Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4+ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4+ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic TEM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with TCM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4+ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4+ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.

T follicular helper (Tfh) cells are an important component of germinal center (GC)-mediated humoral immunity. Yet, how a chronic type 1 versus protective type 2 helminth infection modulates Tfh-GC responses remains poorly understood. Here, we employ the helminth Trichuris muris model and demonstrate that Tfh cell phenotypes and GC are differentially regulated in acute versus chronic infection. The latter failed to induce Tfh-GC B cell responses, with Tfh cells expressing Τ-bet and interferon-γ. In contrast, interleukin-4-producing Tfh cells dominate responses to an acute, resolving infection. Heightened expression and increased chromatin accessibility of T helper (Th)1- and Th2 cell-associated genes are observed in chronic and acute induced Tfh cells, respectively. Blockade of the Th1 cell response by T-cell-intrinsic T-bet deletion promoted Tfh cell expansion during chronic infection, pointing to a correlation between a robust Tfh cell response and protective immunity to parasites. Finally, blockade of Tfh-GC interactions impaired type 2 immunity, revealing the critical protective role of GC-dependent Th2-like Tfh cell responses during acute infection. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic T. muris infection.

Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4(+) T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4(+) T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1(+)CTLA4(+)) cells in blood contain more HIV DNA compared with double-negative (PD1(-) CTLA4(-)) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6- d -ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.

CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.

The dynamics of CD4(+) T cell memory development remain to be examined at genome scale. In malaria-endemic regions, anti-malarial chemoprevention protects long after its cessation and associates with effects on CD4(+) T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (T(H)1) and follicular helper T (T-FH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T(H)1 and T-FH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T-FH and central memory were revealed, with antimalarials modulating these responses and boosting T(H)1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4(+) T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations (http://haquelab.mdhs.unimelb.edu.au/cd4_memory/).

Background Identifying the factors that contribute to chronicity in inflamed colitic tissue is not trivial. However, in mouse models of colitis, we can investigate at preclinical timepoints. We sought to validate murine Trichuris muris infection as a model for identification of factors that promote development of chronic colitis. Methods We compared preclinical changes in mice with a resolving immune response to T. muris (resistant) vs mice that fail to expel the worms and develop chronic colitis (susceptible). Findings were then validated in healthy controls and patients with suspected or confirmed IBD. Results The receptor for advanced glycation end products (RAGE) was highly dysregulated between resistant and susceptible mice before the onset of any pathological signs. Increased soluble RAGE (sRAGE) in the serum and feces of resistant mice correlated with reduced colitis scores. Mouse model findings were validated in a preliminary clinical study: fecal sRAGE was differentially expressed in patients with active IBD compared with IBD in remission, patients with IBD excluded, or healthy controls. Conclusions Preclinical changes in mouse models can identify early pathways in the development of chronic inflammation that human studies cannot. We identified the decoy receptor sRAGE as a potential mechanism for protection against chronic inflammation in colitis in mice and humans. We propose that the RAGE pathway is clinically relevant in the onset of chronic colitis and that further study of sRAGE in IBD may provide a novel diagnostic and therapeutic target.

CD4 + T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th cell lineage commitment and stability, and suggest that inhibition of DOT1L may provide a novel therapeutic strategy to limit type 2 immune responses.

The interaction between micronutrients and the immune system is emerging as a key control point in immune cell function in homeostasis and disease. For example, the vitamin A metabolite all-trans retinoic acid (atRA) has established roles in immune cell migration and effector function. Vitamin A deficiency results in a significant reduction in intestinal immune cells, which is thought to be associated with a lack of intestinal homing receptor expression and reduced migration to the intestine. However, the precise molecular pathways controlling intestinal immune cell homeostasis remains unclear. Here, we show that the atRA-inducible BTB-POZ transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) controls intestinal T cell residency. Mice with a T cell-intrinsic deletion of HIC1 (Hic1ΔT mice) have reduced numbers of CD69+ CD103+ tissue resident memory T (TRM) cells in the intestine, demonstrating that the atRA/HIC1 axis is critical for establishing and maintaining TRM cells. Further, following influenza A virus infection, numbers of TRM cells in the lungs are significantly reduced in Hic1ΔT mice, as well as in mice treated with the pan-retinoic acid receptor inhibitor BMS493, or in mice with a T cell-specific deletion of RARa (RaraΔT mice). Consistent with this, activation of naive CD8+ T cells from mice or humans in the presence of IL-2, IL-15, TGFβ and atRA results in the generation of CD69+ CD103+ ‘TRM-like’ cells that share the transcriptional profile of bona fide TRM cells. Taken together, these results identify atRA as a critical regulator of T cell tissue residency, acting by inducing expression of HIC1 and promoting the tissue residency gene program.

CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th cell lineage commitment and stability, and suggest that inhibition of DOT1L may provide a novel therapeutic strategy to limit type 2 immune responses. SUMMARY Histone methyltransferases comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, Scheer et al. report that DOT1L is a key regulator of the Th cell lineage and integrity, thereby affecting Th cell-dependent responses at mucosal sites.

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.

