Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion.

The thymus contains a diversity of dendritic cells (DCs) that exist in defined locations and have different antigen-processing and -presenting features. This suggests that they play nonredundant roles in mediating thymocyte selection. In an effort to eliminate SIRPα+ classic DC2 subsets, we discovered that a substantial proportion expresses the surface lectin, CD301b, in the thymus. These cells resemble the CD301b+ type 2 immune response promoting DCs that are present in the skin-draining lymph nodes. Transcriptional and phenotypic comparison to other DC subsets in the thymus revealed that thymic CD301b+ cDCs represent an activated state that exhibits enhanced antigen processing and presentation. Furthermore, a CD301b+ cDC2 subset demonstrated a type 2 cytokine signature and required steady-state interleukin-4 receptor signaling. Selective ablation of CD301b+ cDC2 subsets impaired clonal deletion without affecting regulatory T cells (Treg cells). The T cell receptor α repertoire sequencing confirmed that a cDC2 subset promotes deletion of conventional T cells with minimal effect on Treg cell selection. Together, these findings suggest that cytokine-induced activation of DCs in the thymus substantially enforces central tolerance.

Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Eosinophils orchestrate cancer rejection by normalizing tumor vessels and enhancing infiltration of CD8(+) T cells.

Tumor-associated eosinophilia is frequently observed in cancer. However, despite numerous studies of patients with cancer and mouse models of cancer, it has remained uncertain if eosinophils contribute to tumor immunity or are mere bystander cells. Here we report that activated eosinophils were essential for tumor rejection in the presence of tumor-specific CD8(+) T cells. Tumor-homing eosinophils secreted chemoattractants that guided T cells into the tumor, which resulted in tumor eradication and survival. Activated eosinophils initiated substantial changes in the tumor microenvironment, including macrophage polarization and normalization of the tumor vasculature, which are known to promote tumor rejection. Thus, our study presents a new concept for eosinophils in cancer that may lead to novel therapeutic strategies.

Type III interferon drives thymic B cell activation and regulatory T cell generation.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Sterile production of interferons in the thymus affects T cell repertoire selection.

Type I and III interferons (IFNs) are robustly induced during infections and protect cells against viral infection. Both type I and III IFNs are also produced at low levels in the thymus at steady state; however, their role in T cell development and immune tolerance is unclear. Here, we found that both type I and III IFNs were constitutively produced by a very small number of AIRE+ murine thymic epithelial cells, independent of microbial stimulation. Antigen-presenting cells were highly responsive to thymic IFNs, and IFNs were required for the activation and maturation of thymic type 1 conventional dendritic cells, macrophages, and B cells. Loss of IFN sensing led to reduced regulatory T cell selection, reduced T cell receptor (TCR) repertoire diversity, and enhanced autoreactive T cell responses to self-antigens expressed during peripheral IFN signaling. Thus, constitutive exposure to IFNs in the thymus is required for generating a tolerant and diverse TCR repertoire.

An OGT-STAT5 Axis in Regulatory T Cells Controls Energy and Iron Metabolism.

The immunosuppressive regulatory T (Treg) cells exert emerging effects on adipose tissue homeostasis and systemic metabolism. However, the metabolic regulation and effector mechanisms of Treg cells in coping with obesogenic insults are not fully understood. We have previously established an indispensable role of the O-linked N-Acetylglucosamine (O-GlcNAc) signaling in maintaining Treg cell identity and promoting Treg suppressor function, via STAT5 O-GlcNAcylation and activation. Here, we investigate the O-GlcNAc transferase (OGT)-STAT5 axis in driving the immunomodulatory function of Treg cells for metabolic homeostasis. Treg cell-specific OGT deficiency renders mice more vulnerable to high-fat diet (HFD)-induced adiposity and insulin resistance. Conversely, constitutive STAT5 activation in Treg cells confers protection against adipose tissue expansion and impaired glucose and insulin metabolism upon HFD feeding, in part by suppressing adipose lipid uptake and redistributing systemic iron storage. Treg cell function can be augmented by targeting the OGT-STAT5 axis to combat obesity and related metabolic disorders.

Intestinal CD8αα IELs derived from two distinct thymic precursors have staggered ontogeny.

CD8αα intraepithelial lymphocytes (IELs) are abundant T cells that protect the gut epithelium. Their thymic precursors (IELps) include PD-1+ type A and Tbet+ type B populations, which differ in their antigen-receptor specificities. To better understand CD8αα IEL ontogeny, we performed "time-stamp" fate mapping experiments and observed that it seeds the intestine predominantly during a narrow time window in early life. Adoptively transferred IELps parked better in the intestines of young mice than in adults. In young mice, both type A and type B IELps had an S1PR1+ and α4ß7+ emigration- and mucosal-homing competent phenotype, while this was restricted to type A IELps in adults. Only CD8αα IELs established in early life were enriched in cells bearing type B IELp TCR usage. Together, our results suggest that the young intestine facilitates CD8αα IEL establishment and that early IELs are distinct from IELs established after this initial wave. These data provide novel insight into the ontogeny of CD8αα IELs.

The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation.

Regulatory T (Treg) cells control self-tolerance, inflammatory responses and tissue homeostasis. In mature Treg cells, continued expression of FOXP3 maintains lineage identity, while T cell receptor (TCR) signaling and interleukin-2 (IL-2)/STAT5 activation support the suppressive effector function of Treg cells, but how these regulators synergize to control Treg cell homeostasis and function remains unclear. Here we show that TCR-activated posttranslational modification by O-linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. Collectively, our data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells.