Coexpression of Tim-3 and PD-1 of T Lymphocytes Prevents Acute Graft-Versus-Host Disease

T lymphocytes are critical players in acute graft-versus-host disease (aGVHD). TIM3expression on the T cell surface was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 ⁺TIM3 ⁺ double-positive T cells displayed more potent immunosuppression compared to PD1 ⁺ T cells. However, their clinical potential is greatly limited by their low cell number in peripheral blood (PB). We already introduced a novel method to obtain a high number of PD1 ⁺TIM3 ⁺ lymphocytes that culturing CD3 ⁺ cells isolated from G-CSF mobilized peripheral blood stem cells (CD3 ⁺G-PBSC) with human serum albumin (HSA). We also confirmed that cultured PD1 ⁺TIM3 ⁺ lymphocytes are the most potent subset of the CD3 ⁺ cells that inhibit T cell proliferation. In this study, we investigate the immunosuppressive effects of PD1 ⁺TIM3 ⁺ lymphocytes after a 2-day culture with HSA (D2-CD3 ⁺G-PBSC) or after a 4-day culture with HSA (D4-CD3 ⁺G-PBSC). Also, we evaluate the treatment efficacy of PD1 ⁺TIM3 ⁺ lymphocytes in the xenograft mouse aGVHD model.

Methods. The healthy donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3 ⁺ cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. CD3 ⁺ G-PBSC were cultured with 5% HSA for 2 days or 4 days (1x10 ⁵ cells/mL). PD1 and TIM3 expressions were analyzed using flow cytometry (FACS). Sorted T lymphocytes were cultured with irradiated allo-MNCs for 3 days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. aGVHD was induced by intraperitoneal injection of human PB MNCs to the NOD-SCID IL-2Rγ null mouse after exposure of 3Gy irradiation. The treatment efficacy of PD1 ⁺TIM3 ⁺ lymphocytes was analyzed by determining the survival rate after tail vein injection of PD1 ⁺TIM3 ⁺ lymphocytes (1x10 ⁷/kg).

Results. Normally, very few cells (0.2±0.2%) were PD1 ⁺TIM3 ⁺ lymphocytesin PB. The numbers of PD1 ⁺TIM3 ⁺ lymphocytes markedly increased by 74-fold in D2-CD3 ⁺G-PBSC and 370-fold in D4-CD3 ⁺G-PBSC, respectively. CD3 ⁺ G-PBSC were composed of three types of cells such as PD1 ^-TIM3 ^-, PD1 ⁺TIM3 ^-, and PD1 ⁺TIM3 ⁺ lymphocytes. To check the origin of PD1 ⁺TIM3 ⁺ lymphocytes after HSA treatment, PD1 ^-Tim3 ^- and PD1 ⁺Tim3 ^- lymphocytes from CD3 ⁺G-PBSC were sorted and cultured with HSA. It was confirmed that PD1 ⁺TIM3 ⁺ lymphocytes originated from PD1 ^-TIM3 ^- lymphocytes.

To assess the immunosuppressive effects of cultured T lymphocytes, we used CSFE-MLR. D2-CD3 ⁺G-PBSC significantly increased the levels of unstimulated T cells compared to D4-CD3 ⁺G-PBSC (% of unstimulated T cells in D2-CD3 ⁺G-PBSC vs. in D4-CD3 ⁺G-PBSC; 38.9±3.0% vs. 13.9±6.6%). Also, both PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC demonstrated significantly enhanced levels of immunosuppression (% of unstimulated T cells PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC; 31.6±5.5 and 36.6±13.3, respectively). Lastly, we tested the effects of anti-PD1 or anti-TIM3 blocking mAb on the PD1 ⁺TIM3 ⁺ lymphocytes. The immunosuppressive effects of PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC were significantly decreased by anti-PD1 mAb (% of unstimulated T cells in PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC; 21.1±4.5 from 31.6±5.5 and 18.4±7.8 from 36.6±13.3, respectively). However, the immunosuppression of PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC was not decrease by anti-TIM3 mAb (% of unstimulated T cells in PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC; 28.7±3.3 from 31.6±5.5 and 22.7±5.6 from 36.6±13.3, respectively). These data indicated that PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC have the same phenotype as PD1 ⁺TIM3 ⁺ lymphocytes from D4-CD3 ⁺G-PBSC, however, their functions are different.

Next, we used the xenograft mouse aGVHD model to evaluate the treatment efficacy of PD1 ⁺TIM3 ⁺ lymphocytes for preventing aGVHD. In the control group, 14% of the mice survived at 100 days. Mice that were treated with PD1 ⁺TIM3 ⁺ lymphocytes from D2-CD3 ⁺G-PBSC and D4-CD3 ⁺G-PBSC demonstrated 100% and 71% survival rate at 100 days, respectively.

Conclusion. Ex vivo HSA treatment of CD3 ⁺ G-PBSCs for 2 days shows promising therapeutic potential for the treating patients with aGVHD.