Background. CAR T-cell therapy is the standard of care for R/R DLBCL, but more than half of patients fail to achieve long-term remission. Identification of cellular biomarkers in CAR T-cell infusion products (IPs) that predict complete remission beyond six months may guide the development of strategies to improve outcomes. Methods. IPs from 13 R/R DLBCL patients were analyzed using a 39-marker mass cytometry panel, comparing cell populations between long-term responders (R) and non-responders (NR). Both unsupervised and supervised analyses were performed. Longitudinal blood samples were analyzed for 30 days to track CAR T-cell subpopulation dynamics. Results. At a median follow-up of 13.5 months, median progression-free survival (PFS) was 13.3 months (95% CI: 9.7-24.3) in R (n=8) versus 3.5 months (95% CI: 0.5-5.4) in NR (n=5). The HR for PFS was 56.67 (95% CI: 7.3-439.3; P=0.0001). A subset of CD3+CXCR3+CD7+ CAR T-cells found in both CD4+ and CD8+ populations was significantly enriched in R and expressed higher levels of perforin, granzyme B, and NKG2D (restricted to CD8+). NR had more CXCR3+CD7+LAG3+ CAR T-cells. CD3, CD7, CXCR3, and NKG2D cell surface levels were higher in R, whereas LAG3, Ki67, and CD71 were elevated in NR. A predictive cut-off ratio of CD3+CXCR3+CD7+LAG3+CAR+ T-cells <0.83 and CD3+CXCR3+CD7+NKG2D+CAR+ T-cells >1.034 yielded a predictive accuracy of 0.92. Serum CXCL9 and CXCL10 levels were not different between groups. Conclusions. Increased frequency of CAR T-cells expressing CD7, CXCR3 and NKG2D in R versus LAG3 and CD71 in NR emerged as strong correlates of therapeutic outcome.

MO received honoraria and speaker fees from Moderna, Roche and BMS. C.A. holds patents and provisional patent applications in the field of engineered T cell therapies. C.A. receives licensing fees and royalties from Immatics (through previous institution Baylor College of Medicine), participated in advisory boards for Kite/ Gilead, Janssen and Celgene/ BMS, received sponsored travel from Gilead (through current institution Lausanne University Hospital (CHUV). G.C. has received honoraria from Bristol-Myers Squibb. CHUV has received honoraria for advisory services provided by G.C. to Iovance and EVIR. G.C. has received royalties from the University of Pennsylvania for CAR T cell therapy licensed to Novartis and Tmunity Therapeutics. G.C. has received royalties from the Ludwig Institute for Cancer Research, UNIL and CHUV for NeoTIL intellectual property previously licensed to Tigen Pharma. G.C. is inventor in technologies related to T cell expansion and engineering for T cell therapy. RS participated in advisory boards for Janssen, Celgene/ BMS, AbbVie, Takeda and Incyte.

This study did not receive any dedicated funding

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Cantonal Ethics Committee (CER-VD) of the Vaud Canton (Switzerland) gave ethical approval for this work

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