Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.

Nitric oxide (NO) contributes to protection from tuberculosis (TB). It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis (Mtb) growth, which prevents subsequent pathological inflammation. In contrast, we report NO primarily protects mice by repressing an interleukin-1 and 12/15-lipoxygenase dependent neutrophil recruitment cascade that promotes bacterial replication. Using Mtb mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports Mtb growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes TB in patients. The human 12/15 lipoxygenase ortholog, ALOX12, is expressed in cavitary TB lesions, the abundance of its products correlate with the number of airway neutrophils and bacterial burden, and a genetic polymorphism that increases ALOX12 expression is associated with TB risk. These data suggest that Mtb exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

The transmembrane protein Tim-3 has been shown to negatively regulate T-cell-dependent immune responses and was recently demonstrated to be associated with the phenomenon of immune exhaustion, which can occur as a consequence of chronic viral infection. Unlike other negative regulators of T-cell function (e.g., PD-1), Tim-3 does not contain any obvious inhibitory signaling motifs. We have found that ectopic expression of Tim-3 in T cells leads to enhancement of T-cell receptor (TCR)-dependent signaling pathways, which was observed at the level of transcriptional reporters and endogenous cytokine production. We have exploited this observation to dissect what elements within the cytoplasmic tail of Tim-3 are required for coupling to downstream signaling pathways. Here we have demonstrated that two of the more membrane-proximal cytoplasmic tail tyrosines are required for Tim-3 signaling to T-cell activation pathways in a redundant fashion. Furthermore, we show that Tim-3 can directly bind to the Src family tyrosine kinase Fyn and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor. Thus, at least under conditions of short-term stimulation, Tim-3 can augment T-cell activation, although this effect can be blocked by the inclusion of an agonistic antibody to Tim-3. These findings should help further the study of Tim-3 function in other physiological settings, such as those that lead to immune exhaustion.During the expansion phase of an immune response to acute infection, newly activated antigen-specific T cells expand rapidly and acquire effector functions. This is then followed by a period of contraction, where all but 5 to 10% of these effector T cells succumb to apoptosis. The remaining T cells constitute the memory pool-multifunctional T cells that persist in the host in an antigen-independent manner with the ability to respond quickly upon reexposure to viral antigen. However, during chronic viral infection, effector CD8 ϩ T cells generated during the expansion phase fail to develop into memory CD8 ϩ T cells. Instead, these effector CD8 ϩ T cells appear to become exhausted (2, 33).T-cell exhaustion is characterized as the progressive and stepwise loss of the ability to secrete interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥) in response to antigenic stimulation, culminating (in the most extreme cases) in apoptosis (33). This system of clonal deletion has been documented under conditions of persistent antigen stimulation, such as high-grade chronic viral infections in both mouse and human and, most recently, in patients with advanced melanoma. Exhausted CD8 ϩ T cells have a distinct molecular signature that resembles that of effector T cells more than that of memory T cells (34). Of the several hundred genes differentially upregulated in exhausted CD8 ϩ T cells, some are inhibitory receptors, such as CTLA-4, LAG-3, and PD-1. Several studies have confirmed that PD-1 is upregulated on exhausted CD4 ϩ and CD8 ϩ T cells (4, 6, 7, 10). However, blocking the interaction between PD...

HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.

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