Predominance of CD137+ And TNF-α Expressing CD8+ Central Memory T Cells in Mild COVID-19 Recovered Patients Upon SARS-CoV-2 Re-Exposure.

INTRODUCTION: Memory CD8+ T cells are essential for long-term immune protection in viral infections, including COVID-19. METHODS: This study examined the responses of CD8+ TEM, TEMRA, and TCM subsets from unvaccinated individuals who had recovered from mild and severe COVID-19 by flow cytometry. RESULTS AND DISCUSSION: The peptides triggered a higher frequency of CD8+ TCM cells in the recovered mild group. CD8+ TCM and TEM cells showed heterogeneity in CD137 expression between evaluated groups. In addition, a predominance of CD137 expression in naïve CD8+ T cells, TCM, and TEM was observed in the mild recovered group when stimulated with peptides. Furthermore, CD8+ TCM and TEM cell subsets from mild recovered volunteers had higher TNF-α expression. In contrast, the expression partner of IFN-γ, IL-10, and IL-17 indicated an antiviral signature by CD8+ TEMRA cells. These findings underscore the distinct functional capabilities of each memory T cell subset in individuals who have recovered from COVID-19 upon re-exposure to SARS-CoV-2 antigens.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

The ecto-enzyme CD38 modulates CD4T cell immunometabolic responses and participates in HIV pathogenesis.

Despite abundant evidence correlating T cell CD38 expression and HIV infection pathogenesis, its role as a CD4T cell immunometabolic regulator remains unclear. We find that CD38's extracellular glycohydrolase activity restricts metabolic reprogramming after T cell receptor (TCR)-engaging stimulation in Jurkat T CD4 cells, together with functional responses, while reducing intracellular nicotinamide adenine dinucleotide and nicotinamide mononucleotide concentrations. Selective elimination of CD38's ectoenzyme function licenses them to decrease the oxygen consumption rate/extracellular acidification rate ratio upon TCR signaling and to increase cycling, proliferation, survival, and CD40L induction. Pharmacological inhibition of ecto-CD38 catalytic activity in TM cells from chronic HIV-infected patients rescued TCR-triggered responses, including differentiation and effector functions, while reverting abnormally increased basal glycolysis, cycling, and spontaneous proinflammatory cytokine production. Additionally, ecto-CD38 blockage normalized basal and TCR-induced mitochondrial morphofunctionality, while increasing respiratory capacity in cells from HIV+ patients and healthy individuals. Ectoenzyme CD38's immunometabolic restriction of TCR-involving stimulation is relevant to CD4T cell biology and to the deleterious effects of CD38 overexpression in HIV disease.

Central and Effector Memory Human CD4+ and CD8+ T Cells during Cutaneous Leishmaniasis and after In Vitro Stimulation with Leishmania (Viannia) braziliensis Epitopes

Cutaneous Leishmaniasis (CL) is a Neglected Tropical Disease characterized by skin ulcers caused by Leishmania spp. protozoans and there is no safe and effective vaccine to reduce its negative consequences. In a previous work by our group, we identified T cell epitopes of Leishmania (Viannia) braziliensis which stimulated patients' T cells in vitro. In the present work, the peptides were tested as two pools for their ability to rescue memory T cells during natural infection by Leishmania. We analyzed the frequency of central memory (TCM, CD45RA-CD62L+) and effector memory (TEM, CD45RA + CD62L-) cells during active CL and post-treatment. In parallel, we investigated cell proliferation levels and the cytokines produced after stimulation. Interestingly, we observed higher frequencies (%) in CD4+ TEM during CL, and CD8+ TEM and CD8+ TCM during CL and post-treatment. Cell proliferation was increased, and a significant difference in expression was observed on T-bet and RORγT. Besides that, IFN-γ, IL-2, and IL-10 were detected in patient samples. Collectively, this dataset suggests that during CL there is an increase in the frequency of TCM and TEM, especially in the CD8 compartment. These results indicate a potentially immunogenic profile of the peptide pools, which can support the development of anti-Leishmania formulations.

Modulation of the Response to Mycobacterium leprae and Pathogenesis of Leprosy.

