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Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine., (© 2024 Cohn et al.)

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Cystic echinococcosis (CE) is a zoonotic parasitic disease spread worldwide caused by Echinococcus granulosus ( E g), which sometimes causes serious damage; however, in many cases, people are not aware that they are infected. A number of recombinant vaccines based on E g are used to evaluate their effectiveness against the infection. Our previous report showed that recombinant E g.P29 (r E g.P29) has a marvelous immunoprotection and can induce Th1 immune response. Furthermore, data of miRNA microarray in mice spleen CD4 + T cells showed that miR-126a-5p was significantly elevated 1 week after immunization by using r E g.P29. Therefore, in this perspective, we discussed the role of miR-126a-5p in the differentiation of naive CD4 + T cells into Th1/Th2 under r E g.P29 immunization and determined the mechanisms associated with delta-like 1 homolog (DLK1) and Notch1 signaling pathway. One week after P29 immunization of mice, we found that miR-126a-5p was significantly increased and DLK1 expression was decreased, while Notch1 pathway activation was enhanced and Th1 response was significantly stronger. The identical conclusion was obtained by overexpression of mmu-miR-126a-5p in primary naive CD4 + T cells in mice. Intriguingly, mmu-miR-126a-5p was significantly raised in serum from mice infected with protoscolex in the early stages of infection and markedly declined in the late stages of infection, while has-miR-126-5p expression was dramatically reduced in serum from CE patients. Taken together, we show that miR-126a-5p functions as a positive regulator of Notch1-mediated differentiation of CD4 + T cells into Th1 through downregulating DLK1 in vivo and in vitro . Hsa-miR-126-5p is potentially a very promising diagnostic biomarker for CE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Zhu, Zhang, Wang, Tao, Yang, Zhu and Zhao.)

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4 + and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4 + T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4 + /Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission., Competing Interests: AH is named inventor on patent applications covering malaria vaccines and immunization regimens. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zaric, Marini, Nielsen, Gupta, Mekhaiel, Pham, Elias, Taylor, de Graaf, Payne, Li, Silk, Williams, Hill, Long, Miura and Biswas.)

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-β 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., (© 2021 Federation of American Societies for Experimental Biology.)

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP + Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4 + IL-4 + cells and increasing CD8 + IFN-γ + cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plasmacytoid dendritic cells (pDCs) are potent producers of type I IFN (IFN-I) during viral infection and respond to IFN-I in a positive feedback loop that promotes their function. IFN-I shapes dendritic cell responses during helminth infection, impacting their ability to support Th2 responses. However, the role of pDCs in type 2 inflammation is unclear. Previous studies have shown that pDCs are dispensable for hepatic or splenic Th2 responses during the early stages of murine infection with the trematode Schistosoma mansoni at the onset of parasite egg laying. However, during S. mansoni infection, an ongoing Th2 response against mature parasite eggs is required to protect the liver and intestine from acute damage and how pDCs participate in immune responses to eggs and adult worms in various tissues beyond acute infection remains unclear. We now show that pDCs are required for optimal Th2 cytokine production in response to S. mansoni eggs in the intestinal-draining mesenteric lymph nodes throughout infection and for egg-specific IFN-γ at later time points of infection. Further, pDC depletion at chronic stages of infection led to increased hepatic and splenic pathology as well as abrogated Th2 cell cytokine production and activation in the liver. In vitro, mesenteric lymph node pDCs supported Th2 cell responses from infection-experienced CD4 + T cells, a process dependent on pDC IFN-I responsiveness, yet independent of Ag. Together, these data highlight a previously unappreciated role for pDCs and IFN-I in maintaining and reinforcing type 2 immunity in the lymph nodes and inflamed tissue during helminth infection., (Copyright © 2021 The Authors.)

