Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes.

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

Interleukin-27-Producing CD4(+) T Cells Regulate Protective Immunity during Malaria Parasite Infection.

Interleukin-27 (IL-27) is a heterodimeric regulatory cytokine of the IL-12 family, which is produced by macrophages, dendritic cells, and B cells upon stimulation through innate immune receptors. Here, we described regulatory CD4(+) T cells that produce IL-27 in response to T cell receptor stimulation during malaria infection, inhibiting IL-2 production and clonal expansion of other T cells in an IL-27-dependent manner. IL-27-producing CD4(+) T cells were Foxp3(-)CD11a(+)CD49d(+) malaria antigen-specific CD4(+) T cells and were distinct from interferon-γ (IFN-γ) producing Th1 or IL-10 producing Tr1 cells. In mice lacking IL-27 in T cells, IL-2 production was restored and clonal expansion and IFN-γ production by specific CD4(+) T cells were improved, culminating in reduced parasite burden. This study highlights a unique population of IL-27 producing regulatory CD4(+) T cells and their critical role in the regulation of the protective immune response against malaria parasites.

Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection.

Infection of erythrocytes with Plasmodium species induces clinical malaria. Parasite-specific CD4(+) T cells correlate with lower parasite burdens and severity of human malaria and are needed to control blood-stage infection in mice. However, the characteristics of CD4(+) T cells that determine protection or parasite persistence remain unknown. Here we show that infection of humans with Plasmodium falciparum resulted in higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction. In vivo blockade of the PD-1 ligand PD-L1 and the inhibitory receptor LAG-3 restored CD4(+) T cell function, amplified the number of follicular helper T cells and germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, and proper function can be restored by inhibitory therapies to enhance parasite control.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Integral Use of Immunopeptidomics and Immunoinformatics for the Characterization of Antigen Presentation and Rational Identification of BoLA-DR-Presented Peptides and Epitopes.

MHC peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein Ags to elicit functional T cell responses. Liquid chromatography-mass spectrometry analysis of MHC-eluted ligand data has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of Ag presentation have reached a high level of accuracy for both MHC class I and class II. In this study, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte Ag class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by eluted ligand data derived from bovine cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan, available as a Web server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR-restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced mass spectrometry peptidomics with immunoinformatics for characterization of the BoLA-DR Ag presentation system and provide a prediction tool that can be used to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Effector functions of Th17 cells are regulated by IL-35 and TGF-ß in visceral leishmaniasis.

Visceral leishmaniasis (VL) is a debilitating human pathogenesis in which the body's immune functions are severely compromised. Various subsets of T cells, including Th17 cells are important regulators of immune responses observed in various pathologies. The role of Th17 cells and its correlation with immuno-regulatory cytokines are however not well understood in human VL. Herein we studied how IL-17 is associated with the progression of Leishmania donovani infection using murine model of VL. We found induction of a strong IL-17 response at the early phase of infection which progressively reduced to basal level during chronic VL. The mechanistic study of this behavior was found to be linked with the role of regulatory T cells (CD4+ CD25+ T cells) that suppresses the proliferation of the Th17 cell population. Moreover, TGF-ß and IL-35 derived from CD4+ CD25+ T cells are the key mediators for the downregulation of IL-17 during chronic VL. Thus, this study points to an antagonistic effect of Tregs and Th17 cells that can be used for designing better therapeutic and preventive strategies against leishmaniasis.

Thymic stromal lymphopoietin contributes to protection of mice from Strongyloides venezuelensis infection by CD4+ T cell-dependent and -independent pathways.

