Immune Regulation in Mycobacterial and Fungal Infections

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of global mortality due to infectious disease. The only available vaccine for tuberculosis (TB) is Bacillus Calmette-Guerin (BCG), which protects infants against severe forms of TB, but does little to prevent disease in adolescents and adults. A highly effective TB vaccine would have a tremendous beneficial impact on global public health. However, due to the lack of robust correlates of protection, TB vaccine development is largely empirical, which contributes to the slow rate of conceptual and practical progress. A better understanding of the mechanisms of protection could help guide rationale design of TB vaccine candidates. CD4 T cells are particularly critical in control of Mtb infection and are likely to be a major contributor to vaccine-elicited protection. The anti-mycobacterial effect of CD4 T cells is largely attributed to their production of interferon-gamma (IFNγ), as mice deficient in IFNγ or CD4 T cells are highly susceptible to mycobacterial infection. In Mtb-infected mice, CD4 T cell-derived IFNγ acts, at least in part, on infected macrophages (Macs), as Macs lacking MHCII or IFNγ receptor (IFNγR) are unable to suppress growth of Mtb, but its role in driving nitric oxide synthase 2 (NOS2) expression has been thought to be essential for the protective effects of IFNγ during Mtb infection. Thus, data from mice has led to the model where CD4 T cells secrete IFNγ onto Mtb-infected Macs to drive NOS2, which generates reactive free radicals that exert anti-mycobacterial effects as well as control detrimental inflammation. However, there are major differences in the induction of NOS2 between murine and human Macs, and the role of IFNγ at the site of infection in human lungs is poorly understood. It is also clear that CD4 T cells in mice can mediate control of Mtb infection independent of IFNγ production. Our previous studies indicated that CD4 T cell-derived IFNγ is critical in limiting extrapulmonary growth of the bacteria, while IFNγ-independent pathways have a large contribution to T cell-mediated suppression of Mtb growth in the lungs. Children with inborn errors in IFNG, IFNGR1, IFNGR2, or the downstream signaling molecules STAT1 and IRF1, are susceptible to mycobacterial infection (known as Mendelian susceptibility to mycobacterial diseases [MSMD]), however these individuals usually manifest with extrapulmonary non-tuberculous mycobacterial (NTM) infections. Similarly, adults that developed neutralizing autoantibodies (autoAbs) against IFNγ are also highly susceptible to mycobacterial infections, however, they almost all develop extrapulmonary NTM infections rather than typical pulmonary TB. Additional insight into the role of IFNγ in Mtb infection has come from the study of “resisters”, i.e. individuals that have been highly exposed to contagious TB index cases but persistently test negative in the IFNγ release assay (IGRA) blood test. It was found that these individuals are infected by Mtb, but their antigen (Ag)-specific CD4 T cells produce little IFNγ. Instead, their Mtb-specific T cells are skewed towards a Th17 and Treg phenotype. Thus, some individuals are able to contain Mtb infection despite the fact that their Mtb-specific CD4 T cells fail to polarize into IFNγ-secreting Th1 cells. Collectively, these observations suggest there are IFNγ-independent mechanisms of CD4 T cell-mediated control of Mtb infection, but their relative importance is not clear. Here we block IFNγR1 signaling in Mtb-infected rhesus macaques. IFNγ blockade from day 45 to 49 post-infection reduced 18FDG-PET/CT scores of lung inflammation and modulated macrophage gene expression. However, it also increased IFNβ, pDCs, plasma cells, cytotoxic cells and IFN-driven gene expression in neutrophils. Blockade from day 0 to week 13 altered the kinetics of T cell responses and enhanced B cell accumulation in granulomas. Surprisingly, however, it did not impact bacterial loads. We show that patients with αIFNγ neutralizing autoantibodies develop disseminated non-tuberculosis mycobacterial disease but not pulmonary TB despite previous exposure to Mtb. Thus, IFNγ may benefit the host by broadly regulating the class of inflammation during TB, but protection may be much less dependent on generating high levels of IFNγ than currently thought. This work has been submitted for publication. In humans and macaques, most Mtb peptide-specific T cells are characterized by a mixture of Th1 and Th17 characteristics. They produce far more IFNγ than IL-17A, and accordingly they are referred to as Th1* cells. T cells of this polarization fate are associated with protection from TB. Indeed, individuals with deficiencies in TBX21, RORC, IL23R or those with anti-IL23 neutralizing autoantibodies are susceptible to Mtb infection. Although T cell-dependent protection against Mtb has long been attributed to their production of IFNγ, the data presented above, as well as previous data from ourselves and other, indicates that IFNγ-independent mechanisms may be critical in T cell-mediated control of Mtb infection. However, no other effector molecules have been shown to be essential for T cell-mediated control of Mtb infection. This may be in part due to the lack of information on the function and specificity of granuloma T cells. Bulk RNAseq analysis of lung and blood T cells from Mtb infected macaques has identified genes preferentially expressed in granuloma vs circulating T cells, and single cell RNA sequencing analysis has identified several distinct T cell subsets at sites of Mtb infection. Yet, it is not clear from these data which populations of T cells are Mtb-specific and which are bystanders, as several studies have found that only ~1-20% of T cells in granulomas are Mtb peptide-specific. Thus, the mechanisms T cells use to suppress Mtb growth at sites of infection are poorly understood, largely due to the lack of functional data on Mtb peptide-specific T cells in granulomas. Here we characterize the functional profiles of Mtb-specific granuloma T cells stimulated with cognate antigens. Ex vivo stimulation with a pool of several hundred Mtb-derived, T cell antigenic peptides triggers major transcriptional changes in the Ag-specific T cells, making them easily identifiable in scRNAseq data. Upon TCR stimulation, we find that macaque Mtb peptide-specific T cells rapidly express many potentially disease-relevant genes, encompassing a broad array of effector functions. Mtb peptide-specific T cells also drive robust responses in non-specific bystander and unconventional T cells, indicating that conventional peptide-specific T cells may also protect against Mtb infection by promoting effector functions of unconventional T cells. Lastly, we examine the function of granuloma T cells isolated from a surgically resected lung from a TB patient and find a population of T cells that closely matches macaque Mtb peptide-specific T cells. These data give insight into the properties of Mtb peptide-specific T cells and provide novel candidate molecules that may play a role in T cell-mediated protection and pathology in tuberculosis. This work has been submitted for publication and is in revision.