Transplantation of donor grafts with defined ratio of conventional and regulatory T cells in HLA-matched recipients

Abstract

BACKGROUNDIn preclinical murine and early clinical studies of hematopoietic cell transplantation, engineering of donor grafts with defined ratios of CD4+CD25+FoxP3+ Tregs to conventional T cells (Tcons) results in the prevention of graft-versus-host disease and improved immune reconstitution. The use of highly purified primary graft Tregs for direct cell infusion has potential advantages over impure immunomagnetic selection or culture expansion, but has not been tested clinically. We performed a phase I study of the timed addition of CD34-selected hematopoietic stem cells and Tregs, followed by Tcons for the treatment of patients with high-risk hematological malignancies.METHODSWe present interim evaluation of a single-center open phase I/II study of administration of human leukocyte-matched Tregs and CD34-selected hematopoietic cells, followed by infusion of an equal ratio of Tcons in adult patients undergoing myeloablative hematopoietic stem cell transplantation (HCT) for high-risk or active hematological malignancies. Tregs were purified by immunomagnetic selection and high-speed cell sorting.RESULTSHere we report results for the first 12 patients who received Tregs of between 91% and 96% purity. Greater than grade II GVHD was noted in 2 patients in the first cohort of 5 patients, who received cryopreserved Tregs, but neither acute nor chronic GVHD was noted in the second cohort of 7 patients, who received fresh Tregs and single-agent GVHD prophylaxis. Patients in the second cohort appeared to have normal immune reconstitution compared with patients who underwent transplantation and did not develop GVHD.CONCLUSIONOur study shows that the use of highly purified fresh Tregs is clinically feasible and supports continued investigation of the strategy.TRIAL REGISTRATIONClinicalTrials.gov NCT01660607.FUNDINGNIH NHBLI R01 HL114591 and K08HL119590.

Keywords: Bone marrow transplantation; Cellular immune response; Clinical Trials; Transplantation.

Conflict of interest statement

Conflict of interest: The authors have no ownership or income to declare. EHM has a sponsored research agreement with Orca Biosystems Inc. initiated in 2018. EHM and RSN have declared methodology for Treg selection to Stanford University, which submitted a patent application.

Figures

Figure 1. Protocol schema.

The initial protocol utilized frozen Tregs and no GVHD prophylaxis. After the accrual of the first 5 patients between September 2011 and May 2015 (initial protocol), the protocol underwent a review and revision in November 2015 (modified protocol) based on preclinical data that frozen and thawed Tregs do not function as well as freshly isolated cells.

Figure 2. Cell selection and purification of Tregs.

(A) Enrichment of Tregs from peripheral blood G-CSF–mobilized progenitor cells, following depletion of CD34+ cells by immunomagnetic selection (left), selection of CD25+ by immunomagnetic selection (middle), and purification by FACS sorting of CD4+CD127loCD25+ cells (right panel). Representative plots from 1 patient expressed as CD4+ and intracellularly stained FoxP3. (B) Suppression of the MLR by purified Tregs. The percentage of CFSEdim populations of T cell–cultured HLA-mismatched PBMCs with/without Tregs (1:1 ratio) in allo-MLR, analyzed by FACS. Results are mean ± SD from 4 patients evaluated; P < 0.01, 2-tailed Student’s t test.

Figure 3. Reconstitution of immune cell populations following hematopoietic cell transplantation.

Patients on the modified protocol demonstrated improved absolute cell counts in B cells, T cells, and Tregs compared with initial patients 180 days after hematopoietic cell transplantation (HCT). Counts are shown in cells/ml of blood. *P < 0.05, Mann-Whitney U test, between control patients and patients on the modified protocol. Error bars represent mean ± SEM. n = 7 for modified protocol; n = 5 for control; and n = 3 for initial protocol.

Figure 4. Immune reconstitution and TCR diversity of peripheral blood Tregs.

Reconstitution of naive Tregs as quantified by (A) CyTOF and (B) flow cytometric staining, both gated on CD4+CD25+CD127lo. Patients on the modified protocol (n = 6) are compared with standard-of-care controls (n = 5) and initial protocol (n = 5) participants at 60 days after HCT. (C) TCRα and -β CDR3 repertoire diversity of FACS-purified CD4+CD25+CD127lo Tregs at day 90 after HCT for patients on the modified protocol (n = 7) versus standard-of-care controls (n = 5). *P < 0.05 and **P < 0.01 respectively, Mann-Whitney U test, between control patients and patients on the modified protocol. Error bars represent mean ± SEM.