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico , in vitro , and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro , to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro . The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5., Competing Interests: AN, TP, KT, VK, and GG are employees of Leidos, Inc., the prime contractor for the USAID Malaria Vaccine Development Program (MVDP) Contract AID-OAA-C-15-00071. FT, PH, LM, WM, and ADG are employees of EpiVax, Inc., an MVDP subcontractor. PH is a previous employee of EpiVax, Inc. SD, SS, CN, and RA are employees of the University of Oxford, an MVDP subcontractor. SD is a named inventor on patent applications relating to the RH5-based vaccines used in the VAC057 and VAC063 clinical studies., (Copyright © 2021 Kotraiah, Phares, Terry, Hindocha, Silk, Nielsen, Moise, Tucker, Ashfield, Martin, De Groot, Draper, Gutierrez and Noe.)

An effective malaria vaccine must prevent disease in a range of populations living in regions with vastly different transmission rates and protect against genetically-diverse Plasmodium falciparum (Pf) strains. The protective efficacy afforded by the currently licensed malaria vaccine, Mosquirix™, promotes strong humoral responses to Pf circumsporozoite protein (CSP) 3D7 but protection is limited in duration and by strain variation. Helper CD4 T cells are central to development of protective immune responses, playing roles in B cell activation and maturation processes, cytokine production, and stimulation of effector T cells. Therefore, we took advantage of recent in silico modeling advances to predict and analyze human leukocyte antigen (HLA)-restricted class II epitopes from PfCSP - across the entire PfCSP 3D7 sequence as well as in 539 PfCSP sequence variants - with the goal of improving PfCSP-based malaria vaccines. Specifically, we developed a systematic workflow to identify peptide sequences capable of binding HLA-DR in a context relevant to achieving broad human population coverage utilizing cognate T cell help and with limited T regulatory cell activation triggers. Through this workflow, we identified seven predicted class II epitope clusters in the N- and C-terminal regions of PfCSP 3D7 and an additional eight clusters through comparative analysis of 539 PfCSP sequence variants. A subset of these predicted class II epitope clusters was synthesized as peptides and assessed for HLA-DR binding in vitro . Further, we characterized the functional capacity of these peptides to prime and activate human peripheral blood mononuclear cells (PBMCs), by monitoring cytokine response profiles using MIMIC ® technology (Modular IMmune In vitro Construct). Utilizing this decision framework, we found sufficient differential cellular activation and cytokine profiles among HLA-DR-matched PBMC donors to downselect class II epitope clusters for inclusion in a vaccine targeting PfCSP. Importantly, the downselected clusters are not highly conserved across PfCSP variants but rather, they overlap a hypervariable region (TH2R) in the C-terminus of the protein. We recommend assessing these class II epitope clusters within the context of a PfCSP vaccine, employing a test system capable of measuring immunogenicity across a broad set of HLA-DR alleles., Competing Interests: AN, TP, KT, VK, JC, and GG are employees of Leidos, Inc., prime contractor for USAID Malaria Vaccine Development Program (MVDP) Contract AID-OAA-C-15-00071, and hold Leidos stock and/or stock options. FT, LM, WM, and ADG are employees of EpiVax, Inc., an MVDP subcontractor. PH was a previous employee of EpiVax, Inc. BS, ES, and DD are employees of Sanofi Pasteur, an MVDP subcontractor, and hold Sanofi Pasteur stock and/or stock options., (Copyright © 2021 Noe, Terry, Schanen, Sassano, Hindocha, Phares, Moise, Christen, Tucker, Kotraiah, Drake, Martin, De Groot and Gutierrez.)

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., Competing Interests: Declaration of competing interest The authors declare no financial or commercial conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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., (© 2020 The Scandinavian Foundation for Immunology.)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

CD4 + effector lymphocytes (T eff ) are traditionally classified by the cytokines they produce. To determine the states that T eff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic T eff 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 (T H ) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T H 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.

Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus (Eg) infection. Our previous study confirmed that recombinant Eg.P29 (rEg.P29) could protect against echinococcus granulosus secondary infection in sheep and mice. The aim of the study was to investigate the association between immunoprotection of rEg.P29 vaccine and mmu-miR-374b-5p (miR-374b-5p) and study the immunity influence of miR-374b-5p on CD4 + T cells in mice spleen. MiR-374b-5p level was significantly increased after the second-week and the fourth week of vaccination with rEg.P29. Overexpression of miR-374b-5p increased IFN- γ , IL-2, IL-17A mRNA levels and decreased IL-10 mRNA levels in CD4 + T cells. Moreover, the inhibition of miR-374b-5p decreased IFN- γ and IL-17A and increased IL-10 mRNA levels in CD4 + T cells; this was further confirmed by the flow cytometry. The vaccination of rEg.P29 enhanced miR-374b-5p expression that was associated with a higher Th1 and Th17 immune response, a lower IL-10 mRNA production with miR-374b-5p overexpression, a lower Th1 immune response, and a higher IL-10 mRNA levels with miR-374b-5p inhibitions. To sum up, these data suggest that miR-374b-5p may participate in rEg.P29 immunity by regulating Th1 and Th17 differentiation., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Dongjie Li et al.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN- and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro., Competing Interests: Appendix A

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., Competing Interests: The authors have declared that no competing interests exist.

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/AS01 E 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 (r Py CSP), 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., (Copyright © 2019 American Society for Microbiology.)

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., (© 2019 John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

Schistosoma mansoni (Sm) infection has been linked with an increased risk of HIV acquisition in women. Therefore, defining the mechanism(s) by which Sm alters HIV susceptibility might lead to new HIV prevention strategies. Here, we analyze the impact of standard Sm therapy in HIV-uninfected Sm+ Ugandan adult women on genital HIV susceptibility and mucosal and systemic immunology. Schistosomiasis treatment induces a profound reduction of HIV entry into cervical and blood CD4+ T cells that is sustained for up to two months, despite transient systemic and mucosal immune activation and elevated genital IL-1α levels. Genital IFN-α2a levels are also elevated post-treatment, and IFN-α2a blocks HIV entry into primary CD4+ T cells ex vivo. Transcriptomic analysis of blood mononuclear cells post-Sm treatment shows IFN-I pathway up-regulation and partial reversal of Sm-dysregulated interferon signaling. These findings indicate that Sm therapy may reduce HIV susceptibility for women with Sm infection, potentially through de-repression of IFN-I pathways.

Leishmania ( V .) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania ( L .) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4 + T cell-mediated protection against L . ( L .) infantum chagasi infection. Prevention of L . ( L .) amazonensis infection requires in contrast an additional CD8 + T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L . ( L .) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L . ( V .) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ / IL-10 and TNF-α / IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4 + and CD8 + cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4 + and CD8 + IL-2 + TNF-α + IFN-γ + T cells. Also as observed against L . ( L .) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L . ( V .) braziliensis . Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L . ( V .) braziliensis .

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., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR., (Copyright © 2019 The Authors.)

Human CD8 + and CD4 + T cell lines and clones are valuable tools to explore the role of these cells in the context of diseases, especially in cases in which the main underlying actor is the immune response, like Chagas disease. These cell lines and clones provide a good experimental system to address the phenotypic and functional features of specific T cell subpopulations and furthermore settle the framework necessary for analyzing their antigen/peptide specificity.This chapter details a culture method for the establishment of T. cruzi-specific memory T cell lines from mononuclear cells isolated from Chagas disease patients' peripheral blood. The presented protocol comprises (1) enrichment of memory CD4 + T cells, (2) stimulation with parasite lysate, (3) evaluation of specificity, and (4) expansion and maintenance of specific T cell lines.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Early plasmablast induction is a hallmark of Plasmodium infection and is thought to contribute to the control of acute parasite burden. Although long understood to be a T-cell dependent phenomenon, regulation of early plasmablast differentiation, however, is poorly understood. Here, we identify a population of CD4 + T cells that express the innate NK cell marker NK1.1 as an important source of T cell help for early plasmablast and parasite-specific Ab production. Interestingly, NK1.1 + CD4 + T cells arise from conventional, naive NK1.1 - CD4 + T cells, and their generation is independent of CD1d but critically reliant on MHC-II. CD4 + T cells that express NK1.1 early after activation produce IFN-γ and IL-21, and express the follicular helper T (Tfh) cell markers ICOS, PD-1 and CXCR5 more frequently than NK1.1 - CD4 + T cells. Further analysis of this population revealed that NK1.1 + Tfh-like cells were more regularly complexed with plasmablasts than NK1.1 - Tfh-like cells. Ultimately, depletion of NK1.1 + cells impaired class-switched parasite-specific antibody production during early Plasmodium yoelii infection. Together, these data suggest that expression of NK1.1 defines a population of rapidly expanding effector CD4 + T cells that specifically promote plasmablast induction during Plasmodium infection and represent a subset of T cells whose modulation could promote effective vaccine design.

