NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d^−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d^−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection.     

Murine and Bovine �æ� T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ^−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα^−/−, and GMCSF^−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ^−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.     

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4^−/− and Myd88^−/−, but not TRIF^−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.     

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-��

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56^brightCD16⁻ NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

Systemic BCG immunization induces persistent lung mucosal multifunctional CD4 T(EM) cells which expand following virulent mycobacterial challenge

To more closely understand the mechanisms of how BCG vaccination confers immunity would help to rationally design improved tuberculosis vaccines that are urgently required. Given the established central role of CD4 T cells in BCG induced immunity, we sought to characterise the generation of memory CD4 T cell responses to BCG vaccination and M. bovis infection in a murine challenge model. We demonstrate that a single systemic BCG vaccination induces distinct systemic and mucosal populations of T effector memory (T_EM) cells in vaccinated mice. These CD4⁺CD44^hiCD62L^loCD27⁻ T cells concomitantly produce IFN-γ and TNF-α, or IFN-γ, IL-2 and TNF-α and have a higher cytokine median fluorescence intensity MFI or ‘quality of response’ than single cytokine producing cells. These cells are maintained for long periods (>16 months) in BCG protected mice, maintaining a vaccine–specific functionality. Following virulent mycobacterial challenge, these cells underwent significant expansion in the lungs and are, therefore, strongly associated with protection against M. bovis challenge. Our data demonstrate that a persistent mucosal population of T_EM cells can be induced by parenteral immunization, a feature only previously associated with mucosal immunization routes; and that these multifunctional T_EM cells are strongly associated with protection. We propose that these cells mediate protective immunity, and that vaccines designed to increase the number of relevant antigen-specific T_EM in the lung may represent a new generation of TB vaccines.     

Cutting edge: murine cytomegalovirus induces a polyfunctional CD4 T cell response

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.     

Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease

Background

Myocardium damage during Chagas'' disease results from the immunological imbalance between pro- and production of anti-inflammatory cytokines and has been explained based on the Th1–Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas'' disease.

Methodology/Principal Findings

First, we observed CD4⁺IL-17⁺ T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas'' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas'' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-α, IFN-γ and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4⁺IL-17⁺ cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas'' disease patients presented the same frequency of CD4⁺CD25⁺ regulatory T cells. However, CD4⁺CD25⁺ T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-γ levels during chronic Chagas'' disease are inversely correlated to the LVEF (P = 0.007, r = −0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500).

Conclusion/Significance

These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-γ and TNF-α is correlated with the severity of the Chagas'' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.     

Activation of NF-κB in CD8+ dendritic cells Ex Vivo by the γ134.5 null mutant correlates with immunity against herpes simplex virus 1

The γ₁34.5 protein of herpes simplex viruses (HSV) is essential for virulence. Accordingly, an HSV mutant lacking γ₁34.5 is attenuated in vivo. Despite its vaccine potential, the mechanism by which the γ₁34.5 null mutant triggers protective immunity is unknown. In this report we show that vaccination with the γ₁34.5 null mutant protects against lethal challenge from wild-type virus via IκB kinase in dendritic cells (DCs), which sense virus-associated molecular patterns. Unlike mock-treated DCs, DCs primed with the γ₁34.5 null mutant ex vivo mediate resistance to wild-type HSV after adoptive transfer into naïve mice. Furthermore, the γ₁34.5 null mutant activates IκB kinase, which facilitates p65/RelA phosphorylation and nuclear translocation, resulting in DC maturation. While unable to produce infectious virus in DCs, this mutant virus expresses early and late genes. In its abortive infection, the γ₁34.5 null mutant induces protective immunity more effectively in CD8⁺ DCs than in CD8⁻ DCs. This is mirrored by a higher level of interleukin-6 (IL-6) and IL-12 secretion by CD8⁺ DCs than CD8⁻ DCs. Remarkably, inhibition of p65/RelA phosphorylation or nuclear translocation in CD8⁺ DCs disrupts protective immunity. These results suggest that engagement of the γ₁34.5 null mutant with CD8⁺ DCs elicits innate immunity to activate NF-κB, which translates into protective immunity.     

