, 107, 108, 109, 109, 110, 111, 112, 113 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0129] The iNKT population, for example, is approximately 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 24-24, 24-36, 24-42, 24-42, 24-54, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-36, 24-42 ...42, 24-42, 24-42, 24-42, 24-42, 24-42, 24-42, 24- The subject may be administered 6, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0130] In some embodiments, a population of hematopoietic stem and progenitor cells (HSPCs), a population of cells including regulatory T cells (Tregs), a population of conventional T cells (Tcon), and a population of memory T cells (Tmem) are administered to a subject.

[0131] The population of Tmems may be administered to the subject at the same time or at a similar time as the population of HSPCs, hi some embodiments, the population of Tmems is administered to the subject after the population of HSPCs.

[0132] The Tmem population is at least approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, The compound may be administered to the subject more than 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0133] In some embodiments, the population of Tmem is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0134] The Tmem population is approximately 6–96, 12–84, 12–72, 12–66, 12–60, 12–54, 12–48, 12–42, 12–36, 12–30, 12–24, 12–18, 18–72, 18–66, 18–60, 18–54, 18–48, 18–42, 18–36, 18–30, 18–24, 24–72, 24–66, 24–60, 24–54, 24–48, 24–42, 24–36, 24–30, 24–24, 24–28, 24–36, 24–30, 24–28, 24–30, 24–30, 24–40, 24–42, 24–40 ... The subject may be administered within 30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0135] The population of Tmems may be administered to the subject at the same time or at the same time as the population of cells comprising Tregs, hi some embodiments, the population of Tmems is administered to the subject after the population of cells comprising Tregs.

[0136] The Tmem population is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 ...9, The compound may be administered to the subject more than 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later.

[0137] In some embodiments, the population of Tmem is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 110, 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours later, the subject is administered

[0138] The Tmem population is approximately 6–96, 12–84, 12–72, 12–66, 12–60, 12–54, 12–48, 12–42, 12–36, 12–30, 12–24, 12–18, 18–72, 18–66, 18–60, 18–54, 18–48, 18–42, 18–36, 18–30, 18–24, 24–72, 24–66, 24–60, 24–54, 24–48, 24–42, 24–36, The subject may be administered 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours later.

[0139] dose HSPC The population of HPSCs administered to the subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6、1.1×10 6 、1.2×10 6 、1.3×10 6 、1.4×10 6 、1.5×10 6 、1.6×10 6 、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×106 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , 9.5×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of more than one or more cells can be administered.

[0140] In some embodiments, the population of HPSCs administered to a subject is at most about 1×10 HPSCs per kilogram of recipient body weight. 4 , 1×10 5、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、1.1×10 6 、1.2×10 6 、1.3×10 6 、1.4×10 6 、1.5×10 6 、1.6×10 6 、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6, 5.6×10 6 , 5.7×10 6 , 5.8×10 6 , 5.9×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , 9.5×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of one or fewer cells can be administered.

[0141] The population of HPSCs administered to a subject may be, for example, at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6 , 4.1×10 6 , 4.2×10 6 , 4.3×10 6 , 4.4×10 6 , 4.5×10 6 , 4.6×10 6 , 4.7×10 6 , 4.8×10 6 , 4.9×10 6 , 5×106 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8 、5.5×10 8 、6×10 8 、6.5×10 8 、7×10 8 、7.5×10 8 、8×10 8 、8.5×108 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of one or more CD34+ cells may be administered.

[0142] The population of HPSCs administered to the subject will be up to approximately 1 × 10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6 , 4.1×10 6 , 4.2×10 6 , 4.3×10 6 , 4.4×10 6 , 4.5×106 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8 、5.5×10 8 、6×108 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 A dose of one or fewer CD34+ cells may be administered.

[0143] For example, the population of HPSCs administered to a subject may be 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~9×10 6 , 5×10 5 ~8×10 6 , 5×10 5 ~7×10 6 , 5×10 5 ~6×10 6 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1.5×10 7 , 1×10 6 ~1×10 7 , 1×10 6 ~9×10 6 , 1×10 6 ~8×10 6 , 1×10 6 ~7×10 6 , 1×106 ~6×10 6 、1×10 6 ~5×10 6 、1×10 6 ~4×10 6 、1×10 6 ~3×10 6 、1×10 6 ~2×10 6 、1.5×10 6 ~1.5×10 7 、1.5×10 6 ~1×10 7 、1.5×10 6 ~9×10 6 、1.5×10 6 ~8×10 6 、1.5×10 6 ~7×10 6 、1.5×10 6 ~6×10 6 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×10 6 ~3×10 6 、1.5×10 6 ~2×10 6 、2×10 6 ~1.5×10 7 、2×10 6 ~1×10 7 、2×10 6 ~9×10 6 、2×10 6 ~8×10 6 、2×10 6 ~7×10 6 、2×10 6 ~6×10 6 、2×10 6 ~5×10 6 、2×10 6 ~4×10 6 、2×10 6 ~3×10 6 、2.5×10 6 ~1.5×10 7 、2.5×10 6 ~1×10 7 、2.5×10 6 ~9×10 6, 2.5×10 6 ~8×10 6 , 2.5×10 6 ~7×10 6 , 2.5×10 6 ~6×10 6 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The antibody can be administered in doses of cells.

[0144] The population of HPSCs administered to a subject may be, for example, 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~9×10 6 , 5×10 5 ~8×10 6 , 5×10 5 ~7×10 6 , 5×10 5 ~6×10 6 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1.5×10 7 , 1×10 6 ~1×10 7 , 1×10 6 ~9×10 6 , 1×10 6~8×10 6 、1×10 6 ~7×10 6 、1×10 6 ~6×10 6 、1×10 6 ~5×10 6 、1×10 6 ~4×10 6 、1×10 6 ~3×10 6 、1×10 6 ~2×10 6 、1.5×10 6 ~1.5×10 7 、1.5×10 6 ~1×10 7 、1.5×10 6 ~9×10 6 、1.5×10 6 ~8×10 6 、1.5×10 6 ~7×10 6 、1.5×10 6 ~6×10 6 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×10 6 ~3×10 6 、1.5×10 6 ~2×10 6 、2×10 6 ~1.5×10 7 、2×10 6 ~1×10 7 、2×10 6 ~9×10 6 、2×10 6 ~8×10 6 、2×10 6 ~7×10 6 、2×10 6 ~6×10 6 、2×10 6 ~5×10 6 、2×10 6 ~4×10 6 、2×10 6 ~3×10 6 、2.5×10 6 ~1.5×10 7 、2.5×10 6~1×10 7 , 2.5×10 6 ~9×10 6 , 2.5×10 6 ~8×10 6 , 2.5×10 6 ~7×10 6 , 2.5×10 6 ~6×10 6 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The patient may be administered a dose of CD34+ cells.

[0145] The population of HSPCs of the present disclosure can have a defined purity level for CD34+ cells.

[0146] For example, a population of HSPCs disclosed herein may comprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67% or more of CD45+ cells as a percentage of total cells, nucleated cells, or CD45+ cells. , 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more CD34+ cells.

[0147] In some embodiments, a population of HSPCs of the disclosure comprises up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, or 70% of total cells, nucleated cells, or CD45+ cells. Contains 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less CD34+ cells.

