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Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. It is hypothesized that the activation of host immunity by co‐delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)‐β expression using a polyoxazoline (POx)‐poly(lactic‐co‐glycolic) acid (PLGA) nanovaccine, while modulating the tumor‐associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti‐programmed cell death protein 1 (PD‐1) can constitute an alternative approach for cancer immunotherapy. POx‐Mannose (Man) nanovaccines generate antigen‐specific T‐cell responses that control tumor growth to a higher extent than poly(ethylene glycol) (PEG)‐Man nanovaccines. This anti‐tumor effect induced by the POx‐Man nanovaccines is mediated by a CD8+‐T cell‐dependent mechanism, in contrast to the PEG‐Man nanovaccines. POx‐Man nanovaccine combines with pexidartinib, a modulator of the TAM function, restricts the MC38 tumor growth, and synergizes with PD‐1 blockade, controlling MC38 and CT26 tumor growth and survival. This data is further validated in the highly aggressive and poorly immunogenic B16F10 melanoma mouse model. Therefore, the synergistic anti‐tumor effect induced by the combination of nanovaccines with the inhibition of both TAM‐ and PD‐1‐inducing immunosuppression, holds great potential for improving immunotherapy outcomes in solid cancer patients. [ABSTRACT FROM AUTHOR]

Autoimmune diseases are life-threatening disorders that cause increasing disability over time. Systemic lupus erythematosus (SLE) and other autoimmune diseases arise when immune stimuli override mechanisms of self-tolerance. Accumulating evidence has demonstrated that protein glycosylation is substantially altered in autoimmune disease development, but the mechanisms by which glycans trigger these autoreactive immune responses are still largely unclear. In this study, we found that presence of microbial-associated mannose structures at the surface of the kidney triggers the recognition of DC-SIGN–expressing γδ T cells, inducing a pathogenic interleukin-17a (IL-17a)–mediated autoimmune response. Mice lacking Mgat5, which have a higher abundance of mannose structures in the kidney, displayed increased γδ T cell infiltration into the kidney that was associated with spontaneous development of lupus in older mice. N-acetylglucosamine supplementation, which promoted biosynthesis of tolerogenic branched N-glycans in the kidney, was found to inhibit γδ T cell infiltration and control disease development. Together, this work reveals a mannose–γδ T cell–IL-17a axis in SLE immunopathogenesis and highlights glycometabolic reprogramming as a therapeutic strategy for autoimmune disease treatment. Branching out: Differential glycosylation patterns on proteins are becoming increasingly implicated in inflammatory diseases. Here, Alves et al. identified aberrant mannosylation patterns on the surface of kidney cells as a potential driver of lupus nephritis. Mice lacking Mgat5 had more mannosylation on the surface of kidney cells, which was also associated with activation of IL-17a–producing γδ T cells and spontaneous development of lupus-like disease in older mice. Therapeutic supplementation of N-acetylglucosamine, which promoted tolerogenic branched N-glycan synthesis, inhibited γδ T cell-driven pathogenesis, suggesting that glycometabolic reprogramming could be a therapeutic strategy for lupus nephritis. —CM [ABSTRACT FROM AUTHOR]

Within the thymus, thymic epithelial cells (TECs) provide a dedicated niche for the selection of functional T cells expressing a highly variable and self‐tolerant T‐cell receptor (TCR) repertoire. In this minireview, we start by summarizing recent studies that have improved our understanding on the composition of cortical TEC and medullary TEC microenvironments. Next, we focus on the molecular processes that control the function of TECs in T‐cell selection. In particular, we discuss the role of cortical TECs in positive selection and the pathways employed by these cells to generate and present selecting self‐peptides:MHC II complexes. Several studies have underscored the role of the β5t‐containing thymoproteasome in the production of unique MHC I‐bound peptides critical for CD8 T‐cell selection. Contrarily, the identity of the molecular determinants that regulate the generation of MHC II‐bound self‐peptides capable of positive selecting CD4 T cells is far more uncertain. We highlight recent advances that interconnect the autophagy‐lysosomal pathway, the presentation of specific sets of self‐peptide:MHC II complexes, and the diversification of CD4 TCR repertoire. Lastly, we discuss how these findings may open up new avenues for deciphering the identity of the MHC I and MHC II ligandome in the thymus. [ABSTRACT FROM AUTHOR]

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire. Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type. [ABSTRACT FROM AUTHOR]

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi , is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response. [ABSTRACT FROM AUTHOR]

