Deficiency in TLR2 but not in TLR4 impairs dendritic cells derived IL-10 responses to schistosome antigens

The purpose of this study was to observe the diverse functions of Toll-like receptors (TLRs) in responses to specific schistosome antigens. Bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient (TLR2(-/-)) or TLR4-deficient (TLR4(-/-)) mice were activated with soluble schistosomule antigen (SSA) or soluble egg antigen (SEA). TLR2 mRNA expression was significantly increased in B6 BMDCs following SEA stimulation. TLR2-deficient BMDCs showed enhanced MHCII expression following SSA and SEA stimulation. TLR2-deficient but not TLR4-deficient BMDC failed to produce IL-12p70 and IL-10 in response to schistosome antigens. TLR2-deficient BMDCs induced a stronger CD4(+) T cell proliferative response. IL-4 and IL-10 expression was inhibited in CD4(+) T cells primed with TLR2-deficient BMDCs, while enhanced in TLR4-deficient BMDCs-primed CD4(+) T cells. These results suggest that TLR2 is essential for the establishment of the DC production of IL-12p70 and IL-10.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

Specific MDR1 P-glycoprotein blockade inhibits human alloimmune T cell activation in vitro

MDR1 P-glycoprotein (P-gp), the multidrug resistance-associated transmembrane transporter, is physiologically expressed by human peripheral immune cells, but its role in cell-mediated immunity remains poorly understood. Here, we demonstrate a novel role for P-gp in alloantigen-dependent human T cell activation. The pharmacologic P-gp inhibitor tamoxifen (1-10 microM) and the MDR1 P-gp-specific mAb Hyb-241 (1-20 microg/ml), which detected surface P-gp on 21% of human CD3(+) T cells and 84% of CD14(+) APCs in our studies, inhibited alloantigen-dependent, but not mitogen-dependent, T cell proliferation in a dose-dependent manner from 40-90% (p < 0.01). The specific inhibitory effect on alloimmune T cell activation was associated with >85% inhibition (p < 0.01) of IL-2, IFN-gamma, and TNF-alpha production in 48-h MLR coculture supernatants. Addition of recombinant human IL-2 (0.1-10 ng/ml) restored proliferation in tamoxifen-treated cocultures. Pretreatment of purified CD4(+) T cells with Hyb-241 mAb before coculture resulted in inhibition of CD4(+) T cellular IFN-gamma secretion. Also, blockade of P-gp on allogeneic APCs inhibited IL-12 secretion. Taken together these results demonstrate that P-gp is functional on both CD4(+) T cells and CD14(+) APCs, and that P-gp blockade may attenuate both IFN-gamma and IL-12 through a positive feedback loop. Our results define a novel role for P-gp in alloimmunity and thus raise the intriguing possibility that P-gp may represent a novel therapeutic target in allograft rejection.     

A role for immature myeloid cells in immune senescence

The reduced efficiency of the mammalian immune system with aging increases host susceptibility to infectious and autoimmune diseases. However, the mechanisms responsible for these pathologic changes are not well understood. In this study, we demonstrate that the bone marrow, blood, and secondary lymphoid organs of healthy aged mice possess increased numbers of immature myeloid cells that are phenotypically similar to myeloid-derived suppressor cells found in lymphoid organs of mice with progressive tumors and other pathologic conditions associated with chronic inflammation. These cells are characterized by the presence of Gr1 and CD11b markers on their surfaces. Gr1(+)CD11b(+) cells isolated from aged mice possess an ability to suppress T cell proliferation/activation and produce heightened levels of proinflammatory cytokines, both constitutively and upon activation, including IL-12, which promotes an excessive production of IFN-γ. IFN-γ priming is essential for excessive proinflammatory cytokine production and the suppressive activities by Gr1(+)CD11b(+) cells from aged mice. These cells suppress T cell proliferation through an NO-dependent mechanism, as depletion of splenic Gr1(+) cells reduces NO levels and restores T cell proliferation. Insights into mechanisms responsible for the proinflammatory and immune suppressive activities of Gr1(+)CD11b(+) cells from aged mice have uncovered a defective PI3K-Akt signaling pathway, leading to a reduced Akt-dependent inactivation of GSK3β. Our data demonstrate that abnormal activities of the Gr1(+)CD11b(+) myeloid cell population from aged mice could play a significant role in the mechanisms responsible for immune senescence.     

CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.     

Pro-IL-16 regulation in activated murine CD4+ lymphocytes

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4(+) T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G(0)/G(1) arrest by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1). In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27(Kip1) were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4(+) T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4(+) T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4(+) T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Membrane-bound IL-22 after de novo production in tuberculosis and anti-Mycobacterium tuberculosis effector function of IL-22+ CD4+ T cells

The role of IL-22-producing CD4(+) T cells in intracellular pathogen infections is poorly characterized. IL-22-producing CD4(+) T cells may express some effector molecules on the membrane, and therefore synergize or contribute to antimicrobial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22(+) T cells cannot be purified for evaluation due to secretion nature of cytokines. In this study, we surprisingly found that upon activation, CD4(+) T cells in Mycobacterium tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22(+) CD4(+) T effector cells appeared to mature in vivo and sustain membrane distribution in highly inflammatory environments during active M. tuberculosis infection. Near-field scanning optical microscopy/quantum dot-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ～100-200 nm nanoclusters or ～300-600 nm nanodomains for potential interaction with IL-22R. Importantly, purified membrane-bound IL-22(+) CD4(+) T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22-producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 t(1/2) and to exert efficient cell-cell interaction for anti-M. tuberculosis effector function.     

IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+ T cell- and STAT3-dependent manners

The hepatoprotective effect of IL-6 on various forms of liver injury including T cell-mediated hepatitis has been well documented, and it is believed that induction of antiapoptotic proteins is an important mechanism. In this study, we provide evidence suggesting an additional mechanism involved in the protective role of IL-6 in T cell-mediated hepatitis. In NKT cell-depleted mice, Con A-induced liver injury is diminished; this can be restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice. In vitro IL-6 treatment inhibits the ability of mononuclear cells to restore Con A-induced liver injury in NKT-depleted mice, whereas the same treatment does not inhibit purified NKT cells from restoring the injury. The addition of CD3(+) T cells or CD4(+) T cells can restore the inhibitory effect of IL-6 on purified NKT cells, whereas the addition of CD3(+) T cells from CD4-deficient mice fails to restore this inhibitory effect. The expression of IL-6R was detected in 52.6% of hepatic CD3(+) T cells and 32.7% of hepatic CD4(+) T cells, but only in 3.9% of hepatic NK and 1.5% of hepatic NKT cells. Finally, treatment with IL-6 induces STAT3 activation in hepatic lymphocytes and hepatic T cells, and blocking such activation abolishes the inhibitory effect of IL-6 on hepatic lymphocytes to restore liver injury. Taken together, these findings suggest that in addition to its antiapoptotic abilities, as previously well documented, IL-6/STAT3 inhibits NKT cells via targeting CD4(+) T cells and consequently prevents T cell-mediated hepatitis.     

STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.     

IL-12/23p40-dependent clearance of Anaplasma phagocytophilum in the murine model of human anaplasmosis

Human anaplasmosis is an emerging infectious disease transmitted by ticks that can be potentially fatal in the immunocompromised and the elderly. The mechanisms of defense against the causative agent, Anaplasma phagocytophilum, are not completely understood; however, interferon (IFN)-gamma plays an important role in pathogen clearance. Here, we show that IFN-gamma is regulated through an early IL-12/23p40-dependent mechanism. Interleukin (IL)-12/23p40 is regulated in macrophages and dendritic cells after activation by microbial agonists and cytokines and constitutes a subunit of IL-12 and IL-23. IL-12/23p40-deficient mice displayed an increased A. phagocytophilum burden, accelerated thrombocytopenia and increased neutrophil numbers in the spleen at day 6 postinfection. Infection of MyD88- and mitogen-activated kinase kinase 3 (MKK3)-deficient mice suggested that the early susceptibility due to IL-12/23p40 deficiency was not dependent on signaling through MyD88 or MKK3. The lack of IL-12/23p40 reduced IFN-gamma production in both CD4(+) and CD8(+) T cells although the effect was more pronounced in CD4(+) T cells. Our data suggest that the immune response against A. phagocytophilum is a multifactorial and cooperative process. The IL-12/23p40 subunit drives the CD4(+) Th1 immune response in the early phase of infection and IL-12/23p40-independent mechanisms ultimately contribute to pathogen elimination from the host.     

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.