Elevated circulating activated FOXP3+ regulatory T cell subset in patients with advanced chronic lymphocytic leukemia as a marker for severe infections during 1000 days of follow-up Free

Patients with chronic lymphocytic leukemia (CLL) are more susceptible to infections, which remain the leading cause of death in this population. Previous studies have shown elevated levels of FOXP3+ regulatory T cells (Tregs) in CLL, which correlate with impaired T cell responses to microbial antigens. Tregs consist of various subpopulations with different immunosuppressive potentials, among which the CD45RA⁻FOXP3^high activated Tregs (aTregs) are considered the most suppressive. Thus, subclassification of Tregs is essential for understanding their clinical impact.

We conducted a single-centre, prospective cohort study including 37 patients with advanced CLL at the Department of Haematology, University Medical Centre Maribor. Peripheral blood samples were collected at therapy initiation and at multiple time points during treatment. We used conventional and imaging flow cytometry to analyse CD4+FOXP3+ T cells, encompassing both nonsuppressive and suppressive subsets, including CD45RA⁻FOXP3^high aTregs. The first patients were enrolled in 2020, and follow-up continued for up to 1000 days. Severe infections were defined as grade ≥3 according to the Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. Infectious complications were analysed using the Kaplan–Meier method, and group comparisons were performed using the log-rank test. A p-value < 0.05 was considered statistically significant.

Among the 37 included patients (all of Caucasian origin), the mean age was 69 years, and 24 were male. On average, patients had been monitored for CLL for 50 months prior to enrolment. At the time of therapy initiation, 12 patients were in Binet stage B, and 25 in stage C. TP53 mutations were detected in 5 (13.5%) patients, and 19 (51.4%) had an unmutated immunoglobulin heavy-chain variable region (IGHV) gene status. Treatment included chemoimmunotherapy in 12 patients, Bruton tyrosine kinase inhibitors (BTKi) in 19, and venetoclax in 6 patients.Based on our prior studies in healthy controls, we set a threshold of 26% aTregs among CD4+FOXP3+ T cells to define two groups: group 1 (≤26% aTregs, n = 18) and group 2 (>26% aTregs, n = 19). There were no significant differences between the groups in terms of demographics, clinical characteristics, laboratory findings, or therapy type.The most pronounced difference in infection frequency and severity was observed during the first year of follow-up. By day 365, 58% of patients in group 2 had experienced grade ≥3 infections, compared to only 6% in group 1. The rate of grade ≥3 infections per year at day 500 day of the follow-up was significantly higher in group 2 (0.70/year vs. 0.04/year, p = 0.001). Up to 1000 days of follow-up, only one additional serious infection occurred in group 1 (mean grade ≥3 infection rate 0.38/year vs. 0.06/year, p = 0.0021). Furthermore, none of the patients in group 1 and five in group 2 required hospitalisation due to severe COVID-19.There was no statistically significant difference in infection rates between patients treated with chemoimmunotherapy and those receiving BTKi. Overall, 13 patients died during follow-up: 5 due to infections, 5 due to comorbidities, and 3 due to Richter transformation. Among infection-related deaths, 4 occurred in group 2 and only 1 in group 1.

A higher percentage of aTregs among CD4+FOXP3+ T cells was associated with an increased incidence of severe (grade ≥3) infections and infection-related mortality in patients with advanced CLL. The type of therapy (targeted vs. chemoimmunotherapy) did not influence the rate of severe infections. Elevated aTreg levels could serve as a potential marker for identifying patients at higher risk for serious infections, which may inform future decisions regarding prophylactic interventions (e.g., antibiotics, immunoglobulins, antifungals), pending confirmation in larger randomized studies.