Summary: We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex‐determining region Y‐box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T‐box transcription factor TBX21 (T‐bet), programmed cell death protein 1 (PD‐1), programmed‐death ligand 1 (PD‐L1) and CD163 were investigated for all‐cause mortality in 282 patients with MCL and time‐to‐progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3+ T lymphocytes was seen. T‐cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki‐67, morphology and frequency of tumour cells. The all‐cause mortality was higher in patients with PD‐L1‐expression above cut‐off [hazard ratio (HR) 1·97, 95% confidence interval (CI) 1·18–3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163+ cells (continuously, HR 1·51, 95% CI 1·03–2·23, adjusting for age, sex, morphology, Ki‐67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3+ cells (HR 3·22, 95% CI 1·40–7·43) and CD163+ cells (HR 6·09, 95% CI 1·84–20·21), independent of sex and MIPI. When combined a higher frequency of CD163+ macrophages and PD‐L1+ cells or high CD163+ macrophages and FoxP3+ regulatory T cells indicated worse outcome independent of established risk factors. The T‐cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age. [ABSTRACT FROM AUTHOR]

Medullary thymic epithelial cells (mTECs) play a central role in T cell tolerance. However, how the mTEC compartment is maintained remains elusive. We review recent discoveries on new transcription factors involved in mTEC homeostasis and discuss the possibility that their actions might be facilitated by the unique biology of mTECs. [ABSTRACT FROM AUTHOR]

Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life. [ABSTRACT FROM AUTHOR]

Although CD4+ Foxp3+ T cells are largely described in the regulation of CD4+ T cell responses, their role in the suppression of CD8+ T cell priming is much less clear. Because the induction of CD8+ T cells during experimental infection with Trypanosoma cruzi is remarkably delayed and suboptimal, we raised the hypothesis that this protozoan parasite actively induces the regulation of CD8+ T cell priming. Using an in vivo assay that eliminated multiple variables associated with antigen processing and dendritic cell activation, we found that injection of bone marrow-derived dendritic cells exposed to T. cruzi induced regulatory CD4+ Foxp3+ T cells that suppressed the priming of transgenic CD8+ T cells by peptide-loaded BMDC. This newly described suppressive effect on CD8+ T cell priming was independent of IL-10, but partially dependent on CTLA-4 and TGF-β. Accordingly, depletion of Foxp3+ cells in mice infected with T. cruzi enhanced the response of epitope-specific CD8+ T cells. Altogether, our data uncover a mechanism by which T. cruzi suppresses CD8+ T cell responses, an event related to the establishment of chronic infections. [ABSTRACT FROM AUTHOR]

The β1i, β2i and β5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected β1i, β2i and β5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

Background: Recent studies have tried to identify host genetic variants that could explain severe cases and deaths in infection with Influenza A(H1N1)pdm09, especially among children and young adults. CCR5 is a chemokine receptor expressed on T cells, macrophages and dendritic cells, which is an important mediator of leukocyte chemotaxis during the immune response. A deletion mutation (Δ32) in this gene interferes with the response of immune cells, impairing viral clearance. We evaluated the CCR5Δ32 polymorphism (rs333) in individuals of the Brazilian admixed population with a diagnosis of Influenza A(H1N1)pdm09 infection. Methods: A total of 330 subjects with a diagnosis of Influenza A(H1N1)pdm09, evaluated at health services in the northern and northeastern regions of Brazil between June 2009 and August 2010, were genotyped for the Δ32 deletion (rs333). The cases were classified according to the progression of infection into a group of hospitalized patients (n = 156) and a group of non-hospitalized patients (n = 174). Results: No significant differences in the allele or genotype frequencies of the CCR5Δ32 polymorphism were observed between non-hospitalized and hospitalized patients (p = 0.289 and p = 0.431, respectively). Conclusion: The Δ32 deletion in the CCR5 gene is not associated with an unfavorable outcome in patients infected with Influenza A(H1N1)pdm09 in the Brazilian admixed population. [ABSTRACT FROM AUTHOR]

Targeting antigens to dendritic cells (DCs) by using hybrid monoclonal antibodies (mAbs) directed against DC receptors is known to improve activation and support long-lasting T cell responses. In the present work, we used the mAb αDEC205 fused to the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) to identify a region of this protein recognized by specific T cells. The hybrid αDEC-ASP2 mAb was successfully generated and preserved its ability to bind the DEC205 receptor. Immunization of BALB/c mice with the recombinant mAb in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)) specifically enhanced the number of IFN-γ producing cells and CD4+ T cell proliferation when compared to mice immunized with a mAb without receptor affinity or with the non-targeted ASP-2 protein. The strong immune response induced in mice immunized with the hybrid αDEC-ASP2 mAb allowed us to identify an ASP-2-specific CD4+ T cell epitope recognized by the BALB/c MHCII haplotype. We conclude that targeting parasite antigens to DCs is a useful strategy to enhance T cell mediated immune responses facilitating the identification of new T-cell epitopes. [ABSTRACT FROM AUTHOR]