Diagnosis of visceral leishmaniasis (VL) and assessment of treatment response in human immunodeficiency virus (HIV)-coinfected patients still relies on invasive tissue aspiration. This hampers scale-up and decentralization of care in resource-limited settings. Noninvasive diagnostics are urgently needed. KATEX is a frequently used latex agglutination test for Leishmania antigen in urine that has never been evaluated in HIV-coinfected individuals from Leishmania donovani -endemic areas. This was an exploratory sub-study embedded within the screening phase of a trial in highly endemic northwestern Ethiopia. All patients were HIV-positive and aspirate-confirmed VL cases. We assessed diagnostic accuracy of KATEX for VL diagnosis and as test of cure at end of treatment, using tissue aspirate parasite load as reference methods. We also described the evolution of weekly antigen levels during treatment. Most of the 87 included patients were male (84, 97%), young (median age 31 years), and had poor immune status (median cluster of differentiation type 4 count 56 cells/μL). KATEX had moderate sensitivity (84%) for VL diagnosis. KATEX had moderate sensitivity (82%) and a moderate negative predictive value (87%) but only low specificity (49%) and a low positive predictive value (40%) for the assessment of treatment outcomes. Weekly antigen levels showed characteristic patterns during treatment of patients with different initial parasite loads and treatment outcomes. Antigen detection in urine using KATEX can contribute to improved VL diagnosis in HIV-coinfected patients but has limited use for monitoring of treatment response. Better noninvasive diagnostics are needed to reduce reliance on invasive methods and thus to expand and improve clinical care for VL in resource-limited settings.

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.

Manifestations of Leishmania infantum infection range from asymptomatic to symptomatic visceral leishmaniasis (VL). People with symptomatic VL (sVL) have suppressed immune responses against Leishmania antigens that are reversed after clinical cure. The intradermal leishmanin skin test (LST) is negative during sVL, but it becomes positive after treatment. The aim of this study was to compare T cell responses in individuals with sVL, recovered VL (RecVL), and endemic controls. Endemic controls were household contacts of a VL case and they were grouped by their LST results, either positive (LST+) or negative (LST-). Mononuclear cells were studied ex vivo or after stimulation with soluble Leishmania antigens (SLA); cell surface markers and cytokines were determined. T cells, ex vivo, from individuals with sVL and from LST+ individuals presented a higher activation for CD4 + and CD8 + cells expressing CD69. However, lymphocytes from sVL stimulated with SLA had lower percentages of CD4 + and CD8 + cells expressing CD69 and CD8 + cells expressing CD25, with no release of interferon-γ or tumor necrosis factor. sVL subjects had lower percentage of memory cells (CD4 + CD45RO+), ex vivo, without SLA stimulation than RecVL, LST+, or LST- ( P = 0.0022). However, individuals with sVL had fewer regulatory cells after SLA stimulation (CD4 + CD25 HIGH , P = 0.04 and CD4 + FOXP3+, P = 0.02) than RecVL. The decrease in specific memory and activated CD4 + and CD8 + cells, as in response to Leishmania antigens, could explain, in part, the immune impairment during sVL. Finally, protective T cell responses are long lasting because both RecVL or LST+ individuals maintain a specific protective response to Leishmania years after the primary 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., (Published by Elsevier Inc.)

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.

