Predominance of CD4 Th1 and CD8 Tc1 Cells Revealed by Characterization of the Cellular Immune Response Generated by Immunization with a DNA Vaccine Containing a Trypanosoma cruzi Gene

ABSTRACT Immunization with a plasmid DNA containing the gene encoding the catalytic domain of trans-sialidase (TS) elicits protective immune responses against experimental Trypanosoma cruziinfection. As several studies provided strong evidence that during infection CD4 Th1 and CD8 T cytotoxic type 1 (Tc1) cells are important factors in host resistance, the present study was designed to evaluate which T-cell types were activated in DNA-vaccinated BALB/c mice. We found that bulk cells from DNA-immunized mice had CD4 and CD8 T cells that produced gamma interferon (IFN-γ) but not interleukin-4 (IL-4) or IL-10. To characterize the TS-specific T cells at the clonal level, we generated CD4 and CD8 clones. We obtained cytotoxic CD4 clones of the Th1 type that secreted large amounts of IFN-γ but not IL-4 or IL-10. Unexpectedly, we obtained other CD4 clones with a Th2 phenotype, secreting IL-4 and IL-10 but not IFN-γ. All CD8 clones were cytotoxic and produced IFN-γ. IL-4 and IL-10 were not secreted by these cells. Using synthetic peptides, we determined a CD8 epitope recognized by several clones as being represented by amino acids IYNVGQVSI. The antiparasitic activity of a CD4 Th1 and a CD8 Tc1 clone was assessed in vitro. CD4 or CD8 T cells significantly inhibited T. cruzidevelopment in infected macrophages or fibroblasts, respectively. We concluded that DNA vaccine efficiently generates potentially protective CD4 Th1 and CD8 Tc1 cells specific for a T. cruzi antigen, therefore reinforcing the possibility of using this strategy for developing a preventive or therapeutic vaccine against Chagas’ disease.

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The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽

10.1182/blood-2019-125046 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5011-5011

Author(s):

Haiping He ◽

Atsuko Takahashi ◽

Yuki Yamamoto ◽

Akiko Hori ◽

Yuta Miharu ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cd8 T Cells ◽

Research Funding ◽

Complete Medium ◽

Surface Markers ◽

Rt Pcr ◽

Tnf Α ◽

Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

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The effect of Kefir on The Immune Response of Healthy Volunteers In Vitro

Journal of Agromedicine and Medical Sciences ◽

10.19184/ams.v3i2.5067 ◽

2017 ◽

Vol 3 (2) ◽

pp. 28

Author(s):

Desie Dwi Wisudanti

Keyword(s):

Immune Response ◽

T Cells ◽

Immune Responses ◽

Healthy Volunteers ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Fermented Milk ◽

Bioactive Components ◽

Ifn Γ

Kefir is a functional foodstuff of probiotics, made from fermented milk with kefir grains containing various types of beneficial bacteria and yeast. There have been many studies on the effects of oral kefir on the immune system, but few studies have shown the effect of bioactive components from kefir (peptides and exopolysaccharides/ kefiran), on immune responses. The purpose of this study was to prove the effect of kefir supernatant from milk goat on healthy immune volunteer response in vitro. The study was conducted on 15 healthy volunteers, then isolated PBMC from whole blood, then divided into 5 groups (K-, P1, P2, P3 and P4) before culture was done for 4 days. The harvested cells from culture were examined for the percentage of CD4+ T cells, CD8+ T cells, IFN-γ, IL-4 using flowsitometry and IL-2 levels, IL-10 using the ELISA method. The results obtained that kefir do not affect the percentage of CD4+ T cells and CD8+ T cells. The higher the concentration of kefir given, the higher levels of secreted IFN- γ and IL-4, but a decrease in IL-2 levels. Significant enhancement occurred at levels of IL-10 culture PBMC given kefir with various concentrations (p <0.01), especially at concentrations of 1%. These results also show the important effects of kefir bioactive components on immune responses. The conclusion of this study is that kefir can improve the immune response, through stimulation of IL-10 secretion in vitro.