Mast cells are innate immune cells that respond rapidly to infection in barrier tissues such as the skin and intestinal mucosa. Expulsion of parasitic worms in the gut involves a robust type 2 host response, and an acute mastocytosis is often generated at the site of infection. However, the role of mast cells in resistance to worm infections appears to be parasite specific. Mast cells are also involved in tissue repair, but the long‐term contribution of mast cell activation after worm expulsion has not been definitively studied. In this issue of European Journal of Immunology , Sorobetea et al. [Eur. J. Immunol. 2017. 47 : 257–268] demonstrate that activated mast cells persist in the large intestinal lamina propria and intraepithelial compartment long after worm expulsion, resulting in continued local and systemic presence of the mast cell protease mast cell protease 1 (MCPt‐1) and enhanced intestinal permeability. In this commentary, we discuss these findings in the wider context of mast cell function in health and disease.

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor Hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.

In animal-based biomedical research, both the sex and the age of the animals studied affect disease phenotypes by modifying their susceptibility, presentation and response to treatment. The accurate reporting of experimental methods and materials, including the sex and age of animals, is essential so that other researchers can build on the results of such studies. Here we use text mining to study 15,311 research papers in which mice were the focus of the study. We find that the percentage of papers reporting the sex and age of mice has increased over the past two decades: however, only about 50% of the papers published in 2014 reported these two variables. We also compared the quality of reporting in six preclinical research areas and found evidence for different levels of sex-bias in these areas: the strongest male-bias was observed in cardiovascular disease models and the strongest female-bias was found in infectious disease models. These results demonstrate the ability of text mining to contribute to the ongoing debate about the reproducibility of research, and confirm the need to continue efforts to improve the reporting of experimental methods and materials.

Isotype control staining. Representative figures showing isotype control staining in small and large intestine. Scale Bar represents 100Â Iźm. (PDF 2178Â kb)

BACKGROUND:Eosinophils are innate immune cells present in the intestine during steady state conditions. An intestinal eosinophilia is a hallmark of many infections and an accumulation of eosinophils is also observed in the intestine during inflammatory disorders. Classically the function of eosinophils has been associated with tissue destruction, due to the release of cytotoxic granule contents. However, recent evidence has demonstrated that the eosinophil plays a more diverse role in the immune system than previously acknowledged, including shaping adaptive immune responses and providing plasma cell survival factors during the steady state. Importantly, it is known that there are regional differences in the underlying immunology of the small and large intestine, but whether there are differences in context of the intestinal eosinophil in the steady state or inflammation is not known.RESULTS:Our data demonstrates that there are fewer IgA(+) plasma cells in the small intestine of eosinophil-deficient ΔdblGATA-1 mice compared to eosinophil-sufficient wild-type mice, with the difference becoming significant post-infection with Toxoplasma gondii. Remarkably, and in complete contrast, the absence of eosinophils in the inflamed large intestine does not impact on IgA(+) cell numbers during steady state, and is associated with a significant increase in IgA(+) cells post-infection with Trichuris muris compared to wild-type mice. Thus, the intestinal eosinophil appears to be less important in sustaining the IgA(+) cell pool in the large intestine compared to the small intestine, and in fact, our data suggests eosinophils play an inhibitory role. The dichotomy in the influence of the eosinophil over small and large intestinal IgA(+) cells did not depend on differences in plasma cell growth factors, recruitment potential or proliferation within the different regions of the gastrointestinal tract (GIT).CONCLUSIONS:We demonstrate for the first time that there are regional differences in the requirement of eosinophils for maintaining IgA+ cells between the large and small intestine, which are more pronounced during inflammation. This is an important step towards further delineation of the enigmatic functions of gut-resident eosinophils.

BACKGROUND:Current understanding of the onset of inflammatory bowel diseases relies heavily on data derived from animal models of colitis. However, the omission of information concerning the method used makes the interpretation of studies difficult or impossible. We assessed the current quality of methods reporting in 4 animal models of colitis that are used to inform clinical research into inflammatory bowel disease: dextran sulfate sodium, interleukin-10, CD45RB T cell transfer, and 2,4,6-trinitrobenzene sulfonic acid (TNBS). METHODS:We performed a systematic review based on PRISMA guidelines, using a PubMed search (2000-2014) to obtain publications that used a microarray to describe gene expression in colitic tissue. Methods reporting quality was scored against a checklist of essential and desirable criteria. RESULTS:Fifty-eight articles were identified and included in this review (29 dextran sulfate sodium, 15 interleukin-10, 5 T cell transfer, and 16 TNBS; some articles use more than 1 colitis model). A mean of 81.7% (SD = ±7.038) of criteria were reported across all models. Only 1 of the 58 articles reported all essential criteria on our checklist. Animal age, gender, housing conditions, and mortality/morbidity were all poorly reported. CONCLUSIONS:Failure to include all essential criteria is a cause for concern; this failure can have large impact on the quality and replicability of published colitis experiments. We recommend adoption of our checklist as a requirement for publication to improve the quality, comparability, and standardization of colitis studies and will make interpretation and translation of data to human disease more reliable.