The gut-brain-microbiota axis is increasingly recognized as an important regulator of intestinal physiology. Exposure to psychological stress causes activation of the hypothalamic-pituitary-adrenal (HPA) axis and causes altered intestinal barrier function, intestinal dysbiosis, and behavioral changes. The primary aim of this study was to determine whether the effects of psychological stress on intestinal physiology and behavior, including anxiety and memory, are mediated by the adaptive immune system. Furthermore, we wanted to determine whether treatment with probiotics would normalize these effects. Here we demonstrate that B and T cell-deficient Rag1−/− mice displayed altered baseline behaviors, including memory and anxiety, accompanied by an overactive HPA axis, increased intestinal secretory state, dysbiosis, and decreased hippocampal c-Fos expression. Both local (intestinal physiology and microbiota) and central (behavioral and hippocampal c-Fos) changes were normalized by pretreatment with probiotics, indicating an overall benefit on health conferred by changes in the microbiota, independent of lymphocytes. Taken together, these findings indicate a role for adaptive immune cells in maintaining normal intestinal and brain health in mice and show that probiotics can overcome this immune-mediated deficit in the gut-brain-microbiota axis. [ABSTRACT FROM AUTHOR]

Owing to the importance of major histocompatibility complex class Ia-restricted CD8+ T cells for host survival following viral, bacterial, fungal, or parasitic infection, it has become largely accepted that these cells should be considered in the design of a new generation of vaccines. For the past 20 years, solid evidence has been provided that the heterologous prime-boost regimen achieves the best results in terms of induction of long-lived protective CD8+ T cells against a variety of experimental infections. Although this regimen has often been used experimentally, as is the case for many vaccines, the mechanism behind the efficacy of this vaccination regimen is still largely unknown. The main purpose of this review is to examine the characteristics of the protective CD8+ T cells generated by this vaccination regimen. Part of its efficacy certainly relies on the generation and maintenance of large numbers of specific lymphocytes. Other specific characteristics may also be important, and studies on this direction have only recently been initiated. So far, the characterization of these protective, long-lived T cell populations suggests that there is a high frequency of polyfunctional T cells; these cells cover a large breadth and display a T effector memory (TEM) phenotype. These TEM cells are capable of proliferating after an infectious challenge and are highly refractory to apoptosis due to a control of the expression of pro-apoptotic receptors such as CD95. Also, they do not undergo significant long-term immunological erosion. Understanding the mechanisms that control the generation and maintenance of the protective activity of these long-lived TEM cells will certainly provide important insights into the physiology of CD8+ T cells and pave the way for the design of new or improved vaccines. [ABSTRACT FROM AUTHOR]

Owing to the importance of major histocompatibility complex class Ia-restricted CD8+ T cells for host survival following viral, bacterial, fungal, or parasitic infection, it has become largely accepted that these cells should be considered in the design of a new generation of vaccines. For the past 20 years, solid evidence has been provided that the heterologous prime-boost regimen achieves the best results in terms of induction of long-lived protective CD8+ T cells against a variety of experimental infections. Although this regimen has often been used experimentally, as is the case for many vaccines, the mechanism behind the efficacy of this vaccination regimen is still largely unknown. The main purpose of this review is to examine the characteristics of the protective CD8+ T cells generated by this vaccination regimen. Part of its efficacy certainly relies on the generation and maintenance of large numbers of specific lymphocytes. Other specific characteristics may also be important, and studies on this direction have only recently been initiated. So far, the characterization of these protective, long-lived T cell populations suggests that there is a high frequency of polyfunctional T cells; these cells cover a large breadth and display a T effector memory (TEM) phenotype. These TEM cells are capable of proliferating after an infectious challenge and are highly refractory to apoptosis due to a control of the expression of pro-apoptotic receptors such as CD95. Also, they do not undergo significant long-term immunological erosion. Understanding the mechanisms that control the generation and maintenance of the protective activity of these long-lived TEM cells will certainly provide important insights into the physiology of CD8+ T cells and pave the way for the design of new or improved vaccines. [ABSTRACT FROM AUTHOR]

