Helios+ and RORγt+ Treg populations are differentially regulated by MHCII, CD28, and ICOS to shape the intestinal Treg pool.

Foxp3+ regulatory T cells (Tregs) are essential for intestinal homeostasis. Tregs in the small intestine include Helios+ thymus-derived Tregs (tTregs) and RORγt+ Tregs that differentiate in the periphery after antigenic stimulation (pTregs). TCR and costimulatory signals sustain Tregs with effector phenotypes, including those in the intestine, but it is unknown if tTregs and pTregs have similar requirements for these pathways. We previously used mice lacking peripheral expression of MHCII to demonstrate that the small intestine sustains tTregs independently of peripheral antigen. Here, we show that the effector phenotype and tissue-resident signature of tTregs are also MHCII-independent. Using this model, we define the distinct costimulatory requirements of intestinal tTregs and pTregs. Helios+ effector tTregs proliferate through CD28 and require neither ICOS nor MHCII for maintenance. In contrast, RORγt+ pTregs use CD28 and ICOS. Notably, the differential costimulatory utilization allows tTregs and pTregs to dynamically respond to perturbations to support a fixed number of intestinal Tregs. This suggests that the environmental regulation of costimulatory ligands might shape the subpopulations of intestinal Tregs and promote effective homeostasis and defense. Our data reveal new complexity in effector Treg biology and costimulatory signaling of tTregs and pTregs and highlight the importance of analyzing both subpopulations.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

The Women of FOCIS: Promoting Equality and Inclusiveness in a Professional Federation of Clinical Immunology Societies.

The authors of this article, all women who have been deeply committed to the Federation of Clinical Immunology Societies (FOCIS), performed a retrospective analysis of gender equality practices of FOCIS to identify areas for improvement and make recommendations accordingly. Gender data were obtained and analyzed for the period from January 2010 to July 2021. Outcome measures included numbers of men and women across the following categories: membership enrollment, meeting and course faculty and attendees, committee and leadership composition. FOCIS' past and present leaders, steering committee members, FCE directors, individual members, as well as education, annual meeting scientific program and FCE committee members and management staff of FOCIS were surveyed by email questionnaire for feedback on FOCIS policies and practice with respect to gender equality and inclusion. Although women represent 50% of the membership, they have been underrepresented in all leadership, educational, and committee roles within the FOCIS organization. Surveying FOCIS leadership and membership revealed a growing recognition of disparities in female leadership across all FOCIS missions, leading to significant improvement in multiple areas since 2016. We highlight these changes and propose a number of recommendations that can be used by FOCIS to improve gender equality.

A review of signaling and transcriptional control in T follicular helper cell differentiation.

T follicular helper (Tfh) cells are a critical component of adaptive immunity and assist in optimal Ab-mediated defense. Multiple effector functions of Tfh support germinal center B cell survival, Ab class switching, and plasma cell maturation. In the past 2 decades, the phenotype and functional characteristics of GC Tfh have been clarified allowing for robust studies of the Th subset including activation signals and environmental cues controlling Tfh differentiation and migration during an immune response. A unique, 2-step differentiation process of Tfh has been proposed but the mechanisms underlying transition between unstable Tfh precursors and functional mature Tfh remain elusive. Likewise, newly identified transcriptional regulators of Tfh development have not yet been incorporated into our understanding of how these cells might function in disease. Here, we review the signals and downstream transcription factors that shape Tfh differentiation including what is known about the epigenetic processes that maintain Tfh identity. It is proposed that further evaluation of the stepwise differentiation pattern of Tfh will yield greater insights into how these cells become dysregulated in autoimmunity.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.

SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor ß (Tgf-ß) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Inflammatory CD4(+) T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII(+) ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4(+) T cells in the intestine and suggest that this process is dysregulated in human IBD.

Regulatory T cells occupy an isolated niche in the intestine that is antigen independent.

Regulatory T cells (Tregs) are CD4(+) T cells that maintain immune homeostasis and prevent autoimmunity. Like all CD4(+) T cells, Tregs require antigen-specific signals via T cell receptor-major histocompatibility complex class II (TCR-MHCII) interactions for their development. However, the requirement for MHCII in Treg homeostasis in tissues such as intestinal lamina propria (LP) is unknown. We examined LP Treg homeostasis in a transgenic mouse model that lacks peripheral TCR-MHCII interactions and generation of extrathymic Tregs (iTregs). Thymically generated Tregs entered the LP of weanlings and proliferated independently of MHCII to fill the compartment. The adult LP was a closed niche; new thymic Tregs were excluded, and Tregs in parabiotic pairs were LP resident. The isolated LP niche was interleukin-2 (IL-2) independent but dependent on commensal bacteria. Thus, an LP Treg niche can be filled, isolated, and maintained independently of antigen signals and iTregs. This niche may represent a tissue-specific mechanism for maintaining immune tolerance.

Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell?

Dendritic cells, macrophages and B cells are regarded as the classical antigen-presenting cells of the immune system. However, in recent years, there has been a rapid increase in the number of cell types that are suggested to present antigens on MHC class II molecules to CD4(+) T cells. In this Review, we describe the key characteristics that define an antigen-presenting cell by examining the functions of dendritic cells. We then examine the functions of the haematopoietic cells and non-haematopoietic cells that can express MHC class II molecules and that have been suggested to represent 'atypical' antigen-presenting cells. We consider whether any of these cell populations can prime naive CD4(+) T cells and, if not, question the effects that they do have on the development of immune responses.

Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

B cell antigen presentation in the initiation of follicular helper T cell and germinal center differentiation.

