Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

Dynamic balance of pro- and anti-inflammatory signals controls disease and limits pathology.

Immune responses to pathogens are complex and not well understood in many diseases, and this is especially true for infections by persistent pathogens. One mechanism that allows for long-term control of infection while also preventing an over-zealous inflammatory response from causing extensive tissue damage is for the immune system to balance pro- and anti-inflammatory cells and signals. This balance is dynamic and the immune system responds to cues from both host and pathogen, maintaining a steady state across multiple scales through continuous feedback. Identifying the signals, cells, cytokines, and other immune response factors that mediate this balance over time has been difficult using traditional research strategies. Computational modeling studies based on data from traditional systems can identify how this balance contributes to immunity. Here we provide evidence from both experimental and mathematical/computational studies to support the concept of a dynamic balance operating during persistent and other infection scenarios. We focus mainly on tuberculosis, currently the leading cause of death due to infectious disease in the world, and also provide evidence for other infections. A better understanding of the dynamically balanced immune response can help shape treatment strategies that utilize both drugs and host-directed therapies.

Systems biology predicts that fibrosis in tuberculous granulomas may arise through macrophage-to-myofibroblast transformation.

Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB), a disease characterized by development of granulomas. Granulomas consist of activated immune cells that cluster together to limit bacterial growth and restrict dissemination. Control of the TB epidemic has been limited by lengthy drug regimens, antibiotic resistance, and lack of a robustly efficacious vaccine. Fibrosis commonly occurs during treatment and is associated with both positive and negative disease outcomes in TB but little is known about the processes that initiate fibrosis in granulomas. Human and nonhuman primate granulomas undergoing fibrosis can have spindle-shaped macrophages with fibroblast-like morphologies suggesting a relationship between macrophages, fibroblasts, and granuloma fibrosis. This relationship has been difficult to investigate because of the limited availability of human pathology samples, the time scale involved in human TB, and overlap between fibroblast and myeloid cell markers in tissues. To better understand the origins of fibrosis in TB, we used a computational model of TB granuloma biology to identify factors that drive fibrosis over the course of local disease progression. We validated the model with granulomas from nonhuman primates to delineate myeloid cells and lung-resident fibroblasts. Our results suggest that peripheral granuloma fibrosis, which is commonly observed, can arise through macrophage-to-myofibroblast transformation (MMT). Further, we hypothesize that MMT is induced in M1 macrophages through a sequential combination of inflammatory and anti-inflammatory signaling in granuloma macrophages. We predict that MMT may be a mechanism underlying granuloma-associated fibrosis and warrants further investigation into myeloid cells as drivers of fibrotic disease.

Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4ß1-Expressing Immune Cells.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

Immunology studies in non-human primate models of tuberculosis.

Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However, in the last 15 years, this model has been refined substantially to allow careful investigations of the immune response and host-pathogen interactions in Mycobacterium tuberculosis infection. Low-dose challenge with fully virulent strains in cynomolgus macaques result in the full clinical spectrum seen in humans, including latent and active infection. Reagents from humans are usually cross-reactive with macaques, further facilitating the use of this model system to study tuberculosis. Finally, macaques develop the spectrum of granuloma types seen in humans, providing a unique opportunity to investigate bacterial and host factors at the local (lung and lymph node) level. Here, we review the past decade of immunology and pathology studies in macaque models of tuberculosis.

Computational modeling predicts IL-10 control of lesion sterilization by balancing early host immunity-mediated antimicrobial responses with caseation during mycobacterium tuberculosis infection.

Although almost a third of the world's population is infected with the bacterial pathogen Mycobacterium tuberculosis, our understanding of the functions of many immune factors involved in fighting infection is limited. Determining the role of the immunosuppressive cytokine IL-10 at the level of the granuloma has proven difficult because of lesional heterogeneity and the limitations of animal models. In this study, we take an in silico approach and, through a series of virtual experiments, we predict several novel roles for IL-10 in tuberculosis granulomas: 1) decreased levels of IL-10 lead to increased numbers of sterile lesions, but at the cost of early increased caseation; 2) small increases in early antimicrobial activity cause this increased lesion sterility; 3) IL-10 produced by activated macrophages is a major mediator of early antimicrobial activity and early host-induced caseation; and 4) increasing levels of infected macrophage derived IL-10 promotes bacterial persistence by limiting the early antimicrobial response and preventing lesion sterilization. Our findings, currently only accessible using an in silico approach, suggest that IL-10 at the individual granuloma scale is a critical regulator of lesion outcome. These predictions suggest IL-10-related mechanisms that could be used as adjunctive therapies during tuberculosis.

Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques.

The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M.tuberculosis bacilli, M.tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.

Macrophage polarization drives granuloma outcome during Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), induces formation of granulomas, structures in which immune cells and bacteria colocalize. Macrophages are among the most abundant cell types in granulomas and have been shown to serve as both critical bactericidal cells and targets for M. tuberculosis infection and proliferation throughout the course of infection. Very little is known about how these processes are regulated, what controls macrophage microenvironment-specific polarization and plasticity, or why some granulomas control bacteria and others permit bacterial dissemination. We take a computational-biology approach to investigate mechanisms that drive macrophage polarization, function, and bacterial control in granulomas. We define a "macrophage polarization ratio" as a metric to understand how cytokine signaling translates into polarization of single macrophages in a granuloma, which in turn modulates cellular functions, including antimicrobial activity and cytokine production. Ultimately, we extend this macrophage ratio to the tissue scale and define a "granuloma polarization ratio" describing mean polarization measures for entire granulomas. Here we coupled experimental data from nonhuman primate TB granulomas to our computational model, and we predict two novel and testable hypotheses regarding macrophage profiles in TB outcomes. First, the temporal dynamics of granuloma polarization ratios are predictive of granuloma outcome. Second, stable necrotic granulomas with low CFU counts and limited inflammation are characterized by short NF-κB signal activation intervals. These results suggest that the dynamics of NF-κB signaling is a viable therapeutic target to promote M1 polarization early during infection and to improve outcome.

