CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling

The present study addressed the modulatory role of CC chemokine receptor 4 (CCR4) in Toll-like receptor (TLR) 9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMPhi) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IkappaB kinase (IKK)/NF-kappaB were impaired compared to wild-type (WT) cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-kappaB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMPhi exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMPhi skewed to an alternatively activated phenotype. These results indicate that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.     

Adenoid-derived TH2 cells reactive to allergen and recall antigen express CC chemokine receptor 4

BACKGROUND: Interrupting recruitment of allergen-specific T(H)2 cells to the airway is an attractive potential therapeutic strategy for allergic disease. CC chemokine receptor 4 (CCR4) is preferentially expressed on T(H)2 cells, and CCR4-expressing cells have been described at sites of allergic inflammation. However, whether selective recruitment of allergen-specific T(H)2 cells to the airways occurs through CCR4 or other chemokine receptors remains controversial. OBJECTIVE: We investigated the expression of the T(H)2-associated chemokine receptors (CCR3, CCR4, and CCR8) by primary antigen-specific human airway T(H)2 cells. METHODS: Children undergoing elective adenoidectomy were recruited, and their atopic status was determined. Adenoid cells were cultured with allergen or recall antigen. Flow cytometric analyses permitted identification of T(H) cells proliferating in response to antigen and characterization of chemokine receptor and cytokine expression. RESULTS: An increased proportion of airway CD4(+) T cells proliferated to allergen in atopic children (n = 6, of which 4 were given diagnoses of asthma or rhinitis) compared with nonatopic children (P =.0004). These cells were 44.7% (32.6% to 50.0%) IL-4(+) and only 2.5% (0.6% to 3.3%) IFN-(gamma) and showed a greater than 5-fold upregulation of CCR4 expression to 54.0% (40.7% to 67.8%) after culture, whereas CCR3 was expressed on 9.7% (7.4% to 18.9%) of allergen-reactive cells and CCR8 on less than 1%. Interestingly, increased expansion of recall antigen-specific cells was also seen in atopic children, and these cells were also predominantly of a T(H)2 CCR4(+) phenotype. CONCLUSION: We conclude that airway allergen-specific T(H)2 cells are CCR4(+), but in the atopic child CCR4 does not distinguish between recall antigen and allergen specificity.     

Cytotoxic T-cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis

Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+gammadelta and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies.     

CD4 T cell responses to influenza infection

Immune responses to viral infections involve a complex orchestration between innate signals and adaptive responses of specific T and B cells. Anti-viral CD4 cells can direct CD8 responses by secreting a Type 1 panel of cytokines including IFN-gamma, IL-2 and TNF-alpha and can drive B cell production of IgG2a to neutralize infective viral particles. This review will focus specifically on the role of CD4 cells in the immune response to influenza, an acute, localized respiratory viral infection. We suggest that CD4 cells act as direct effectors in protection against influenza, may contribute to immunopathology and generate functionally distinct memory subsets.     

Targeting cellular fatty acid synthesis limits T helper and innate lymphoid cell function during intestinal inflammation and infection

CD4⁺ T cells contribute critically to a protective immune response during intestinal infections, but have also been implicated in the aggravation of intestinal inflammatory pathology. Previous studies suggested that T helper type (Th)1 and Th17 cells depend on de novo fatty acid (FA) synthesis for their development and effector function. Here, we report that T-cell-specific targeting of the enzyme acetyl-CoA carboxylase 1 (ACC1), a major checkpoint controlling FA synthesis, impaired intestinal Th1 and Th17 responses by limiting CD4⁺ T-cell expansion and infiltration into the lamina propria in murine models of colitis and infection-associated intestinal inflammation. Importantly, pharmacological inhibition of ACC1 by the natural compound soraphen A mirrored the anti-inflammatory effects of T-cell-specific targeting, but also enhanced susceptibility toward infection with C. rodentium. Further analysis revealed that deletion of ACC1 in RORγt⁺ innate lymphoid cells (ILC), but not dendritic cells or macrophages, decreased resistance to infection by interfering with IL-22 production and intestinal barrier function. Together, our study suggests pharmacological targeting of ACC1 as an effective approach for metabolic immune modulation of T-cell-driven intestinal inflammatory responses, but also reveals an important role of ACC1-mediated lipogenesis for the function of RORγt⁺ ILC.     