IL-27 Receptor Signalling Restricts the Formation of Pathogenic,Terminally Differentiated Th1 Cells during Malaria Infection by Repressing IL-12 Dependent Signals

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1^−/− and IL-10^−/− mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1^−/− mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.     

Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses

Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (20 to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ⁺ IL-2^+/− TNF-α⁺) and CD4 (CD40L^+/− IFN-γ⁺ IL-2⁺ TNF-α⁺) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people.     

Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3⁻CD16⁺ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4⁺ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4⁺ T cells and absent in peripheral blood leukocyte (PBL) CD4⁺ T cells, was down-regulated in LN CD4⁺ T cells and up-regulated in PBL CD4⁺ T cells immediately after infection. CD8⁺ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4⁺ T cells but not in LN CD4⁺ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection.The immune system of higher vertebrates consists of innate and adaptive components. Innate immunity exhibits immediate recognition and response without prior sensitization. Cells of the innate immune system (i.e., monocytes/macrophages, natural killer [NK] cells, and polymorphonuclear leukocytes) recognize pathogen-associated molecular patterns and activate events such as phagocytosis, induction of the synthesis of antimicrobial peptides, expression of inflammatory and effector cytokines and chemokines, induction of nitric oxide synthase in macrophages, and expression of costimulatory molecules on antigen-presenting cells. The adaptive immune system uses somatically generated antigen receptors that are clonally distributed on T and B lymphocytes. Generally, adaptive immune recognition in the absence of innate immune recognition results in inactivation of lymphocytes that express receptors involved in the identification events (20). Thus, innate immune responses have critical consequences in adaptive immune responses.Little is known of the contribution of the innate immune system during infection with the human immunodeficiency virus (HIV). Based on similarities of biologic and genetic features, simian immunodeficiency virus (SIV) infection of rhesus macaques provides the best animal model of HIV infection and AIDS. Accordingly, this animal model is critical for the elucidation of mechanisms of pathogenesis and for the development of vaccines and antiviral therapies (12). As with almost all viral infections, the innate immune system is thought to be the first component of the immune system that recognizes SIV infection. However, few studies have methodically analyzed the changes induced in cell phenotype and cytokine levels by SIV infection. Recent studies have demonstrated that SIV infection results in a generalized increase in lymphocyte turnover (23) and that the primary site for viral replication is activated memory CD4⁺ T cells that are present in the intestinal lamina propia (46). Although cellular changes are not that dramatic at this early stage in peripheral lymphoid tissue, peripheral blood (PB) and lymph nodes (LN) still reflect the pathologic changes induced by the viral infection and are readily available for longitudinal studies.To analyze changes in the activation state of cells from the innate and adaptive immune system after SIV infection, we evaluated NK activity, cytokine levels in plasma, and changes in activation markers on lymphoid cells of rhesus macaques after infection with pathogenic SIVmac251. We found the sequential appearance in plasma of interferon-α/β (IFN-α/β) interleukin-18 (IL-18) and IL-12, whereas IL-4, IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) remained undetectable. We also found transient activation of NK cells during the peak of viral replication, and this activation was not predictive of disease progression. Finally, we observed that after SIV infection, both CD4⁺ and CD8⁺ T cells became activated in the absence of markers for proliferation, suggesting that the increased turnover of these cells reflects activation-induced cell death rather than differential compartmentalization.     

