Elucidating the Role of the Humoral Response in Mycobacterium tuberculosis infected Cynomolgus Macaques

Mycobacterium tuberculosis remains as a global burden today with an estimated one-third of the global population being infected and at risk of developing active infection. Most studies in the immunology and pathogenesis of tuberculosis have revealed the importance of cellular immunity in controlling the infection. However, the role of the humoral immune response is poorly understood, particularly in the primate models of M. tuberculosis infection. The primary goal of this thesis was to understand how B cells and antibody contribute towards containment of M. tuberculosis within the cynomolgus macaque model of infection. The thesis starts off by characterizing B cells and antibody profiles within the granulomas of M. tuberculosis infected cynomolgus macaques. B cells were noted organize themselves into clusters that resemble germinal centers found in lymphoid organs or in chronic autoimmune conditions, within the granuloma. The effect of B cell depletion on the outcome of M. tuberculosis infection in cynomolgus macaques was also performed. The study findings suggest that B cells and antibody contribute very little in terms of disease control in the early stages of natural M. tuberculosis infection. However, subtle differences such as a slight increase in bacterial burden within individual granulomas and altered cytokine correlations within individual granulomas were noted. Macrophage behavior in the absence of B cells and antibody was also studied using granuloma tissue derived from the B cell depletion study. No differences were found within the macrophages in the absence of B cells at least at the acute stage of infection. The experiments detailed in this thesis suggest that the humoral response is not crucial towards M. tuberculosis control in the early stages of natural infection. However, the findings in this thesis suggest that B cells and antibody may play a role in long term chronic control of tuberculosis infection

Reactivation of Latent Tuberculosis in Cynomolgus Macaques Infected with SIV Is Associated with Early Peripheral T Cell Depletion and Not Virus Load

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

Variability in Tuberculosis Granuloma T Cell Responses Exists, but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

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

The proportion of T cells with cytokine response of IFN-γ (A), IL-2 (B), TNF (C), IL-17 (D), T-1/T-17 (E) and IL-10 (F).

<p>Each symbol represents a granuloma. Each color represents an animal. Granulomas are grouped based on the clinical status of the animals. Solid line indicates median response. (*: p<0.05; **: p<0.001, ***: p<0.0001. Dunn’s multiple test comparison).</p

Correlation of pro-inflammatory T cell cytokine response to the bacterial burden (CFU per granuloma) in each of the individual granulomas.

<p>Significant inverse correlation was observed between frequencies of TNF (<b>A</b>) and T-1/T-17 (<b>B</b>) producing T cells and bacterial burden. CFU per granuloma value of zero were transformed to “1” and included in this analysis. Each symbol represents a granuloma. Line indicates the slope. The clinical status of an animal is represented by the color of the granuloma. Animals with active disease are in red, latent infection in blue and those with ∼11 weeks of infection are in green.</p

CD3+ T cells making single (one) or multiple (two, three or four) cytokines from individual granulomas.

<p>Non-sterile granulomas are in open circles (red) and sterile granulomas are represented as open squares (blue). Solid line indicates median response. The table on the X axis reflects the groups positive for each cytokine represented by a dot. There was no significant difference between the multiple cytokine pattern of response between sterile and non-sterile granulomas or between various clinical states of the animal. (n = 121).</p

Bacterial burden of individual granulomas.

<p>Bacterial burden is represented as the colony forming units (CFU) in logarithmic scale, per granuloma. Greyed area represents sterile granulomas (indicated as log₁₀ 1), which were assigned arbitrary range of values so that all granulomas can be seen in the graph. Each symbol represents a granuloma and a color represents granulomas from one animal. Granulomas are grouped as per the clinical status of the animal, where those animals that were infected for ∼11 weeks are in shades of green, active disease in shades of warm colors and latent infection in shades of cool colors. (****: p<0.0001, Dunn’s multiple test comparison).</p

Pairwise occurrence matrix and sterilization frequency of T cell cytokines.

<p><b>A</b> is an example figure: For each cytokine variable of the granuloma, continuous percentage values were binned into one of four categories depending on quartile distribution (bins 1, 2, 3, or 4). Number of granulomas (number of times particular combination of binned cytokines occurred) in each of the bins were counted, for example, low IFN-γ (bin 1) and high IL-2 (bin 4) had 10 granulomas and summarized in a 4×4 co-occurrence matrix. For each pairwise cytokine comparison, the frequency of occurrence was summarized in heat-map form. <b>B</b> is the frequency of occurrence for all pairwise combination of T cell cytokines from individual granulomas. The scale represents the frequency of occurrence ranging from none/low to high. <b>C</b> is the sterilization frequency where matrices were constructed in a similar fashion by calculating the number of times sterilization occurred in a given particular cytokine combination and magnitude. Each square in each 4×4 heat-map therefore represents the frequency of sterilization under that particular combination of feature bins (# of sterilizing granulomas with cytokine A at level X and cytokine B at level Y out of total # of granulomas with cytokine A at level X and cytokine B at level Y). For each pairwise cytokine comparison, the frequency of sterilization under that particular combination of cytokines and levels is plotted and summarized in heat-map form. White squares indicate that granulomas with particular combination of cytokines at those levels did not occur.</p