The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae-specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic changes, including the formation of lipid droplets, that are associated with macrophage M2 phenotype and the production of mediators that facilitate the differentiation of specific T cells for M. leprae-expressed antigens to a memory regulatory phenotype. Here, we discuss the possible initials steps of M. leprae infection that may lead to active disease onset, mainly focusing on events prior to the manifestation of the established clinical forms of leprosy. We hypothesize that the progressive differentiation of T cells to the Tregs phenotype inhibits effector function against the bacillus, allowing an increase in the bacillary load and evolution of the infection to active disease. Epigenetic and metabolic mechanisms described in other chronic inflammatory diseases are evaluated for potential application to the understanding of leprosy pathogenesis. A potential role for post-exposure prophylaxis of leprosy in reducing M. leprae-induced anti-inflammatory mediators and, in consequence, Treg/T effector ratios is proposed.

Tumor-Specific CD8+ T-Cell Responses Induced by DNA Vaccination.

DNA vaccines assisted by electroporation efficiently trigger antitumor cytotoxic CD8+ T cell responses in preclinical cancer models and hold potential for human use. They can be easily engineered to express either tumor-associated self-antigens, which are broadly expressed among tumor patients but also in healthy tissue, or tumor-specific neoantigens, which are uniquely expressed in tumors and differ among patients. Recently, it has been demonstrated that DNA vaccination generates both circulating and tissue-resident compartments of CD8+ T cells, which act concertedly against tumors. Here we describe the steps to obtain and test DNA vaccines against models of self-antigens and neoantigens in mice. It includes the evaluation of effector and memory CD8+ T cell responses, as well as assessing the antitumor potential in vivo using transplantable syngeneic tumor models.

Induction of Progenitor Exhausted Tissue-Resident Memory CD8+ T Cells Upon Salmonella Typhi Porins Adjuvant Immunization Correlates With Melanoma Control and Anti-PD-1 Immunotherapy Cooperation.

Immunotherapy has improved the clinical response in melanoma patients, although a relevant percentage of patients still cannot be salvaged. The search for the immune populations that provide the best tumor control and that can be coaxed by immunotherapy strategies is a hot topic in cancer research nowadays. Tumor-infiltrating TCF-1+ progenitor exhausted CD8+ T cells seem to grant the best melanoma prognosis and also efficiently respond to anti-PD-1 immunotherapy, giving rise to a TIM-3+ terminally exhausted population with heightened effector activity. We tested Porins from Salmonella Typhi as a pathogen associated molecular pattern adjuvant of natural or model antigen in prophylactic and therapeutic immunization approaches against murine melanoma. Porins induced protection against melanomas, even upon re-challenging of tumor-free mice. Porins efficiently expanded IFN-γ-producing CD8+ T cells and induced central and effector memory in lymph nodes and tissue-resident (Trm) T cells in the skin and tumors. Porins induced TCF-1+ PD-1+ CD8+ Trm T cells in the tumor stroma and the presence of this population correlated with melanoma growth protection in mice. Porins immunization also cooperated with anti-PD-1 immunotherapy to hamper melanoma growth. Importantly, the potentially protective Trm populations induced by Porins in the murine model were also observed in melanoma patients in which their presence also correlated with disease control. Our data support the use of cancer vaccination to sculpt the tumor stroma with efficient and lasting Trm T cells with effector activities, highlighting the use of Porins as an adjuvant. Furthermore, our data place CD8+ Trm T cells with a progenitor exhausted phenotype as an important population for melanoma control, either independently or in cooperation with anti-PD-1 immunotherapy.

Chimeric Vaccines Designed by Immunoinformatics-Activated Polyfunctional and Memory T Cells That Trigger Protection against Experimental Visceral Leishmaniasis.