[0148] The population of HSPCs of the present disclosure can have a defined level of contaminating CD3+ cells.

[0149] For example, at least about 1 × 10 per kilogram of recipient body weight. 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 More than 1,000,000, or more CD3+ cells are present in the population of HSPCs of the present disclosure.

[0150] In some embodiments, up to about 1×10 per kilogram of recipient body weight. 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 or fewer CD3+ cells are present in the population of HSPCs of the present disclosure.

[0151] A population of HSPCs of the present disclosure may comprise at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 10% of total cells, nucleated cells, or CD45+ cells. The patient may contain more than 8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more CD3+ cells.

[0152] In some embodiments, a population of HSPCs of the disclosure comprises at most about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 0.9%, 0.1%, 0.4%, 0.5%, 0.7%, 0.8%, 0.9%, 0.1%, 0.5%, 0.8%, 0.9%, 0.1%, 0.6%, 0.7%, 0.8%, 0.9%, 0.1 ... 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or less of CD3+ cells.

[0153] Treg The population of Treg-containing cells administered to the subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 2×10 5、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、1.1×10 6 、1.2×10 6 、1.3×10 6 、1.4×10 6 、1.5×10 6 、1.6×10 6 、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8 、5.5×10 8 、6×10 8 、6.5×10 8 、7×10 8 、7.5×10 8 、8×10 8 、8.5×10 8 、9×10 8 、9.5×10 8 、1×10 9Doses of more than one or more cells can be administered.

[0154] In some embodiments, the population of cells comprising Tregs administered to a subject is up to about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6 , 4.1×10 6 , 4.2×10 6 , 4.3×10 6 , 4.4×10 6 , 4.5×106 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8 、5.5×10 8 、6×108 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of one or fewer cells can be administered.

[0155] The population of cells containing Tregs administered to a subject may be, for example, at least about 1×10 per kilogram of recipient body weight. 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×106 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8, 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of more than one or more Tregs may be administered, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0156] The population of cells, including Tregs, administered to a subject may be, for example, up to about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7, 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 The present invention can be administered in doses of 10 or less Tregs, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0157] The population of Treg-containing cells administered to the subject is 1 × 10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~1×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1×10 7 , 1×10 6 ~5×10 6 , 1×10 6 ~4×10 6 , 1×10 6 ~3×10 6 , 1×10 6 ~2×10 6 , 1×10 6 ~1.5×10 6 , 1.5×10 6 ~1×10 7 , 1.5×10 6 ~5×10 6 , 1.5×10 6 ~4×10 6 , 1.5×10 6 ~3×10 6 , 1.5×10 6 ~2×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×106 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~2×10 6 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The antibody can be administered in doses of cells.

[0158] In some embodiments, the population of cells comprising Tregs administered to a subject is about 1×10 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~1×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1×10 7 , 1×10 6 ~5×10 6 , 1×10 6 ~4×10 6 , 1×10 6 ~3×10 6 , 1×10 6 ~2×10 6 , 1×10 6 ~1.5×10 6 , 1.5×10 6 ~1×10 7 , 1.5×10 6 ~5×10 6, 1.5×10 6 ~4×10 6 , 1.5×10 6 ~3×10 6 , 1.5×10 6 ~2×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~2×10 6 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The therapeutic agent may be administered in a dose of Tregs, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0159] A population of cells comprising Tregs of the present disclosure can comprise, for example, greater than at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more FOXP3+ cells as a percentage of total cells, nucleated cells, or CD45+ cells.

[0160] In some embodiments, a population of cells comprising Tregs of the present disclosure comprises up to about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less FOXP3+ cells as a percentage of total cells, nucleated cells, or CD45+ cells.

[0161] A population of cells comprising Tregs of the present disclosure may comprise, for example, at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... The patient may contain greater than 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more CD4+CD25+CD127dim cells.

[0162] In some embodiments, the population of cells comprising Tregs of the present disclosure is at most about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%. Contains 1%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less CD4+CD25+CD127dim cells.

[0163] For example, a population of cells comprising Tregs of the present disclosure can comprise greater than at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Tregs as a percentage of total cells, nucleated cells, or CD45+ cells. Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0164] In some embodiments, a population of cells comprising Tregs of the present disclosure comprises up to about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less Tregs as a percentage of total cells, nucleated cells, or CD45+ cells. Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+CD4+CD25+CD127dimFOXP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0165] The population of cells comprising Tregs of the present disclosure may be expressed as a percentage of total cells, nucleated cells, or CD45+ cells, for example, 50%-100%, 60%-100%, 70%-100%, 75%-100%, 80%-100%, 81%-100%, 82%-100%, 83%-100%, 84%-100%, 84%-100%, 86%-100%, 87%-100%, 88%-100%, 89%-100%, 90%-91%, 92%-100%, 93%-100%, 94%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, 99%-100%, 99.5%~100%, 50%~99%, 60%~99%, 70%~99%, 80%~99%, 81%~99%, 82%~99%, 83%~99%, 84%~99%, 85%~99%, 86%~99%, 87%~99%, 88%~99%, 89%~99%, 90%~99%, 91%~99%, 92%~99%, 94%~99%, 95%~99%, 96%~97%, 98%~99%, 50%~98%, 60%~98%, 70%~98%, 80% ~98%, 81%~98%, 82%~98%, 83%~98%, 84%~98%, 85%~98%, 86%~98%, 87%~98%, 88%~98%, 89%~98%, 90%~98%, 91%~98%, 92%~98%, 94%~98%, 95%~98%, 96%~97%, 98%~98%, 50%~97%, 60%~97%, 70%~97%, 80%~97%, 81%~97%, 82%~97%, 83%~97%, 84%~97 %, 85%~97%, 86%~97%, 87%~97%, 88%~97%, 89%~97%, 90%~97%, 91%~97%, 92%~97%, 94%~97%, 95%~97%, 96%~97%, 50%~96%, 60%~96%, 70%~96%, 80%~96%, 81%~96%, 82%~96%, 83%~96%, 84%~96%, 85%~96%, 86%~96%, 87%~96%, 88%~96%, 89%~96%, 9 The culture may contain 0%-96%, 91%-96%, 92%-96%, 94%-96%, 95%-96%, 50%-95%, 60%-95%, 70%-95%, 80%-95%, 81%-95%, 82%-95%, 83%-95%, 84%-95%, 85%-95%, 86%-95%, 87%-95%, 88%-95%, 89%-95%, 90%-95%, 91%-95%, 92%-95%, or 94%-95% FOXP3+ cells.

