Background: Bcl6 is required for the development of T follicular helper and regulatory (Tfh, Tfr) cells that regulate germinal center responses. Bcl6 also impacts the function of regulatory T (Treg) cells. Objective:The goal of this study is to define the functions of Bcl6 in Treg cells including Tfr cells in the context of allergic airway inflammation (AAI). Methods: We employed a model of house dust mite (HDM) sensitization to challenge wild type, Bcl6 fl/fl Foxp3-Cre and Prdm1(Blimp1) fl/fl Foxp3-Cre mice to study the reciprocal roles of Bcl6 and Blimp1 in AAI. Results: In the HDM model, Tfr cells repress the production of IgE and Bcl6+ Treg cells suppress the generation of type 2 cytokine producing cells in the lungs. In mice with Bcl6deficient Treg cells, twice as many ST2 (IL-33R) + Tregs develop as observed in wild type mice. ST2 + Tregs in the context of AAI are Blimp1-dependent, express type 2 cytokines, and share features of visceral adipose tissue Treg cells. Bcl6-deficient Tregs are more susceptible, and Blimp1-deficient Tregs are resistant, to acquiring the ST2 + Treg cell phenotype in vitro and in vivo in response to IL-33. Bcl6-deficient ST2+ Tregs but not Bcl6-deficient ST2+ T conventional cells strongly promote AAI when transferred into recipient mice. Lastly, ST2 is required for the exacerbated AAI in Bcl6 fl/fl Foxp3-Cre mice. Conclusions: During AAI, Bcl6 and Blimp1 play dual roles in regulating Tfr activity in the germinal center and in the development of ST2 + Tregs that promote type 2 cytokine responses. Koh page 4 4 Key Messages: • Tfr cells limit IgE production in mice challenged by airway allergen • Bcl6 and Blimp1 reciprocally regulate ST2 + Treg development • ST2 + Tregs promote allergic airway inflammation Capsule Summary: Bcl6 attenuates allergic disease by promoting Tfr cell development to repress the allergen-specific humoral response and by limiting expansion of ST2 + Tregs.

Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4 + T cells that secreted IL-9 as an obligate effector cytokine. IL-9–secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall–specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4 + T cell population was required for an allergen recall response.

Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell-independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice are acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 is expressed in neutrophils and activated by IL-12 via a Jak2-dependent pathway. We demonstrate that STAT4 is required for multiple neutrophil functions including IL-12-induced ROS production, chemotaxis, and production of the neutrophil extracellular traps.Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 results in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic tnf-α + CD8 + T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ + CD4 + T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet + CD4 + t cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.

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