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Mycobacterium tuberculosis (Mtb) is the leading cause of mortality worldwide due to a single infectious agent. Mtb resides in pulmonary macrophages, neutrophils and phagocytes, which are also key effector cells that limit bacterial growth. The role of other granulocytic phagocytes during Mtb infection is largely unknown. Eosinophils have comparable phagocytic function to neutrophils and an overlapping repertoire of granular contents capable of limiting bacterial growth. Here we characterized eosinophil responses to Mtb in vitro and in vivo. Eosinophils from healthy human donors responded to Mtb in vitro by CD69 up-regulation and CD62L down-regulation. Mtb induced the release of the eosinophil-specific granule proteins EDN and ECP. Mtb exposure resulted in secretion of IL-1a, TNFa, MIP-1a, MIP-1b, and IL-8. These results suggest that Mtb directly affects eosinophil degranulation, and that eosinophils are able to contribute to Mtb-driven inflammation. To investigate whether eosinophils actively participate in the cellular immune response to Mtb in vivo we followed the eosinophilic response after pulmonary Mtb infection in non-human primates. Eosinophils were significantly increased in the BAL fluid of Mtb infected rhesus macaques, providing evidence that eosinophils are being actively recruited to the lung in response to Mtb in vivo. Indeed, when we analyzed Mtb infected lung tissue from patients who had undergone lung resection, eosinophils were enriched in fibrotic TB consolidations, cavity wall and necrotic caseum. We are currently investigating the biological relevance of the eosinophilic response during Mtb infection in the mouse model. This work was supported by the intramural research programs of NIAID, NIH.

CD4 T cells promote memory immune responses, but it is unclear whether vaccine-induced CD4 T cells contribute to immune protection. We interrogated if virus-specific CD4 T cells would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that induced CD4 T cell responses resulted in mortality following infection with persistent strains of LCMV. Immunopathology was associated with high levels of virus-specific CD4 T cells, generalized inflammation and collapse of adaptive immunity. Interestingly, virus-specific CD8 T cells or antibodies abrogated these lethal effects of CD4 T cell vaccines. Our data demonstrate the importance of tightly regulating CD4 T cell responses following vaccination, and suggest that CD4 T cells in the absence of effective antiviral immune responses could induce lethal immunopathology.

Anti-retroviral therapy (ART) of HIV-infected individuals usually leads to clinical improvement, but some individuals develop a worsening of symptoms following ART, referred to as Immune Reconstitution Inflammatory Syndrome (IRIS). CD4 T cells are thought to be a primary mediator of this paradoxical disease and recent work in patients has correlated activated monocytes with the development of IRIS. Here we use a mouse model of Mycobacterium avium associated IRIS to examine myeloid cell responses in IRIS. We find that CD4 T cell reconstitution of chronically infected TCRaKO mice leads to large increases in neutrophils and Ly6C+ CCR2+ monocytes. Prior to CD4 T cell transfer, myeloid cells in chronically infected TCRaKO mice expressed less iNOS but higher TNF compared to WT mice. Within ~12 days after CD4 T cell reconstitution, multiple myeloid cell subsets dramatically upregulated iNOS and TNF reaching levels far above what is ever observed in WT mice at any time during infection. Transfer of IFNgKO CD4 T cells into chronically infected TCRaKO mice failed to drive myeloid cell expansion or increased function, and mice displayed greatly reduced signs of illness. These data indicate that progression of a chronic mycobacterial infection in the absence of CD4 T cell help predisposes myeloid cells to over respond to T cell stimulation when CD4 T cells are later introduced, indicating myeloid cell hyper responsiveness may underly the immunopathogenesis of IRIS.

PD-1 is an inhibitory receptor expressed on the surface of activated T cells. PD-1 previously has been shown to play a significant role in regulating effector T cell responses against viral infections and tumors. However, its role in the control of CD4 T cell responses during infection with fungal pathogens remains poorly understood. Here we examine the role of PD-1 in regulating host responses and resistance to the opportunistic fungal pathogen Cryptococcus neoformans. We find that in wild-type mice C. neoformans elicits Th1, Th2, and Th17 responses, and PD-1 is expressed on each of these effector T cell subsets. In PD-1 deficient mice, the frequency of IFN-γ-producing and t-bet expressing CD4+ T cells were increased, while CD4+ T cells making IL-13, IL-5, and IL-4 were decreased and Th17 responses were unchanged. Also, PD-1 deficient mice show a decrease in eosinophils in the lung, and in fungal burden. These data suggest that PD-1 impairs host resistance to C. neoformans by selectively inhibiting Th1 responses. The work was supported by the NIH Intramural Research Program.

CD4 T cells are important for promoting functional adaptive immune responses following viral infections, but the specific contribution of vaccine-elicited CD4 T cell responses to immune protection remains unclear. We interrogated whether selective induction of virus-specific CD4 T cell responses by vaccination, in the absence of other adaptive immune responses, would improve immune protection against a chronic lymphocytic choriomeningitis virus (LCMV) challenge in mice. Surprisingly, we observed that immunization of mice with a vaccine that induced predominantly CD4 T cell responses resulted in severe immunopathology and mortality following chronic LCMV clone 13 challenge. Vaccinated mice exhibited impaired adaptive responses, severe inflammation, accelerated destruction of lymphoid tissues, and fulminant mortality as compared with naïve mice following challenge. This immunopathology was caused by uncontrolled antigen-driven stimulation of memory CD4 T cells that resulted in cytokine storm and widespread inflammation. The adverse effects of vaccination were abrogated by co-immunization with vaccines that generated CD8 T cell responses, adoptive transfer of virus-specific CD8 T cells, or passive immunization with virus-specific immunoglobulins. Altogether, these data raise important concerns for vaccines that predominantly elicit CD4 T cell responses.

