Publications

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.

Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-β, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.

BACKGROUND: Although BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity.

METHODS: Here, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL).

RESULTS: Prior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection.

CONCLUSIONS: Overall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.

Mycobacterium tuberculosis (Mtb) has developed specialized mechanisms to parasitize its host cell, the macrophage. These mechanisms allow it to overcome killing by oxidative burst and persist in the wake of an inflammatory response. Mtb infection in the majority of those exposed is controlled in an asymptomatic form referred to as latent tuberculosis infection (LTBI). HIV is a well-known catalyst of reactivation of LTBI to active TB infection (ATB). Through the use of nonhuman primates (NHPs) co-infected with Mtb and Simian Immunodeficiency Virus (Mtb/SIV), we are able to simulate human progression of TB/AIDS comorbidity. The advantage of NHP models is that they recapitulate the breadth of human TB outcomes, including immune control of infection, and loss of this control due to SIV co-infection. Identifying correlates of immune control of infection is important for both vaccine and therapeutics development. Using macaques infected with Mtb or Mtb/SIV and with different clinical outcomes we attempted to identify signatures between those that progress to active infection after SIV challenge (reactivators) and those that control the infection (non-reactivators). We particularly focused on pathways relevant to myeloid origin cells such as macrophages, as these innate immunocytes have an important contribution to the initial control or the lack thereof, following Mtb infection. Using bacterial burden, C-reactive protein (CRP), and other clinical indicators of disease severity as a guide, we were able to establish gene signatures of host disease state and progression. In addition to gene signatures, clustering algorithms were used to differentiate between host disease states and identify relationships between genes. This allowed us to identify clusters of genes which exhibited differential expression profiles between the three groups of macaques: ATB, LTBI and Mtb/SIV. The gene signatures were associated with pathways relevant to apoptosis, ATP production, phagocytosis, cell migration, and Type I interferon (IFN), which are related to macrophage function. Our results suggest novel macrophage functions that may play roles in the control of Mtb infection with and without co-infection with SIV. These results particularly point towards an interplay between Type I IFN signaling and IFN-γ signaling, and the resulting impact on lung macrophages as an important determinant of progression to TB.

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy. IMPORTANCE Myeloid cells are immunocytes of innate origin that orchestrate the first response toward pathogens via immune surveillance (uptake and killing), antigen presentation, and initiation of adaptive immunity by T cell stimulation. However, MDSCs are a subset of innate immunocytes that deviate to an immunoregulatory phenotype. MDSCs possess strong immunosuppressive capabilities that are induced in autoimmune, malignant neoplastic, and chronic inflammatory diseases. Induction of MDSCs has been found in peripheral blood, bronchoalveolar lavage (BAL) fluid, and pleural effusions of active TB patients, but their precise localization in lung tissue and in TB granulomas remains unclear due to challenges associated with sampling lungs and granulomas from active TB patients. Nonhuman primates (NHPs) are an important animal model with TB granulomas that closely mimic those found in humans and can therefore be used for studies that are otherwise challenging with patient material. Herein, we study MDSC localization in the lungs of NHPs exhibiting latent and active TB. Our findings reveal that MDSCs localize and exert their immunosuppressive roles at the periphery rather than in the core of TB granulomas.

Smoking is known to be an added risk factor for tuberculosis (TB), with nearly a quarter of the TB cases attributed to cigarette smokers in the 22 countries with the highest TB burden. Many studies have indicated a link between risk of active TB and cigarette smoke. Smoking is also known to significantly decrease TB cure and treatment completion rate and increase mortality rates. Cigarette smoke contains thousands of volatile compounds including carcinogens, toxins, reactive solids, and oxidants in both particulate and gaseous phase. Yet, to date, limited studies have analyzed the impact of cigarette smoke components on Mycobacterium tuberculosis (Mtb), the causative agent of TB. Here we report the impact of cigarette smoke condensate (CSC) on survival, mutation frequency, and gene expression of Mtb in vitro. We show that exposure of virulent Mtb to cigarette smoke increases the mutation frequency of the pathogen and strongly induces the expression of the regulon controlled by SigH-a global transcriptional regulator of oxidative stress. SigH has previously been shown to be required for Mtb to respond to oxidative stress, survival, and granuloma formation in vivo. A high-SigH expression phenotype is known to be associated with greater virulence of Mtb. In patients with pulmonary TB who smoke, these changes may therefore play an important, yet unexplored, role in the treatment efficacy by potentially enhancing the virulence of tubercle bacilli.