Higher helios⁺ treg levels pre- and post-CAR T infusion are associated with inferior outcomes in patients with LBCL

Abstract Background Regulatory T cells (Tregs) are key immunosuppressive cells in the tumor microenvironment that may impair CAR T-cell expansion and antitumor efficacy (Tao, Blood Cancer J 2024). Helios, a transcription factor expressed by thymic-derived Tregs, marks a more stable and suppressive Treg subset (Zhou, Nat Immunol 2019). However, the clinical relevance of the presence of Helios+ and Helios- Tregs in the setting of CAR T-cell therapy (CART) has not been characterized. We examined the relationship between Helios-defined Treg subsets, clinical outcomes and CAR T expansion in patients with large B-cell lymphoma (LBCL) treated with CD19-directed CART. Methods Patients ≥18 years with refractory/refractory LBCL after ≥1 prior therapy who received axicabtagene ciloleucel (axi-cel) at the University Medical Center Groningen were included if peripheral blood mononuclear cells (PBMCs) were available in the Oncological Life Study (OncoLifeS) biobank. Clinical data were obtained from the Follow-that-CAR registry. Flow cytometry was performed on PBMCs collected at baseline (day -28 to -6), pre-CAR T infusion (day 0) and post-CAR T infusion (day 7). Tregs were defined as CD4⁺CD25⁺CD127lowFOXP3⁺ and further subdivided into Helios⁺ and Helios⁻ subsets. Levels of Tregs and Helios⁺/Helios⁻ subsets are expressed as % of total CD4⁺ T-cells. Patients were stratified into high and low groups based on the median. CAR T expansion was measured on days 7, 14, 21 and 28 by flow cytometry using a biotinylated anti-CAR19 antibody and reported as cells/µL and area under the curve from day 0 to 28 (AUCd0-28). CAR T subsets (naive, central memory, effector memory and terminally differentiated effector memory) were expressed as % of CAR+ cells. Endpoints included best overall response and progression free survival (PFS). Results Forty LBCL patients (median age 63, range 34–80) were included in this study. Median number of prior lines of therapy was 2 (range 1-3) and 7 patients (18%) had undergone autologous stem cell transplantation. Bridging therapy was administered in 75% of cases, primarily radiotherapy (67%), systemic therapy (13%) or both (20%). PBMCs were available for 28 patients at baseline, 33 pre-CAR T infusion and 29 on day 7. Median Helios⁺ Tregs increased from 5.4% (IQR 3.7-6.9%) at baseline to 8.8% (6.0-11.4%) pre-CAR T infusion and 9.4% (4.3-13.5%) post-CAR T infusion, while Helios⁻ Tregs increased from 2.5% (1.9-3.5%) to 6.9% (4.3-9.8%) pre-CAR T infusion, before declining to 4.3% (2.9-6.7%) post-CAR T infusion. Patients with low (<8.8%) pre-CAR T infusion Helios⁺ Tregs achieved complete remission (CR) as best overall response in 75% of cases vs 35% with high (≥8.8%) pre-CAR T infusion Helios⁺ Tregs (p=0.022). Patients with low pre-CAR T infusion Helios⁺ Tregs had a significantly better 1-year PFS than patients with high levels of Helios⁺ Tregs (54% vs 30%, p=0.033). Pre-CAR T infusion Helios⁻ Tregs showed no significant association with response or PFS. CAR T expansion data was available for 29 patients. Median CAR T expansion was 37 cells/μL (IQR 15-122 cells/μL) on day 7, 4.8 cells/μL (2.3-13.6 cells/μL) on day 28, with a median AUCd0–28 of 131 cells/μL×days (37-540 cells/μL×days). While pre-CAR T infusion Treg levels did not correlate with CAR T expansion, post-CAR T infusion Helios⁺ Tregs were negatively correlated with CAR T expansion on days 7, 28, and AUCd0-28 (Spearman's r = -0.37 to -0.59, p ≤ 0.036), an effect not seen with Helios⁻ Tregs. Furthermore, low (<9.4%) post-CAR T infusion Helios⁺ Tregs were associated with increased central memory (CM) CAR T-cell subsets on days 7 and 28 (day 7 CD4⁺ CM: 17% vs 6.5%, p=0.035; day 7 CD8+ CM: 5.3% vs 3.0%, p=0.12; day 28 CD4⁺ CM: 17% vs 6.5%, p=0.023 and day 28 CD8⁺ CM: 2.1% vs 0.4%, p=0.044). ConclusionThis study demonstrates, for the first time, that higher pre-CAR T infusion Helios⁺ Tregs are associated with lower CR rates and shorter PFS. Post-infusion Helios⁺ Tregs inversely correlate with CAR T expansion and low post-infusion Helios⁺ Treg levels are linked to a more favorable central memory-like CAR T phenotype. The increase in Treg levels from baseline to pre-CAR T infusion suggests relative resistance to lymphodepleting chemotherapy. These findings identify Helios⁺ Tregs as a potential barrier to effective CAR T-cell therapy and, while warranting validation in larger cohorts, support exploring strategies to modulate these cells, or refine lymphodepletion to improve outcomes.