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The critical immunosuppressive effect of MDSC-derived exosomes in the tumor microenvironment

10.1101/2020.03.05.979195 ◽

2020 ◽

Cited By ~ 3

Author(s):

Mohammad H. Rashid ◽

Thaiz F. Borin ◽

Roxan Ara ◽

Raziye Piranlioglu ◽

Bhagelu R. Achyut ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Tumor Growth ◽

Cd8 T Cells ◽

Suppressor Cells ◽

Cell Types ◽

Biological Macromolecules

AbstractMyeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

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An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

Infection and Immunity ◽

10.1128/iai.66.10.4981-4988.1998 ◽

1998 ◽

Vol 66 (10) ◽

pp. 4981-4988 ◽

Cited By ~ 33

Author(s):

Irina Lyadova ◽

Vladimir Yeremeev ◽

Konstantin Majorov ◽

Boris Nikonenko ◽

Sergei Khaidukov ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antimycobacterial Activity ◽

Enzymatic Digestion ◽

Interleukin 4 ◽

T Cell Clones ◽

Cell Clones ◽

Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

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Differences between Exhausted CD8+ T cells in Hepatocellular Carcinoma Patients with and without Uremia

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2019-0641 ◽

2020 ◽

Author(s):

Xiaohong Chen ◽

Jianzhou Zou ◽

Bo Shen ◽

Wenlv Lv ◽

Xuesen Cao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Cd8 T Cells ◽

Ifn Γ ◽

Uremic Patients ◽

Anti Tumor Activity

Purpose: To explore the differences between exhausted CD8+ T cells in HCC patients with and without uremia. Methods: We enrolled 45 uremic patients who were recently diagnosed with HCC into the HCC & uremia cohort. We also enrolled similar patients with HCC but without uremia; this was the HCC only cohort. Lymphocytes were obtained from the two cohorts and exhausted CD8+ T cells, comprising PD-1+CD8+, TIM-3+CD8+, and LAG-3+CD8+ T cells, were sorted and expanded in vitro. Results: The proportions of PD-1+CD8+, TIM-3+CD8+, and LAG-3+CD8+ T cells after expansion were significantly higher in the HCC only cohort as compared to those in the HCC & uremia cohort. CD8+ T cells expressing PD-1, TIM-3, or LAG-3 showed increased tumor reactivity and release of IFN-γ in vitro; however, these cells demonstrated weaker anti-tumor activity in HCC & uremia patients than those in HCC patients without uremia. Among the expanded lymphocytes, only the decreased proportion of PD-1+CD8+ T cells correlated with the HCC & uremia cohort (OR: 2.731, p=0.009). Conclusions: Peripheral CD8+ T cells expressing PD-1, TIM-3, or LAG-3 from the HCC & uremia cohort were dysfunctional in vitro. Among these populations, PD-1+CD8+ T cells were the most predominant in HCC patients with uremia.

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Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽

10.1182/blood.v112.11.206.206 ◽

2008 ◽

Vol 112 (11) ◽

pp. 206-206 ◽

Cited By ~ 1

Author(s):

Sonja Schmucker ◽

Mario Assenmacher ◽

Jurgen Schmitz ◽

Anne Richter

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Peptide Pool ◽

Adoptive T Cell Therapy ◽

T Cell Therapy ◽

A Cell ◽

Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

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Phagocytosis May Be a Regulatory Mechanism for Myeloid Dendritic Cells to Terminate Type 1 CD8+ T Cells.

Blood ◽

10.1182/blood.v114.22.1654.1654 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1654-1654

Author(s):

Young-June Kim ◽

Hal E. Broxmeyer

Keyword(s):