Many vaccine candidates against visceral leishmaniasis (VL) have been proposed; however, to date, none of them have been efficacious for the human or canine disease. On this basis, the design of leishmaniasis vaccines has been constantly changing, and the use of approaches to select specific epitopes seems to be crucial in this scenario. The ability to predict T cell-specific epitopes makes immunoinformatics an even more necessary approach, as in VL an efficient immune response against the parasite is triggered by T lymphocytes in response to Leishmania spp. immunogenic antigens. Moreover, the success of vaccines depends on the capacity to generate long-lasting memory and polyfunctional cells that are able to eliminate the parasite. In this sense, our study used a combination of different approaches to develop potential chimera candidate vaccines against VL. The first point was to identify the most immunogenic epitopes of Leishmania infantum proteins and construct chimeras composed of Major histocompatibility complex (MHC) class I and II epitopes. For this, we used immunoinformatics features. Following this, we validated these chimeras in a murine model in a thorough memory study and multifunctionality of T cells that contribute to a better elucidation of the immunological protective mechanisms of polyepitope vaccines (chimera A and B) using multicolor flow cytometry. Our results showed that in silico-designed chimeras can elicit polyfunctional T cells producing T helper (Th)1 cytokines, a strong immune response against Leishmania antigen, and the generation of central and effector memory T cells in the spleen cells of vaccinated animals that was able to reduce the parasite burden in this organ. These findings contribute two potential candidate vaccines against VL that can be used in further studies, and help in this complex field of vaccine development against this challenging parasite.

Dormant tumor cells interact with memory CD8+ T cells in RET transgenic mouse melanoma model.

Melanoma is an aggressive form of skin-cancer. Melanoma cells are characterized by their plasticity, resulting in therapy resistance. Using RET transgenic mouse melanoma model, we characterized dormant tumor cells accumulated in the bone marrow (BM) and investigated their interaction with effector memory CD8+ T cells. We found that cells expressing melanoma-associated antigen tyrosinase related protein (TRP)-2 and stemness marker CD133 represented less than 1.5% of all melanoma cells in primary skin lesions and metastatic lymph nodes. The majority of these cells were negative for the proliferation marker Ki67. In the BM, CD133+TRP-2+ melanoma cells displayed an aberrant expression of p16, p27, Ki67 and PCNA proteins, suggesting their dormant phenotype. Moreover, these cells were characterized by an elevated expression of various molecules characterized stemness, metastatic, angiogenic and immunosuppressive properties such as CD271, CD34, HIF-1α, CXCR3, CXCR4, VEGR2, PD-L1, CTLA-4, CD39 and CCR4 as compared to their CD133- counterparts. Disseminated BM dormant TRP-2+ tumor cells were found to be co-localized with memory CD8+ T cells. Our data suggest that these dormant melanoma cells in the BM could play an important role in the maintenance of memory T cells in the BM.

Synthetic Peptides Elicit Strong Cellular Immunity in Visceral Leishmaniasis Natural Reservoir and Contribute to Long-Lasting Polyfunctional T-Cells in BALB/c Mice.

Reverse vaccinology or immunoinformatics is a computational methodology which integrates data from in silico epitope prediction, associated to other important information as, for example, the predicted subcellular location of the proteins used in the design of the context of vaccine development. This approach has the potential to search for new targets for vaccine development in the predicted proteome of pathogenic organisms. To date, there is no effective vaccine employed in vaccination campaigns against visceral leishmaniasis (VL). For the first time, herein, an in silico, in vitro, and in vivo peptide screening was performed, and immunogenic peptides were selected to constitute VL peptide-based vaccines. Firstly, the screening of in silico potential peptides using dogs naturally infected by L. infantum was conducted and the peptides with the best performance were selected. The mentioned peptides were used to compose Cockt-1 (cocktail 1) and Cockt-2 (cocktail 2) in combination with saponin as the adjuvant. Therefore, tests for immunogenicity, polyfunctional T-cells, and the ability to induce central and effector memory in T-lymphocytes capacity in reducing the parasite load on the spleen for Cockt-1 and Cockt-2 were performed. Among the vaccines under study, Cockt-1 showed the best results, eliciting CD4+ and CD8+ polyfunctional T-cells, with a reduction in spleen parasitism that correlates to the generation of T CD4+ central memory and T CD8+ effector memory cells. In this way, our findings corroborate the use of immunoinformatics as a tool for the development of future vaccines against VL.

Characterization of memory T cells in individuals resistant to Schistosoma mansoni infection.

