IL-17; bacteria; inflammation; immunity
While the nasal mucosa is a potentially useful site for human immunization, toxin-based nasal adjuvants are generally unsafe and less effective in humans. Safe mucosal adjuvants that activate protective immunity via mucosal administration are highly dependent on barrier antigen sampling by epithelial and dendritic cells. Here, we demonstrate that protein antigens formulated in unique oil-in-water nanoemulsions results in unique trans-cellular antigen uptake in ciliated nasal epithelial cells, leading to delivery into nasal associated lymphoid tissue. Nanoemulsion formulation also enhances MHC class II expression in epithelial cells and DC activation/trafficking to regional lymphoid tissues in mice. These materials appear to induce local epithelial cell apoptosis and heterogeneous cytokine production by mucosal epithelial cells and mixed nasal tissues, including G-CSF, GM-CSF, IL-1a, IL-1b, IL-5, IL-6, IL-12, IP-10, KC, MIP-1a, TGF-β, and TSLP. This is the first observation of a nasal adjuvant that activates calreticulin-associated apoptosis of ciliated nasal epithelial cells to generate broad cytokine/chemokine responses in mucosal tissue.
ciliated epithelial cells; dendritic cells; antigen sampling; IL-6; nanoemulsion; mucosal vaccines
Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4+CD8+ thymocyte using an Ikaros null CD4−CD8− mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry non-functional rearrangements on both alleles of their TCR α loci. Retroviral re-introduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR+ cells. The process is RAG dependent, requires SWI/SNF chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/NuRD complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR+ CD4+CD8+ thymocyte transition.
T Cells; Transcription Factors; T Cell Receptor Genes; VDJ Recombination
IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3), and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent anti-tumor activity via activation of a variety of cellular components, including anti-tumor CD8+ T-cell responses. However, the exact mechanisms of how IL-27 enhances anti-tumor CD8+ T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen specific CD8+ T cells in vitro and in vivo, and programs tumor antigen-specific CD8+ T cells into memory precursor (MPC)-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to MPC phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances anti-tumor CTL responses via programming tumor antigen-specific CD8+ T cells into a unique memory precursor-type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.
CD19-deficient mice were used as a model to study FDC activation because these mice have normal numbers of FDC-containing primary follicles, but lack the ability to activate FDC or form GC. It was hypothesized that CD19 expression is necessary for B cell activation and upregulation of membrane-lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM-1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19+ wild-type B cells into CD19-deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19+ donor B cells lacking mLT were unable to induce VCAM-1 expression on FDC and FcγRII/III upregulation was impaired. VCAM-1 expression on FDC, but not FcγRII/III, was rescued when CD19-deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19+, mLT-deficient B cells, suggesting that FDC activation requires the CD19-dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.
Follicular Dendritic Cell; Germinal Center; CD19; Membrane Lymphotoxin
Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Accumulation of regulatory T cells (Tregs) and up-regulation of inhibitory signaling pathways (such as Tim-3/Gal-9) play pivotal roles in suppressing antiviral effector T cells (Teffs) that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teffs, their role in regulating Tregs is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg development, in this study we provide pilot data showing that HCV-infected hepatocytes express higher levels of Gal-9 and TGF-β, and up-regulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Tregs. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Tregs in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate Foxp3+ Treg development and function during HCV infection.
Tim-3; Gal-9; Foxp3; HCV; regulatory T cells; hepatocytes; immune modulation
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of SLE, lack an inhibitor of B- and myeloid cell activation. This results in B-cell hyperresponsiveness, plasma cell (PC) accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn−/− mice. We find that IL-21 mRNA is reduced in the spleens of lyn−/−IL-6−/− and lyn−/−Btklo mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for PC accumulation and IgM autoantibodies in lyn−/− mice, it is required for anti-DNA IgG antibodies and some aspects of T cell activation. Surprisingly, GN still develops in lyn−/−IL-21−/− mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA antigens. These studies identify a specific role for IL-21 in the class switching of anti-DNA B-cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn−/− mice.
autoimmunity; Lyn; IL-21; IL-6; autoantibody; glomerulonephritis
Increased susceptibility to tuberculosis following HIV-1 seroconversion contributes significantly to the tuberculosis epidemic in sub-saharan Africa. Lung specific mechanisms underlying the interaction between HIV-1 and Mycobacterium (M.) tuberculosis infection are incompletely understood. This study addressed the effect of HIV-1 and latent M. tuberculosis infection on viral-entry receptors and ligands in bronchoalveolar lavage (BAL). Median fluorescence intensity (MFI) of entry receptor expression was measured by multiparameter flow cytometry and chemokine expression by multiplex bead array.