T Cells ◽

Immune Responses ◽

Cd8 T Cells ◽

Phagocytic Activity ◽

Regulatory Mechanism ◽

Gm Csf ◽

T Cell Immune Responses ◽

Ifn Γ

Abstract Abstract 1654 Poster Board I-680 CD8+ cytotoxic T cells are often ‘exhausted’ by programmed death-1 (PD-1) signaling, and subsequently the functions of these cells are terminated especially in a tumor environment or upon chronic HIV or HCV infection. Subsets of myeloid cells referred to as myeloid derived suppressor cells (MDSC) or regulatory dendritic cells (DCs) have been implicated in inducing exhaustion or termination of effector CD8+ T cells. To this end, we developed various myeloid-derived dendritic cell (DC) types in vitro from human CD14+ monocytes using M-CSF or GM-CSF in the presence of IL-4 with/without other cytokines, and characterized these DCs with respect to their capacity to induce PD-1 expression on and exhaustion of CD8+ T cells. We then assessed their impact on longevity of CD8+ T cells following coculture. Myeloid DCs developed in vitro with M-CSF and IL-4 for 5 days (referred to as M-DC) did not express ligand for PD-1 (PD-L1) nor did they induce PD-1 on CD8+ T cells. Thus, using M-DCs as starting cells, we sought determinant factors that could modulate M-DCs to express PD-L1 and thereby induce exhaustion of CD8+ T cells. In order to better monitor exhaustion processes, we incubated human peripheral CD8+ T cells for 5 days in the presence of IL-15, an important cytokine for maintaining viability, before coculture. M-DCs showed little impact on exhaustion or longevity of the CD8+ T cells. IL-10 converted M-DC into a distinct myeloid DC subset (referred to as M-DC/IL-10) with an ability to express PD-L1 as well as to induce PD-1 on cocultured CD8+ T cells. M-DC/IL-10 cells markedly suppressed proliferation of cocultured CD8+ T cells. M-DC/IL-10 cells were morphologically unique with many granules and filamentous structures around the cell periphery. These IL-10 effects on M-DC were completely abrogated in the presence of TNF-á. M-DC/IL-10 cells could be further differentiated into another myeloid DC subset in the presence of IFN-γ (referred to as M-DC/IL-10/IFN-γ) with an ability to express even higher levels of PD-L1 compared to M-DC/IL-10 cells. The most remarkable effect of M-DC/IL-10/IFN-γ cells on cocultured CD8+ T cells was a dramatic loss of CD8+ T cells. Light and confocal microscopic observations indicated that loss of CD8+ T cells was due to phagocytosis by M-DC/IL-10/IFN-γ cells. As IFN-γ, a type 1 cytokine which is induced in CD8+ T cells by IL-12 is essential for phagocytosis, we tested whether IL-12 treatment of CD8+ T cells could further enhance phagocytosis induced by M-DC/IL-10/IFN-γ cells. Indeed, IL-12 treatment greatly increased numbers of phagocytosed CD8+ T cells. In contrast, IL-4 treated CD8+ T cells became resistant to phagocytosis, suggesting IFN-γ producing (type1) CD8+ T cells may be primary target cells for the M-DC/IL-10 cells mediated phagocytosis. CD4+ T cells were not as susceptible as CD8+ T cells to phagocytosis. We failed to detect such phagocytic activity induced by prototype DCs generated with GM-CSF and IL-4. Phagocytic activity was not inhibited by various arginase-1 inhibitors suggesting that nitric oxide signaling may not mediate phagocytic activity. Neutralizing antibody to PD-L1 slightly but significantly lowered phagocytic activity suggesting that PD-L1/PD-1 interaction may be partially involved in this process. Myeloid DCs are thought to be immunogenic, actively inducing T cell immune responses. Our results demonstrate that myeloid DCs may play suppressive roles as well through induction of phagocytic activity, especially against IFN-γ producing CD8+ T cells. This may serve as a regulatory mechanism for type 1 CD8+ T cell immune responses in an IL-10 enriched microenvironment. Disclosures No relevant conflicts of interest to declare.

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Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Blood ◽

10.1182/blood.v114.22.711.711 ◽

2009 ◽

Vol 114 (22) ◽

pp. 711-711

Author(s):

Srimoyee Ghosh ◽

Sergei B Koralov ◽

Irena Stevanovic ◽

Mark S Sundrud ◽

Yoshiteru Sasaki ◽

...

Keyword(s):

T Cells ◽

Transgenic Mice ◽

Cd4 T Cells ◽

Th17 Cells ◽

Autoimmune Encephalitis ◽

Cell Types ◽

Wild Type ◽

Ifn Γ

Abstract Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

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Critical role for perforin and Fas-dependent killing of dendritic cells in the control of inflammation

Blood ◽

10.1182/blood-2011-06-363994 ◽

2012 ◽

Vol 119 (1) ◽

pp. 127-136 ◽

Cited By ~ 42

Author(s):

Min Chen ◽

Kumar Felix ◽

Jin Wang

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

Inflammatory Responses ◽

Critical Role ◽

Cell Types ◽

Activated T Cells ◽

Ifn Γ

AbstractAfter stimulation of antigen-specific T cells, dendritic cell (DCs) are susceptible to killing by these activated T cells that involve perforin and Fas-dependent mechanisms. Fas-dependent DC apoptosis has been shown to limit DC accumulation and prevent the development of autoimmunity. However, a role for perforin in the maintenance of DC homeostasis for immune regulation remains to be determined. Here we show that perforin deficiency in mice, together with the deletion of Fas in DCs (perforin−/−DC-Fas−/−), led to DC accumulation, uncontrolled T-cell activation, and IFN-γ production by CD8+ T cells, resulting in the development of lethal hemophagocytic lymphohistiocytosis. Consistently, adoptive transfer of Fas−/− DCs induced over-activation and IFN-γ production in perforin−/− CD8+ T cells. Neutralization of IFN-γ prevented the spreading of inflammatory responses to different cell types and protected the survival of perforin−/−DC-Fas−/− mice. Our data suggest that perforin and Fas synergize in the maintenance of DC homeostasis to limit T cell activation, and prevent the initiation of an inflammatory cascade.

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