Immune profiling reveals favourable phenotypes among relapsed/refractory large B-cell lymphoma patients undergoing CAR T-cell therapy in second line

Supported by clinical trials showing improved outcomes, Chimeric Antigen Receptor (CAR) T-cell therapy is increasingly being introduced earlier in the treatment of relapsed/refractory large B-cell lymphomas. In current clinical practice, CAR T-cell therapy is used in the second-line setting for patients eligible for autologous stem cell transplantation. However, the biological mechanisms underlying early CAR T-cell administration remain insufficiently defined, and a comprehensive analysis of immunophenotypes between patients treated in second-line versus later-line settings are still lacking. Therefore, this study aimed to characterize patient clinical features, investigate CAR T-cell expansion kinetics, and evaluate phenotypic differences in T cells, CAR T cells, and myeloid populations in patients treated with axicabtagene ciloleucel in the second-line (n=68) and later-line (n=223) settings. Clinical characteristics of patients treated in the second and later line settings were compared using GraphPad Prism (v10.6.1). Depending on sample availability, leukapheresis, infusion products, and peripheral blood samples from both cohorts were immune profiled using the MACSQuant® Analyzer 16. Dimensionality reduction and clustering were performed using the OMIQ platform. Patients treated in the second-line setting were older than those treated in later lines (median age 63.5 vs 60 years; p<0.05). A higher proportion of complete responders was observed in the second line cohort compared with later lines (75% vs 58.6%; p=0.056). At one year, relapse-free rates were 40% and 44% in the second and later line groups, respectively. Leukapheresis products from second-line patients were enriched in CD8⁺ T stem cell memory cells (median 1.45% vs 0.38%; p<0.05) and showed lower frequencies of CD8⁺ exhausted T cells (median 0.48% vs 1.96%; p<0.05) as well as intermediate monocytes (median 9.8% vs 17.5%; p<0.05). In infusion products, second-line patients exhibited a higher proportion of CAR⁺ CD8⁺ central memory cells (median 28.7% vs 12.6%; p<0.01) and a lower CAR⁺ CD4/CD8 ratio (median 0.63 vs 1.2; p<0.05). Furthermore, CAR T-cell expansion was greater in the second-line cohort, as reflected by a higher area under the curve within the first 30 days post-infusion (435 vs 295; p<0.05), along with higher circulating CAR T-cell levels at day 10 (32 vs 18.4 cells/µL; p<0.05). Despite being older, patients treated in the second-line setting exhibited a more favourable immune profile across multiple time points during CAR T-cell therapy. As less differentiated T cells, enhanced CAR T expansion, and lower frequencies of myeloid populations are associated with improved CAR T-cell efficacy, these findings suggest a possible biological advantage for earlier administration of axicabtagene ciloleucel in relapsed/refractory large B-cell lymphomas, potentially explaining the higher response rates observed in second-line patients. Ongoing work aims to leverage spectral flow cytometry for a 40-color T cell panel and to perform Single Cell Energetic metabolism by profiling Translation inhibition (SCENITH) assay. This will enable a more detailed characterization of the phenotypic and metabolic differences between the two cohorts.