[0166] The population of cells containing Tregs of the present disclosure may be 50% to 100%, 60% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84% to 100%, 84% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 91%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100% as a percentage of total cells, nucleated cells, or CD45+ cells. , 99.5%~100%, 50%~99%, 60%~99%, 70%~99%, 80%~99%, 81%~99%, 82%~99%, 83%~99%, 84%~99%, 85%~99%, 86%~99%, 87%~99%, 88%~99%, 89%~99%, 90%~99%, 91%~99%, 92%~99%, 94%~99%, 95%~99%, 96%~97%, 98%~99%, 50%~98%, 60%~98%, 70%~98%, 80%~98%, 81%~98%, 82%~98%, 83%~98%, 84%~98%, 85%~98%, 86%~9 8%, 87%~98%, 88%~98%, 89%~98%, 90%~98%, 91%~98%, 92%~98%, 94%~98%, 95%~98%, 96%~97%, 98%~98%, 50%~97%, 60%~97%, 70%~97%, 80%~97%, 81%~97%, 8 2%~97%, 83%~97%, 84%~97%, 85%~97%, 86%~97%, 87%~97%, 88%~97%, 89%~97%, 90%~97%, 91%~97%, 92%~97%, 94%~97%, 95%~97%, 96%~97%, 50%~96%, 60%~96 %, 70%~96%, 80%~96%, 81%~96%, 82%~96%, 83%~96%, 84%~96%, 85%~96%, 86%~96%, 87%~96%, 88%~96%, 89%~96%, 90%~96%, 91%~96%, 92%~96%, 94%~96%, 95% ~96%, 50%~95%, 60%~95%, 70%~95%, 80%~95%, 81%~95%, 82%~95%, 83%~95%, 84%~95%, 85%~95%, 86%~95%, 87%~95%, 88%~95%, 89%~95%, 90%~95%, 91%~95%,The Tregs may comprise 92%-95%, or 94%-95% Tregs, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+CD4+CD25+CD127dim, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+CD127dim.

[0167] The population of cells comprising Tregs of the present disclosure can have a defined level of contaminating non-Treg cells.

[0168] For example, at least about 1 × 10 per kilogram of recipient body weight. 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5More than one, or more, non-Treg cells can be present in a population of Treg-containing cells of the present disclosure, where the non-Treg cells are FOXP3- or CD127+ / bright.

[0169] In some embodiments, up to about 1×10 per kilogram of recipient body weight. 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , or fewer, non-Treg cells can be present in a population of Treg-containing cells of the present disclosure, where the non-Treg cells are FOXP3- or CD127+ / bright.

[0170] A population of cells comprising Tregs of the present disclosure may comprise, for example, at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or 2% of total cells, nucleated cells, or CD45+ cells. The tumor may comprise greater than 1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more non-Treg cells, wherein the non-Treg cells are FOXP3- or CD127+ / bright.

[0171] In some embodiments, a population of cells comprising Tregs of the present disclosure comprises, for example, at most about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or less of non-Treg cells, and non-Treg cells are FOXP3- or CD127+ / bright.

[0172] Tcon The population of Tcon administered to a subject is, for example, at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6 , 4.1×10 6 , 4.2×10 6 , 4.3×10 6 , 4.4×10 6 , 4.5×10 6 , 4.6×10 6 , 4.7×10 6 , 4.8×10 6、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8 、5.5×10 8 、6×10 8 、6.5×10 8 、7×10 8 、7.5×10 8 、8×108 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of more than one or more cells can be administered.

[0173] In some embodiments, the population of Tcon administered to a subject is up to about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×108 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 The antibody may be administered in doses of one or fewer cells.

[0174] The mass of Tcon administered to the subject is at least about 1×10 per kilogram of recipient body weight. 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×107 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 More than one or more doses of Tcon may be administered, where Tcon is defined as CD3+.

[0175] The mass of Tcon administered to the subject is up to approximately 1 × 10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×107 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 The antibody may be administered at a dose of 100,000,000, or less than 100,000 Tcon, where Tcon is defined as CD3+.

[0176] In some embodiments, the population of Tcon administered to a subject is 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~1×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1×10 7 , 1×106 ~5×10 6 , 1×10 6 ~4×10 6 , 1×10 6 ~3×10 6 , 1×10 6 ~2×10 6 , 1×10 6 ~1.5×10 6 , 1.5×10 6 ~1×10 7 , 1.5×10 6 ~5×10 6 , 1.5×10 6 ~4×10 6 , 1.5×10 6 ~3×10 6 , 1.5×10 6 ~2×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~1×10 7 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2×10 6 ~2×10 6 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The antibody can be administered in doses of cells.

[0177] In some embodiments, the population of Tcon administered to a subject is 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5~1×10 8 、1×10 5 ~1×10 7 、5×10 5 ~1×10 7 、5×10 5 ~5×10 6 、5×10 5 ~4×10 6 、5×10 5 ~3×10 6 、5×10 5 ~2×10 6 、5×10 5 ~1×10 6 、1×10 6 ~1×10 7 、1×10 6 ~5×10 6 、1×10 6 ~4×10 6 、1×10 6 ~3×10 6 、1×10 6 ~2×10 6 、1×10 6 ~1.5×10 6 、1.5×10 6 ~1×10 7 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×10 6 ~3×10 6 、1.5×10 6 ~2×10 6 、2×10 6 ~1×10 7 、2×10 6 ~5×10 6 、2×10 6 ~4×10 6 、2×10 6 ~3×10 6 、2×10 6 ~1×10 7 、2×10 6 ~5×10 6 、2×10 6 ~4×10 6 、2×10 6 ~3×10 6 、2×10 6 ~2×10 6, 2.5×10 6 ~1×10 7 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 It can be administered in a volume of Tcon, where Tcon is defined as CD3+.

[0178] The Tcon population of the present disclosure is at least about 5%, 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, The antibody may contain greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more CD3+ cells.

[0179] The Tcon population of the present disclosure may be up to about 5%, 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, It may contain 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less CD3+ cells.

[0180] For example, a population of Tcon of the present disclosure may be present as a percentage of total cells, nucleated cells, or CD45+ cells, and may be present in a range of about 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-80%, 30%-90%, 30%-100%, 30%-90%, 30%-80%, 30%-10 ... The antibody may contain 0%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% CD3+ cells.

[0181] iNKT The population of iNKT administered to the subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×106 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×107 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of more than one or more cells can be administered.

[0182] The iNKT population administered to the subject is approximately 1 × 10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×106 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×107 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 The antibody may be administered in doses of one or fewer cells.

[0183] In some embodiments, the population of iNKT administered to a subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×106 , 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 More than or equal to CD3 + Vα24Jα18 + The dose may be in the form of a cell.

[0184] In some embodiments, the population of iNKT administered to a subject is up to about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×105 、1×10 6 、1.1×10 6 、1.2×10 6 、1.3×10 6 、1.4×10 6 、1.5×10 6 、1.6×10 6 、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 、2.1×10 6 、2.2×10 6 、2.3×10 6 、2.4×10 6 、2.5×10 6 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6, 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , 9.5×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 or less CD3 + Vα24Jα18 + The dose may be in the form of a cell.

[0185] The population of iNKT administered to a subject may be, for example, 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~9×10 6 , 5×10 5 ~8×10 6 , 5×10 5 ~7×10 6 , 5×10 5 ~6×10 6 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1.5×10 7 , 1×10 6 ~1×10 7 , 1×10 6 ~9×10 6 , 1×10 6 ~8×10 6 , 1×10 6 ~7×10 6 , 1×10 6 ~6×10 6 , 1×10 6 ~5×10 6 , 1×10 6 ~4×10 6 , 1×10 6 ~3×10 6 , 1×10 6 ~2×10 6 , 1.5×10 6 ~1.5×10 7 , 1.5×10 6~1×10 7 , 1.5×10 6 ~9×10 6 , 1.5×10 6 ~8×10 6 , 1.5×10 6 ~7×10 6 , 1.5×10 6 ~6×10 6 , 1.5×10 6 ~5×10 6 , 1.5×10 6 ~4×10 6 , 1.5×10 6 ~3×10 6 , 1.5×10 6 ~2×10 6 , 2×10 6 ~1.5×10 7 , 2×10 6 ~1×10 7 , 2×10 6 ~9×10 6 , 2×10 6 ~8×10 6 , 2×10 6 ~7×10 6 , 2×10 6 ~6×10 6 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2.5×10 6 ~1.5×10 7 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~9×10 6 , 2.5×10 6 ~8×10 6 , 2.5×10 6 ~7×10 6 , 2.5×10 6 ~6×10 6 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6The antibody can be administered in doses of cells.

