The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.

CircPTPN22 modulates T-cell activation by sponging miR-4689 to regulate S1PR1 expression in patients with systemic lupus erythematosus.

BACKGROUND: Circular RNAs are involved in autoimmune disease pathogenesis. Our previous study indicated that circPTPN22 is involved in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the underlying mechanisms remain unclear. METHODS: First, the expression of circPTPN22 was detected by real-time PCR and western blotting. After overexpression or knockdown of circPTPN22, the proliferation of Jurkat cells was detected by the CCK-8 assay, and the apoptosis of Jurkat cells was detected by flow cytometry. In addition, the relationship between circPTPN22-miR-4689-S1PR1 was confirmed by bioinformatic analyses, fluorescence in situ hybridization assays, RNA-binding protein immunoprecipitation, and dual luciferase reporter assays. RESULTS: We found that circPTPN22 expression was downregulated in the PBMCs of SLE patients compared to those of healthy controls. Overexpression of circPTPN22 increased proliferation and inhibited apoptosis of Jurkat T cells, whereas knockdown of circPTPN22 exerted the opposite effects. CircPTPN22 acts as a miR-4689 sponge, and S1PR1 is a direct target of miR-4689. Importantly, the circPTPN22/miR-4689/S1PR1 axis inhibited the secretion of TNF-α and IL-6 in Jurkat T cells. CONCLUSIONS: CircPTPN22 acts as a miR-4689 sponge to regulate T-cell activation by targeting S1PR1, providing a novel mechanism for the pathogenesis of SLE.

Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.

HIV-1 DNA and Immune Activation Levels Differ for Long-Lived T-Cells in Lymph Nodes, Compared with Peripheral Blood, during Antiretroviral Therapy.

Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4+ and CD8+ T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4+ T-cell subsets. Immune activation was significantly higher in all CD8+ T-cell subsets, and memory CD8+ T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8+ T-cells. The differences seen in CM and TM CD4+ T-cell subsets were more pronounced among participants with CD4+ T-cell counts of <500 cells/µL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4+ T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4+ and CD8+ T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4+ T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4+ and CD8+ T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.

VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance.

V domain immunoglobulin suppressor of T-cell activation (VISTA) is a premier target for cancer treatment due to its broad expression in many cancer types and enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colorectal cancer model, monotherapy of small tumors with anti-VISTA resulted in slowed tumor growth. In a combination therapy setting, large CT26 tumors showed complete adaptive resistance to anti-PD-1/CTLA-4, but inclusion of anti-VISTA led to rejection of half the tumors. Mechanisms of enhanced antitumor immunity were investigated using single-cell RNA sequencing (scRNA-seq), multiplex image analysis, and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated antigen presentation pathways and reduced myeloid-mediated suppression. Imaging revealed an anti-VISTA stimulated increase in contacts between T cells and myeloid cells, further supporting the notion of increased antigen presentation. scRNA-seq of tumor-specific CD8+ T cells revealed that anti-VISTA therapy induced T-cell pathways highly distinct from and complementary to those induced by anti-PD-1 therapy. Whereas anti-CTLA-4/PD-1 expanded progenitor exhausted CD8+ T-cell subsets, anti-VISTA promoted costimulatory genes and reduced regulators of T-cell quiescence. Notably, this is the first report of a checkpoint regulator impacting CD8+ T-cell quiescence, and the first indication that quiescence may be a target in the context of T-cell exhaustion and in cancer. This study builds a foundation for all future studies on the role of anti-VISTA in the development of antitumor immunity and provides important mechanistic insights that strongly support use of anti-VISTA to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4. See related Spotlight by Wei, p. 3.

LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

Surveying Mechanisms of Immune Checkpoint Blockade from a VISTA.

Defining the mechanisms of action of immune checkpoint blockade therapies is essential for effectively designing combination therapeutic approaches and developing the next generation of immunotherapies. In this issue, Schaafsma and colleagues report that V-domain immunoglobulin suppressor of T-cell activation antagonism acts through mechanisms distinct from anti-CTLA-4 and anti-programmed cell death protein 1 via remodeling of the myeloid-cell compartment and modulating T-cell quiescence. See related article by Schaafsma et al. p. 38 (1).

A Morbillivirus Infection Shifts DC Maturation Toward a Tolerogenic Phenotype to Suppress T Cell Activation.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Monocyte subsets, T cell activation profiles, and stroke in men and women: The Multi-Ethnic Study of Atherosclerosis and Cardiovascular Health Study.