The dynamics of mouse cytomegalovirus-specific CD4 T cell responses during acute and latent infection

The dynamics of mouse cytomegalovirus (MCMV)-specific CD4 T cell responses and the mechanisms by which these cells contribute to viral control are not well understood, mainly due to lack of appropriate tools to characterize MCMV-specific CD4 T cells. We therefore generated MCMV-specific CD4 T cell hybridomas, then used an MCMV expression library and overlapping peptides to identify CD4 T cell epitopes. We used these novel tools to study the long-term kinetics and organ distribution of MCMV-specific CD4 T cells in comparison to MCMV-specific CD8 T cell responses. We demonstrate that the overall MCMV-specific CD4 T cell response stabilizes during the latent stage, which stands in contrast to subpopulations of MCMV-specific CD8 T cells and HCMV-specific CD4 T cells which accumulate over the course of CMV latency. Furthermore, MCMV-specific CD4 T cells displayed a Th1 phenotype, secreting high levels of IFN-gamma and TNF-alpha and to some extent IL-2, cytokines which are involved in protection from CMV disease.     

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.     

Persistent humoral immune responses in the CNS limit recovery of reactivated murine cytomegalovirus

Background

Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8⁺ T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection.

Methodology/Principal Findings

Utilizing our MCMV brain infection model, we investigated B-lymphocyte-lineage cells and assessed their role in controlling the recovery of reactivated virus from latently infected brain tissue. Brain infiltrating leukocytes were first phenotyped using markers indicative of B-lymphocytes and plasma cells. Results obtained during these studies showed a steady increase in the recruitment of B-lymphocyte-lineage cells into the brain throughout the time-course of viral infection. Further, MCMV-specific antibody secreting cells (ASC) were detected within the infiltrating leukocyte population using an ELISPOT assay. Immunohistochemical studies of brain sections revealed co-localization of CD138⁺ cells with either IgG or IgM. Additional immunohistochemical staining for MCMV early antigen 1 (E1, m112–113), a reported marker of viral latency in neurons, confirmed its expression in the brain during latent infection. Finally, using B-cell deficient (Jh^−/−) mice we demonstrated that B-lymphocytes control recovery of reactivated virus from latently-infected brain tissue. A significantly higher rate of reactivated virus was recovered from the brains of Jh^−/− mice when compared to Wt animals.

Conclusion

Taken together, these results demonstrate that MCMV infection triggers accumulation and persistence of B-lymphocyte-lineage cells within the brain, which produce antibodies and play a significant role in controlling reactivated virus.     

Intranodal interaction with dendritic cells dynamically regulates surface expression of the co-stimulatory receptor CD226 protein on murine T cells

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system. Depending on their maturation status, they prime T cells to induce adaptive immunity or tolerance. DCs express CD155, an immunoglobulin-like receptor binding CD226 present on T and natural killer (NK) cells. CD226 represents an important co-stimulator during T cell priming but also serves as an activating receptor on cytotoxic T and NK cells. Here, we report that cells of the T and NK cell lineage of CD155^−/− mice express markedly elevated protein levels of CD226 compared with wild type (WT). On heterozygous CD155^+/− T cells, CD226 up-regulation is half-maximal, implying an inverse gene-dosis effect. Moreover, CD226 up-regulation is independent of antigen-driven activation because it occurs already in thymocytes and naïve peripheral T cells. In vivo, neutralizing anti-CD155 antibody elicits up-regulation of CD226 on T cells demonstrating, that the observed modulation can be triggered by interrupting CD155-CD226 contacts. Adoptive transfers of WT or CD155^−/− T cells into CD155^−/− or WT recipients, respectively, revealed that CD226 modulation is accomplished in trans. Analysis of bone marrow chimeras showed that regulators in trans are of hematopoietic origin. We demonstrate that DCs are capable of manipulating CD226 levels on T cells in vivo but not in vitro, suggesting that the process of T cells actively scanning antigen-presenting DCs inside secondary lymphoid organs is required for CD226 modulation. Hence, a CD226 level divergent from WT may be exploited as a sensor to detect abnormal DC/T-cell cross-talk as illustrated for T cells in mice lacking CCR7.     

