CD7-negative T cell immune reconstitution after CD7-targted CAR-T cell therapy

Abstract

Abstract Background Recently, CD7-targeted CAR-T cells have shown both safety and efficacy in early-stage clinical studies. More than 95% of T-cell malignancies and 30% of acute myeloid leukemia cells express CD7, rendering CD7 CAR-T cells to become a promising candidate for the development of next-generation cellular therapy. However, since >90% of normal T cells also express CD7, the potential of compromised T cell function following CD7 CAR-T cell therapy has become a major concern. We and others have reported the expansion of CD7-negative (CD7-) T cells in patients after CD7 CAR-T cell therapy. However, reactivation of EB virus (EBV) or cytomegalovirus (CMV) infection after CD7 CAR-T cell therapy has also been reported. It still remains largely unknown about the origin and function of these post-therapy CD7- T cells, which is critical for understanding the immune reconstitution of these patients. Methods Peripheral blood mononuclear cell (PBMC) samples from 10 patients underwent flow cytometry, single-cell RNA sequencing (scRNA-seq), and TCR sequencing (scTCR-seq). Comparative TCR profiling was performed on flow-sorted pre-treatment CD7+ T cells, pre-treatment CD7- T cells, and post-treatment CD7- T cells from three patients. Mechanistic studies employed immunofluorescence, Western blot, pharmacological inhibitors, and CD7 promoter methylation analysis (-300 to +200 region) via bisulfite sequencing PCR. Functional assessments included PMA/ionomycin and viral peptide (EBV/CMV) stimulation of PBMCs (pre-treatment, 4-week, and 4-month post-treatment timepoints), with evaluation of proliferation capacity, immunophenotypic shifts, and cytokine secretion profiles. Results Longitudinal analysis of 10 CD7 CAR-T cell responders revealed transcriptional and surface CD7 expression in endogenous T cells progressively declined post-infusion, with a transient surface CD7-negative and cytoplasmic CD7-positive (sCD7-cyCD7+) population peaking at 2 weeks before sCD7-cyCD7- T cells dominated by 4 months. TCR sequencing revealed post-treatment CD7- T cells originated from both pre-existing CD7+ and CD7- clones, demonstrating CD7 downregulation from CD7+ precursors as a key immune reconstitution mechanism after CD7 CAR-T cell therapy. Mechanistically, scRNA-seq identified clathrin-dependent endocytosis enrichment in endogenous T cells at 2 weeks post-infusion. In vitro modeling confirmed that upon contact with CD7 CAR-T cells, surface CD7 internalization via clathrin-mediated endocytosis and degradation through lysosome. Persistent CD7- T cells exhibited CD7 gene -300 to +200 region hypermethylation without alternative splicing, indicating epigenetic silencing enabled T cell escape from CAR-T cell-mediated killing. Functionally, single-cell analysis revealed CD7- T cells post-CAR-T cell therapy displayed memory/cytotoxic features with distinct granzyme/cytokine profiles. Despite reduced TCR diversity, patients maintained EBV/CMV-specific TCRs, though EBV-related complications occurred in one case. CD7+-derived clones exhibited enhanced TNF signaling and reduced exhaustion versus CD7--derived counterparts. Despite impaired costimulatory molecule expression (CD25/CD27/CD28), CD7- T cells maintained activation markers (CD69/CD137) and cytokine production (IFNγ/TNFα/IL1β) upon PMA/ionomycin and viral peptide stimulation, confirming preserved immune responsiveness. Conclusions This study was the first to elucidate the dynamic CD7 expression, mechanisms and function of endogenous T cells following CD7 CAR-T cell therapy. Our results have together revealed that normal T cells can escape the killing of CD7 CAR-T cells through clathrin-dependent endocytosis pathway and lysosomal degradation of CD7, followed by hyper-methylation at the CD7 gene locus. We have also shown that the immune function is partially retained in these CD7- T cells. We believe that this study has greatly contributed to the understanding of post-CAR-T cell immune reconstitution, which will be attractive to broad readers in the fields of immunology and oncology.