CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells

{"Background": "Chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD). CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. While CD57+ CD4+ T cells are normally rare in blood, their phenotype remains controversial\u2014they have been described as exhausted, senescent, or cytotoxic. CTLA4 haploinsufficiency is a Mendelian syndrome of T-cell hyperactivation and autoimmunity that often results in hypogammaglobulinemia and reduced B cells. The authors hypothesized that analysis of T cells from PAD patients could provide insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects.", "Methods": "Blood samples were analyzed from 27 healthy donors, 24 patients with PAD, and 11 CTLA4+/- individuals. Paired blood and tonsil samples were obtained from donors undergoing routine tonsillectomy. Flow cytometry was used to quantify CD57+ CD4+ T cells and granzyme expression. Bulk RNA sequencing was performed on sorted CD57+ CD4+ TEM, CD57+ CD4+ TEMRA, CD57- CD4+ TEM, and CD57+ CD8+ T cells from three healthy donors. Single-cell RNA-seq and TCR sequencing were performed on matched blood and tonsillar CD57+ and CD57- CD4+ T cells. In vitro cytotoxicity assays were conducted using Raji cells and primary B cells cocultured with CD4cyt. In vivo experiments used C57BL/6 mice infected with vaccinia virus (VACV) and treated with anti-CTLA4 antibody or isotype control. Cancer patients (n=7) receiving ipilimumab/nivolumab combination therapy were analyzed before and after treatment.", "Key Results": "Circulating CD57+ CD4+ T cells were significantly increased in PAD patients (mean 5.9% vs. 2.7% in healthy donors) and markedly increased in CTLA4 haploinsufficiency (mean 20.2%). Blood CD57+ CD4+ T cells expressed a comprehensive cytotoxic transcriptome similar to CD8+ effector cells, with 193 genes differentially expressed between CD57+ CD4+ TEM and CD57- CD4+ TEM (FDR<0.05), while only 6 genes differed between CD57+ CD4+ TEM and CD57+ CD8+ cells. Approximately 50% of blood CD57+ CD4+ T cells expressed granzyme A and B, compared to only 1-4% of tonsillar CD57+ CD4+ T cells. Single-cell RNA-seq revealed that tonsillar CD57+ CD4+ T cells exhibited a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression with constitutive high-level CTLA4 expression, while blood CD4cyt showed features of terminal effector cells (KLRG1, PRDM1, TBX21). TCR analysis showed >80% of CD4cyt expressed TCRs from substantially expanded clones. CD4cyt mediated dose-dependent killing of SEB-pulsed Raji cells and primary B cells via granzyme B, and reduced plasmablast formation. In CTLA4+/- individuals, almost all CD4cyt were GzmB+, with increased CD107a expression and reduced TCF1 expression. CTLA4 blockade in VACV-infected mice significantly increased GzmA and GzmB expression in CD4+ T cells but not CD8+ T cells. In cancer patients, GzmA and GzmB expression in CD4cyt increased after anti-CTLA4/PD-1 therapy.", "Clinical Implications": "CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, restraining the formation of cytotoxic CD4+ T cells. The pathological CD4cyt phenotype is accentuated by infection and CTLA4 deficiency. CD4cyt can kill B cells and inhibit B-cell responses, suggesting they may contribute to the B-cell defects and hypogammaglobulinemia observed in CTLA4 haploinsufficiency. The inverse relationship between CD4cyt and memory B cells in PAD patients supports this mechanism. These findings provide new insights into immune pathology arising from gene-environment interactions and may explain the otherwise unexplained immune deficiency in CTLA4 haploinsufficiency and other immune dysregulation syndromes. The study also demonstrates that CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalized between blood and germinal centers, with GC-Tfh remaining refractory to becoming cytotoxic even after CTLA4 inhibition."}