Signatures of T cells as correlates of immunity to Francisella tularensis

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4⁺ and/or CD8⁺ T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naïve). Significant differences were detected between either of the immune donor groups and naïve individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, MCP-1, and MIP-1β. Expression of IFN-γ, MIP-1β, and CD107a by CD4⁺CD45RO⁺ or CD8⁺CD45RO⁺ T cells correlated to antigen concentrations. In particular, IFN-γ and MIP-1β strongly discriminated between immune and naïve individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-α-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.     

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T_H2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺ iNKT cells are present in the thymic CD44^+/− NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻ iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺ iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺ iNKT cells produce T_H2 (IL-13), T_H9 (IL-9 and IL-10), and T_H17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺ iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T_H17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺ iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺ iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.     

SHIP-deficient dendritic cells, unlike wild type dendritic cells, suppress T cell proliferation via a nitric oxide-independent mechanism

Background

Dendritic cells (DCs) not only play a crucial role in activating immune cells but also suppressing them. We recently investigated SHIP''s role in murine DCs in terms of immune cell activation and found that TLR agonist-stimulated SHIP−/− GM-CSF-derived DCs (GM-DCs) were far less capable than wild type (WT, SHIP+/+) GM-DCs at activating T cell proliferation. This was most likely because SHIP−/− GM-DCs could not up-regulate MHCII and/or co-stimulatory receptors following TLR stimulation. However, the role of SHIP in DC-induced T cell suppression was not investigated.

Methodology/Principal Findings

In this study we examined SHIP''s role in DC-induced T cell suppression by co-culturing WT and SHIP−/− murine DCs, derived under different conditions or isolated from spleens, with αCD3+ αCD28 activated WT T cells and determined the relative suppressive abilities of the different DC subsets. We found that, in contrast to SHIP+/+ and −/− splenic or Flt3L-derived DCs, which do not suppress T cell proliferation in vitro, both SHIP+/+ and −/− GM-DCs were capable of potently suppressing T cell proliferation. However, WT GM-DC suppression appeared to be mediated, at least in part, by nitric oxide (NO) production while SHIP−/− GM-DCs expressed high levels of arginase 1 and did not produce NO. Following exhaustive studies to ascertain the mechanism of SHIP−/− DC-mediated suppression, we could conclude that cell-cell contact was required and the mechanism may be related to their relative immaturity, compared to SHIP+/+ GM-DCs.

Conclusions

These findings suggest that although both SHIP+/+ and −/− GM-DCs suppress T cell proliferation, the mechanism(s) employed are different. WT GM-DCs suppress, at least in part, via IFNγ-induced NO production while SHIP−/− GM-DCs do not produce NO and suppression can only be alleviated when contact is prevented.     

Assessing the role of CD103 in immunity to an intestinal helminth parasite

Background

In the intestine, the integrin CD103 is expressed on a subset of T regulatory (T_reg) cells and a population of dendritic cells (DCs) that produce retinoic acid and promote immune homeostasis. However, the role of CD103 during intestinal helminth infection has not been tested.

Methodology/Principal Findings

We demonstrate that CD103 is dispensable for the development of protective immunity to the helminth parasite Trichuris muris. While we observed an increase in the frequency of CD103⁺ DCs in the lamina propria (LP) following acute high-dose infection with Trichuris, lack of CD103 had no effect on the frequency of CD11c⁺ DCs in the LP or mesenteric lymph nodes (mLN). CD103-deficient (CD103^−/−) mice develop a slightly increased and earlier T cell response but resolve infection with similar kinetics to control mice. Similarly, low-dose chronic infection of CD103^−/− mice with Trichuris resulted in no significant difference in immunity or parasite burden. Absence of CD103 also had no effect on the frequency of CD4⁺CD25⁺Foxp3⁺ T_reg cells in the mLN or LP.

Conclusions/Significance

These results suggest that CD103 is dispensable for intestinal immunity during helminth infection. Furthermore, lack of CD103 had no effect on DC or T_reg recruitment or retention within the large intestine.     

IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection

In this study, B cell function in protective T_H2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα^−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4⁺ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88^−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4⁺ T cell-mediated protective immunity against N. brasiliensis infection.