[0186] The iNKT population administered to the subject was 1 × 10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~9×10 6 , 5×10 5 ~8×10 6 , 5×10 5 ~7×10 6 , 5×10 5 ~6×10 6 , 5×10 5 ~5×10 6 , 5×10 5 ~4×10 6 , 5×10 5 ~3×10 6 , 5×10 5 ~2×10 6 , 5×10 5 ~1×10 6 , 1×10 6 ~1.5×10 7 , 1×10 6 ~1×10 7 , 1×10 6 ~9×10 6 , 1×10 6 ~8×10 6 , 1×10 6 ~7×10 6 , 1×10 6 ~6×10 6 , 1×10 6 ~5×10 6 , 1×10 6 ~4×10 6 , 1×10 6 ~3×10 6 , 1×10 6 ~2×10 6、1.5×10 6 ~1.5×10 7 、1.5×10 6 ~1×10 7 、1.5×10 6 ~9×10 6 、1.5×10 6 ~8×10 6 、1.5×10 6 ~7×10 6 、1.5×10 6 ~6×10 6 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×10 6 ~3×10 6 、1.5×10 6 ~2×10 6 、2×10 6 ~1.5×10 7 、2×10 6 ~1×10 7 、2×10 6 ~9×10 6 、2×10 6 ~8×10 6 、2×10 6 ~7×10 6 、2×10 6 ~6×10 6 、2×10 6 ~5×10 6 、2×10 6 ~4×10 6 、2×10 6 ~3×10 6 、2.5×10 6 ~1.5×10 7 、2.5×10 6 ~1×10 7 、2.5×10 6 ~9×10 6 、2.5×10 6 ~8×10 6 、2.5×10 6 ~7×10 6 、2.5×10 6 ~6×10 6 、2.5×10 6 ~5×10 6 、2.5×10 6 ~4×106 , or 2.5 × 10 6 ~3×10 6 CD3 + Vα24Jα18 + The dose may be in the form of a cell.

[0187] Tmem The population of Tmem administered to the subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6 , 3.9×10 6 , 4×10 6 , 4.1×10 6 , 4.2×10 6 , 4.3×10 6、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、3×10 8 、3.5×10 8 、4×10 7 、4.5×10 8 、5×10 8, 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 Doses of more than one or more cells can be administered.

[0188] The population of Tmem administered to subjects is up to approximately 1 × 10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×10 6 , 3.4×10 6 , 3.5×10 6 , 3.6×10 6 , 3.7×10 6 , 3.8×10 6、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7 、9.5×10 7 、1×10 8 、1×10 8 、1.5×10 8 、2×10 8 、2.5×108 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 The antibody may be administered in doses of one or fewer cells.

[0189] For example, the population of Tmem administered to a subject is at least about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×10 6 , 2.6×10 6 , 2.7×10 6 , 2.8×10 6 , 2.9×10 6 , 3×10 6 , 3.1×10 6 , 3.2×10 6 , 3.3×106 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、5.5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7 、9×10 7, 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 More than or equal to CD3 + CD45RA - CD45RO + The dose may be in the form of a cell.

[0190] In some embodiments, the population of Tmem administered to a subject is at most about 1×10 per kilogram of recipient body weight. 4 , 1×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 , 2.1×10 6 , 2.2×10 6 , 2.3×10 6 , 2.4×10 6 , 2.5×106 、2.6×10 6 、2.7×10 6 、2.8×10 6 、2.9×10 6 、3×10 6 、3.1×10 6 、3.2×10 6 、3.3×10 6 、3.4×10 6 、3.5×10 6 、3.6×10 6 、3.7×10 6 、3.8×10 6 、3.9×10 6 、4×10 6 、4.1×10 6 、4.2×10 6 、4.3×10 6 、4.4×10 6 、4.5×10 6 、4.6×10 6 、4.7×10 6 、4.8×10 6 、4.9×10 6 、5×10 6 、5.1×10 6 、5.2×10 6 、5.3×10 6 、5.4×10 6 、5.5×10 6 、5.6×10 6 、5.7×10 6 、5.8×10 6 、5.9×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×107 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1×10 8 , 1.5×10 8 , 2×10 8 , 2.5×10 8 , 3×10 8 , 3.5×10 8 , 4×10 7 , 4.5×10 8 , 5×10 8 , 5.5×10 8 , 6×10 8 , 6.5×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 8.5×10 8 , 9×10 8 , 9.5×10 8 , 1×10 9 or less CD3 + CD45RA - CD45RO + The dose may be in the form of a cell.

[0191] In some embodiments, the population of Tmem administered to a subject is about 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7 , 5×10 5 ~9×10 6 , 5×10 5~8×10 6 、5×10 5 ~7×10 6 、5×10 5 ~6×10 6 、5×10 5 ~5×10 6 、5×10 5 ~4×10 6 、5×10 5 ~3×10 6 、5×10 5 ~2×10 6 、5×10 5 ~1×10 6 、1×10 6 ~1.5×10 7 、1×10 6 ~1×10 7 、1×10 6 ~9×10 6 、1×10 6 ~8×10 6 、1×10 6 ~7×10 6 、1×10 6 ~6×10 6 、1×10 6 ~5×10 6 、1×10 6 ~4×10 6 、1×10 6 ~3×10 6 、1×10 6 ~2×10 6 、1.5×10 6 ~1.5×10 7 、1.5×10 6 ~1×10 7 、1.5×10 6 ~9×10 6 、1.5×10 6 ~8×10 6 、1.5×10 6 ~7×10 6 、1.5×10 6 ~6×10 6 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×10 6 ~3×10 6 、1.5×10 6 ~2×106 , 2×10 6 ~1.5×10 7 , 2×10 6 ~1×10 7 , 2×10 6 ~9×10 6 , 2×10 6 ~8×10 6 , 2×10 6 ~7×10 6 , 2×10 6 ~6×10 6 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2.5×10 6 ~1.5×10 7 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~9×10 6 , 2.5×10 6 ~8×10 6 , 2.5×10 6 ~7×10 6 , 2.5×10 6 ~6×10 6 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 The antibody can be administered in doses of cells.

