# Phenotypic and functional comparisons between cryopreserved and freshly isolated peripheral blood mononuclear cells with or without red blood cell lysate (ACK) treatment with special focus on regulatory T cells

## TL;DR

This study compares the effects of cryopreservation on immune cells, focusing on regulatory T cells, and finds that their function is preserved after freezing.

## Contribution

The study provides new insights into the impact of cryopreservation and ACK treatment on Treg function and viability.

## Key findings

- ACK-treated PBMCs showed increased IFN-γ-producing cells when stimulated with viral peptides.
- Cryopreservation reduced cell viability, CD4+ T-cell population, and FoxP3 expression but preserved Treg function.
- Tregs from both fresh and frozen PBMCs equally suppressed PBMC proliferation.

## Abstract

Adaptive transfer of autologous regulatory T cells (Treg), or ex vivo-generated immunomodulatory cells, has shown promise in reducing/withdrawing immunosuppression after organ transplantation. The effect of cryopreserving such cells is still unclear. This study aims to evaluate the effects of cryopreservation on the immunomodulatory functions of peripheral blood mononuclear cells (PBMCs) with or without pretreatment with red blood cell (RBC) lysate (ACK). Human PBMCs enriched from buffy coats of healthy blood donors were treated either with ACK or phosphate-buffered saline (PBS). Thereafter, a batch of the PBS-control subset was cryopreserved with 10% dimethyl sulfoxide (DMSO) and subsequently examined for phenotype, functionality, and relative gene expression. We found that ACK-treated PBMCs exhibited higher numbers of interferon gamma (IFN-γ)-producing cells when stimulated with viral peptides (p = 0.0078), indicating that ACK treatment may improve the antigen sensitivity of memory T cells. After cryopreservation, contaminated RBCs and granulocytes, cell viability, and CD4+ T-cell population decreased (p = 0.0078); IL-1β expression increased; and FoxP3 expression decreased (p = 0.0312), where the Treg population remained otherwise unchanged. Enriched Tregs from both fresh and frozen PBMCs suppressed the proliferation of anti-CD3/CD28-antibody-stimulated PBMCs equally. In conclusion, the preservation of Treg function following cryopreservation highlights its potential utility in tolerance-induction trials, providing experimental flexibility and simplified logistics.

Graphical Abstract