When animals are infected with helminthic parasites, resistant hosts mount type II helper T (Th2) immune responses to expel worms. Recent studies have clearly shown that epithelial cell-derived cytokines contribute to the induction of Th2 immune responses. Here we demonstrate the role of endogenous thymic stromal lymphopoietin (TSLP) for protection against Strongyloides venezuelensis (S. venezuelensis) infection, utilizing TSLP receptor-deficient Crlf2-/- mice. The number of eggs per gram of feces (EPG) and worm burden were significantly higher in Crlf2-/- mice than in wild type (WT) mice. S. venezuelensis infection induced Tslp mRNA expression in the skin, lung, and intestine and also facilitated the accumulation of mast cells in the intestine in a TSLP-dependent manner. Furthermore, CD4+ T cells from S. venezuelensis-infected Crlf2-/- mice showed diminished capacity to produce Th2 cytokines in the early stage of infection. Finally, CD4+ cell-depleted Crlf2-/- mice still showed higher EPG counts and worm burden than CD4+ cell-depleted WT mice, indicating that TSLP contributes to protecting mice against S. venezuelensis infection in both CD4+ T cell-dependent and -independent manners.

Trickle infection and immunity to Trichuris muris.

The majority of experiments investigating the immune response to gastrointestinal helminth infection use a single bolus infection. However, in situ individuals are repeatedly infected with low doses. Therefore, to model natural infection, mice were repeatedly infected (trickle infection) with low doses of Trichuris muris. Trickle infection resulted in the slow acquisition of immunity reflected by a gradual increase in worm burden followed by partial expulsion. Flow cytometry revealed that the CD4+ T cell response shifted from Th1 dominated to Th2 dominated, which coincided with an increase in Type 2 cytokines. The development of resistance following trickle infection was associated with increased worm expulsion effector mechanisms including goblet cell hyperplasia, Muc5ac production and increased epithelial cell turn over. Depletion of CD4+ T cells reversed resistance confirming their importance in protective immunity following trickle infection. In contrast, depletion of group 2 innate lymphoid cells did not alter protective immunity. T. muris trickle infection resulted in a dysbiotic mircrobiota which began to recover alpha diversity following the development of resistance. These data establish trickle infection as a robust and informative model for analysis of immunity to chronic intestinal helminth infection more akin to that observed under natural infection conditions and confirms the importance of CD4+ T cell adaptive immunity in host protection.

TLR11-independent inflammasome activation is critical for CD4+ T cell-derived IFN-γ production and host resistance to Toxoplasma gondii.

Innate recognition of invading intracellular pathogens is essential for regulating robust and rapid CD4+ T cell effector function, which is critical for host-mediated immunity. The intracellular apicomplexan parasite, Toxoplasma gondii, is capable of infecting almost any nucleated cell of warm-blooded animals, including humans, and establishing tissue cysts that persist throughout the lifetime of the host. Recognition of T. gondii by TLRs is essential for robust IL-12 and IFN-γ production, two major cytokines involved in host resistance to the parasite. In the murine model of infection, robust IL-12 and IFN-γ production have been largely attributed to T. gondii profilin recognition by the TLR11 and TLR12 heterodimer complex, resulting in Myd88-dependent IL-12 production. However, TLR11 or TLR12 deficiency failed to recapitulate the acute susceptibility to T. gondii infection seen in Myd88-/- mice. T. gondii triggers inflammasome activation in a caspase-1-dependent manner resulting in cytokine release; however, it remains undetermined if parasite-mediated inflammasome activation impacts IFN-γ production and host resistance to the parasite. Using mice which lack different inflammasome components, we observed that the inflammasome played a limited role in host resistance when TLR11 remained functional. Strikingly, in the absence of TLR11, caspase-1 and -11 played a significant role for robust CD4+ TH1-derived IFN-γ responses and host survival. Moreover, we demonstrated that in the absence of TLR11, production of the caspase-1-dependent cytokine IL-18 was sufficient and necessary for CD4+ T cell-derived IFN-γ responses. Mechanistically, we established that T. gondii-mediated activation of the inflammasome and IL-18 were critical to maintain robust CD4+ TH1 IFN-γ responses during parasite infection in the absence of TLR11.

A review on the interactions between dendritic cells, filarial parasite and parasite-derived molecules in regulating the host immune responses.