[0192] The population of Tmem administered to a subject may be, for example, 1×10 per kilogram of recipient body weight. 4 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~2×10 7 , 5×10 5 ~2×10 7 , 5×10 5 ~1.5×10 7 , 5×10 5 ~1×10 7、5×10 5 ~9×10 6 、5×10 5 ~8×10 6 、5×10 5 ~7×10 6 、5×10 5 ~6×10 6 、5×10 5 ~5×10 6 、5×10 5 ~4×10 6 、5×10 5 ~3×10 6 、5×10 5 ~2×10 6 、5×10 5 ~1×10 6 、1×10 6 ~1.5×10 7 、1×10 6 ~1×10 7 、1×10 6 ~9×10 6 、1×10 6 ~8×10 6 、1×10 6 ~7×10 6 、1×10 6 ~6×10 6 、1×10 6 ~5×10 6 、1×10 6 ~4×10 6 、1×10 6 ~3×10 6 、1×10 6 ~2×10 6 、1.5×10 6 ~1.5×10 7 、1.5×10 6 ~1×10 7 、1.5×10 6 ~9×10 6 、1.5×10 6 ~8×10 6 、1.5×10 6 ~7×10 6 、1.5×10 6 ~6×10 6 、1.5×10 6 ~5×10 6 、1.5×10 6 ~4×10 6 、1.5×106 ~3×10 6 , 1.5×10 6 ~2×10 6 , 2×10 6 ~1.5×10 7 , 2×10 6 ~1×10 7 , 2×10 6 ~9×10 6 , 2×10 6 ~8×10 6 , 2×10 6 ~7×10 6 , 2×10 6 ~6×10 6 , 2×10 6 ~5×10 6 , 2×10 6 ~4×10 6 , 2×10 6 ~3×10 6 , 2.5×10 6 ~1.5×10 7 , 2.5×10 6 ~1×10 7 , 2.5×10 6 ~9×10 6 , 2.5×10 6 ~8×10 6 , 2.5×10 6 ~7×10 6 , 2.5×10 6 ~6×10 6 , 2.5×10 6 ~5×10 6 , 2.5×10 6 ~4×10 6 , or 2.5 × 10 6 ~3×10 6 CD3 + CD45RA - CD45RO + The dose may be in the form of a cell.

[0193] Treg to Tcon ratio In the methods of the disclosure, the ratio of Tcon:Treg administered to a subject can be, for example, about 1:100, 1:50, 1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1.5:2, 1:1.5, 1:1, 1.5:1, 2:1, 2:1.5, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100:1.

[0194] Conditioning regimens, GVHD prophylaxis, and additional agents In some embodiments of the methods of the present disclosure, the subject may be treated with myeloablative conditioning prior to infusion of the populations of cells described herein.

[0195] In some embodiments, the methods of the disclosure may include administration of one or more immunosuppressants or other agents to a subject.

[0196] Immunosuppressants can be administered to subjects, for example, to prevent rejection of transplant by recipient immune system, or to prevent or reduce GVHD caused by transplant attacking recipient cells.Immunosuppressants include, for example, calcineurin inhibitors combined with binding proteins to inhibit calcineurin activity, and monoclonal antibodies.

[0197] Calcineurin inhibitors include, for example, tacrolimus, cyclosporine A, etc. Adjuvant agents can be combined with calcineurin inhibitors, including, for example, steroids, azathioprine, mycophenolate mofetil, and sirolimus. Monoclonal antibodies useful as immunosuppressants include, for example, antagonist antibodies (e.g., antibodies that antagonize IL-2R, such as basiliximab and daclizumab), and antibodies that deplete populations of immune cells by antibody-dependent cellular cytotoxicity (e.g., anti-CD3 antibodies for T cell depletion, such as muromonab-CD3).

[0198] The method of the present disclosure can further comprise administering an antitumor agent, or a pharmaceutically acceptable salt or prodrug thereof, to a subject in need thereof. Examples of antitumor agents include, but are not limited to, antitumor alkylating agents, antitumor metabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor organoplatinum compounds, antitumor camptothecin derivatives, antitumor tyrosine kinase inhibitors, monoclonal antibodies, interferons, biological response modifiers, and other agents with antitumor activity, or a pharmaceutically acceptable salt thereof.

[0199] Exemplary embodiments In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 a) a population of cells comprising regulatory T cells (Treg), said population of cells comprising Treg having not been cryopreserved prior to administration of said population of cells comprising Treg; and b) a population of conventional T cells (Tcon), said Tcon comprising CD3 + and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon) comprising one or more Tregs (Tcon), wherein the population of HSPCs and a population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon.

[0200] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 a) a population of cells comprising regulatory T cells (Treg), said population of cells comprising Treg having not been cryopreserved prior to administration of said population of cells comprising Treg; and b) a population of conventional T cells (Tcon), said Tcon comprising CD3 + and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon) comprising a Tcon (a Tcon), and d) a graft-versus-host disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon, and wherein the human subject does not develop graft-versus-host disease (GVHD) within 30 days (preferably within 1 year) after administration of the population of Tcon.

[0201] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 Tregs), and the population of cells containing Tregs is + cells, + a) a population of cells comprising regulatory T cells (Treg), wherein more than 50% (preferably more than 90%) of the cells are Treg, and said population of cells comprising Treg has not been cryopreserved prior to administration of said population of cells comprising Treg; and b) a population of conventional T cells (Tcon), wherein said Tcon is a CD3 + and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5~4×10 6 Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon), comprising one or more Tcon (Tcon), wherein the population of Tcon has been cryopreserved prior to administration of the population of Tcon, wherein the population of HSPCs and a population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon.

[0202] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 a) a population of cells comprising regulatory T cells (Treg), said population of cells comprising Treg having not been cryopreserved prior to administration of said population of cells comprising Treg; b) a population of conventional T cells (Tcon), said Tcon comprising CD3 + and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon), comprising administering to the human subject a population of conventional T cells (Tcon), wherein the population of Tcon is cryopreserved prior to administration of the population of Tcon, and d) a graft-versus-host disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to the population of Tcon (preferably 36-60 hours prior), and wherein the human subject does not develop graft-versus-host disease (GVHD) within 30 days (preferably within 1 year) after administration of the population of Tcon.

[0203] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 Tregs), and the population of cells containing Tregs is + cells, + a population of cells comprising regulatory T cells (Treg), wherein more than 50% (preferably more than 90%) of the cells are Treg, and said population of cells comprising Treg has not been cryopreserved prior to administration of said population of cells comprising Treg; c) a population of conventional T cells (Tcon), wherein said Tcon is a CD3 + and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7Less than Tcon (preferably 5×10 5 ~4×10 6 Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon) comprising a Tcon (a Tcon), and d) a graft-versus-host disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon, and wherein the human subject does not develop graft-versus-host disease (GVHD) within 30 days (preferably within 1 year) after administration of the population of Tcon.

[0204] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 Tregs), and the population of cells containing Tregs is + cells, + a) a population of cells comprising regulatory T cells (Treg), wherein more than 50% (preferably more than 90%) of the cells are Treg, and said population of cells comprising Treg has not been cryopreserved prior to administration of said population of cells comprising Treg; and b) a population of conventional T cells (Tcon), wherein said Tcon is a CD3 +and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6 Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon), the population of Tcon comprising a plurality of T cells (Tcon), the population of Tcon being cryopreserved prior to administration of the population of Tcon, wherein the population of HSPCs and a population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon, and the human subject does not develop graft-versus-host disease (GVHD) within 30 days (preferably within 1 year) after administration of the population of Tcon.

