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Both acute and chronic phases of Trypanosoma cruzi (T. cruzi) infection are characterized by tissue inflammation, mainly in the heart. A key step in the inflammatory process is the transmigration of inflammatory cells across the endothelium to underlying infected tissues. We observed increased arachidonic acid release and platelet-activating factor (PAF) production in human coronary artery endothelial cells (HCAEC) at up to 96 h of T. cruzi infection. Arachidonic acid release is mediated by activation of the calcium-independent phospholipase A2 (iPLA2) isoforms iPLA2 β and iPLA2 γ, whereas PAF production was dependent upon iPLA2 β activation alone. Trypanosoma cruzi infection also resulted in increased cell surface expression of adhesion molecules. Increased adherence of inflammatory cells to T. cruzi-infected endothelium was blocked by inhibition of endothelial cell iPLA2 β or by blocking the PAF receptor on inflammatory cells. This suggests that PAF, in combination with adhesion molecules, might contribute to parasite clearing in the heart by recruiting inflammatory cells to the endothelium.

Cardiomyopathy is a serious complication of Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi. The parasite often infects cardiac myocytes, causing the release of inflammatory mediators, including eicosanoids. A recent study from our laboratory demonstrated that calcium-independent phospholipase A2γ (iPLA2γ) accounts for the majority of PLA2 activity in rabbit ventricular myocytes and is responsible for arachidonic acid (AA) and prostaglandin E2 (PGE2) release. Thus, we hypothesized that cardiac iPLA2γ contributes to eicosanoid production in T. cruzi infection. Inhibition of the isoform iPLA2γ or iPLA2β, with the R or S enantiomer of bromoenol lactone (BEL), respectively, demonstrated that iPLA2γ is the predominant isoform in immortalized mouse cardiac myocytes (HL-1 cells). Stimulation of HL-1 cells with thrombin, a serine protease associated with microthrombus formation in Chagas' disease and a known activator of iPLA2, increased AA and PGE2 release, accompanied by platelet-activating factor (PAF) production. Similarly, T. cruzi infection resulted in increased AA and PGE2 release over time that was inhibited by pretreatment with (R)-BEL. Further, T. cruzi-infected iPLA2γ-knockout (KO) mice had lower survival rates and increased tissue parasitism compared to wild-type (WT) mice, suggesting that iPLA2γ-KO mice were more susceptible to infection than WT mice. A significant increase in iPLA2 activity was observed in WT mice following infection, whereas iPLA2γ-KO mice showed no alteration in cardiac iPLA2 activity and produced less PGE2. In summary, these studies demonstrate that T. cruzi infection activates cardiac myocyte iPLA2γ, resulting in increased AA and PGE2 release, mediators that may be essential for host survival during acute infection. Thus, these studies suggest that iPLA2γ plays a cardioprotective role during the acute stage of Chagas' disease.

IL-23 plays an essential role in maintenance of IL-17-producing Th17 cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ-mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3'-untranslated region (3'UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3'UTR-dependent luciferase activity. Additionally, deletion of TTP completely abolishes IFN-γ-mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation, and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ- and LPS-induced p19 mRNA expression, whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3'UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3'UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ, and IL-23, indicating that IFN-γ-mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition.

Understanding the regulation of human immune responses is critical for vaccine development and treating infectious diseases. We have previously shown that simultaneous engagement of the T cell receptor (TCR) and complement regulator CD46 on human CD4(+) T cells in the presence of interleukin-2 (IL-2) induces potent secretion of the immunomodulatory cytokine IL-10. These T cells mediate IL-10-dependent suppression of bystander CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 costimulation, reflecting a T regulatory type 1 (Tr1)-like phenotype. However, CD46-mediated negative regulation of pathogen-specific T cells has not been described. Therefore, we studied the ability of CD46-activated human CD4(+) T cells to suppress T cell responses to Mycobacterium bovis BCG, the live vaccine that provides infants protection against the major human pathogen Mycobacterium tuberculosis. Our results demonstrate that soluble factors secreted by CD46-activated human CD4(+) T cells suppress mycobacterium-specific CD4(+), CD8(+), and γ(9)δ(2) TCR(+) T cells. Dendritic cell functions were not downregulated in our experiments, indicating that CD46-triggered factors directly suppress pathogen-specific T cells. Interestingly, IL-10 appeared to play a less pronounced role in our system, especially in the suppression of γ(9)δ(2) TCR(+) T cells, suggesting the presence of additional undiscovered soluble immunoregulatory factors. Blocking endogenous CD46 signaling 3 days after mycobacterial infection enhanced BCG-specific T cell responses in a subset of volunteers. Taken together, these results indicate that CD46-dependent negative regulatory mechanisms can impair T cell responses vital for immune defense against mycobacteria. Therefore, modulating CD46-induced immune regulation could be integral to the development of improved tuberculosis therapeutics or vaccines.

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases, their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients, but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study, we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers, suggesting that development of tumor-infiltrating CD4(+) Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment, we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs, demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments, and provide new insights relevant for the development of novel cancer immunotherapeutic approaches.

Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) induce potent expansions of human memory Vgamma(9)(+)Vdelta(2)(+) T cells capable of IFN-gamma production, cytolytic activity, and mycobacterial growth inhibition. Certain phosphoantigens expressed by mycobacteria can stimulate gamma(9)delta(2) T cell expansions, suggesting that purified or synthetic forms of these phosphoantigens may be useful alone or as components of new vaccines or immunotherapeutics. However, we show that while mycobacteria-activated gamma(9)delta(2) T cells potently inhibit intracellular mycobacterial growth, phosphoantigen-activated gamma(9)delta(2) T cells fail to inhibit mycobacteria, although both develop similar effector cytokine and cytolytic functional capacities. gamma(9)delta(2) T cells receiving TLR-mediated costimulation during phosphoantigen activation also failed to inhibit mycobacterial growth. We hypothesized that mycobacteria express Ags, other than the previously identified phosphoantigens, that induce protective subsets of gamma(9)delta(2) T cells. Testing this hypothesis, we compared the TCR sequence diversity of gamma(9)delta(2) T cells expanded with BCG-infected vs phosphoantigen-treated dendritic cells. BCG-stimulated gamma(9)delta(2) T cells displayed a more restricted TCR diversity than phosphoantigen-activated gamma(9)delta(2) T cells. In addition, only a subset of phosphoantigen-activated gamma(9)delta(2) T cells functionally responded to mycobacteria-infected dendritic cells. Furthermore, differential inhibitory functions of BCG- and phosphoantigen-activated gamma(9)delta(2) T cells were confirmed at the clonal level and were not due to differences in TCR avidity. Our results demonstrate that BCG infection can activate and expand protective subsets of phosphoantigen-responsive gamma(9)delta(2) T cells, and provide the first indication that gamma(9)delta(2) T cells can develop pathogen specificity similar to alphabeta T cells. Specific targeting of protective gamma(9)delta(2) T cell subsets will be important for future tuberculosis vaccines.

We developed a carboxyfluorescein succinimidyl ester (CFSE)-based flow-cytometric assay that can detect different subsets of vaccinia-specific T cells capable of both antigen-specific expansion and protective effector functions. Proliferation and effector functions were detected by CFSE dilution and intracellular staining, respectively. Absolute numbers of CD4(+)/CFSE(lo)/interferon (IFN)- gamma (+), CD8(+)/CFSE(lo)/IFN- gamma (+), CD8(+)/CFSE(lo)/granzyme A(+), and CD8(+)/CFSE(lo)/CD107a(+) T cells present after in vitro stimulation with live vaccinia were significantly higher in immunized individuals (P<.05). These CD4(+) and CD8(+) T cell increases were >2 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8(+)/CFSE(lo)/IFN- gamma (+) and granzyme A(+) T cell responses were significantly correlated with the results of standard (51)Cr-release cytolytic assays (P<.05). Furthermore, vaccinia induced antigen-specific memory gamma delta T cells. We demonstrate that vaccinia induces robust memory effector CD4(+), CD8(+), and gamma delta T cells, all of which are relevant for protection against smallpox. CFSE-based flow-cytometric assays will be useful in evaluating cell-mediated immune responses induced by new smallpox vaccines.

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. The parasite is transmitted mucosally, with both intra- and extracellular life stages in the human host. Cruzipain, the major cysteinyl proteinase of T. cruzi, has been shown to be antigenic in both humans and mice during infection with the parasite. We extend these observations, showing here that multiple murine immune subsets of potential importance for vaccine-induced protection can be induced by cruzipain. Cruzipain-specific serum IgG responses were induced during chronic infection with T. cruzi. In addition, T. cruzi mucosal infection stimulated the development of cruzipain-specific secretory IgA detectable in fecal extracts from infected mice. Cruzipain-specific type 1 cytokine responses characterized by the production of IFN-gamma but not IL-4 were also detectable during murine infection. Furthermore, immunization of mice with a DNA vaccine encoding cruzipain was shown to stimulate cytotoxic T lymphocyte (CTL) responses capable of recognizing and lysing T. cruzi-infected cells. The induction of serum antibody, mucosal IgA, Th1 cytokine and CTL responses by cruzipain in mice supports the use of this parasite protein for further efforts in T. cruzi vaccine development.

The development of new tuberculosis (TB) vaccines will require the identification of correlates of human protection. This study examined the balance between immunity and virulence in a whole blood infection model in which intracellular mycobacterial survival was measured using BACTEC. In the blood of tuberculin-negative donors, counts of Mycobacterium tuberculosis H(37)Ra organisms fell by 0.14 log(10) CFU during 96 h of whole blood culture, whereas counts of Mycobacterium bovis BCG, M. tuberculosis H(37)Rv, and a clinical TB isolate's organisms increased by 0.13, 0.43, and 1.04 log(10) CFU, respectively (P < 0.001), consistent with their relative virulence. Inhibition of tumor necrosis factor alpha by the addition of methylprednisolone or pentoxifylline or removal of CD4(+) or CD8(+) T cells by magnetic beads had deleterious effects on immune control of intracellular growth only in the blood of tuberculin-positive donors. Repeated vaccination of eight tuberculin-negative volunteers with M. bovis BCG resulted in a 0.3 log (50%) reduction in BCG CFU counts in the model compared to baseline values (P < 0.05). Three of the volunteers responded only after the second vaccination. These experiments indicate that whole blood culture may be used to measure immunity to M. tuberculosis and that further studies of repeated BCG vaccination are warranted.