CAR-T TEMRA and CAR-Tregs in vivo expansion are both associated with a poor response after CD19.CAR-T therapy

Chimeric antigen receptor T-cell (CAR-T) therapy has proven significant efficacy in B-cell malignancies with a subset of patients that fails to respond [ 1 , 2 , 3 ]. Several mechanisms have been proposed to account for the failure of CAR-T cell therapy, including T cell exhaustion and apoptosis [ 4 ], antigen loss [ 5 ] and resistance of cancer cells to apoptosis [ 6 ]. Furthermore, a recent study has demonstrated how the higher composition of the infusion product in terms of CD8 with memory features correlates with a favorable clinical response 3 months after the infusion [ 7 ]. A subsequent study showed, using single-cell transcriptome analysis performed at day 7 post infusion, that the expansion of memory-like CD8 + CAR-T cells occurred in patients responsive to the treatment [ 8 ]. In our study, we longitudinally monitored the expansion and exhaustion profiles of anti-CD19 CAR-T cells. In particular, we studied CD4 + CD19.CAR-T (CD4 + CAR-T) and CD8 + CD19.CAR-T (CD8 + CAR-T) lymphocytes for up to two years following CAR-T infusion and investigated their possible correlation with the clinical response. Thirty-seven patients with relapsed or refractory B-cell lymphomas treated with CD19-directed CAR T-cell therapy were included. The cohort consisted of 18 patients with diffuse large B-cell lymphoma (DLBCL), 7 with primary mediastinal B-cell lymphoma (PMBCL), 4 with transformed follicular lymphoma (tFL), 1 with transformed lymphoplasmocytic lymphoma (tLPL) and 7 with mantle cell lymphoma (MCL). Patients received axicabtagene ciloleucel ( n = 23), tisagenlecleucel ( n = 7), or brexucabtagene autoleucel ( n = 7). The median age at CAR-T infusion was 62 years (interquartile range, 52–68), and 56.8% of patients were male. Most patients presented advanced-stage disease (stage III–IV, 86.5%). Bridging therapy was administered in 78.4% of cases before CAR-T infusion. The study was approved by the local institutional ethics committee (RA/0404367) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before sample collection and analysis. Patients were classified as responders (complete or partial response) and non-responders (stable or progressive disease) based on clinical evaluation at the first response assessment after CAR-T infusion. Clinical outcomes and baseline characteristics of the cohort are summarized in Supplementary Table 1 . Peripheral blood (PB) samples were analyzed by multiparametric flow cytometry to assess the frequency and phenotype of circulating CAR-T cells and their subsets, as already reported [ 9 ]. The flow cytometry reagent list is shown in Supplementary Table 2 . In particular, CAR-T cells were identified as CD3⁺CAR⁺ lymphocytes. Within this population, CD4⁺ (CD3⁺CAR⁺CD4⁺) and CD8⁺ (CD3⁺CAR⁺CD8⁺) CAR-T subsets were quantified and expressed as percentage of CD3⁺CAR⁺ cells. The CD8⁺TEMRA CAR-T subset was defined as CD3⁺CAR⁺CD8⁺CD45RA⁺CCR7⁻ and expressed as percentage of CD3⁺CAR⁺CD8⁺ cells, while the frequency of total CD8⁺T cells was calculated within the CD3⁺CAR⁺ lymphocyte gate. Regulatory CAR-T cells (CAR-Tregs) were identified as CD3 + CAR + CD4 + CD25 + CD127 dim/− FOXP3 + lymphocytes. In addition, the expression of PD-1, LAG3, and TIM3 was evaluated within CD4⁺ and CD8⁺CAR-T cell subsets.