Ex Vivo IL-2 Treatment Increases the Immunosuppressive Subset of T Lymphocytes to Suppress Allo-Immune Response

T lymphocytes are critical players in graft-versus-host disease (GVHD); however, their specific roles and mechanisms in GVHD have not been clearly defined. Co-inhibitory receptors including PD1, TIM3, TIGIT, and LAG3 play a key role in regulating T cell responses and maintaining immune homeostasis. TIM3 expression on lymphocytes 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, despite the strong immunosuppression exerted by PD1 ⁺TIM3 ⁺ double-positive T cells, their clinical potential is greatly limited by their low cell number in peripheral blood. In this study, we introduce a novel method to isolate CD3 ⁺ cells from G-CSF mobilized peripheral blood stem cells (G-PBSCs), and culture CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes to treat GVHD.

Methods. The 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. Then, the highly purified CD3 ⁺ cells were isolated by positive selection with magnetic-activated cell sorting (MACS) from G-PBSCs using CD3 Dynabeads™. Isolated CD3 ⁺ cells were cultured with a low concentration of IL-2 (50U/mL), and PD1, TIM3, LAG3, and TIGIT expressions were assessed using flow cytometry (FACS). CD3 ⁺PD1 ⁺TIM3 ⁺, CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ^- and CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ⁺cells are sorted and cultured with irradiated allo-MNCs for 4 days (Mixed Lymphocyte Reaction; MLR). We used 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure of T cells proliferation.

Results. In normal, untreated G-PBSCs, very low percentages of cells are CD3 ⁺PD1 ⁺TIM3 ⁺, CD3 ⁺PD1 ⁺LAG3 ⁺ and CD3 ⁺PD1 ⁺TIGIT ⁺ (0.6±0.4, 0.3±0.2 and 1.3±0.5% in G-PBSC, respectively). However, after treating G-PBSC cells with a low concentration of IL-2 (50 U/mL), we discovered the percentages of CD3 ⁺PD1 ⁺TIM3 ⁺, CD3 ⁺PD1 ⁺LAG3 ⁺, and CD3 ⁺PD1 ⁺TIGT ⁺ lymphocytes in G-PBSCs markedly increased by 19.6±5.9, 18.5±4.3, 17.7±6.5%, respectively. Of note, there was a very small change (~1.2 fold increase) in the total number of CD3 ⁺ lymphocytes, which indicates that the low dose IL-2 therapy alters subpopulations of T lymphocytes, rather than increasing the proliferation rate. Next, we tested the immunosuppressive capacity of the cultured CD3 ⁺PD1 ⁺TIM3 ⁺cells. To do this, we used CSFE-MLR to measure T cell proliferation. CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes were depleted in CD3 ⁺ G-PBSCs by flow cytometry-based cell sorting. Then we cultured the CD3 ⁺ G-PBSCs with allo-MNCs and discovered that the CD3 ⁺ G-PBSCs that lacked CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes demonstrated a significantly increased level of T-cell proliferation compared to CD3 ⁺ G-PBSCs, and thereby confirming the immunosuppressive function of CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes in inhibiting T cell proliferation (% of unstimulated T cells in CD3 ⁺ G-PBSCs vs. in CD3 ⁺PD1 ⁺TIM3 ⁺ depleted G-PBSC CD3 ⁺ cells; 33.5±7.5% vs. 6.3±4.2%).

After confirming the immunosuppressive function of the IL-2 treated CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes, we investigated expressions of other co-inhibitory surface markers such as LAG3 and TIGIT. Amongst the cultured CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes, 96.2±3.3% of the CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes were LAG3 ⁺, 28.9±5.4% of the CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes were TIGIT ⁺, and 63.7±7.8% of the cells were TIGIT ^-. Interestingly, CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ^- lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ⁺ lymphocytes (% of unstimulated T cells in CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ^- lymphocytes vs. in CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ⁺ lymphocytes, 53.8±5.5% vs. 23.2±7.4%).

Conclusion.

We demonstrate that treating CD3 ⁺ G-PBSCs with low-dose IL-2 markedly increases the percentage of CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes, which is known to exert strong immunosuppression. Also, we suggest that CD3 ⁺PD1 ⁺TIM3 ⁺LAG3 ⁺TIGIT ^- lymphocytes are the subpopulation within CD3 ⁺PD1 ⁺TIM3 ⁺ cells that demonstrate the most potent immunosuppression compared to other subpopulations of CD3 ⁺PD1 ⁺TIM3 ⁺ lymphocytes. Taken together, such findings suggest that the low-dose IL-2 therapy has therapeutic potential to treat patients with GVHD.