Asthma is a chronic disease characterized by airway inflammation, obstruction and hyperresponsiveness. Severe asthma affects a small proportion of subjects but results in most of the morbidity, costs and mortality associated with the disease. Studies have suggested that Schistosoma mansoni infection reduces the severity of asthma and prevent atopy., Objective: We evaluated the ability of S. mansoni antigens, Sm29 and Sm29TSP-2 to modulate lymphocyte activation status in response to the allergen of the mite Dermatophagoides pteronyssinus (Der p1) in cell cultures of individuals with asthma., Methods: Thirty four patients were enrolled in this study: seventeen patients with severe asthma (SA group), seventeen patients with mild asthma (MA group) and six controls with no asthma. Peripheral blood mononuclear cells (PBMC) were obtained and stimulated with Sm29 and Sm29TSP-2 in the presence or absence of Der p1. The expression of surface markers and cytokines on lymphocytes was evaluated by flow cytometry and the levels of IL-10 in the culture supernatant were determined by ELISA., Results: The addition of Sm29 and Sm29TSP-2 antigens to PBMC cultures from both groups of subjects with asthma stimulated with Der p1 reduced the frequency of CD4 + CD25 low cells whereas and increased frequency of CD4 + CD25 high population was observed compared to unstimulated cultures. Moreover, cultures stimulated with Sm29TSP-2 showed a reduction in the frequency of T cells expressing CD69, IFN-γ, TNF and TGF-β in the MA group and an increase in the frequency of CD4 + TSLPR + T cells in the SA group. The addition of Sm29 to the cultures reduced the frequency of CD4 + CD69 + and CD4 + IL-5 + T cells in all asthmatic groups, and reduced the frequency of CD4 + T cells expressing IL-13 in the MA group. The cultures stimulated with Sm29 and Sm29TSP-2 showed an increase in the level of IL-10 in the supernatants., Conclusion: These results suggest that the addition of Sm29 and Sm29TSP-2 to the cells cultures from subjects with asthma reduced cell activation markers and altered the cytokine production pattern in a way that can potentialy control the inflammatory response associated with asthma., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tuberculosis (TB) causes significant morbidity and mortality on a global scale. The African region has 24% of the world's TB cases. TB overlaps with other infectious diseases such as malaria and HIV, which are also highly prevalent in the African region. TB is a leading cause of death among HIV-positive patients and co-infection with HIV and TB has been described as a syndemic. In view of the overlapping epidemiology of these diseases, it is important to understand the dynamics of the immune response to TB in the context of co-infection. We investigated the cytokine response to purified protein derivative (PPD) in peripheral blood mononuclear cells from TB patients co-infected with HIV or malaria and compared it to that of malaria- and HIV-free TB patients. A total of 231 subjects were recruited for this study and classified into six groups; untreated TB-positive, TB positive subjects on TB drugs, TB- and HIV-positive, TB- and malaria-positive, latent TB and apparently healthy control subjects. Our results demonstrate maintenance of interferon (IFN)-γ production in HIV and malaria co-infected TB patients in spite of lower CD4 counts in the HIV-infected cohort. Malaria co-infection caused an increase in the production of the T helper type 2 (Th2)-associated cytokine interleukin (IL)-4 and the anti-inflammatory cytokine IL-10 in PPD-stimulated cultures. These results suggest that malaria co-infection diverts immune response against M. tuberculosis towards a Th-2/anti-inflammatory response which might have important consequences for disease progression., (© 2016 British Society for Immunology.)

Background: The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls., Results: Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling., Conclusion: Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