Schistosomiasis affects about 240 million people worldwide and is estimated that about 700 million people live in areas at risk of infection. In the context of immune response associated with infection by Schistosoma mansoni, the role of memory T cells is not well understood. AIM: To evaluate the frequency of memory CD4+ and CD8+ T cells from individuals resistant and susceptible to Schistosoma mansoni infection. METHODS AND RESULTS: We selected individuals with low (resistant) and high (susceptible) parasite burden using databases generated during previous studies carried out in the same endemic area. The cell surface markers were performed using flow cytometry. In this study, the resistant individuals showed an increase in the CD4+ memory T-cell pool associated with an increase in the central memory cell (TCM) and a decrease in the effector memory cell (TEM ). Individuals susceptible to infection had higher frequencies of effector memory cells compared to resistant individuals. CONCLUSIONS: These data suggest that resistance to S mansoni infection may be associated with an increase in the number of CD4+ memory T cells and susceptibility to infection is associated with a decrease in the central memory cell as well as high proportions of effector memory cells.

Contribution of Resident Memory CD8+ T Cells to Protective Immunity Against Respiratory Syncytial Virus and Their Impact on Vaccine Design.

Worldwide, human respiratory syncytial virus (RSV) is the most common etiological agent for acute lower respiratory tract infections (ALRI). RSV-ALRI is the major cause of hospital admissions in young children, and it can cause in-hospital deaths in children younger than six months old. Therefore, RSV remains one of the pathogens deemed most important for the generation of a vaccine. On the other hand, the effectiveness of a vaccine depends on the development of immunological memory against the pathogenic agent of interest. This memory is achieved by long-lived memory T cells, based on the establishment of an effective immune response to viral infections when subsequent exposures to the pathogen take place. Memory T cells can be classified into three subsets according to their expression of lymphoid homing receptors: central memory cells (TCM), effector memory cells (TEM) and resident memory T cells (TRM). The latter subset consists of cells that are permanently found in non-lymphoid tissues and are capable of recognizing antigens and mounting an effective immune response at those sites. TRM cells activate both innate and adaptive immune responses, thus establishing a robust and rapid response characterized by the production of large amounts of effector molecules. TRM cells can also recognize antigenically unrelated pathogens and trigger an innate-like alarm with the recruitment of other immune cells. It is noteworthy that this rapid and effective immune response induced by TRM cells make these cells an interesting aim in the design of vaccination strategies in order to establish TRM cell populations to prevent respiratory infectious diseases. Here, we discuss the biogenesis of TRM cells, their contribution to the resolution of respiratory viral infections and the induction of TRM cells, which should be considered for the rational design of new vaccines against RSV.

Physical fitness modulates the expression of CD39 and CD73 on CD4+ CD25- and CD4+ CD25+ T cells following high intensity interval exercise.

AIM: To investigate the impact of physical fitness on the mobilization of CD4+ CD25 - CD39 + and CD4 + CD25 + CD39 + T cells in response to acute exercise. METHODS: Fifteen high physical fitness (25.3 ± 1.4 years) and 15 low physical fitness (26.1 ± 1.9 years) men performed a single bout of high-intensity interval exercise (HIIE, 10 bouts of 60 seconds at 85% HRmax intercepted by 75 seconds of recovery at 50% HRmax). Blood lymphocytes were isolated before, immediately after and 1 hour after exercise for assessment of cell surface expression of CD25, CD39, and CD73 on CD4+ T cells. Effector memory T cells (mTeff) were identified by CD4 + CD25 - CD39 + coexpression, and memory regulatory T cells (mTReg) were defined as CD4 + CD25 + CD39 + T cells. RESULTS: Exercise increased CD4+ and CD4 + CD25 + T cell frequencies immediately after followed by a decrease bellow to baseline values at 1 hour after the bout in both low and high physical fitness groups. At baseline, the proportions of mTeff were higher, while mTreg were lower in low physical fitness individuals. The frequency of mTreg increased immediately after HIIE in both groups, and remained higher 1 hour after the bout. However, high physical fitness individuals presented higher mTreg frequency in all periods evaluated. A significantly mobilization of mTeff cells was identified in both groups immediately after HIIE. High physical fitness individuals displayed a decrease in mTeff cells bellow to baseline, while the frequency of mTeff remained higher in low physical fitness group 1 hour after the bout. The peripheral frequency of CD4 + CD25 + CD73 + T cells increased in a similar way immediately after the bout in both groups, returning to the baseline values 1 hour after exercise. No differences in CD4 + CD25 - CD73 + T cells were observed after HIIE in both groups. CONCLUSION: Our results highlight the impact of physical activity status in the redistribution of CD4+ T cells expressing ectonucleotidases in response to HIIE.