Irrespective of HIV-1 status, BAL T-cells expressed higher MFI for the beta-chemokine receptor (CCR)5 than peripheral blood T-cells (p<0.001), in particular the CD8+ T-cells of HIV-1 infected persons showed elevated CCR5 expression (p=0.026). The concentration of BAL CCR5 ligands, regulated upon activation normal T-cell expressed and secreted (RANTES; p<0.001) and macrophage inflammatory protein (MIP)-1β (p=0.004) were elevated in the BAL of HIV-1 infected persons compared to controls. CCR5 expression and RANTES concentration correlated strongly with HIV-1 viral load in BAL. By contrast these alterations were not associated with M. tuberculosis sensitization in vivo nor did M. tuberculosis infection of BAL cells ex vivo change RANTES expression.
These data suggest ongoing HIV-1 replication predominantly drives local pulmonary CCR5+ T-cell activation in HIV/latent M. tuberculosis co-infection.
BAL; CCR5; RANTES; TB; viral load
The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells.
NK cells; PVR; TIGIT
The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B-cell survival and selection in the GC. Here, we have used mice with B-cell-specific deletion of the 5′-inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag- and interferon (IFN)-induced activation, resulting in augmented negative selection in the BM and enhanced B-cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen-egg lysozyme Ag in the MDA4 transgenic model. SHIP-deficient B cells spontaneously produce isotype-switched antibodies; however, they are poor responders in immunization and infection models. While SHIP-deficient B cells form GCs and undergo mutation, they are not properly selected for high-affinity antibodies. These results illustrate the importance of negative regulation of B-cell responses, as lower thresholds for B-cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation.
Affinity maturation; B cell; Negative selection; SHIP
Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adaptor protein SLP-76 in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α SDF-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in this T cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking.
SLP-76; Chemokine; T lymphocyte; Firm arrest; Shear flow; LFA-1
Inflammasomes are cytosolic multi-protein complexes that form in response to infectious or injurious challenges. Inflammasomes control the activity of caspase-1, which is essential for the maturation and release of IL-1b family cytokines. The NLRP1, IPAF and AIM2 inflammasomes recognize specific substances, while the NLRP3 inflammasome responds to many structurally and chemically diverse triggers. Here, we discuss the critical roles of priming and lysosomal damage in NLRP3 inflammasome activation.
Inflammasome; NLRP3; IL-1b
Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6 deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage, and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.
Adaptive Immunity; Heterosubtypic Immunity; IL-6; Innate Immunity; Pulmonary Damage
IFN-γ is generally believed to be important in the autoimmune pathogenesis of type 1 diabetes (T1D). However, the development of spontaneous β cell autoimmunity is unaffected in NOD mice lacking expression of IFN-γ or the IFN-γ receptor (IFNγR), bringing into question the role IFN-γ has in T1D. In the current study an adoptive transfer model was employed to define the contribution of IFN-γ in CD4+ versus CD8+ T cell-mediated β cell autoimmunity. NOD.scid mice lacking expression of the IFNγR β chain (NOD.scid.IFNγRBnull) developed diabetes following transfer of β cell-specific CD8+ T cells alone. In contrast, β cell-specific CD4+ T cells alone failed to induce diabetes despite significant infiltration of the islets in NOD.scid.IFNγRBnull recipients. The lack of pathogenicity of CD4+ T cell effectors was due to the resistance of IFNγR-deficient β cells to inflammatory cytokine-induced cell death. On the other hand, CD4+ T cells indirectly promoted β cell destruction by providing help to CD8+ T cells in NOD.scid.IFNγRBnull recipients. These results demonstrate that IFN-γR may play a key role in CD4+ T cell-mediated β cell destruction.
IFN-γ; NOD mice; Type 1 diabetes
MicroRNAs (miRNAs) play important roles in the regulation of immune responses. There is evidence that miRNAs also participate in the pathogenesis of multiple sclerosis (MS), but how the miRNAs regulate the pathogenesis of MS is still under investigation. The identification of new members of the miRNA family associated with the pathogenesis of MS could facilitate early diagnosis and treatment. Here we show that the level of miRNA let-7e is significantly upregulated in experimental autoimmune encephalomyelitis (EAE), an animal model of MS using miRNA array and quantitative real-time PCR. The expression of let-7e was mainly in CD4+ T cells and infiltrated mononuclear cells of central nervous system, and highly correlated with the development of EAE. We found that let-7e silencing in vivo inhibited encephalitogenic Th1 and Th17 cells and attenuated EAE, with reciprocal increase of Th2 cells; overexpression of let-7e enhanced Th1 and Th17 cells and aggravated EAE. We also identified IL-10 as one of the functional targets of let-7e. Together, we propose that let-7e is a new miRNA involved in the regulation of encephalitogenic T-cell differentiation and the pathogenesis of EAE.
