CD8 T lymphocytes from B-1 cell-deficient mice down-regulates fungicidal activity of macrophages challenged with E. Cuniculi.

BACKGROUND: Encephalitozoon cuniculi is an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+ T cells lymphocytes. However, lower resistance to experimental infection with E. cuniculi was found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the profile of CD8+ T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged with E. cuniculi. METHODS: Naïve CD8 T lymphocytes from WT or Xid mice uninfected and primed CD8 T lymphocytes from WT or Xid mice infected with E cuniculi were co-cultured with macrophages previously challenged with E. cuniculi. We evaluated macrophages viability and microbicidal activity, and CD8 T lymphocytes viability and presence of activating molecules (CD62L, CD69, and CD107a). RESULTS: Macrophages co-cultured with naïve CD8 T lymphocytes from WT demonstrated high microbicidal activity. Naïve CD8 T lymphocytes obtained from WT mice had a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+ T lymphocytes. Primed CD8 T lymphocytes from Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, naïve CD8+ T lymphocytes from Xid mice, deficient in B-1 cells, they had reduced expression of activation molecules and cytotoxic activity.

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.

Adenosine Increases the Immunosuppressive Capacity of Cervical Cancer Cells by Increasing PD-L1 Expression and TGF-ß Production through Its Interaction with A2AR/A2BR.

The present study provides evidence showing that adenosine (Ado) increases the expression of programmed death ligand 1 (PD-L1) in cervical cancer (CeCa) cells by interacting with A2AR/A2BR and that TGF-ß1 acts in an autocrine manner to induce PD-L1 expression, enhancing the immunosuppressive effects of CeCa cells on activated T lymphocytes (ATLs) and CD8+ cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from E6 and E7 proteins of HPV-16. Interestingly, the addition of the antagonists ZM241385 and MRS1754, which are specific for A2AR and A2BR, respectively, or SB-505124, which is a selective TGF-ß1 receptor inhibitor, to CeCa cell cultures significantly inhibited PD-L1 expression. In addition, supernatants from CeCa cells that were treated with Ado (CeCa-Ado Sup) increased the expression of PD-1, TGF-ß1, and IL-10 and decreased the expression of IFN-γ in ATLs. Interestingly, the addition of an anti-TGF-ß neutralizing antibody strongly reversed the effect of CeCa-Ado Sup on PD-1 expression in ATLs. These results strongly suggest the presence of a feedback mechanism that involves the adenosinergic pathway, the production of TGF-ß1, and the upregulation of PD-L1 expression in CeCa cells that suppresses the antitumor response of CTLs. The findings of this study suggest that this pathway may be clinically important and may be a therapeutic target.

Adrenocortical Carcinoma Steroid Profiles: In Silico Pan-Cancer Analysis of TCGA Data Uncovers Immunotherapy Targets for Potential Improved Outcomes.

Despite progress in understanding the biology of adrenocortical carcinoma (ACC), treatment options have not dramatically changed in the last three decades, nor have we learned how to avoid some of its long-term side effects. Our goal was to improve the understanding of immune pathways that may include druggable targets to enhance immune responses of patients with ACC, focusing on immune evasion and the activation of immune cells against ACC. Our strategy was aimed at improving insight regarding gene expression without steroid interference. Using approaches based on high and low steroid phenotypes (HSP and LSP, respectively), we characterized immune pathways using The Cancer Genome Atlas (TCGA) ACC cohort data. Although previous studies have suggested that patients with ACC receive minimal benefit from immunotherapy, high expression of immune modulators was noted in patients with LSP, suggesting the activation of these biomarkers may be an important adjuvant therapy target after clearance of excess glucocorticoids. In addition, patients with LSP ACC had higher immune cell infiltration than patients with HSP ACC and other cancer subtypes. Our findings can be summarized as follows (1): we confirmed and improved the definition of two immune response pathways to ACC (HSP and LSP) based on in silico transcriptome analysis (2), we demonstrated the steroid profile should be considered, otherwise analyses of ACC immune characteristics can generate confounding results (3), among the overexpressed immunotherapy targets, we demonstrated that LSP was rich in PDCD1LG2 (PD-L2) and both HSP and LSP overexpressed CD276 (B7-H3), which was associated with resistance to anti-PD1 therapy and may have accounted for the modest results of previous clinical trials, and (4) identification of patients with LSP or HSP ACC can be used to help determine whether immunotherapy should be used. In conclusion, we highlighted the differences between LSP and HSP, drawing attention to potential therapeutic targets (CD276, PDCD1, and PDCD1LG2). Treatments to reduce immune evasion, as well as the use of other natural and pharmacological immune activators, should include prior pharmacological inhibition of steroidogenesis. Attempts to combine these with tumor cell proliferation inhibitors, if they do not affect cells of the immune system, may produce interesting results.