High-affinity class-switched Abs and memory B cells are products of the germinal center (GC). The CD4+ T cell help required for the development and maintenance of the GC is delivered by follicular Th cells (T(FH)), a CD4+ Th cell subset characterized by expression of Bcl-6 and secretion of IL-21. The cellular interactions that mediate differentiation of TFH and GC B cells remain an important area of investigation. We previously showed that MHC class II (MHCII)-dependent dendritic cell Ag presentation is sufficient for the differentiation of a T(FH) intermediate (termed pre-T(FH)), characterized by Bcl-6 expression but lacking IL-21 secretion. In this article, we examine the contributions of MHCII Ag presentation by B cells to T(FH) differentiation and GC responses in several contexts. B cells alone do not efficiently prime naive CD4+ T cells or induce T(FH) after protein immunization; however, during lymphocytic choriomeningitis virus infection, B cells induce T(FH) differentiation despite the lack of effector CD4+ T cell generation. Still, MHCII+ dendritic cells and B cells cooperate for optimal T(FH) and GC B cell differentiation in response to both model Ags and viral infection. This study highlights the roles for B cells in both CD4+ T cell priming and T(FH) differentiation, and demonstrates that different APC subsets work in tandem to mediate the GC response.

Dendritic cell-MHC class II and Itk regulate functional development of regulatory innate memory CD4+ T cells in bone marrow transplantation.

MHC class II (MHCII)-influenced CD4(+) T cell differentiation and function play critical roles in regulating the development of autoimmunity. The lack of hematopoietic MHCII causes autoimmune disease that leads to severe wasting in syngeneic recipients. Using murine models of bone marrow transplantation (BMT), we find that MHCII(-/-)→wild-type BMT developed disease, with defective development of innate memory phenotype (IMP, CD44(hi)/CD62L(lo)) CD4(+) T cells. Whereas conventional regulatory T cells are unable to suppress pathogenesis, IMP CD4(+) T cells, which include conventional regulatory T cells, can suppress pathogenesis in MHCII(-/-)→wild-type chimeras. The functional development of IMP CD4(+) T cells requires hematopoietic but not thymic MHCII. B cells and hematopoietic CD80/86 regulate the population size, whereas MHCII expression by dendritic cells is sufficient for IMP CD4(+) T cell functional development and prevention of pathogenesis. Furthermore, the absence of Tec kinase IL-2-inducible T cell kinase in MHCII(-/-) donors leads to preferential development of IMP CD4(+) T cells and partially prevents pathogenesis. We conclude that dendritic cells-MHCII and IL-2-inducible T cell kinase regulate the functional development of IMP CD4(+) T cells, which suppresses the development of autoimmune disorder in syngeneic BMTs.

Cutting edge: Conditional MHC class II expression reveals a limited role for B cell antigen presentation in primary and secondary CD4 T cell responses.

The activation, differentiation, and subsequent effector functions of CD4 T cells depend on interactions with a multitude of MHC class II (MHCII)-expressing APCs. To evaluate the individual contribution of various APCs to CD4 T cell function, we have designed a new murine tool for selective in vivo expression of MHCII in subsets of APCs. Conditional expression of MHCII in B cells was achieved using a cre-loxP approach. After i.v. or s.c. priming, partial proliferation and activation of CD4 T cells was observed in mice expressing MHCII only by B cells. Restricting MHCII expression to B cells constrained secondary CD4 T cell responses in vivo, as demonstrated in a CD4 T cell-dependent model of autoimmunity, experimental autoimmune encephalomyelitis. These results highlight the limitations of B cell Ag presentation during initiation and propagation of CD4 T cell function in vivo using a novel system to study individual APCs by the conditional expression of MHCII.

Adaptor protein-3 in dendritic cells facilitates phagosomal toll-like receptor signaling and antigen presentation to CD4(+) T cells.

Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic Toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4(+) T cell activation and Th1 effector cell function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores.

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold.

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

Asymmetric B cell division in the germinal center reaction.

Lifelong antibody responses to vaccination require reorganization of lymphoid tissue and dynamic intercellular communication called the germinal center reaction. B lymphocytes undergo cellular polarization during antigen stimulation, acquisition, and presentation, which are critical steps for initiating humoral immunity. Here, we show that germinal center B lymphocytes asymmetrically segregate the transcriptional regulator Bcl6, the receptor for interleukin-21, and the ancestral polarity protein atypical protein kinase C to one side of the plane of division, generating unequal inheritance of fate-altering molecules by daughter cells. Germinal center B lymphocytes from mice with a defect in leukocyte adhesion fail to divide asymmetrically. These results suggest that motile cells lacking constitutive attachment can receive provisional polarity cues from the microenvironment to generate daughter cell diversity and self-renewal.

Cutting edge: dendritic cell-restricted antigen presentation initiates the follicular helper T cell program but cannot complete ultimate effector differentiation.

Follicular helper T (T(FH)) cells are critical for germinal center (GC) formation. The processes that drive their generation and effector potential remain unclear. In this study, we define requirements for MHC class II APCs in murine T(FH) cell formation by either transiently ablating or restricting Ag presentation to dendritic cells (DCs). We find that cognate interactions with DCs are necessary and sufficient to prime CD4(+) T cells toward a CXCR5(+)ICOS(+)Bcl6(+) T(FH) cell intermediate. However, in the absence of additional APCs, these T(FH) cells fail to produce IL-21. Furthermore, in vitro priming of naive T cells by B cells engenders optimal production of IL-21, which induces a GC B cell transcriptional profile. These results support a multistep model for effector T(FH) cell priming and GC initiation, in which DCs are necessary and sufficient to induce a T(FH) cell intermediate that requires additional interactions with distinct APCs for full effector function.