Induction of T helper 1- and T helper 2-type immune responses during Haemonchus contortus infection in sheep

The production of cytokines by lymphoid cells, isolated from non-infected and Haemonchus contortus-infected lambs, was investigated. Particular attention was paid to differences in T helper 1- (Th1) and Th2-type immune profiles between genetically resistant and random-bred animal groups. Non-infected resistant and random-bred lambs produced equivalent levels of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), from isolated abomasal lymph node cells (ALN), mesenteric lymph node cells (MLN) and spleen cells (SC), in response to in vitro stimulation with T-cell mitogen (concanavalin A) or larval parasite antigen. ALN and MLN cells derived from infected resistant and random-bred lambs produced relatively lower levels of IFN-gamma, following in vitro stimulation with parasite antigen, when compared with their uninfected counterparts. In contrast, infected lambs of both groups showed enhanced mitogen- and antigen-stimulated production of IL-5, in comparison with uninfected controls, at days 5 and 28 postinfection (p.i.). Mitogen- and antigen-stimulated IL-5 responses were higher among resistant lambs compared with random-bred lambs, with the highest overall production of IL-5 by parasite antigen-stimulated ALN and MLN cells. Among day 28 p.i. lambs, levels of cell culture-derived parasite-specific immunoglobulin G1 (IgG1) and IgE antibodies were higher in resistant lambs than in random-bred lambs, following in vitro stimulation of SC or ALN cells with parasite antigen. Finally, after 28 days p.i., histological examination of abomasal tissue revealed higher densities of mast cells and eosinophils in the mucosa of resistant lambs than in random-bred lambs. Taken together, these data support the notion of a strong Th2-type immune response to Haemonchus infection in genetically resistant sheep, and support the claim for a Th1/Th2 dichotomy in ruminants.     

The murine chemokine receptor CXCR4 is tightly regulated during T cell development and activation

We have characterized the murine homolog of the HIV-co-receptor CXCR4 during T cell development and activation. Our data demonstrate that this chemokine receptor, although highly conserved between human and mouse, is differently expressed and regulated in both species. Mitogenic activation resulted in an increase of surface CXCR4 on murine T cells within 2 days, whereas the receptor was strongly down-regulated on human T cells during this period. Furthermore, intraperitoneal immunization of mice resulted in a strong increase of splenic and mesenteric cytotoxic T cells co-expressing CXCR4. It is interesting that, on thymocytes, expression of CXCR4 is restricted to CD4+CD8+ cells. Stromal cell-derived factor-1alpha, a natural ligand of CXCR4, induced chemotaxis of thymocytes and was found to counteract dexamethasone-induced apoptosis to a certain extent in these cells. Thus, our data show that expression of CXCR4 is tightly controlled on murine T cells and indicate that this highly conserved chemokine receptor might serve different functions in humans and mice.     

Mycobacterium bovis BCG-infected neutrophils and dendritic cells cooperate to induce specific T cell responses in humans and mice

Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil-DC partnership in the response to Mycobacterium bovis BCG-the vaccine used against tuberculosis. We compared neutrophil-DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG-enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non-apoptotic BCG-infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG-infected DC produced interleukin (IL)-10, an inhibitory cytokine, whereas DC exposed to BCG-infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG-infected neutrophils induced sustained IL-2 production by DC. Human DC exposed to BCG-infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalbumin (OVA)-producing BCG (rBCG(ova)) elicited proliferation of TCR-OVA-transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCG(ova)-infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross-present BCG antigens to T cells. Our results suggest that this "ménage à trois" involving neutrophils, DC and T cells plays a major role in the immune response to BCG.     