CD4 + T cells that produce IFN-γ are the source of host-protective IL-10 during primary infection with a number of different pathogens, including Plasmodium spp. The fate of these CD4 + IFN-γ + IL-10 + T cells following clearance of primary infection and their subsequent influence on the course of repeated infections is, however, presently unknown. In this study, utilizing IFN-γ-yellow fluorescent protein (YFP) and IL-10-GFP dual reporter mice, we show that primary malaria infection-induced CD4 + YFP + GFP + T cells have limited memory potential, do not stably express IL-10, and are disproportionately lost from the Ag-experienced CD4 + T cell memory population during the maintenance phase postinfection. CD4 + YFP + GFP + T cells generally exhibited a short-lived effector rather than effector memory T cell phenotype postinfection and expressed high levels of PD-1, Lag-3, and TIGIT, indicative of cellular exhaustion. Consistently, the surviving CD4 + YFP + GFP + T cell-derived cells were unresponsive and failed to proliferate during the early phase of secondary infection. In contrast, CD4 + YFP + GFP - T cell-derived cells expanded rapidly and upregulated IL-10 expression during secondary infection. Correspondingly, CD4 + T cells were the major producers within an accelerated and amplified IL-10 response during the early stage of secondary malaria infection. Notably, IL-10 exerted quantitatively stronger regulatory effects on innate and CD4 + T cell responses during primary and secondary infections, respectively. The results in this study significantly improve our understanding of the durability of IL-10-producing CD4 + T cells postinfection and provide information on how IL-10 may contribute to optimized parasite control and prevention of immune-mediated pathology during repeated malaria infections., (Copyright © 2016 The Authors.)

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL., (© 2016 British Society for Immunology.)

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. CD8 T-lymphocytes help to control infection, but apoptosis of CD8 T cells disrupts immunity and efferocytosis can enhance parasite infection within macrophages. Here, we investigate how apoptosis of activated CD8 T cells affects M1 and M2 macrophage phenotypes. First, we found that CD8 T-lymphocytes and inflammatory monocytes/macrophages infiltrate peritoneum during acute T. cruzi infection. We show that treatment with anti-Fas ligand (FasL) prevents lymphocyte apoptosis, upregulates type-1 responses to parasite antigens, and reduces infection in macrophages cocultured with activated CD8 T cells. Anti-FasL skews mixed M1/M2 macrophage profiles into polarized M1 phenotype, both in vitro and following injection in infected mice. Moreover, inhibition of T-cell apoptosis induces a broad reprogramming of cytokine responses and improves macrophage-mediated immunity to T. cruzi. The results indicate that disposal of apoptotic CD8 T cells increases M2-macrophage differentiation and contributes to parasite persistence.

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(+)αβ 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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4+FoxP3+T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4+FoxP3+T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4+FoxP3+T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.

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.

Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L(-/-) mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L(-/-) mice infected with T. gondii displayed an expansion of CD8α(+) and CD11b(lo)CD8α(-) DCs. Infection also induced an expansion of parasite-specific CD4(+) and CD8(+) T cells in Flt3L(-/-) mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L(-/-) mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming., (Copyright © 2015 by The American Association of Immunologists, Inc.)

It is well established that CD8(+) T cells play an important role in protective immunity against protozoan infections. However, their role in the course of Neospora caninum infection has not been fully elucidated. Here we report that CD8-deficient mice infected with N. caninum presented higher parasitic loads in the brain and lungs and lower spleen and brain immunity-related GTPases than their wild-type counterparts. Moreover, adoptive transfer of splenic CD8(+) T cells sorted from N. caninum-primed immunosufficient C57BL/10 ScSn mice prolonged the survival of infected IL-12-unresponsive C57BL/10 ScCr recipients. In both C57BL/6 and C57BL/10 ScSn mice CD8(+) T cells are activated and produce interferon-γ (IFN-γ) upon challenged with N. caninum. The host protective role of IFN-γ produced by CD8(+) T cells was confirmed in N. caninum-infected RAG2-deficient mice reconstituted with CD8(+) T cells obtained from either IFN-γ-deficient or wild-type donors. Mice receiving IFN-γ-expressing CD8(+) T cells presented lower parasitic burdens than counterparts having IFN-γ-deficient CD8(+) T cells. Moreover, we observed that N. caninum-infected perforin-deficient mice presented parasitic burdens similar to those of infected wild-type controls. Altogether these results demonstrate that production of IFN-γ is a predominant protective mechanism conferred by CD8(+) T cells in the course of neosporosis.