Neuroendocrine apendicopathy in morphologically normal appendices of patients with diagnosis of acute appendicitis: Diagnostic study.

BACKGROUND: About 15%-25% of appendices removed to treat acute appendicitis present normal macro- and macroscopic morphology. The objective of this study was to verify an association of proinflammatory, neuroendocrine and immune mediators with morphologically normal appendices removed from patients with clinical laboratorial and imaging characteristics of acute appendicitis. MATERIALS AND METHODS: Appendices removed from 121 adult patients of both genders were distributed into three groups according to their following characteristics: group 1: 53 macro- and microscopically normal appendices from patients with clinical, laboratorial and imaging diagnosis of acute appendicitis; group 2: 24 inflamed appendices from patients with clinical, laboratorial, imaging and histopathological diagnosis of acute appendicitis; group 3: 44 normal appendices from patients submitted to right colectomy to treat localized ascending colon adenocarcinoma. All appendices were immunohistochemically studied for gastrin inhibitor peptide, mast cell tryptase, vascular endothelial growth factor; intestinal vasoactive peptide, tumor necrosis factor alpha, interleukin 1, prostaglandin E2, gene-protein product 9.5, CD8 T lymphocytes, synaptophysine, enolase, and S100 protein. RESULTS: The group 1 revealed increased levels of synaptophysine, enolase, mast cell tryptase and PGP-9.5 comparing with the other two groups. The group 2 presented increased levels of interleukin 1, CD8 T lymphocytes and prostaglandin E2 comparing with the other two groups. The group 3 confirmed the normal levels of all these neuroendocrine, immune and proinflammatory mediators. CONCLUSIONS: Morphologically normal appendices removed from patients with clinical and complementary exams indicating acute appendicitis have appendicular neuroimmunoendocrine disorder associated with the mediators synaptophysin, enolase, mast cell-related tryptase and gene-protein product 9.5.

Characterization of CD40+ leukocytes in flounder (Paralichthys olivaceus) and its response after Hirame novirhabdovirus infection and immunization.

CD40 is a crucial signal mediating factor in T-dependent B cell responses and involved in many aspects of cellular and humoral immunity. In this paper, recombinant protein of CD40 in flounder (Paralichthys olivaceus) and its antibodies (Abs) were produced, native CD40 molecules in flounder tissues were identified, then the CD40+ leukocytes in T/B lymphocytes were characterized, and the variations of CD40+ leukocytes in flounder after Hirame novirhabdovirus (HIRRV) infection and immunization were investigated, respectively. The results showed that the Abs could specifically recognize native flounder CD40 molecule at 32 kDa. The proportions of CD40+ leukocytes were varied by flounder tissues. CD40+/IgM+ B lymphocytes, CD40+/CD4-1+ T lymphocytes, CD40+/CD4-2+ T lymphocytes and CD40+/CD8+ T lymphocytes in peripheral blood leukocytes (PBLs) were 1.18 ± 0.27%, 0.69 ± 0.17%, 0.75 ± 0.14% and 0.25 ± 0.14%; were 2.80 ± 0.32%, 0.71 ± 0.19%, 0.88 ± 0.23% and 0.33 ± 0.17% in spleen; 4.11 ± 0.47%, 0.92 ± 0.18%, 1.09 ± 0.17% and 0.9 ± 0.17% in head kidney; 1.92 ± 0.39%, 1.02 ± 0.23%, 1.33 ± 0.38% and 0.67 ± 0.24% in intestine; 1.24 ± 0.36%, 1.21 ± 0.24%, 1.70 ± 0.3% and 0.97 ± 0.21% in gill, respectively. The percentages of CD40+ leukocytes in PBLs were significantly increased in both HIRRV infection and immunization groups, and reached their peak levels at 3rd day with 5.70 ± 0.16% and 6.40 ± 0.13%, respectively. Concluded with our previous study, these data first reported that CD40 molecules were expressed on both B and T lymphocytes in teleost, and had a coordination with T and B lymphocytes in immune responses.