[0205] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 Tregs), and the population of cells containing Tregs is + cells, + a population of cells comprising regulatory T cells (Treg), wherein more than 50% (preferably more than 90%) of the cells are Treg, and said population of cells comprising Treg has not been cryopreserved prior to administration of said population of cells comprising Treg; c) a population of conventional T cells (Tcon), wherein said Tcon is a CD3+ and the population of Tcon is 1×10 per kilogram per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6 Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon), comprising administering to the human subject a population of conventional T cells (Tcon), wherein the population of Tcon has been cryopreserved prior to administration of the population of Tcon, and d) a graft-versus-host disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to (preferably 36-60 hours prior to) the population of Tcon.

[0206] In some embodiments, there is provided a method of treating a human subject in need thereof, comprising: a) administering to the human subject a population of hematopoietic stem and progenitor cells (HSPCs), the population of HSPCs being at least 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 HSPCs (preferably 5 × 10 5 ~2×10 7 HSPCs), wherein the hPSCs are CD34 + a) a population of hematopoietic stem and progenitor cells (HSPCs) which are: + CD25 + CD127 dim or CD4 + FOXP3 + and the population of cells comprising Tregs is 1×10 per kilogram of body weight of the human subject. 5 More than 5 × 10 Tregs 5 ~4×10 6 Tregs), and the population of cells containing Tregs is + cells, + a population of cells comprising regulatory T cells (Treg), wherein more than 50% (preferably more than 90%) of the cells are Treg, and said population of cells comprising Treg has not been cryopreserved prior to administration of said population of cells comprising Treg; c) a population of conventional T cells (Tcon), wherein said Tcon is a CD3+ and the population of Tcon is 1×10 per kilogram of body weight of the human subject. 7 Less than Tcon (preferably 5×10 5 ~4×10 6 Disclosed herein is a method comprising administering to the human subject a population of conventional T cells (Tcon), comprising administering to the human subject a population of conventional T cells (Tcon), wherein the population of Tcon is cryopreserved prior to administration of the population of Tcon, and d) a graft-versus-host disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to the population of Tcon (preferably 36-60 hours prior), and wherein the human subject does not develop graft-versus-host disease (GVHD) within 30 days (preferably within 1 year) after administration of the population of Tcon. EXAMPLES

[0207] Example 1: Clinical Trial Including Administration of HSPCs and Tregs Prior to Tcon This example illustrates the utility of the methods disclosed herein for treating subjects with cancer, including achieving immune reconstitution and GVT in the absence of GVHD.

[0208] The study included 12 patients with high-risk malignancies with HLA-matched sibling donors. The study was approved by the Stanford Institutional Review Board (NCT 01660607) and FDA IND 14686. Written informed consent was obtained for all patients and donors in accordance with the Declaration of Helsinki.

[0209] research design Patient characteristics are provided in Figure 1, with abbreviations as follows: TBI: total body irradiation, Cy: cytoxan, VP-16: etoposide, Bu: busulfan, AML: acute myeloid leukemia, CR: complete remission, MDS: myelodysplastic syndromes, RAEB2: refractory anemia with excess blasts 2, NHL: non-Hodgkin's lymphoma, ETP-ALL: early T-cell precursor acute lymphoblastic leukemia, MRD: minimal residual disease, CML: chronic myeloid leukemia, MF: myelofibrosis.

[0210] Following myeloablative conditioning, subjects received a population of HSPCs and a population of Tregs on day 0, followed by a population of Tcon on day 2. The primary objectives of this study were to determine the efficacy, safety, and feasibility of this technique, determine the maximum tolerated dose of infused Tregs and Tcon, and determine EFS at 1 year after HCT. Safety was assessed as any grade 2 or greater serious adverse event attributable to the treatment, and specific dose-limiting toxicities were defined as acute GVHD > grade 2, grade 4 neutropenia persisting up to 28 days after HCT, and grade 3-5 cytokine / release syndrome / acute infusion reactions.

[0211] Inclusion criteria for enrollment were acute leukemia (primary refractory, minimal residual disease (MRD) positive after CR1 or CR1 with high-risk features), chronic myeloid leukemia (accelerated or blast phase), myelodysplastic syndrome (intermediate risk 2 or high risk), myeloproliferative disorder, non-Hodgkin lymphoma, with high-risk features unsuitable for autologous HCT, age ≤60 years, availability of an HLA-matched related donor, and no prior myeloablative therapy or HCT.

[0212] The original clinical protocol scheme is shown in Figure 2A. Tregs were administered together with the HSPC donor grafts 2 days before Tcon administration. 。 After securing the first cohort of five patients for the original protocol, GVHD was noted. The protocol was modified to utilize fresh rather than frozen Treg cells and to introduce targeted doses of single-agent tacrolimus (4–6 ng / ml) or sirolimus (6–8 ng / ml) GVHD prophylaxis (Figure 2B).

[0213] Conditioning regimens and supportive care The clinical protocol supported the use of different myeloablative conditioning regimens depending on the disease characteristics of the enrolled subjects. For acute leukemia, high-risk CML, myelodysplastic syndromes, and myeloproliferative disorders, conditioning included fractionated total body irradiation: fTBI (1320 cGy, fractionated over 4 days), VP16 (60 mg / kg, as a single infusion) and cyclophosphamide (60 mg / kg, as a single infusion) or busulfan (initially 3.6 mg / kg q24, infused over 4 days, targeting a busulfan level of 800-900 nM), as well as cyclophosphamide (60 mg / kg per infusion, 2 infusions). For non-Hodgkin's lymphoma, conditioning consisted of carmustine (300 mg / m2), VP16 (60 mg / kg), and cyclophosphamide (100 mg / kg).

[0214] Subjects in the original protocol did not receive GVHD prophylaxis, but after a protocol amendment, subjects could receive either tacrolimus or sirolimus based on preclinical evidence that sirolimus promotes Treg function. After one case of hepatic veno-occlusive disease (SOS) in a BU / Cy-conditioned subject, sirolimus GVHD prophylaxis was restricted to only TBI-conditioned subjects.

[0215] Infectious disease prophylaxis consisted of acyclovir 400 mg TID PO (1 year), single strength bactrim (days 30-60), and ciprofloxacin 500 mg PO BID (day -2 until engraftment). Viral surveillance by quantitative PCR for EBV (every 2 weeks) and CMV (weekly) was performed starting on day +14 through day +100.

[0216] Transplant Engineering Donor cells were obtained by apheresis from volunteer donors at Stanford Health Care using a continuous flow cell separator (SPECTRA; Cobe BCT) after 10 mcg / kg rhG-CSF (Neupogen, Amgen, Thousand Oaks, CA) administered five times daily. Two consecutive apheresis collections were performed on days 4 and 5, and the cell products were combined. CD34+ cells were collected using either an Isolex 300i (Baxter healthcare, Deerfield, IL) or a CliniMACS Cell Selection System (Miltenyi Biotec, Bergish Gladbach, Germany). The CD34-depleted (flow-through) fraction was retained and used for isolation of donor Tregs. For cell selection, clinical grade reagents were used under Good Manufacturing Practice Conditions within the BMT Cell Therapy facility.