MicroRNA; mir-let-7e; EAE/MS; Th1/Th2 cells; Cell differentiation
We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed “suppressed immunization”) could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that in the spleen and peripheral lymph nodes, dexamethasone dose-dependently enriches CD11cloCD40lo macrophages by depleting all other CD11c+CD40+ cells including dendritic cells. The enriched macrophages display a distinct MHC IIloCD86hi phenotype. Upon activation by a recall antigen in vivo, they upregulate IL-10, a classic marker for tolerogenic antigen-presenting cells, and elicit a serum IL-10 response. When presenting a recall antigen in vivo, they do not elicit recall responses of memory T cells, but rather stimulate the expansion of antigen-specific regulatory T cells. Moreover, depletion of CD11cloCD40lo macrophages during suppressed immunization diminishes the latter’s tolerogenic efficacy. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11cloCD40lo tolerogenic macrophages.
Tolerance; Dexamethasone; Macrophage
MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease.
Antigen presentation/processing; Antigen presenting cells; B cells; Cathepsin S; Gamma-interferon-inducible lysosomal thiol reductase (GILT)
Mast cells play a critical role in the pathogenesis of allergic diseases. How mast cell function is regulated is still not well understood. Both phosphatidic acid (PA) and diacylglycerol (DAG) are important second messengers involved in mast cell activation. Lipin1 is a phosphatidate phosphatase that hydrolyzes PA to produce DAG. The role of lipin1 in mast cell function has been unknown. In this report, we show that lipin1 is an important and selective inhibitor of mast cell degranulation. Lipin1 deficiency enhanced FcεRI-mediated β-hexosaminidase and prostaglandin D2 release from mast cells in vitro and exacerbated the passive systemic anaphylaxis reaction in vivo. However, Lipin1 deficiency did not exert obvious effects on IL-6 or TNF-α production following FcεRI engagement. FcεRI-induced PKC and SNAP-23 phosphorylation was augmented in the lipin1-deficient mast cells. Moreover, inhibition of PKC activity reduced SNAP-23 phosphorylation and mast cell degranulation in lipin1 deficient mast cells. Together, our findings suggest that lipin1 may negatively control mast cell degranulation and anaphylactic response through inhibiting the PKC-SNAP-23 pathway.
Mast cells; lipin1; Phosphatidic acid; PKC; SNAP-23
Antiviral RNA silencing has been recognized as an important defense mechanism in arthropods against RNA viruses. However, the role of this pathway in DNA virus infection remains largely unexplored. A report in this issue of the European Journal of Immunology [Eur J Immunol 2012. XXXX] provides new insight into the role of RNA silencing in antiviral defense against DNA viruses. Huang and Zhang found that the dsDNA virus White Spot Syndrome virus, an agriculturally important pathogen of shrimp, is targeted by the shrimp RNA silencing machinery via the production of virus-derived siRNAs. Furthermore, the authors show that the RNA silencing pathway, and crucially, Dicer-2, is important for restricting viral infection. This study provides novel insights not only into shrimp antiviral defenses but also potentially into antiviral immunity against DNA viruses in a larger spectrum of hosts, as discussed in this Commentary. Furthermore, this study may contribute to the future development of immune-based therapeutics to combat viral pathogens, not only in aquaculture, but also in insect vectors of human diseases.
Of the three nonclassical class I antigens expressed in humans, HLA-F has been least characterized with regard to expression or function. In this study we examined HLA-F expression focusing on lymphoid cells where previous work with homologous cell lines had demonstrated surface HLA-F expression. HLA-F protein expression was observed by western analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA-F intracellularly showed an induction of surface HLA-F protein. An examination of peripheral blood from individuals genetically deficient for TAP and Tapasin expression demonstrated the same activation expression profiles for HLA-F but with altered kinetics post activation. Further analysis of CD4+, CD25+ regulatory T cells showed HLA-F was not upregulated on the major fraction of these cells when they were activated, whereas CD4+, CD25- T cells showed strong expression of surface HLA-F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA-F and towards the potential clinical use of HLA-F as a marker of an activated immune response.
