TFH subsets in patients after allogenic transplant are dysregulated prior to chronic GVHD development

Abstract

Abstract Background: Chronic graft-versus-host disease (cGvHD) remains a major cause of late morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Aberrant germinal center (GC) activity, driven by dysregulated T-B cell interactions, contributes to disease pathogenesis. PD-1+ CXCR5+ follicular helper T (TFH) cells orchestrate GC responses by promoting memory B cell and plasmablast formation, and elevated circulating (c) TFH frequencies have been linked to cGvHD severity. Here, we report the identification of a cytotoxic TFH subset, previously published by us and referred to as killer TFH or TFK cells, co-expressing canonical TFH markers with high levels of the cytotoxic effector molecules NKG-7 and granzyme B (GRZB). This dual helper–killer phenotype suggests a unique capacity to both disrupt GC homeostasis and mediate direct tissue injury on MHC-II-upregulating epithelia. Integrating these cells into the immunopathogenic framework of cGvHD offers a novel perspective on disease initiation and progression. Methods: In a prospective trial, peripheral blood from 80 allo-HSCT patients was collected on median day +113 (range +91 to +190). In 24 patients, additional samples were obtained at cGvHD onset (median day +187, range +98 to +410) and 4 and 8 weeks later (9 de novo, 15 quiescent/progressive cases). CD4+ T cell subsets, particularly cTFH cells, were analyzed by flow cytometry. Results: At a median of day +113 post allo-HSCT, patients without current or future GvHD exhibited significantly higher frequencies of PD-1+ CXCR5+ cTFH cells (median 7.98%, range 16.92%) compared to those who later developed cGvHD (median 3.81%, range 21.36%, p<0.002). In the latter group, cTFH frequencies were already reduced at day +113 and remained low following disease onset.Subtype analysis revealed that cTFH1 (CXCR3+CCR6-) and cTFH1/17 (CXCR3+CCR6+) frequencies at day +113 were markedly elevated in patients who never developed GvHD (cTFH1: median 4.38%, range 13.25%; cTFH1/17: median 1.18%, range 4.2%) compared to those with subsequent disease (cTFH1: median 2.53%, range 9.96%, p<0.03; cTFH1/17: median 0.31%, range 2.02%, p<0.0002). cTFH2 (CXCR3-CCR6-) and cTFH17 (CXCR3-CCR6+) frequencies declined from day +113 to disease onset (week 0) in untreated cGvHD patients.A prominent difference was also observed for cytotoxic NKG-7+ (anti-TIA-1) GRZB+ CD4+ T cells: Patients not developing any GvHD later on displayed significantly higher levels of NKG-7+ and NKG-7+ GRZB+ T cells at day +113 (NKG-7+: 24.56%, range 64.85%; NKG-7+ GRZB+: median 6.03%, range 23.00%, p<0.03) than patients who later developed cGvHD (NKG-7+: median 12.07%, range 51.98%; NKG-7+ GRZB+: median 0.99%, range 25.95%). A trend mirrored in the TFH compartment, where killer cTFH cells (NKG-7+ GRZB+) were most abundant in patients without developing any form of GvHD.Receiver operating characteristic (ROC) analysis assessed on day +113 identified several populations with strong prognostic potential subsequent for cGvHD: cTFH cells (AUC = 0.77; p = 0.0019), NKG-7+ GRZB+ CD4+ cells (AUC = 0.73; p = 0.0081), cTFH1 cells (AUC = 0.73; p = 0.0072), and cTFH1/17 cells (AUC = 0.84; p<0.0001). Conclusion: Our findings reveal that early post-transplant preservation of cTFH subsets, particularly cTFH1, cTFH1/17, and killer cTFH cells, can be found in patients without future or present cGvHD, whereas their decline in peripheral blood precedes disease onset. The identification of a killer TFH phenotype with high GRZB and NKG-7 expression adds a novel layer to the immunopathogenesis of cGvHD. Furthermore, the predictive value of the cytotoxic NKG-7+ GRZB+ CD4+T cells indicates a potential benefit for risk stratification and will be correlated further with clinical parameters.