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviralvaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination. [ABSTRACT FROM AUTHOR]

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development. [ABSTRACT FROM AUTHOR]

Background: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone. Methodology: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8+ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation. Conclusion: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi. Author Summary: Over 11 million individuals are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which kills >50,000 people annually. Although recent vector control efforts and increased use and effectiveness of chemotherapeutic drugs including benznidazole have reduced infection rates and mortality, a safe, effective vaccine is needed. Vaccination with the T. cruzi trans-sialidase (TS) has been used effectively in mice to reduce mortality and chronic disease, however, the establishment of vaccine-induced long-term protective immunity remains elusive. Co-immunization strategies utilizing immune regulators such as interleukin-12 (IL-12) and interleukin-15 (IL-15) can be used to enhance antigen-specific T cell responses and prolong protective immunity. In the present report, we show that genetic vaccination of BALB/c mice with plasmid DNA encoding both TS and IL-15 compared with plasmid DNA encoding TS alone significantly enhanced CD4+ and CD8+ T cell responses including increased TNF-α, IFN-γ, and IL-2 production, and long-term protection against lethal systemic parasite challenge. [ABSTRACT FROM AUTHOR]

Introduction: Cancer stems from mutations in specific genes that induce uncontrolled cell proliferation. Dendritic cells (DCs) are important immunologic cells and play a crucial role in the induction of an antitumour response. Patients and methods: We examined the immune response mediated by T lymphocytes, helper T cells, cytotoxic T cells, and regulatory T cells, as well as the cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-10], produced by these cell populations, in cancer patients (N = 7) undergoing immunotheraphy with autologous DCs. Results: We observed an initial increase in T helper cells (CD4+) expressing IL-2, IFN-γ, IL-12, TNF-α, and IL-10 after initiation of treatment, with statistically significant for the cytokines IL-2, TNF-α and IL-10. A similar significant effect was observed for IL-2-expressing cytotoxic T cells (CD8+). The percentage of total T cells (CD3+) remained elevated throughout immunotherapy. Regulatory T cells (CD25+/FOXP3+) only showed high percentage of their maximum value when analyzed the pretreatment levels, with statistically significant. Conclusion: Immunotherapy with DCs stimulated the immune response, as evidenced by an increase in percent fluorescence of most cell populations investigated during the specified treatment period. [ABSTRACT FROM AUTHOR]

Background: The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. Results: Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. Conclusions: We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response. [ABSTRACT FROM AUTHOR]

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-c secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2-/-, Tlr4-/-, Tlr9-/- or Myd88-/- mice generated both specific cytotoxic responses and IFN-c secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-c+CD4+ cells was diminished in infected Myd88-/- mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-c, TNF-a and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4-/- mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi. [ABSTRACT FROM AUTHOR]

Oral tolerance is a T-cell mediated phenomenon defined by inhibition of immune responsiveness to a protein previously contacted by the oral route. Oral tolerance may prevent autoimmune and allergic diseases that involve the recruitment and/or activation of different cell types including mast cells, neutrophils, eosinophils, monocytes and lymphocytes. The mechanisms by which oral tolerance avoids these immunological disorders are still controversial. Herein we used a murine model of ovalbumin (OVA)-induced peritonitis to investigate the effect of oral tolerance on allergic inflammation. Frequency of leucocyte subpopulations was evaluated by global and differential cell counts in peritoneal lavage fluid, peripheral blood, and bone marrow. Changes on lymphocyte subsets and adhesion molecules expression by these cells were analysed by flow cytometry. As compared with OVA-immune mice, intraperitoneal challenge of tolerant animals with OVA resulted in a significantly milder peritonitis, mostly affecting neutrophils and eosinophils; a concomitant reduction in total white blood cell counts was also observed, mainly because of lower neutrophil and eosinophil counts. Eosinophils, but not neutrophils, were also reduced in the bone-marrow of OVA-challenged tolerant mice. No changes occurred in total peritoneal lymphocyte counts in OVA-tolerant mice, however, there was a significant decrease in CD3+ CD8+ T cells and an increase in B cells (CD45R+) in these animals as compared to immune OVA-challenged animals. Altered expression of CD18 and CD54, respectively, in blood and peritoneal lymphocytes was also noted. These results suggest that, in addition to local specific effects, oral tolerance has systemic effects on the mobilization of leucocytes and bone-marrow eosinopoiesis. [ABSTRACT FROM AUTHOR]