BACKGROUND AND AIMS: Despite mechanistic data implicating unresolving inflammation in stroke pathogenesis, data regarding circulating immune cell phenotypes - key determinants of inflammation propagation versus resolution - and incident stroke are lacking. Therefore, we aimed to comprehensively define associations of circulating immune phenotypes and activation profiles with incident stroke. METHODS: We investigated circulating leukocyte phenotypes and activation profiles with incident adjudicated stroke in 2104 diverse adults from the Multi-Ethnic Study of Atherosclerosis (MESA) followed over a median of 16.6 years. Cryopreserved cells from the MESA baseline examination were thawed and myeloid and lymphoid lineage cell subsets were measured using polychromatic flow cytometry and intracellular cytokine activation staining. We analyzed multivariable-adjusted associations of cell phenotypes, as a proportion of parent cell subsets, with incident stroke (overall) and ischemic stroke using Cox regression models. RESULTS: We observed associations of intermediate monocytes, early-activated CD4+ T cells, and both CD4+ and CD8+ T cells producing interleukin-4 after cytokine stimulation (Th2 and Tc2, respectively) with higher risk for incident stroke; effect sizes ranged from 35% to 62% relative increases in risk for stroke. Meanwhile, differentiated and memory T cell phenotypes were associated with lower risk for incident stroke. In sex-stratified analyses, positive and negative associations were especially strong among men but null among women. CONCLUSIONS: Circulating IL-4 producing T cells and intermediate monocytes were significantly associated with incident stroke over nearly two decades of follow-up. These associations were stronger among men and not among women. Further translational studies are warranted to define more precise targets for prognosis and intervention.

Negative times negative equals positive, THEMIS sets the rule on thymic selection and peripheral T cell responses.

The activity of T cells is finely controlled by a set of negative regulators of T-cell antigen receptor (TCR)-mediated signaling. However, how those negative regulators are themselves controlled to prevent ineffective TCR-mediated responses remain poorly understood. Thymocyte-expressed molecule involved in selection (THEMIS) has been characterized over a decade ago as an important player of T cell development. Although the molecular function of THEMIS has long remained puzzling and subject to controversies, latest investigations suggest that THEMIS stimulates TCR-mediated signaling by repressing the tyrosine phosphatases SHP-1 and SHP-2 which exert regulatory function on T cell activation. Recent evidences also point to a role for THEMIS in peripheral T cells beyond its role on thymic selection. Here, we present an overview of the past research on THEMIS in the context of T cell development and peripheral T cell function and discuss the possible implication of THEMIS-based mechanisms on TCR-dependent and independent signaling outcomes.

Contribution of Dendritic Cell Subsets to T Cell-Dependent Responses in Mice.

BATF3-deficient mice that lack CD8+ dendritic cells (DCs) showed an exacerbation of chronic graft-versus-host disease (cGVHD), including T follicular helper (Tfh) cell and autoantibody responses, whereas mice carrying the Sle2c2 lupus-suppressive locus with a mutation in the G-CSFR showed an expansion of CD8+ DCs and a poor mobilization of plasmacytoid DCs (pDCs) and responded poorly to cGVHD induction. Here, we investigated the contribution of CD8+ DCs and pDCs to the humoral response to protein immunization, where CD8neg DCs are thought to represent the major inducers. Both BATF3-/- and Sle2c2 mice had reduced humoral and germinal center (GC) responses compared with C57BL/6 (B6) controls. We showed that B6-derived CD4+ DCs are the major early producers of IL-6, followed by CD4-CD8- DCs. Surprisingly, IL-6 production and CD80 expression also increased in CD8+ DCs after immunization, and B6-derived CD8+ DCs rescued Ag-specific adaptive responses in BATF3-/- mice. In addition, inflammatory pDCs (ipDCs) produced more IL-6 than all conventional DCs combined. Interestingly, G-CSFR is highly expressed on pDCs. G-CSF expanded pDC and CD8+ DC numbers and IL-6 production by ipDCs and CD4+ DCs, and it improved the quality of Ab response, increasing the localization of Ag-specific T cells to the GC. Finally, G-CSF activated STAT3 in early G-CSFR+ common lymphoid progenitors of cDCs/pDCs but not in mature cells. In conclusion, we showed a multilayered role of DC subsets in priming Tfh cells in protein immunization, and we unveiled the importance of G-CSFR signaling in the development and function pDCs.

Dynamic Number and Function of IL-10-Producing Regulatory B Cells in the Immune Microenvironment at Distinct Stages of Type 1 Diabetes.

The critical role of IL-10-producing B cells (B10 cells) with a unique CD1dhiCD5+ phenotype in suppressing autoimmune responses and relieving inflammation has been demonstrated in several models of autoimmune diseases. However, the regulatory role of B10 cells in T cell-mediated autoimmune responses during the natural history of type 1 diabetes is unclear. In this study, we used the NOD mouse model of autoimmune diabetes to clarify the changes and potential mechanisms of B10 cells for disease. Compared with B10 cells present in the 4-wk-old normoglycemic NOD mice, the frequency of B10 cells was increased in the insulitis and diabetic NOD mice, with the highest proportion in the insulitis NOD mice. The changes in the relative number of B10 cells were most pronounced in the pancreas-draining lymph nodes. The pathogenic T cells, including Th1 and Th17 cells, remarkably increased. The assays in vitro showed that B10 cells in the NOD mice did not inhibit the proliferation of CD4+CD25- T cells. They also had no regulatory effect on IFN-γ and IL-4 secretion or on Foxp3 expression of T cells. B10 cells suppressed T cell-mediated autoimmune responses via an IL-10-dependent pathway. In contrast, B10 cells in the NOD mice exhibited a significant reduction in IL-10 production. In summary, a defect in the number and function of B10 cells may participate in the development and progression of type 1 diabetes.