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4⁺ T regulatory cells (T_regs) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2⁺ cells increased over time for both CD4⁺ and CD8⁺ T cells. The T_reg subsets CD25^highCD127^low, CD25^highCD147⁺⁺ and CD25^highCD127^lowCD161⁺ expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25^highCD127^lowCD39⁺ T_regs remained unchanged. The fraction of CD25^highCD127^low T_regs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of T_regs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4⁺ and CD8⁺ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.     

Evidence for CD4+ T cell responses common to Plasmodium falciparum and recall antigens

In individual donors which have never been exposed to malaria parasites, the CD4+ T cell precursor frequencies for tetanus toxoid (TT) and Plasmodium falciparum responses are similar (range 1:850- 1:4800). Limiting dilution cultures set up in response to P. falciparum trophozoites can be re-stimulated with the same stage of the parasite or TT and respond with similar frequencies. A substantial overlap in the responses to different agents was confirmed in suicide selection experiments where cells responding to malaria parasite, TT or influenza virus antigens were deleted using the cell cycle inhibitor cytosine arabinoside (Ara-C). The responses of the remaining cells to P. falciparum were almost completely abrogated and only weak responses were observed to different recall antigens (0.2-21% of untreated control). Little or no effect was observed on the responses to superantigen or mitogen. Furthermore, in contrast to superantigen, the observed responses to TT and Plasmodium were polyclonal, the blastoid cells generated reacting with a range of anti-TCR Vbeta antibodies with little preferential usage.      

DAP10 contributes to CD8(+) T cell-mediated cytotoxic effector mechanisms during Mycobacterium tuberculosis infection

The activating C-type lectin-like receptor NKG2D, which is expressed by mouse NK cells and activated CD8 T cells, was previously demonstrated to be involved in tumor rejection and as a defense mechanism against viral and bacterial infections. Because CD8 T cells are important for protective immune responses during chronic Mycobacterium tuberculosis (Mtb) infection and represent a promising target for new vaccine strategies to prevent human pulmonary tuberculosis (TB), we studied the immune response in mice deficient for the NKG2D adapter molecule DAP10 during experimental TB. After aerosol infection, DAP10-defcient mice displayed an unimpaired recruitment, activation and development of antigen-specific CD8 T cells. Whereas the frequency of interferon-gamma-producing CD8 T cells from Mtb-infected DAP10-defcient mice was not affected, CD8 T cell-mediated cytotoxicity was significantly reduced in the absence of DAP10. The loss of cytotoxic activity in DAP10-deficient CD8 T cells was associated with an impaired release of cytotoxic granules. Together, our results suggest that during Mtb infection DAP10 is required for maximal cytolytic activity of CD8 T cells.     

RIP2 contributes to Nod signaling but is not essential for T cell proliferation, T helper differentiation or TLR responses

Receptor-interacting protein 2 (RIP2), also known as CARDIAK and RICK, has been reported to play a role in both adaptive T cell responses and innate immunity as a mediator in TLR signaling and nucleotide-binding oligomerization domain (Nod) signaling. Because initial reports remain controversial, we have further examined both innate and adaptive immune responses in RIP2-deficient mice on the C57BL/6 background. Despite the up-regulation of RIP2 after T cell activation, we could not detect any defect in T cell proliferation or Th1/Th2 responses in RIP2-KO mice. Furthermore, we found that TLR responses in RIP2-deficient macrophages were normal. However, our analysis showed that Nod signaling was impaired in macrophages from RIP2-deficient mice. In conclusion, our data demonstrate a critical role for RIP2 in Nod signaling, while T cell proliferation, T helper differentiation and TLR responses were unaffected by the absence of RIP2.     

Triggering of toll-like receptor signaling pathways in T cells contributes to the anti-tumor efficacy of T cell responses

Traditionally, expression of toll-like receptors (TLRs) has been associated with innate immune cells in particular professional antigen presenting cells and natural killer cells. This led to the concept that the adjuvant effects of ligation of TLR in a host occur mainly in innate immune cells. However, this concept has been challenged by recent studies including ours demonstrating that T cells express appreciated levels of different TLRs, which can serve as costimulatory co-receptors during polyclonal and antigen-specific stimulation of T cells. Because T cells express low levels of TLRs as compared to innate immune cells, increasing the expression levels of TLRs in T cells can significantly maximize their responses to the costimulatory effects of TLR ligation. This review article focuses on the potential role of TLR expression in T cells in their responses to vaccination regimen containing TLR agonists and how it can be modulated to optimize anti-tumor immunity.     

Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS

NK cells play an important role in controlling viral infections. They can kill virus-infected cells directly as well as indirectly via antibody-dependent, cell-mediated cytotoxicity. They need no prior sensitization and expansion for this killing. NK cells are also considered as important regulators of antiviral immune responses. They do so by secreting a multitude of soluble mediators and by directly interacting with other immune cells, e.g., dendritic cells. NK cells do not possess a single well-defined receptor to recognize antigens on target cells. Instead, they express an array of inhibitory and activating receptors and coreceptors, which bind to their cognate ligands expressed on the surface of target cells. These ligands include classical and nonclassical MHC class I antigens, MHC-like proteins, and a variety of other self- and virus-derived molecules. They may be expressed constitutively and/or de novo on the surface of virus-infected cells. NK cell receptors (NKRs) of the killer-cell Ig-like receptor (KIR) family, like their MHC class I ligands, are highly polymorphic. Several recent studies suggest that epistatic interactions between certain KIR and MHC class I genes may determine innate resistance of the host to viral infections, including HIV. In the first part of this review article, we provide an overview of the current state of knowledge of NK cell immunobiology and describe how NKR genes, alone and in combination with HLA genes, may determine genetic resistance/susceptibilty to HIV infection and the development of AIDS in humans.     

Evidence for lack of cross-genotype protection of CD4+ T cell responses during chronic hepatitis C virus infection

CD4+ T lymphocyte responses are thought to play a major role in control of the hepatitis C virus (HCV). Few, however, have been mapped down to the level of peptide and HLA restriction. Furthermore, the ability of such T cells to respond to viruses which differ in genotype has not been addressed in detail. In most cases of persistent infection with HCV, CD4 proliferative responses are weak or absent. From a large cohort of persistently infected patients, we identified an individual with unusually robust and persistent responses in the face of chronic infection. We firstly mapped two peptide epitopes to regions of the nonstructural protein NS4 (aa1686-1705 and aa 1746-1765). However, in contrast to the genotype 1a derived antigens used for mapping, the infecting virus was identified as genotype 3a. Strikingly, the patient's CD4 response to these epitopes were specific only for the genotype 1a sequence, and did not recognize genotype 3a synthetic peptides. Serologic assays indicated that prior exposure to HCV of genotype 1 had occurred. This patient therefore maintains strong CD4 proliferative responses which are genotype specific and not cross-reactive. The apparent 'misdirection' of these nonprotective responses has important implications for the role of natural and vaccine induced CD4 responses in the face of variable viruses.     

IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.     

XCL1 (lymphotactin) chemokine produced by activated CD8 T cells during the chronic stage of infection with Mycobacterium tuberculosis negatively affects production of IFN-gamma by CD4 T cells and participates in granuloma stability

CD8 T cell immune responses are known not to be essential during the initial stages of infection with Mycobacterium tuberculosis (Mtb), but their presence becomes important as the chronic infection ensues. The basis of this is still not clear. In previous studies, we showed that CD8 T cells have a distinctive positioning in the architecture of the granuloma lesion, with further changes throughout the course of the chronic infection. We have also hypothesized that further movement of lymphocytes once they are within the lung lesions could be associated with the levels of expression of the chemokine XCL1 (lymphotactin). XCL1 is produced mainly by activated CD8 T cells, and its chemotactic activity seems primarily controlling movement of CD4 and CD8 T cells. In this study, using a murine low-dose aerosol infection model coupled with antibody depletion of T cell subsets, we investigated the role of CD8 T cells in the control of the bacterial growth and in the pathogenesis of the disease in mice at early, mid, or late stages of the chronic disease state. Additionally, we also describe for the first time that during Mtb infection, activated CD8 T cells in the lungs produce XCL1 and that this chemokine is capable of controlling IFN-gamma production by CD4 T cells.