T cells responding to Trypanosoma cruzi detected by membrane TNF-α and CD154 in chagasic patients.

INTRODUCTION: Chagas disease is a parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. Variability in cytokine secretion among chronic Trypanosoma cruzi-infected patients might preclude the identification of the pool of antigen specific T cells. The goal of this study was to determine the fraction of T cells responding to T. cruzi antigen measured by the expression of membrane TNF-α and CD154. METHODS: A total of 21 chagasic patients, 11 healthy and 5 non-chagasic cardiomyopathy controls were analyzed. PBMCs were short-term cultured in the presence of anti-CD28, anti-CD49d, anti-TNF-α, and TACE (TNF-α converting enzyme) inhibitor either under T. cruzi-lysate or polyclonal stimuli. Cells were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD154, and analyzed with flow cytometry. RESULTS: CD4+ and CD8+ T cells in chagasic patients displayed higher percentages of membrane-bound TNF-α+ and CD154+ compared with controls after T. cruzi-antigen stimulation. Both markers displayed a positive correlation in the T cell subpopulations analyzed. Symptomatic chagasic patients were differentiated from asymptomatic patients based on the expression of CD154 and membrane TNF-α in TCD4+ and TCD8+ compartments, respectively. CONCLUSIONS: These results show that both markers could be useful for assessing the pool of antigen-specific T cells in chronic chagasic patients.

Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1ß) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1ß production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1. METHODS: C57BL/6 (wild type, WT) and TLR2/9-/- mice were infected by the intranasal route with HSV-1 (1 × 106 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1ß, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1ß and granzyme B were quantified by real time PCR. RESULTS: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1ß and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8+ T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9-/- infected mice. CONCLUSION: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8+ T lymphocytes through IL-1ß, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

Diminished effector and memory CD8+ circulating T lymphocytes in patients with severe influenza caused by the AH1N1 pdm09 virus.

The T cell immune response to viral infection includes the expansion of naïve T cells, effector cell differentiation and the induction of long-lived memory cells. We compared the differentiation of CD8+ T cells in patients with severe or mild pneumonia induced by influenza infection occurring during the 2009 influenza outbreak and compared their T cell subsets with those in blood samples obtained from healthy volunteers before the AH1N1 influenza outbreak in Mexico. Patients with severe influenza exhibited significantly lower numbers of effector memory CD8+CD26 high CD45RO+CCR7+ phenotype and lower numbers of central memory CD8+CD26high CD62L+CCR7+, CD26 high CD62L+CD127+ or CD26 high CD45RO+CD57 low phenotypes than patients with mild influenza or unexposed healthy subjects. Effector T cells with CD8+CD26CD62L low CD57+ phenotype were significantly diminished in severe influenza patients compared to those in patients with mild influenza or unexposed healthy subjects. These results suggest that low levels of circulating CD8+ T effector and central memory cells are associated with influenza severity.

NFAT2 Regulates Generation of Innate-Like CD8+ T Lymphocytes and CD8+ T Lymphocytes Responses.

