STAT3 activation hallmarks human atypical T-betpos B cells and reveals IL-21–driven proliferative potential

Abstract Background Atypical B cells, epitomized by the CD21low T-betpos CD11cpos phenotype, expand in several immunological disorders. These putatively pathogenic cells have gained remarkable clinical interest, calling for better knowledge of molecular pathways leading to their generation and expansion. Recent studies unveil a role for toll-like receptors (TLRs), B-cell receptor (BCR), and IFN-γ and IL-21 JAK/STAT pathways, all concurring to their differentiation and expansion. While it is well established that IFN-γ is the main inducer of T-bet, thereby priming B cells toward atypical differentiation, the role of IL-21 is still unclear. IL-21 is a well-known activator of STAT3. Overactivation of STAT3 is linked with autoimmunity and lymphoproliferation, and germline gain-of-function mutations of the STAT3 gene associate with expansion of atypical CD21low T-betpos B cells. Moreover, constitutive activation of STAT3 in haematological malignancies is associated with uncontrolled proliferation. Aim The overarching aim of this study is to investigate atypical T-betpos B cells from patients with immune- dysregulation, including common variable immunodeficiency (CVID) and mixed cryoglobulinemia (MC), and healthy donors (HDs), focusing on basal and IL-21-mediated STAT3 activation. A further objective is to explore the effects of IL-21-mediated STAT3 activation on T-betpos B cells generated in vitro and to assess its functional significance. Methods and results PBMCs from CVID and MC patients, and from HDs, were processed with different fixation/permeabilization protocols to investigate, in parallel, T-bet, phosphorylated STAT3 (pSTAT3), and other markers, including atypical B cells hallmarks, such as CD21 and CD11c. We identified two distinct populations, a T-bethi subset with high CD11c and negative/low CD21 expression and a T-betdim subset with low CD11c and low/positive CD21 expression. Furthermore, we found that T-bethi B cells from 3 CVID patients were more frequently Ki-67pos than T-betdim B cells, suggesting a higher proliferative activity. This observation parallels a higher constitutive STAT3 activation in T-bethigh B cells compared to T-betneg and T-betdim B cells across all study groups (17 CVID, 7 MC and 15 HDs). Strikingly, short-term stimulation with IL-21 robustly increased pSTAT3 levels in T-betpos B cell subsets, with the most pronounced response observed in the T-bethigh population. In vitro, IFN-γ + anti-IgM co-stimulation of purified B cells from HDs induced high frequencies of T-betpos B cells, and the addition of IL-21 led to the generation of T-betpos pSTAT3pos B cell population mirroring those found in vivo. By labelling B cells with Cell Trace Violet (CTV) to monitor cell divisions, we observed that T-bet induction by anti-IgM + IFN-γ alone did not promote proliferation, whereas the combination of anti-IgM + IFN-γ + IL-21 led to robust STAT3 phosphorylation and significantly enhanced proliferative activity. Conclusions Our findings highlight a central role for IL-21-driven STAT3 activation in the generation, and expansion, of atypical T-betpos B cells bona fide identical to those found in vivo. STAT3 overactivation and proliferative activity are highest in T-bethi B cells, emphasizing these cells as the most IL-21-responsive and dynamically expanding population within the atypical B-cell compartment. Finally, these findings endorse the suggestion of targeting JAK/STAT3 signalling to control the accumulation of these cells in disease.