Human FOXP3⁺ regulatory T (T_reg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve T_reg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate T_reg cells into naïve, activated, and effector stages, and resolve the HLA-DR^hi, LIMS1^hi, highly suppressive FOXP3^hi, and highly proliferative MKI67^hi effector subsets. Trajectory analysis assembles T_reg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3^hi and MKI67^hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3^hi and MKI67^hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of T_reg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of T_reg complexity in health and disease.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen–antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.

Regulatory T (Treg) cells play central roles in maintaining immune homeostasis and self-tolerance. However, the molecular mechanisms underlying Treg cell homeostasis and suppressive function are still not fully understood. Here, we report that the deletion of another P subfamily members of the forkhead box (Foxp) subfamily member Foxp1 in Treg cells led to increased numbers of activated Treg (aTreg) cells at the expense of quiescent Treg cells, and also resulted in impaired Treg suppressive function. Mice with Foxp1-deficient Treg cells developed spontaneous inflammatory disease with age; they also had more severe inflammatory disease in colitis and experimental autoimmune encephalomyelitis (EAE) models. Mechanistically, we found that Foxp1 bound to the conserved noncoding sequence 2 (CNS2) element of the Foxp3 locus and helped maintain Treg suppressive function by stabilizing the Foxp3 expression. Furthermore, we found that Foxp1 and Foxp3 coordinated the regulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression levels. Taken together, our study demonstrates that Foxp1 plays critical roles in both maintaining Treg cell quiescence during homeostasis and regulating Treg suppressive function.

Previously we have shown that transcription factor Foxp1 plays an essential role in maintaining naive T cell quiescence; in the absence of Foxp1, mature naive CD8⁺ T cells proliferate in direct response to homeostatic cytokine IL-7. In this study, we report that the deletion of Foxp1 in naive CD8⁺ T cells leads to enhanced activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway and its downstream cell growth and metabolism targets in response to IL-7. We found that Foxp1 directly regulates PI3K interacting protein 1, a negative regulator of PI3K. Additionally, we found that deletion of Foxp1 in naive CD8⁺ T cells results in increased expression levels of E2fs, the critical components for cell cycle progression and proliferation, in a manner that is not associated with increased phosphorylation of retinoblastoma protein. Taken together, our studies suggest that Foxp1 enforces naive CD8⁺ T cell quiescence by simultaneously repressing key pathways in both cellular metabolism and cell cycle progression.

Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses.

Significance: T _FH cell differentiation and antigen-specific antibody production is critical for humoral responses. In this work, we show that extracellular matrix protein 1 (ECM1) is a critical positive regulator in T-follicular helper (T _FH ) differentiation by repressing IL-2–STAT5–Bcl6 signaling pathway. Importantly, ECM1 effectively enhanced T _FH differentiation, germinal center responses, and neutralizing antibody production both in antigen-immunized conditions and influenza infection, which indicate ECM1 may serve as a positive regulator for humoral responses in vivo.