Abstract
Background: CD19-directed CART cell (CART19) therapy is standard of care for relapsed/refractory large B-cell lymphomas (r/r LBCL), but only one-third of patients (pts) maintain remission beyond 5 years. Mechanisms of resistance to CART range from antigen-negative escape to CART cell dysfunction. Recent studies have shown that regulatory or exhausted CART phenotypes correlate with poor outcomes. Emerging data also suggest that the myeloid compartment actively participates in shaping CART functionality. However, a comprehensive understanding of the longitudinal interactions between myeloid cells and CART cells remains largely unexplored.
Method: We conducted a longitudinal study of 15 r/r LBCL pts who received third-line or later 4-1BB–based CART19 (CTL019 [NCT02030834] or Tisa-cel [UPCC37418]). We selected eight patients who achieved a complete response (CR) that lasted over a year and seven who experienced progressive disease (PD). CR and PD pt groups were balanced for histology, age, prior therapy, CART19 type, and toxicities. The median follow-up was 4.9 years. A total of 67 peripheral blood mononuclear cell samples obtained at apheresis, prior to infusion, peak expansion, and day 28 post-infusion were sequenced using single-cell (sc) CITE-Seq and scTCR sequencing. We analyzed differentially expressed genes (DEGs) in annotated myeloid and lymphocyte cell types comparing CR and PD pts, followed by gene set enrichment analysis (GSEA). We also analyzed TCR clonality, cell-cell communication (CCC) using LIANA, and longitudinal cell fate with Moscot.
Results: A total of 65,618 CART cells and 298,855 non-CAR cells were sequenced. Although infusion products were enriched for CD4+ T cells, CR pts exhibited significant post-infusion expansion of CD8+ effector memory (EM) CART cells. TCR clonal dynamics revealed that CART19 infusion products primarily contain singleton clones. High proportions of longitudinally shared TCR clones associate with preserved lymphocyte populations after lymphodepletion, not response, suggesting clonal persistence associates with immune resilience. In myeloid lineages on day 0, blood dendritic cells (DCs) were more abundant in CR pts. After infusion, we observed increased CD16+ monocytes and a subset of ISG15+ CD14+ monocytes in PD pts.
DEG analysis in CD8+ CART cells showed genes in the interferon (IFN)-induced (IFI) and GTPase (GIMAP) families were highly expressed at multiple time points in PD pts. GSEA with these genes confirmed that CD8+ CART cells in PD pts exhibited increased IFN signaling after infusion. In contrast, CR pts showed increased tumor necrosis factor signaling, suggesting distinct immune-activated pathways depending on clinical outcome. Interestingly, in monocytes and DCs, DEG analysis revealed a similar but stronger IFN gene expression signature (IFI44L, ISG15, IFITM1, MX1, IRF7) in PD pts from day 0 to 28. This finding is further supported by GSEA, which shows that IFN signaling pathways are the most significant in PD pts across myeloid lineages. At peak expansion, higher levels of IFNγ were also detected in the serum of PD pts in an additional cohort of 39 CART19-treated LBCL pts. Altogether, this suggests a complex interaction among immune cells via IFN signaling that begins at day 0 and continues after CART infusion.
Supporting this interaction, cell fate trajectory analysis revealed that IFN-associated genes, as well as activation/checkpoint inhibitor genes (CD38, TIGIT, LAG3), drive the transcriptomic evolution towards CD8+ EM CART cells in PD pts post-infusion. Notably, this gene signature was diminished in CR pts. In contrast, memory-associated genes (TCF7, SELL) were positively correlated with the CD8+ EM transition in CR pts. CCC analysis between monocytes and DCs with CD8+ EM CART cells confirmed baseline IFNγ signaling (IFNG/IFNGR) in all pts post-infusion. This suggests that the strength of signaling, rather than its mere presence, contributes to CART19 therapy outcomes. We also observed an increase in IFNγ-stimulated interactions (HLA-DR/LAG3) between these myeloid lineages and CD8+ EM CART cells in PD pts.
Conclusions: Elevated IFNγ signaling in myeloid and CAR T cell compartments, pre- and post-infusion, correlates with CART19 resistance in r/r LBCL. This dysfunctional IFN signaling leads to increased HLA-DR expression, which could enhance HLA-DR and LAG3 signaling between myeloid cells and CD8+ EM CART cells, influencing CART cell function and treatment response.