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4+CTLA4+Foxp3+ cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion. depletion of TCD4+CTLA4+Foxp3+ cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 ± 50.04; PC61: 187.23 ± 3138: DTA-1: 64.53 ± 25.65, mg/dL p<0.05). These data were corroborated by histopathology. We observed an increase of HO-I expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61(anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. [ABSTRACT FROM AUTHOR]

Despite the well-documented effect of castration in thymic regeneration, the singular contribution of the bone marrow (BM) versus the thymus to this process remains unclear. The chief role of IL-7 in pre- and intrathymic stages of T lymphopoiesis led us to investigate the impact of disrupting this cytokine during thymic rebound induced by androgen blockade. We found that castration promoted thymopoiesis in young and aged wild-type mice. In contrast, only young germline IL-7-deficient (Il7-/-) mice consistently augmented thymopoiesis after castration. The increase in T cell production was accompanied by the expansion of the sparse medullary thymic epithelial cell and the peripheral T cell compartment in young Il7-/- mice. In contrast to young Il7-/- and wild-type mice, the poor thymic response of aged Il7-/- mice after castration was associated with a defect in the expansion of BM hematopoietic progenitors. These findings suggest that BM-derived T cell precursors contribute to thymic rebound driven by androgen blockade. To assess the role of IL-7 within the thymus, we generated mice with conditional deletion of IL-7 (Il7 conditional knockout [cKO]) in thymic epithelial cells. As expected, Il7cKO mice presented a profound defect in T cell development while maintaining an intact BM hematopoietic compartment across life. Unlike Il7-/- mice, castration promoted the expansion of BM precursors and enhanced thymic activity in Il7cKO mice independently of age. Our findings suggest that the mobilization of BM precursors acts as a prime catalyst of castration-driven thymopoiesis. [ABSTRACT FROM AUTHOR]

Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8+ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8+ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1High CD27Low CD43Low CD183Low T-betHigh EomesLow phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8+ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8+ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8+ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity. [ABSTRACT FROM AUTHOR]

Abstract: Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2d-restricted CD8+ T cell-specific epitope (CS280–288) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS280–288 peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS280–288 peptide. The results showed that CS280–288-specific cytotoxic CD8+ T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280–288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280–288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8+ T cell responses without the need of a heterologous booster immunization. The CD8+ T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c+ dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. [Copyright &y& Elsevier]

Abstract: Immunisation with Amastigote Surface Protein 2 (asp-2) and trans-sialidase (ts) genes induces protective immunity in highly susceptible A/Sn mice, against infection with parasites of the Y strain of Trypanosoma cruzi. Based on immunological and biological strain variations in T. cruzi parasites, our goal was to validate our vaccination results using different parasite strains. Due to the importance of the CD8+ T cells in protective immunity, we initially determined which strains expressed the immunodominant H-2Kk-restricted epitope TEWETGQI. We tested eight strains, four of which elicited immune responses to this epitope (Y, G, Colombian and Colombia). We selected the Colombian and Colombia strains for our studies. A/Sn mice were immunised with different regimens using both T. cruzi genes (asp-2 and ts) simultaneously and subsequently challenged with blood trypomastigotes. Immune responses before the challenge were confirmed by the presence of specific antibodies and peptide-specific T cells. Genetic vaccination did not confer protective immunity against acute infection with a lethal dose of the Colombian strain. In contrast, we observed a drastic reduction in parasitemia and a significant increase in survival, following challenge with an otherwise lethal dose of the Colombia strain. In many surviving animals with late-stage chronic infection, we observed alterations in the heart''s electrical conductivity, compared to naive mice. In summary, we concluded that immunity against T. cruzi antigens, similar to viruses and bacteria, may be strain-specific and have a negative impact on vaccine development. [Copyright &y& Elsevier]

Abstract: We recently described that vaccination of mice with a gluthatione S transferase fusion protein representing amino acids 261–500 of the Amastigote Surface Protein-2 efficiently cross-primed protective CD8+ T cells against a lethal challenge with the human protozoan parasite Trypanosoma cruzi. In this study, we initially established that this protective immunity was long lived. Subsequently, we studied the importance of TLR9 agonist CpG ODN 1826, TLR4 and CD4+ T cells for the generation of these protective CD8+ T cells. We found that: (i) the TLR9 agonist CpG ODN 1826 improved the efficiency of protective immunity; (ii) TLR4 is not relevant for priming of specific CD8+ T cells; (iii) CD4+ T cells are critical for priming of memory/protective CD8+ T cells. [Copyright &y& Elsevier]