# Increased Frequency of Circulating Activated FOXP3+ Regulatory T Cell Subset in Patients with Chronic Lymphocytic Leukemia Is Associated with the Estimate of the Size of the Tumor Mass, STAT5 Signaling and Disease Course during Follow-Up of Patients on Therapy

## TL;DR

This study finds that higher levels of a specific type of regulatory T cell in chronic lymphocytic leukemia patients are linked to larger tumor size, STAT5 signaling, and more severe disease progression.

## Contribution

The study identifies aTreg expansion and STAT5 signaling as potential markers for severe disease progression in CLL patients.

## Key findings

- Increased aTreg frequency correlates with advanced CLL stages and larger tumor mass.
- Higher aTreg levels are associated with more severe infections during follow-up.
- STAT5 phosphorylation is significantly linked to aTreg expansion in CLL patients.

## Abstract

Patients with chronic lymphocytic leukemia (CLL) are more susceptible to infections, which are also the most common cause of death in these patients. Previous studies in patients with CLL described elevated levels of FOXP3+ regulatory T cells (Tregs), which also correlated with decreased T cell responses to microbial antigens. As the activation of the STAT5 transcription factor induces the expression of FOXP3 and human CD4+FOXP3+ T cells that also contain nonsuppressive T cells, we analyzed STAT5 phosphorylation (pSTAT5) and suppressive subpopulations, including activated Tregs (aTregs). We found a significantly increased frequency of aTregs in patients with advanced stages, which significantly correlated with the total tumor mass score. aTreg expansion in vitro was associated with significantly higher aTreg pSTAT5 responses to SARS-CoV-2 antigen-specific stimulation in vitro. Finally, a subgroup of patients characterised by an increased aTreg percentage among CD4+FOXP3+ T cells experienced a more severe disease course with serious grade ≥3 infections during follow-up.

Introduction: Advanced chronic lymphocytic leukemia (CLL) is accompanied by increased circulating regulatory T cells (Tregs) and increased susceptibility to severe infections, which were also shown to entail a striking induction of FOXP3 expression in Tregs. As homeostasis of the most suppressive CD45RA−FOXP3high activated Treg (aTreg) subset differs, it is critical to analyse homeostatic signalling in Treg subsets. Materials and Methods: In this study, by using conventional and imaging flow cytometry, we monitored STAT5 signalling/phosphorylation (pSTAT5) and investigated Treg subsets in relation to the Binet stage, the total tumor mass score (TTM) and the disease course during a follow-up of 37 patients with CLL. Results: The aTreg percentage was significantly increased among CD4+ T cells from patients with advanced disease and significantly correlated with the TTM. A subgroup of patients with higher aTreg percentages among CD4+FOXP3+ T cells at the start of therapy was characterised by more frequent episodes of severe infections during follow-up. Conclusions: The results suggesting that an aTreg fraction could represent a possible marker of a severe disease course with infectious complications. Augmented homeostatic STAT5 signalling could support aTreg expansion, as higher pSTAT5 levels were significantly correlated with an increased aTreg frequency among CD4+FOXP3+ T cells during the follow-up of patients on therapy, as well as following SARS-CoV-2 antigen-specific stimulation in vitro.