Ex Vivo IL-2 Treatment Increases the Immunosuppressive Subset of T Lymphocytes to Suppress Allo-Immune Response
Authors: Kim Jeong-A , KIM Jinhang , Yang Misuk , Park Youngrok , Kwak Jae-Yong 
Published: November 2021

Abstract
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.

Disclosures
No relevant conflicts of interest to declare.