The Study on the expansion and function of Tregs from human PBMCs

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The Study on the expansion and function of Tregs from human PBMCs

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Co-author 1

Ye Na Kim 67920@naver.com 1Gospel Hospital, Kosin University College of Medicine, Busan, South Korea,2Transplantation Research Institute, Kosin University College of Medicine, Busan, South Korea Renal Division, Department of Internal Medicine Busan Korea (Republic of) *

Co-author 2

Woosik Yang 67920@naver.com 1Gospel Hospital, Kosin University College of Medicine, Busan, South Korea,2Transplantation Research Institute, Kosin University College of Medicine, Busan, South Korea Renal Division, Department of Internal Medicine Busan Korea (Republic of) -

Co-author 3

Ho Sik Shin 67920@naver.com 1Gospel Hospital, Kosin University College of Medicine, Busan, South Korea,2Transplantation Research Institute, Kosin University College of Medicine, Busan, South Korea Renal Division, Department of Internal Medicine Busan Korea (Republic of) -

Co-author 4

Yeonsoon Jung 67920@naver.com 1Gospel Hospital, Kosin University College of Medicine, Busan, South Korea,2Transplantation Research Institute, Kosin University College of Medicine, Busan, South Korea Renal Division, Department of Internal Medicine Busan Korea (Republic of) -

Co-author 5

Hark Rim 67920@naver.com 1Gospel Hospital, Kosin University College of Medicine, Busan, South Korea,2Transplantation Research Institute, Kosin University College of Medicine, Busan, South Korea Renal Division, Department of Internal Medicine Busan Korea (Republic of) -

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Introduction

Regulatory T cells (Tregs) constitute a vital subgroup within CD4+ T cells. These cells play a pivotal role in inducing immune tolerance, preserving immune homeostasis, and mitigating autoimmune diseases. Typically, Tregs are present in small numbers under normal physiological conditions. The current study aims to devise an effective method for expanding human peripheral blood Tregs in vitro and subsequently analyzing the phenotype, purity, and function of these expanded Treg cells.

Methods

Peripheral blood samples were collected from 11 healthy donors. Tregs were isolated from peripheral blood mononuclear cells (PBMCs) using magnetic-activated cell sorting (MACS) based on the expression of CD4+ and CD25+ markers. An optimized culture system was employed for the amplification of Tregs. The in vitro amplification efficiency of Treg cells was assessed to evaluate the expression levels and purity of Treg cell-specific surface markers across various culture cycles. The analysis of Tregs involved using Flow cytometry to analyze various target markers, and Treg-related genes were analyzed using RT-PCR methods.

Results

Treg cells were successfully isolated via magnetic activated cell sorting. Following 11 days of in vitro culture, there was a significant difference in the mean Tregs/PBMC ratio between the younger group (< 40 years) and the group older than 60 years. The proliferated Treg cells exhibited a clustering phenomenon of small cells, and cell expansion increased geometrically from day 7 onwards. Flow cytometry analysis revealed that, compared to PBMCs, the percentages of CD4+/CD25+ and CD25+/Foxp3+ cells increased to 157 and 60 fold, respectively, by in Treg cells after 11 days of culture. Expanded Treg cells upregulated gene expression of CD25, Foxp3, IL-10, and CTLA-4 at the RNA level. Another interesting finding is the observed increase in CD25+, Foxp3+, and CTLA4+ expression in Tregs treated with Adipose-derived stem cells (ASC) secretome. Further investigation into the effects of secretome on Treg proliferation would be necessary.

Conclusion

We have successfully developed a technical protocol for generating a substantial quantity of Tregs with high efficiency in vitro. These expanded Tregs consistently maintain FOXP3 expression and demonstrate potent immune suppression. This finding holds significant promise for adoptive Treg therapy in the treatment of graft-versus-host disease and autoimmune disorders.

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