[0217] CD25+ cells were then selected from the CD34-depleted fraction using bead purification (Miltenyi). Tcon was obtained from the negative fraction, and the positive fraction was used for Treg purification. CD4+CD25+CD127dim cells were further selected by FACS using a BD Influx cell sorter (BD Biosciences, San Jose CA). A representative analysis of cells collected after CD34+ selection, CD25+ cell selection, and CD4+CD127+ cell sorting analyzed for CD4 and FoxP3 expression is shown in Figure 3. Enrichment of Tregs after depletion of CD34+ cells by immunomagnetic selection (left panel), selection of CD25+ by immunomagnetic selection (middle panel), and purification by FACS sorting of CD4+CD127lowCD25+ cells (right panel) is shown. Representative plots are provided from one patient. As can be seen, highly pure Tregs were obtained. These cells were highly suppressive in mixed lymphocyte reactions (MLR). FIG. 4A shows CFSE of T cell-cultured HLA-mismatched PBMCs with / without Tregs in allo-MLR (1:1 ratio) analyzed by FACS. dimFor the MLR, donor peripheral blood or apheresis-derived enriched CD4 + and CD8 + cells (2×10 5 Responders were set up in triplicate in 96-well flat-bottom plates using CellTrace CFSE-labeled (Invitrogen, Molecular Probes) on day 0 according to the manufacturer's instructions, or 1 μCi / well [ 3 The stimulant was a combination of nine different buffy coats from healthy donors, pulsed with a cesium irradiator ( 157 Cs) at 33 Gy and set in responder to stimulator (R:S) ratios of 1:0, 1:1, 1:2, and 1:4. Tregs were added as suppressors in responder to suppressor (R:SUP) ratios of 1:1, 1:0.5, 1:0.25, and 1:10. Culture medium (cRPMI) was supplemented with OKT3 (Miltenyi Biotech). Results were read on day 5 by flow cytometry (LSRII BD) or Beta counting reader (Tomtec, Wallac). Further results are shown in Figure 4B-C.

[0218] For clinical screening, antibodies were repurified on a Protein A (or G) column with a 30 minute hold at low pH. Antibodies were conjugated with FITC and Alexafluor647, sterile filtered, and stored in single-use aliquots.

[0219] Dose escalation and graft characteristics Following myeloablative conditioning, subjects received a population of HSPCs and a population of Tregs on day 0, followed by a population of Tcon on day 2.

[0220] The study included a dose escalation scheme as shown in Figure 5. Treg dose escalation was established as a ratio of Treg:Tcon, with the initial cohort target dose being 1 × 10 in the first cohort of 3 patients. 6Tregs / kg and 3 × 10 6 The CD3+Tcon / kg was 1 × 10 6 Tregs / kg and 1 × 10 6 The group was expanded to three additional subjects with a 1:1 Treg:Tcon ratio at 1 Tcon / kg. No dose-limiting toxicity was observed in these additional subjects, so the target dose was increased to 3 × 10 6 Tregs / kg and 3 × 10 6 After no dose-limiting toxicity was observed in three additional patients, the maximum tolerated target dose was established, which was also the maximal technical dose of Tregs that could be harvested from two apheresis collections, and three additional patients were secured at this dose level. In most patients, the dose was increased to 3×10 6 Achieving 10 Treg / kg was not feasible, so whatever cell dose was achieved was administered.

[0221] Figure 6 provides a schematic of graft generation and administration of the correction protocol with fresh Tregs. 6 All subjects at the 1.0×10 Treg / kg dose received this target dose of Tregs, but all subjects received 3.0×10 6 None of the subjects received the target dose of 2.5×10 Tregs / kg. 6 Tregs / kg (range 2.4–3.0 × 10 6 Figure 1 shows cell dose yields for subjects receiving >2.0 × 10 Tregs / kg. Median Treg recovery was 67% (range 50.7-87.8%) and median Treg purity was 94% (range 91-96%) based on FOXP3+ expression by intracellular cytokine staining. All but one patient received CD34+ doses >2.0 × 10 6 cells / kg, with a median dose of 3.9 × 10 6 The range was 1.2–15.9 × 10 6 Administration details are provided in Figure 8.

[0222] Cell infusion, engraftment, and clinical outcomes Clinical outcomes are summarized in Figure 9. No infusion reactions were observed. All 12 subjects achieved primary complete donor engraftment, except for the lowest dose level (1 × 10 6 Tregs / kg and 1 × 10 6 One subject who received 100 CD3+Tcon / kg had secondary graft failure. Including this subject, neutrophil counts were 1.0 × 10 at a median of 11 days (range, 10–16). 9 The platelet count reached 50 × 10 9 / L. Before the protocol modification, 3 of 5 treated patients achieved complete remission (2 of which remained in remission for >1 year). After the protocol modification (to fresh Tregs with GVHD prophylaxis), 5 of 7 patients achieved complete remission (4 of which remained in remission for >1 year) and no GVHD was observed.

[0223] In early patients who received Treg:Tcon at a ratio of 1:3, acute GVHD grade III was observed. Therefore, for both Treg and Tcon, 6 At a dose of 100 mg / kg, the ratio was adjusted to 1:1 per protocol. Of the first three subjects treated at this dose level, one case of acute GVHD was observed (grade III). Two additional patients were treated at this ratio. One additional patient developed acute grade I cutaneous GVHD that was steroid-responsive.

[0224] The protocol was modified to use only fresh Tregs and to include low-dose single-agent GVHD prophylaxis with sirolimus (as this agent has been shown to protect Tregs) (Figure 2). In clinical situations where the risk of hepatic veno-occlusive disease may be increased with sirolimus, low-dose tacrolimus was substituted. After protocol modification, no additional cases of GVHD were observed in the subsequent seven patients, although one subject had transaminemia and was treated for possible hepatic GVHD or drug intolerance, and rapid resolution of transaminemia was observed after the drug was changed.

[0225] A total of 11 patients achieved survival >6 months and were evaluable for chronic GVHD. At a median follow-up of 481 days (range 212-1887 days), 2 of the 11 subjects had developed chronic GVHD, both in the initial protocol cohort. Both had previous grade III acute GVHD, and the chronic GVHD manifestations were limited to the skin and steroid-responsive. After protocol amendment, no patients in the second cohort developed chronic GVHD.

[0226] After a protocol amendment, one patient who was pretreated with busulfan and cyclophosphamide and received sirolimus for GVHD prophylaxis had mild SOS that resolved after being treated with defibrotide and supportive care.Eleven of 12 patients experienced mucositis (four grade 1, three grade 2, and four grade 3).

[0227] immune reconstitution To assess immune reconstitution in patients enrolled in this study, a comparative standard of care control cohort of five concurrent patients who underwent myeloablative HCT and did not have acute or chronic GVHD or relapse was identified. Using flow cytometry, immune cell subsets were quantified from frozen and thawed PBMCs at standardized time points post-HCT (Figure 10).

[0228] PBMCs from BMT patients with or without prior infusion of Tregs were harvested from liquid nitrogen. PBMCs were washed with MACS buffer and incubated with either a pan-PBMC panel or T reg The cells were stained with the activation panel for 1 hour at 4°C. After washing with MACS buffer, the stained cells were fixed with 4% formaldehyde overnight at 4°C. Flow cytometry analysis was performed on an LSR II and data were analyzed by FlowJo software. Viability staining was performed at room temperature for 5 minutes using 0.5 μM cisplatinum (Fluidigm catalog number 201064). A cell surface antibody master mix was prepared and filtered through a 0.2 μm filter. The cells were resuspended in the master mix and incubated for 30 minutes. The cells were washed and incubated in fixation solution (eBiosciences catalog number 00-5523) prepared according to the manufacturer's recommendations. The cells were fixed for 1 hour at room temperature, washed, and then incubated with the intracellular antibody mix in permeabilization buffer (eBiosciences catalog number 00-5523) for 1 hour.

