Immune Regulation in Mycobacterial and Fungal Infections

National Institute Of Allergy And Infectious Diseases

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Abstract

Tuberculosis (TB) is the leading cause of death due to a single infectious agent. The only vaccine currently available for TB, Bacillus Calmette-Guerin (BCG), provides little protection from TB beyond infancy as currently utilized, and new vaccination strategies are greatly needed. Anti-tubercular chemotherapy is effective in treating infection with drug-susceptible strains of Mycobacterium tuberculosis (Mtb), but new approaches in treating TB disease are required to meet the growing threat posed by drug resistant Mtb. Methodologies that manipulate host immune responses to treat TB, host-directed therapies (HDTs), hold great promise but none have been approved for clinical use. A better understanding of host protective immune cells and molecules may provide insight into targets for the development of novel vaccines and treatments for TB. In this reporting period we have examined two distinct approaches to targeting T cell responses to enhance control of Mtb infection using non-human primates. In the first, we tested if blocking the inhibitory receptor PD-1, which is expressed by activated CD4 and CD8 T cells in granulomas, could lead to enhanced control of Mtb infection. In the second, we tested if stimulating Mucosal Associated Invariant T cells, population of unconventional T cells, with their TCR ligand would lead to enhanced control of Mtb infection. PD-1 blockade is used in the treatment of various forms of cancer and there is a long-standing interest in developing PD-1 targeting strategies for the treatment of chronic infectious diseases, including tuberculosis. However, as we have previously reported data in the mouse model of tuberculosis showed that PD-1 KO mice succumb rapidly following Mtb exposure with extensive lung necrosis and high bacterial loads. There are also anecdotal case reports of cancer patients developing tuberculosis after PD-1 blockade. However, mice do not recapitulate many features of human TB, and it is not possible to establish causality based on sporadic the case reports in cancer patients. Therefore, we decided to test the hypothesis that PD-1 is host protective during Mtb infection using the non-human primate model, widely considered the gold standard model of tuberculosis. Rhesus macaques (n=6/group) were infected with Mtb and treated with isotype control of anti-PD-1 starting two weeks post infection. All isotype control treated macaques remained asymptomatic until the pre-determine endpoint at 16 weeks post-infection. In contrast, three of the six anti-PD-1 treated animals developed signs of severe disease. At necropsy, we found 4 of the 6 anti-PD-1 treated animals had significantly elevated bacterial loads in granulomas. The increased bacterial loads were associated with several pro-inflammatory cytokines, in particular IL-1b, IL-18 and IFNg, and increased Mtb-specific CD8 T cell responses. We used a novel live imaging approach to examine the trafficking of CD4 T cells in thick section granuloma explants and found that PD-1 blockade was also associated with decreased intralesional CD4 T cell trafficking. Overall, these data showed control of Mtb infection requires PD-1, highlighting the importance of negative regulation during tuberculosis. Moreover, these data indicate that TB should be considered a potential risk factor during PD-1 blockade for cancer immunotherapy. Several types of T cells restricted by class Ib molecules recognize mycobacterial antigens and have a hypothetical advantage over conventional T cells as vaccine and therapy targets due to the relatively non-polymorphic nature of the restriction elements and abundance of these cells at mucosal surfaces. MAIT cells are a particularly interesting potential target for TB vaccination and HDT. MAIT cells express a semi-invariant TCR specific for the riboflavin metabolite derivative 5-OP-RU presented by MR1. In support of their potential role in Mtb infection, MAIT cells are reduced in circulation and enriched in the airways of individuals with active TB disease compare to healthy donors. MAIT cells represent the majority of human PBMCs that produce IFN- after in vitro restimulation with BCG, and BCG revaccination of Mtb-infected individuals after isoniazid preventative therapy boosts MAIT frequencies. Intradermal BCG vaccination of macaques results in the upregulation of activation markers on MAIT cells, and intravenous BCG vaccination induces pulmonary MAIT expansion in rhesus macaques. In the mouse model, MAIT TCR transgenic mice displayed transiently reduced pulmonary Mtb loads during early infection. Finally, in the previous reporting period, we found in mice that stimulation of MAIT cells through administration of 5-OP-RU leads to an 100 fold expansion of MAIT cells and an IL-17A-dependent reduction of lung bacterial loads. Given these positive data, we tested the hypothesis that stimulating MAIT cells would enhance control of Mtb infection in non-human primates. Rhesus macaques were infected with Mtb and six weeks post-infection treated with either PBS or 5-OP-RU, a major TCR ligand for MAIT cells (n=5/group). 5-OP-RU treatment had no beneficial impact on lung disease as measured by PET-CT imaging or bacterial loads in individual granulomas. MAIT cells became activated after 5-OP-RU treatment as indicated by PD-1 and Ki67 expression, but failed to increase in number in BAL fluid, lung tissue, granulomas or any of the lymphoid tissues we examined. In fact, we found that MAIT cells rapidly entered a dysfunctional state resembling T cell exhaustion after 5-OP-RU treatment. This functional inactivation was observed in a second cohort of macaques that were not infected, indicating that the Mtb infection did not result in the failure of the MAIT cells to respond. Moreover, PD-1 blockade at the time of 5-OP-RU treatment did not enhance MAIT cell expansion. Therefore, unlike what was observed in mice, stimulating MAIT cells failed to enhance control of Mtb infection and instead resulted in their functional inactivation. This highlights an important feature of basic MAIT cell in vivo biology in non-human primates. It also indicates that in order to explore the therapeutic value of MAIT cells in non-human primates, approaches for driving MAIT cell expansion in vivo must be developed.