Activated Lymphocytes; Human; MHC; HLA-F
The analysis of the human memory B cell repertoire is of both fundamental and practical significance. We developed a simple method for the selective activation of memory B cells in total fresh or frozen PBMC using a combination of R848 and IL-2. In these conditions 30–40% of memory B cells generated clones producing on average 200 ng IgG in 10 days. This method was used to measure the frequency of antigen specific memory B cells as well as the fine specificity, crossreactivity and neutralizing activity of the secreted antibodies. Following influenza vaccination, specific B cells expanded dramatically, reaching up to 50% of total clonable memory B cells on day 14. Specific B cell expansions were detected also in individuals that did not show a significant serological response. Dynamic changes and persistence of B cells specific for a variety of pathogens were documented in serial PBMC samples collected over almost two decades. These results reveal novel aspects of memory B cell kinetics and provide a powerful tool to monitor immune responses following infection and vaccination.
memory B cells; Vaccines; antibody
The development of an effective vaccine against HIV/AIDS has been hampered, in part, by a poor understanding of the rules governing helper T-cell epitope immunodominance. Studies in mice have shown that antigen structure modulates epitope immunodominance by affecting the processing and subsequent presentation of helper T-cell epitopes. Previous epitope mapping studies showed that the immunodominant helper T-cell epitopes in mice immunized with gp120 were found flanking flexible loops of the protein. In this paper, we show that helper T-cell epitopes against gp120 in humans infected with HIV are also found flanking flexible loops. Immunodominant epitopes were found to be located primarily in the outer domain, an average of 13 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of 5 residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops. These results show that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in humans. This relationship between antigen structure and helper T-cell epitope immunodominance may prove to be useful in the development of rationally designed vaccines against pathogens such as HIV.
HIV envelope protein gp120; antigen processing; protein structure; helper T lymphocyte; MHC class II
Macrophages (MF) are becoming increasingly recognized as key cellular players in intestinal immune homeostasis. However, differentiating MF from dendritic cells (DC) is often difficult, and finding a specific phenotypic signature for intestinal MF identification has remained elusive. In this issue of European Journal of Immunology Tamoutounour et al. identify CD64 as a specific MF marker capable of discriminating DC from MF in the murine small and large bowel, and under steady state and inflammatory conditions. The authors also propose a sequential ‘monocyte (Mo)-waterfall’ model for intestinal MF differentiation, with implications for immune tolerance and inflammation at the gut mucosal interface.
Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contrast to this innate immune deficit, CGD patients and animal models display predisposition to autoimmune disease and enhanced response to H. pylori and influenza infection. These data imply an altered, perhaps augmented, adaptive immune response in CGD. Previous data demonstrated functional NOX2 expression in T cells, and the goal was to determine if NOX2-deficient T cells are inherently altered in their responses. Activation of purified naive CD4+ T cells from NOX2-deficient mice led to augmented IFN-γ and diminished IL-4 production and an increased ratio of expression of the TH1-specific transcription factor T-bet versus the TH2-specfic transcription factor GATA-3, consistent with a TH1 skewing of naïve T cells. Selective inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Exposure to anti-oxidants inhibited, while pro-oxidants augmented TH2 cytokine secretion and STAT5 phosphorylation, supporting the redox dependence of these signaling changes. These data suggest that TCR-induced ROS generation from NOX2 activation can regulate the adaptive immune response in a T cell inherent fashion, and propose a possible role for redox signaling in T helper differentiation.
chronic granulomatous disease; NADPH oxidase; reactive oxygen species; STAT5
Animals lacking the inducible nitric oxide synthase gene (nos2−/−) are less susceptible to M. avium strain 25291 and lack nitric oxide-mediated immunomodulation of CD4+ T cells. Here we show that the absence of nos2 results in increased accumulation of neutrophils and both CD4+ and CD8+ T cells within the M. avium-containing granuloma. Examination of the T-cell phenotype in M. avium-infected mice demonstrated that CD4+CD44hi effector T cells expressing the Th1 transcriptional regulator T-bet (T-bet+) were specifically reduced by the presence of nitric oxide. Importantly, the T-bet+ effector population could be separated into CD69hi and CD69lo populations, with the CD69lo population only able to accumulate during chronic infection within infected nos2−/− mice. Transcriptomic comparison between CD4+CD44hiCD69hi and CD4+CD44hiCD69lo populations revealed that CD4+CD44hiCD69lo cells had higher expression of the integrin itgb1/itga4 (VLA-4, CD49d/CD29). Inhibition of Nos2 activity allowed increased accumulation of the CD4+CD44hiT-bet+CD69lo population in WT mice as well as increased expression of VLA-4. These data support the hypothesis that effector T cells in mycobacterial granulomata are not a uniform effector population but exist in distinct subsets with differential susceptibility to the regulatory effects of nitric oxide.
CD4 T cells; cell differentiation; immunopathology; inflammation; bacterial infection