[0229] Patients in the initial cohort showed relatively low T, B, NK, and Treg reconstitution compared to patients treated under modified protocols or standard care (Figure 10). Patients treated with modified protocols using fresh Tregs appeared to have a generally similar degree of immune reconstitution in T, B, NK, DC, and monocyte populations compared to standard care controls. Absolute numbers of iNKT cells were significantly lower in the first 6 months in both the initial and modified patient cohorts when compared to controls (two-tailed Student's T test, p<0.01).

[0230] Patients treated with the modified protocol using fresh Tregs appeared to have a similar degree of immune reconstitution in percentage and absolute number of Tregs after HCT compared to standard care controls who did not develop acute or chronic GVHD (two-tailed Student's T-test, p=0.01). Phenotypic characterization of Tregs by flow cytometry showed no statistical differences in expression of surface receptors CTLA-4, PD-1, CD62L, CD45RO, or FOXP3. This was confirmed by CyTOF, where it was additionally observed that ex vivo, without stimulation, there appeared to be no increase in intracellular IFN-γ, TNF-α expression. Figure 11A shows the characteristics of Tregs (gated at CD4+CD25+CD127dim) quantified by flow cytometry at day 60 after HCT. Figure 11B shows the characteristics of Tregs (gated at CD4+CD25+CD127dim) quantified by CyTOF at day 60 after HCT.

[0231] Treg TCR repertoire analysis To assess TCR repertoire reconstitution of the Treg compartment after HCT, CD4+CD25+CD127dim Tregs were purified from patients at approximately day 90 post-transplant using FACS. For TCR repertoire analysis, Tregs were enriched from patients' PBMCs by a two-step isolation procedure. CD25+ cells were isolated from PBMCs using CD25 MicroBeads II (Miltenyi Biotech) according to the manufacturer's instructions. After staining with anti-CD3 (OKT3, BioLegend), -CD4 (OKT4, BioLegend), -CD8 (RPA-T8, BioLegend), -CD25 (BC96, BioLegend), and -CD127 (A019D5, BioLegend) antibodies, CD3+, CD4+, CD8-, CD25+, CD127dim cells were isolated, lysed by TRIzol® Plus reagent, and stored at -80°C. 2,700-48,000 Treg cells were isolated.

[0232] TCR Rep-Seq was performed using 5'RACE methodology with modified gene-specific primers in TCRA or TCRB constant regions using RNA as template. Purified 5'RACE PCR products were processed to generate sequencing libraries using KAPA Hyper Prep kit. Sequencing was performed using Illumina MiSeq Reagent 500 Cycle V2 kit with paired-end 250x2 cycles. Paired-end reads from MiSeq were submitted to MixCR for TCRA and TCRB rearrangement analysis. Unique CDR3 amino acid sequences (CDR3 clones) in each sample were summarized based on MixCR results. Single copy CDR3 clones were removed. Clonotype frequency was calculated by dividing the copy number of clonotype by the total copy number of all clonotypes in the sample. Data were subjected to bioinformatics analysis. There was no statistical difference in Treg diversity after standard care or modified clinical trial protocol where fresh Tregs were administered (Figure 11C).

[0233] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 628,105, filed February 8, 2018, which is incorporated herein by reference in its entirety.

Claims

1. 1. A pharmaceutical composition for use in a method of hematopoietic stem cell transplantation (HCT) in a human subject, comprising: The pharmaceutical composition comprises a population of hematopoietic stem and progenitor cells (HSPCs), a population of cells including freshly isolated regulatory T cells (Tregs), and isolated CD3 ＋ Conventional T cells (CD3 ＋ and a single graft-versus-host disease (GVHD) prophylactic agent, The method comprises administering to the human subject: a) a population of HSPCs; b) a fresh population of Treg-containing cells; c) a population of CD3 + Tcon; and d) A single graft-versus-host disease (GVHD) prophylactic agent and administering to the subject a the population of HSPCs and the population of Treg-containing cells are administered prior to the population of CD3 + Tcon, the fresh population of Treg-containing cells is not cryopreserved, and the population of HSPCs comprises less than 2% CD3 + cells. Pharmaceutical compositions.

2. The pharmaceutical composition described in claim 1, wherein the population of fresh Treg-containing cells comprises CD45 + cells, of which more than 75% are Treg.

3. The pharmaceutical composition of claim 1, wherein the fresh Treg-containing cells are CD4 + CD25 + CD127 dim or CD4 + FOXP3 + .

4. The pharmaceutical composition according to claim 1, wherein the population of CD3 + Tcon is cryopreserved.

5. A pharmaceutical composition described in any one of claims 1 to 4, wherein the single GVHD prophylactic agent is sirolimus or tacrolimus.

6. The pharmaceutical composition of claim 1, wherein the ratio of fresh Treg to CD3 + Tcon is about 1:1 to about 1:

3.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the population of HSPCs comprises greater than 1 x 10 5 HSPCs per kilogram of body weight of the human subject.

8. The pharmaceutical composition of claim 7, wherein the population of HSPCs comprises 5×10 5 to 2×10 7 HSPCs per kilogram of body weight of the human subject.

9. The pharmaceutical composition of claim 8, wherein the population of HSPCs comprises 1.2×10 6 to 1.59×10 7 HSPCs per kilogram of body weight of the human subject.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the population of fresh Treg-containing cells comprises more than 1 x 10 5 Tregs per kilogram of body weight of the human subject.

11. The pharmaceutical composition according to claim 10, wherein the population of fresh Treg-containing cells comprises 5×10 5 to 4×10 6 Tregs per kilogram of body weight of the human subject.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the population of CD3 + Tcon comprises less than 1 x 10 7 CD3 + Tcon per kilogram of body weight of the human subject.

13. The pharmaceutical composition of claim 12, wherein the population of CD3 + Tcon comprises 5×10 5 to 4×10 6 CD3 + Tcon per kilogram of body weight of the human subject.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the population of HSPCs is obtained from a donor that is HLA-matched to the human subject.

15. A pharmaceutical composition described in any one of claims 1 to 14, wherein the human subject exhibits mixed donor chimerism after administration of the population of cells.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the population of HSPCs is administered separately from the population of cells containing fresh Tregs.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the method comprises administering the population of CD3 + Tcon at least 12 hours after administration of the population of HSPCs or at least 12 hours after administration of the population of cells comprising fresh Tregs.

18. The pharmaceutical composition of claim 17, wherein the method comprises administering the population of CD3 + Tcon at least 12 hours after administration of the population of HSPCs and at least 12 hours after administration of the population of cells comprising fresh Tregs.

19. The pharmaceutical composition described in claim 18, wherein the human subject does not develop a stage 2 or higher GVHD response within 30 days after administration of the CD3 + Tcon population, or does not develop chronic GVHD within 100 days after administration of the CD3 + Tcon population.

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