Loss of T-Cell Multifunctionality and TCR-Vß Repertoire Against Epstein-Barr Virus Is Associated With Worse Prognosis and Clinical Parameters in HIV+ Patients.

Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of aggressive and poor-prognosis B-cell lymphomas in patients infected with human immunodeficiency virus (HIV+ patients). The most important risk factors for these malignancies include immune dysfunction, chronic immune activation, and loss of T-cell receptor (TCR) repertoire. The combination of all these factors can favor the reactivation of EBV, malignant cell transformation, and clinical progression toward B-cell lymphomas. The overarching aim of this study was to evaluate the frequency, phenotype, functionality, and distribution of TCR clonotypes for EBV-specific T-cell subpopulations in HIV+ patients at different clinical stages and for HIV+ patients with B-cell lymphoma, as well as to establish their association with clinical variables of prognostic value. Factors were studied in 56 HIV+ patients at different clinical stages and in six HIV+ subjects with diagnosed B-cell lymphoma. We found a significant decrease in all subpopulations of EBV-specific CD4+ T cells from HIV+ patients at stage 3 and with B-cell lymphoma. EBV-specific effector CD8+ T cells, particularly effector memory cells, were also reduced in HIV+ patients with B-cell lymphoma. Interestingly, these cells were unable to produce IFN-γ and lacked multifunctionality in HIV+ patients. The TCR-Vß repertoire, which is key for protection against EBV in healthy individuals, was less diverse in HIV+ patients due to a lower frequency of TCR-Vß2+, Vß4+, Vß7.1+, Vß9+, Vß13.6+, Vß14+, Vß17+, Vß22+ CD4+, Vß14+, and Vß17+ CD8+ T cells. HIV+ patients with positive plasma EBV loads (EBV+HIV+) had a noteworthy decrease in the levels of both TNF-α+ and multifunctional TNF-α+/IL-2+ and TNF-α+/IFN-γ+ CD8+ T cells. Altogether, our findings demonstrate that HIV+ patients have significant alterations in the immune response to EBV (poor-quality immunity) that can favor viral reactivation, escalating the risk for developing EBV-associated B-cell lymphomas.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study.

BACKGROUND: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. OBJECTIVE: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. METHODS: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. RESULTS: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. CONCLUSIONS: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study

ABSTRACT Background: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. Objective: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. Methods: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. Results: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. Conclusions: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

Kv1.3 channel blockade with the Vm24 scorpion toxin attenuates the CD4+ effector memory T cell response to TCR stimulation.

BACKGROUND: In T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases. METHODS: We combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin. RESULTS: The peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-γ and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. CONCLUSIONS: The Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease.

BACKGROUND: Systemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD. METHODS: Patients with nasal polyposis and asthma with AERD (n=20) and without (n=18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F2, and leukotriene E4 (uLTE4) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges. RESULTS: AERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE4 and percentage of CD4(+)CD25(+)CD127(pos) and CD4(+)CD45RA(-)CD45RO(+) but decreased levels of TGF-ß1 and number of CD4(+)CD25(+)CD127(neg) cells. Aspirin challenge induced the release of uLTE4, IL-6 and increased the number of CD4(+)CD45RA(-)CD45RO(+) memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD20ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal). CONCLUSIONS: AERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

The sterile and tolerogenic fetal niche does not restrict the generation of CD4 T memory cells.

T-cell activation requires a sequence of signals derived from co-stimulatory receptors and from immunogens that may or may not be of infectious origin. This scenario provides the threshold of inflammatory stimulus needed for the induction of antigen-specific T cell responses. One of the dogmas of immunology stipulates that the activation of T lymphocytes is prevented in immunosuppressed or tolerogenic environments. However, it was shown recently that a healthy uterine environment that is considered sterile, therefore not exposed to infection, is capable of generating T memory cells with the capacity to differentiate lineage-specific inflammatory effector T-cell responses. The implications of this finding are discussed in this editorial.