Lymphatic filariasis (LF) is the second leading cause of parasitic disabilities that affects millions of people in India and several other tropical countries. The complexity of this disease is endorsed by various immunopathological consequences such as lymphangitis, lymphadenitis and elephantiasis. The immune evasion strategies that a filarial parasite usually follows are chiefly initiated with the communication between the invaded parasites and parasite-derived molecules, with the Toll-like receptors (TLRs) present on the surface of the antigen-presenting cells (APCs). Classically, the filarial parasites interact with the DCs resulting in lowering of CD4+ T-cell responses. These CD4+ T-cell responses are the key players behind the immune-mediated pathologies associated with LF. In chronic stage, the canonical pro-inflammatory immune responses are shifted towards an anti-inflammatory subtype, which is favouring the parasite survivability within the host. The central theme of this review article is to present the overall immune response elicited when an APC, particularly a DC, encounters a filarial parasite.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses.

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage-derived EVs of the proliferative response of CD4+ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin-specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF-1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF- and EF-1α-deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF-1α alone or in combination with HRF conferred a long-lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei-derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV-mediated immune suppression in malaria-infected individuals.

Early Th1 cell differentiation is marked by a Tfh cell-like transition.

Follicular helper T (Tfh) cells comprise an important subset of helper T cells; however, their relationship with other helper lineages is incompletely understood. Herein, we showed interleukin-12 acting via the transcription factor STAT4 induced both Il21 and Bcl6 genes, generating cells with features of both Tfh and Th1 cells. However, STAT4 also induced the transcription factor T-bet. With ChIP-seq, we defined the genome-wide targets of T-bet and found that it repressed Bcl6 and other markers of Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. Our data argue that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation.

Antibody-Dependent, Gamma Interferon-Independent Sterilizing Immunity Induced by a Subunit Malaria Vaccine.

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4+ and CD8+ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4+ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4+ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Skin-resident CD4+ T cells protect against Leishmania major by recruiting and activating inflammatory monocytes.

Tissue-resident memory T cells are required for establishing protective immunity against a variety of different pathogens, although the mechanisms mediating protection by CD4+ resident memory T cells are still being defined. In this study we addressed this issue with a population of protective skin-resident, IFNγ-producing CD4+ memory T cells generated following Leishmania major infection. We previously found that resident memory T cells recruit circulating effector T cells to enhance immunity. Here we show that resident memory CD4+ T cells mediate the delayed-hypersensitivity response observed in immune mice and provide protection without circulating T cells. This protection occurs rapidly after challenge, and requires the recruitment and activation of inflammatory monocytes, which limit parasites by production of both reactive oxygen species and nitric oxide. Overall, these data highlight a novel role for tissue-resident memory cells in recruiting and activating inflammatory monocytes, and underscore the central role that skin-resident T cells play in immunity to cutaneous leishmaniasis.

IL-17A/F in Leishmania major-resistant C57BL/6 mice.

Proinflammatory IL-17 plays an important role in various diseases and defence against extracellular microorganisms. Healing of leishmaniasis is promoted by Th1/Tc1 cells, whereas Th2/Treg are associated with worsened disease outcome. In addition, high expression of IL-17A in Leishmania-susceptible BALB/c and artificial overexpression of IL-17A in T cells in resistant C57BL/6 mice worsened disease outcome. Since C57BL/6 mice lacking only IL-17A exhibited no phenotype, and IL-17A and IL-17F share similar receptors, but differentially regulate chemokine secretion, we studied mice lacking both IL-17A and IL-17F (IL-17A/F-/- ) in infections with Leishmania major. Interestingly, lesion volumes and parasite burdens were comparable to controls, IL-17A/F-/- mice developed a Th1/Tc1 phenotype, and exhibited normal lesion resolution. Thus, in C57BL/6 mice, secretion of IL-17A and IL-17F does not influence disease progression. It appears that-depending on the genetic background-cytokines of the IL-17 family might be responsible for disease progression primarily in susceptible mice.

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer.

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4(+)and CD8(+)αß T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4(+)CD8(+)double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.

Immunomodulating role of IL-10-producing B cells in Leishmania amazonensis infection.

This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10.

The Extracellular Domains of IgG1 and T Cell-Derived IL-4/IL-13 Are Critical for the Polyclonal Memory IgE Response In Vivo.

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE+ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.