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas’ Disease, which affects ~12 million people in Latin America despite coordinated international control efforts. Cardiomyopathy due to chronic inflammation and fibrosis is the most common and serious presentation of Chagas’ Disease, but its pathogenesis is poorly understood. Several inflammatory chemokines and chemokine receptors have been implicated in mouse models of T. cruzi infection; however, roles for homeostatic chemokines have not previously been reported. Here, we found that mRNA for the homeostatic chemokine Ccl19 and its receptor Ccr7 were both increased in the heart during the acute phase after infection of WT C57BL/6 mice. Consistent with an important functional role, infection was uniformly fatal during the acute phase in infected Ccr7-/- mice, whereas mortality was only 10% in WT control mice. Death in infected KO mice was associated with uncontrolled parasitemia and increased parasite burden in both heart and liver, whereas infected WT mice controlled parasite burden in all 3 compartments. Leukocyte infiltration was also greater in the hearts of infected KO mice compared to infected WT controls, and was characterized by a lower frequency of CD8+ T cells and a greater frequency of Gr-1hiCD11b+IA- cells. Thus Ccr7 is a critical factor for survival and control of T. cruzi infection in C57BL/6 mice. The mechanisms of resistance conferred by Ccr7 are currently under investigation.

The innate immune system recognizes pathogens/antigens via several families of pattern recognition receptors (PRRs). Often, several PRRs will be triggered, resulting in an innate immune response via a complex integration of signals. However, the importance of different PRR signaling pathways in determining adaptive responses such as helper T cell polarization is not fully understood. We studied the role of two pathways—TLRs and the inflammasome—in promoting helper T cell polarization in response to complete Freund’s adjuvant (CFA). Mice immunized with ovalbumin (OVA) in CFA developed an OVA-specific Th1/Th17 response that was dependent on the signaling adaptor MyD88. TLRs were only partially required for this response, whereas interleukin 1 (IL-1) receptor signaling and the inflammasome were critical. We found that heat-killed M. tuberculosis H37Ra, a component of CFA, induces mature IL-1β production in vitro in a process that requires potassium efflux and reactive oxygen species and is mediated by the NLRP3 receptor. We further showed that the major inflammasome-triggering component of H37Ra is the peptidoglycan. However, the peptidoglycan-sensing receptors NOD1 and NOD2 were not needed for this response. Our data suggest that NOD1/2-independent recognition of mycobacterial peptidoglycan by the NLRP3 inflammasome induces IL-1β production and may play an important role in CFA-induced helper T cell responses. This work was supported by the NIAID Intramural Research Program.

Approximately 10-30% of HIV patients develop a paradoxical inflammatory disease shortly after beginning anti-retroviral therapy (ART), termed Immune Reconstitution Inflammatory Syndrome (IRIS), that is emerging as a major problem in the clinical management of the HIV pandemic. Patients with the lowest CD4 counts, the most rapid drop in viral loads after ART and an AIDS defining illness, such as mycobacterial infection, are at highest risk. This indicates that T cell reconstitution itself may be pathogenic rather than beneficial when CD4 T cells suddenly repopulate a lymphopenic host harboring an opportunistic infection. Here we demonstrate that a rapidly fatal wasting occurs in mice when CD4 T cells are transferred into Mycobacterium avium infected TCRαKO animals. This reconstitution disease was dependent on IFNγ and TNFα but not IL-4 or IL-17A. Non-specific T cells induced no disease, but transfer of T cells specific for bacterial or endogenous antigens was rapidly lethal. Importantly, M. avium infected TCRαKO and ovalbumin-specific TCR Tg mice were susceptible hosts, whereas M. avium-specific TCR Tg mice were resistant to reconstitution disease. These data demonstrate that during M. avium infection of lymphopenic hosts the lack of bacteria-specific T cells, rather than the lymphopenic environment per se, predisposes the host to develop reconstitution disease when repopulating CD4 T cells encounter Ag. This work was supported by the NIAID intramural research program.

The PDL-1:PD-1 pathway is involved in T cell exhaustion after chronic viral infection. PD-1 can bind to either PDL-1 or PDL-2, whereas PDL-1 can bind to either PD-1 or B7.1. We tested the effect of combining standard aPDL-1 blockade with a4-1BB (3H3 agonistic antibody) treatment to determine T cell restoration effects in a model of chronic infection with lymphocytic choriomeningitis virus (LCMV). We chose 4-1BB because it has been shown to be an important costimulatory pathway. It appears that a4-1BB treatment enhances the effect of aPDL-1 blockade if given in a low dose (50 μg, once). Conversely, if a4-1BB is given in a high dose (200 μg, five times), it results in sudden increase of antigen-specific T cells by day 7, but massive T cell death by day 14 post treatment. These results show the importance for 4-1BB costimulation in aPDL-1 mediated T cell restoration.