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Introduction: Understanding the factors that delineate the efficacy of T cell responses towards pathogens is crucial for our ability to develop potent therapies against infectious diseases. Multidimensional evaluation of T cell functionality at the single-cell level enables exhaustive analysis of combinatorial functional properties, hence polyfunctionality. We have recently invented an algorithm that quantifies polyfunctionality, the Polyfunctionality Index (Larsen et al. PLoS One 2012). Here we demonstrate that quantitative assessment of T cell polyfunctionality correlates with T cell efficacy measured as the capacity to kill target cells in vitro and control infection in vivo., Methods: We employed the polyfunctionality index on two datasets selected for their unique ability to evaluate the polyfunctional imprint on T cell efficacy. 1) HIV-specific CD8+ T cells and 2) Leishmania major-specific CD4+ T cells were analysed for their capacity to secrete multiple effector molecules, kill target cells and control infection. Briefly, employing the Polyfunctionality Index algorithm we determined the parameter estimates resulting in optimal correlation between T cell polyfunctionality and T cell efficacy., Results: T cell polyfunctionality is correlated with T cell efficacy measured as 1) target killing (r=0.807, P<0.0001) and 2) lesion size upon challenge with Leishmania major (r=-0.50, P=0.004). Contrary to an approach relying on the Polyfunctionality Index algorithm, quantitative evaluation of T cell polyfunctionality traditionally ignores the gradual contribution of more or less polyfunctional T cells. Indeed, comparing both approaches we show that optimal description of T cell efficacy is obtained when gradually integrating all levels of polyfunctionality in accordance with the Polyfunctionality Index., Conclusions: Our study presents a generalizable methodology to objectively evaluate the impact of polyfunctionality on T cell efficacy. We show that T cell polyfunctionality is a superior correlate of T cell efficacy both in vitro and in vivo as compared with response size. Therefore, future immunotherapies should aim to increase T cell polyfunctionality.

In order to test the influence of the sympathetic nervous system on Leishmania mexicana infection, groups of female BALB/c mice were treated (i.p.) with the non-selective β-adrenergic receptor (β-AR) antagonist (S)-propranolol (5mg/kg thrice a day), the β2-AR agonist clenbuterol (1mg/kg once a day) or the α2-AR antagonist yohimbine (2mg/kg twice a day) during 5days. During the second day of treatments, mice were inoculated in the footpad with 1×10(6) or 1×10(3) metacyclic promastigotes of L. mexicana mexicana (LV4). The lesion size was measured weekly, and parasite burden on week 12. In mice treated with (S)-propranolol, the percentage of splenic T lymphocytes producing IFN-γ after antigen challenge was determined by flow cytometry. In mice infected with 1×10(6) parasites, only (S)-propranolol caused a reduction of footpad swelling (p<0.05, weeks 11-12), without effects on parasite burden, or in the percentage of IFN-γ-immunopositive CD4(+) or CD8(+) T lymphocytes. In mice infected with 1×10(3) parasites, the effects of treatments vs. control group were as follows: (a) inhibition of footpad swelling by (S)-propranolol (p<0.01, weeks 3-12), clenbuterol (p<0.05, weeks 7-10), and yohimbine (p<0.01, week 7); (b) a decrease of the parasite burden by (S)-propranolol (p<0.01) and yohimbine (p<0.05); (c) in control mice the percentage of CD4(+) T-cells producing IFN-γ was 6.2±0.5%, while in those treated with (S)-propranolol it increased to 8.7±0.6% (p<0.01); (d) in control mice the percentage of CD8(+) T-cells producing IFN-γ was 3.1±0.4%, while in those treated with (S)-propranolol it increased to 10.4±0.2% (p<0.01). These results indicate that the blockade of β-ARs during infection of BALB/c mice with an inoculum of L. mexicana mexicana similar to that delivered by the bite of a sand fly produces a Th1 bias in the immune response, favoring an increment of T lymphocytes secreting IFN-γ, which correlated with a reduced parasite burden (p<0.05, Spearman's test). We suggest that β-AR antagonists could be of therapeutic value, either as treatment or as adjuvant of vaccines for L. mexicana., (Copyright © 2014. Published by Elsevier B.V.)

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)