Mucosa-Associated Invariant T Cell Hypersensitivity to Staphylococcus aureus Leukocidin ED and Its Modulation by Activation.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib-related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57+ NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Rationally targeted anti-VISTA antibody that blockades the C-C' loop region can reverse VISTA immune suppression and remodel the immune microenvironment to potently inhibit tumor growth in an Fc independent manner.

BACKGROUND: Despite significant progress in cancer immunotherapy in recent years, resistance to existing immune checkpoint therapies (ICT) is common. V-domain Ig suppressor of T cell activation (VISTA), a predominantly myeloid immune checkpoint regulator, represents a promising therapeutic target due to its role in suppressing proinflammatory antitumor responses in myeloid-enriched tumor microenvironments. However, uncertainty around the cognate VISTA ligand has made the development of effective anti-VISTA antibodies challenging. The expression of VISTA on normal immune cell subtypes argues for a neutralizing non-depleting antibody, however, previous reported anti-VISTA antibodies use IgG1 Fc isotypes that deplete VISTA+ cells by antibody dependent cellular cytotoxicity/complement dependent cytotoxicity and these antibodies have shown fast serum clearance and immune toxicities. METHOD: Here we used a rational antibody discovery approach to develop the first Fc-independent anti-VISTA antibody, HMBD-002, that binds a computationally predicted functional epitope within the C-C-loop, distinct from other known anti-VISTA antibodies. This epitope is species-conserved allowing robust in vitro and in vivo testing of HMBD-002 in human and murine models of immune activation and cancer including humanized mouse models. RESULTS: We demonstrate here that blockade by HMBD-002 inhibits VISTA binding to potential partners, including V-Set and Immunoglobulin domain containing 3, to reduce myeloid-derived suppression of T cell activity and prevent neutrophil migration. Analysis of immune cell milieu suggests that HMBD-002 treatment stimulates a proinflammatory phenotype characterized by a Th1/Th17 response, recapitulating a phenotype previously noted in VISTA knockout models. This mechanism of action is further supported by immune-competent syngenic and humanized mouse models of colorectal, breast and lung cancer where neutralizing VISTA, without depleting VISTA expressing cells, significantly inhibited tumor growth while decreasing infiltration of suppressive myeloid cells and increasing T cell activity. Finally, we did not observe either the fast serum clearance or immune toxicities that have been reported for IgG1 antibodies. CONCLUSION: In conclusion, we have shown that VISTA-induced immune suppression can be reversed by blockade of the functional C-C' loop region of VISTA with a first-in-class rationally targeted and non-depleting IgG4 isotype anti-VISTA antibody, HMBD-002. This antibody represents a highly promising novel therapy in the VISTA-suppressed ICT non-responder population.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

An Artifact in Intracellular Cytokine Staining for Studying T Cell Responses and Its Alleviation.

Intracellular cytokine staining (ICS) is a widely employed ex vivo method for quantitative determination of the activation status of immune cells, most often applied to T cells. ICS test samples are commonly prepared from animal or human tissues as unpurified cell mixtures, and cell-specific cytokine signals are subsequently discriminated by gating strategies using flow cytometry. Here, we show that when ICS samples contain Ly6G+ neutrophils, neutrophils are ex vivo activated by an ICS reagent - phorbol myristate acetate (PMA) - which leads to hydrogen peroxide (H2O2) release and death of cytokine-expressing T cells. This artifact is likely to result in overinterpretation of the degree of T cell suppression, misleading immunological research related to cancer, infection, and inflammation. We accordingly devised easily implementable improvements to the ICS method and propose alternative methods for assessing or confirming cellular cytokine expression.

The Discovery of Chicken Foxp3 Demands Redefinition of Avian Regulatory T Cells.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Early alveolar macrophage response and IL-1R-dependent T cell priming determine transmissibility of Mycobacterium tuberculosis strains.

Mechanisms underlying variability in transmission of Mycobacterium tuberculosis strains remain undefined. By characterizing high and low transmission strains of M.tuberculosis in mice, we show here that high transmission M.tuberculosis strain induce rapid IL-1R-dependent alveolar macrophage migration from the alveolar space into the interstitium and that this action is key to subsequent temporal events of early dissemination of bacteria to the lymph nodes, Th1 priming, granulomatous response and bacterial control. In contrast, IL-1R-dependent alveolar macrophage migration and early dissemination of bacteria to lymph nodes is significantly impeded in infection with low transmission M.tuberculosis strain; these events promote the development of Th17 immunity, fostering neutrophilic inflammation and increased bacterial replication. Our results suggest that by inducing granulomas with the potential to develop into cavitary lesions that aids bacterial escape into the airways, high transmission M.tuberculosis strain is poised for greater transmissibility. These findings implicate bacterial heterogeneity as an important modifier of TB disease manifestations and transmission.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.