Nuclear factor of activated T cells (NFAT) 2 null mutant mice die in utero of cardiac failure, precluding analysis of the role of NFAT2 in lymphocyte responses. Only the NFAT2-/-/Rag-1-/- chimeric mice model gave insight into the role of NFAT2 transcription factor in T lymphocyte development, activation, and differentiation. As reports are mainly focused on the role of NFAT2 in CD4+ T lymphocytes activation and differentiation, we decided to investigate NFAT2's impact on CD8+ T lymphocyte responses. We report that NFAT2 is phosphorylated and inactive in the cytoplasm of naive CD8+ T cells, and upon TCR stimulation, it is dephosphorylated and translocated into the nucleus. To study the role of NFAT2 in CD8+ T responses, we employed NFAT2fl/flCD4-Cre mice with NFAT2 deletion specifically in T cells. Interestingly, the absence of NFAT2 in T cells resulted in increased percentage of non-conventional innate-like CD8+ T cells. These cells were CD122+, rapid producer of interferon gamma (IFN-γ) and had characteristics of conventional memory CD8+ T cells. We also observed an expansion of PLZF+ expressing CD3+ thymocyte population in the absence of NFAT2 and increased IL-4 production. Furthermore, we found that CD8+ T lymphocytes deficient in NFAT2 had reduced activation, proliferation, and IFN-γ and IL-2 production at suboptimal TCR strength. NFAT2 absence did not significantly influence differentiation of CD8+ T cells into cytotoxic effector cells but reduced their IFN-γ production. This work documents NFAT2 as a negative regulator of innate-like CD8+ T cells development. NFAT2 is required for complete CD8+ T cell responses at suboptimal TCR stimulation and regulates IFN-γ production by cytotoxic CD8+ T cells in vitro.

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Inmunofenotipificación basal en personas diagnosticadas con VIH-1 en una unidad regional de inmunología (URIC), Venezuela, 2004-2006 / Basal immunephenotyping in HIV-1 diagnosed persons in a regional immunology unit (URIC), Venezuela 2004-2006

En el curso de la infección por el VIH-1, la inmunofenotipificación de los individuos infectados es importante para determinar el estadio inmunológico basal así como para comenzar y monitorear la terapia. Para la inmunofenotipificación basal de individuos recién diagnosticados con infección por el VIH-1 se llevó a cabo un estudio descriptivo y retrospectivo en la URIC de Maracay, Venezuela, de enero 2004 a diciembre 2006. A 238 pacientes (70,6% hombres y 29,4% mujeres) se les determinó el recuento de linfocitos T CD4/CD8 por citometría de flujo. El rango de edad fue de 2 a 74 años y el mayor número de casos (42,0%) se observó entre los 25 y 34 años. Se registraron recuentos de CD4+ menores de 200 cels/mm³ en 39,7% de los individuos; 35,0% presentaron valores entre 200 y 499 cels/mm³ y sólo 25,2% tuvieron valores sobre 500 cels/mm³. Los recuentos de CD8+ en 52,6% de los pacientes fueron mayores de 900 cels/mm³ y en 15,8% menores 500 cels/mm³. El índice CD4/CD8 resultó menor de 1.0 en 94,9% de los individuos, evidenciándose una tendencia creciente a diagnosticar la infección por VIH en una etapa tardía, como indican los recuentos disminuidos de CD4+ encontrados, pero se observó un incremento en el diagnóstico de los casos nuevos en los pacientes menores de 35 años y en las mujeres.

During the course of HIV-1 infection immunephenotyping of infected individuals is important to determine the basal immunological status, as well for initiating and monitoring therapy, for the basal immunephenotyping of recentlyHIV-1 diagnosed individuals we carried out a descriptive and retrospective study at the regional immunology unit (URIC) in Maracay, Venezuela, between January 2004 and December 2006. In the 238 patients included (70.6% men and 29.4% women) we studied CD4/CD8 T lymphocyte numbers through flow cytometry. Age range varied between 2-74 years, and the largest number of cases corresponded to the 25-34 age group. We found CD+ counts under 200 Cells/mm³ in 39.7% individuals; 35.0% had values between 200-499 cells/mm³; and only 25.2% had values over 500 cells/mm³. CD8+ counts in 52.6% of patients were higher than 900 cells/mm³ and 15.8% had counts under 500 cells/mm³. The CD4/CD8 index was lower than 1.0 in 94.9% of the individuals, demonstrating a growing tendency to diagnose HIV infection at a late stage, as shown by the decreased CD4+ counts found. Nevertheless, an increase in the diagnosis of new cases in patients under 35 years and women was also determined.