In-Depth Immune and Metabolic Profiling of GAIA/CLL13 Patients to Uncover Treatment-Specific Changes to the T-Cell Compartment
Authors: Jacobs Chaja Feige , D'Hargues Yannick , Robrecht Sandra , Fürstenau Moritz , Fischer Kirsten , Fink Anna Maria , Stilgenbauer Stephan , Schneider Christof , Tausch Eugen , Niemann Carsten Utoft , da Cunha-Bang Caspar , Dubois Julie , Van Der Spek Ellen , Vlot Andre , Sandberg Yorick , Levin Mark-David , Hallek Michael , Eichhorst Barbara F. , Kater Arnon P. , Peters Fleur S. 
Published: December 2024

Introduction
Chronic lymphocytic leukemia (CLL) induces T-cell dysfunction through tumor cell interactions, affecting phenotype and function. Associations between disease-specific features and T-cell characteristics remain unclear. Additionally, the correlation between pre-treatment T-cell parameters and responses to chemoimmunotherapy (CIT) or venetoclax-based treatments has not been systematically studied. Further research is needed to elucidate these relationships and optimize therapeutic outcomes, particularly to enhance the efficacy of autologous T-cell-based therapies.
This study evaluates baseline and post-treatment T cell characteristics in relation to clinical parameters and outcomes in patients (pts) treated with CIT or venetoclax-based time-limited combinations within the GAIA/CLL13 trial, examining-cell phenotype, metabolism, and function.
Methods
A subset of samples from the GAIA/CLL13 phase 3 trial was analyzed, comparing three different time-limited venetoclax-CD20 antibody based combinations with CIT in treatment-naive fit CLL pts without TP53 aberration. Mass cytometry (CyTOF) was employed to examine baseline (n=54) and post-treatment samples obtained at month 15 (n=39), from all treatment arms. Twelve age-matched healthy donors (HD) were analyzed as reference. T-cell surface and metabolic markers were measured to generate FlowSOM clusters. The abundance of 14 identified clusters was compared between pre- and post-treatment samples and assessed in relation to clinical parameters, including baseline tumor characteristics, tumor burden, and outcome measures.
Results
The median age of the 54 included pts was 62 years, of whom 32 (59.3%) had unmutated IGHV status and 38 (71.7%) had a non-complex karyotype (&lt;3 aberrations). Median time from initial diagnosis to study entry was 24.8 months (interquartile range 4.5-64.1).
The analysis of pre-treatment samples confirmed previously reported T-cell skewing in CLL pts compared to HD, with reduced CD4+ (mean of 15.6 to 27% of total T cells) and CD8+ (7.8 to 3.6%) naive T cells and increased terminally differentiated CD8+ (3.4 to 8.7%) and CD4+ regulatory T cells (3.6 to 5.5%).
Comparing T-cell subsets in samples obtained prior to treatment with clinical characteristics revealed increased CD4+ effector cells (3.1 to 5%) in pts with large lymph nodes (≥5 cm), whereas CD4+ memory T cells were diminished in these pts (8.4 to 4.8%). Analysis of baseline T-cell cluster abundance and treatment outcomes revealed that pts achieving undetectable minimal residual disease at month 15 exhibited higher levels of CD4+ stem-cell memory (SCM) T cells prior to treatment (0.2 to 0.6%).
Comparing T-cell subsets in pre- and post-treatment samples across all arms revealed decreased terminally differentiated CD8+ T cells (8.7 to 4.8 %), CD4+ regulatory T cells (5.5 to 3.6 %), and CD4+ senescent (CD27-CD28-KLRG1+CD57+) (6.6 to 2.8%) T cells after treatment. Notably, a previously undescribed expansion of CD8+ senescent T cells was observed post-treatment (12.4 to 20.1%). Treatment-specific effects on T-cell subsets were also observed, with CIT-treated pts showing persistently low levels of CD4+ naive T cells (27 to 7.7%) compared to HD. In contrast, venetoclax-based treatments led to substantial recovery of CD4+ naive cells (15.3 to 23.6%) and reduction of CD8+ exhausted T cells (8.8 to 5.2%), compared to pre-treatment samples, with the most prominent reduction in the GIVe (venetoclax, obinutuzumab, and ibrutinib) treatment arm.
Quantification of metabolic enzyme expression levels revealed treatment-specific changes in T cells. Venetoclax-based treatment led to a metabolic profile more closely resembling that of healthy donors with a lower GLUT1, TOMM20 and CPT1α expression.
Conclusions
Our observations suggest a relationship between baseline CD4+ SCM T-cell phenotypes and clinical outcomes. Furthermore, this study provides insights into the impact of various CLL treatments on T-cell phenotypes and metabolism, potentially informing future therapeutic strategies and patient management.
A comprehensive analysis of these results, along with functional T-cell responses, is anticipated to be presented at the ASH 2024 conference.