A computational tool integrating host immunity with antibiotic dynamics to study tuberculosis treatment.

While active tuberculosis (TB) is a treatable disease, many complex factors prevent its global elimination. Part of the difficulty in developing optimal therapies is the large design space of antibiotic doses, regimens and combinations. Computational models that capture the spatial and temporal dynamics of antibiotics at the site of infection can aid in reducing the design space of costly and time-consuming animal pre-clinical and human clinical trials. The site of infection in TB is the granuloma, a collection of immune cells and bacteria that form in the lung, and new data suggest that penetration of drugs throughout granulomas is problematic. Here we integrate our computational model of granuloma formation and function with models for plasma pharmacokinetics, lung tissue pharmacokinetics and pharmacodynamics for two first line anti-TB antibiotics. The integrated model is calibrated to animal data. We make four predictions. First, antibiotics are frequently below effective concentrations inside granulomas, leading to bacterial growth between doses and contributing to the long treatment periods required for TB. Second, antibiotic concentration gradients form within granulomas, with lower concentrations toward their centers. Third, during antibiotic treatment, bacterial subpopulations are similar for INH and RIF treatment: mostly intracellular with extracellular bacteria located in areas non-permissive for replication (hypoxic areas), presenting a slowly increasing target population over time. Finally, we find that on an individual granuloma basis, pre-treatment infection severity (including bacterial burden, host cell activation and host cell death) is predictive of treatment outcome.

Monocyte-derived IL-5 reduces TNF production by Mycobacterium tuberculosis-specific CD4 T cells during SIV/M. tuberculosis coinfection.

HIV-infected individuals are significantly more susceptible to tuberculosis (TB) than uninfected individuals. Although it is established that HIV reduces Mycobacterium tuberculosis-specific T cell responses, the causes of this dysfunction are not known. We used the cynomolgus macaque model of TB to demonstrate that ex vivo SIV reduces the frequency of M. tuberculosis-specific TNF and IFN-γ-producing T cells within 24 h after infection. In vivo, T cell IFN-γ responses in granulomas from animals with SIV/M. tuberculosis coinfection were lower than SIV-negative animals with active TB. The SIV effects on the inhibition of T cell responses were primarily on APCs and not the T cells directly. Specifically, reductions in the frequency of TNF-producing M. tuberculosis-specific CD4 T cells were caused, at least in part, by SIV-induced production of monocyte derived IL-5.

Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms.

Macrophages in granulomas are both antimycobacterial effector and host cell for Mycobacterium tuberculosis, yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial NO synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared with nongranulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, whereas epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68, and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1, and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS/Arg1 expression in epithelioid macrophages as compared with cells in the lymphocyte cuff. iNOS, Arg1, and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas, whereas the inner regions were more likely to contain macrophages with proinflammatory, presumably bactericidal, phenotypes. Together, these data support the concept that granulomas have organized microenvironments that balance antimicrobial anti-inflammatory responses to limit pathology in the lungs.

CD38+ Alveolar macrophages mediate early control of M. tuberculosis proliferation in the lung.

Tuberculosis, caused by M.tuberculosis (Mtb), remains an enduring global health challenge, especially given the limited efficacy of current therapeutic interventions. Much of existing research has focused on immune failure as a driver of tuberculosis. However, the crucial role of host macrophage biology in controlling the disease remains underappreciated. While we have gained deeper insights into how alveolar macrophages (AMs) interact with Mtb, the precise AM subsets that mediate protection and potentially prevent tuberculosis progression have yet to be identified. In this study, we employed multi-modal scRNA-seq analyses to evaluate the functional roles of diverse macrophage subpopulations across different infection timepoints, allowing us to delineate the dynamic landscape of controller and permissive AM populations during the course of infection. Our analyses at specific time-intervals post-Mtb challenge revealed macrophage populations transitioning between distinct anti- and pro-inflammatory states. Notably, early in Mtb infection, CD38- AMs showed a muted response. As infection progressed, we observed a phenotypic shift in AMs, with CD38+ monocyte-derived AMs (moAMs) and a subset of tissue-resident AMs (TR-AMs) emerging as significant controllers of bacterial growth. Furthermore, scATAC-seq analysis of naïve lungs demonstrated that CD38+ TR-AMs possessed a distinct chromatin signature prior to infection, indicative of epigenetic priming and predisposition to a pro-inflammatory response. BCG intranasal immunization increased the numbers of CD38+ macrophages, substantially enhancing their capability to restrict Mtb growth. Collectively, our findings emphasize the pivotal, dynamic roles of different macrophage subsets in TB infection and reveal rational pathways for the development of improved vaccines and immunotherapeutic strategies.

The frequency of CD38+ alveolar macrophages correlates with early control of M. tuberculosis in the murine lung.

Tuberculosis, caused by Mycobacterium tuberculosis, remains an enduring global health challenge due to the limited efficacy of existing treatments. Although much research has focused on immune failure, the role of host macrophage biology in controlling the disease remains underappreciated. Here we show, through multi-modal single-cell RNA sequencing in a murine model, that different alveolar macrophage subsets play distinct roles in either advancing or controlling the disease. Initially, alveolar macrophages that are negative for the CD38 marker are the main infected population. As the infection progresses, CD38+ monocyte-derived and tissue-resident alveolar macrophages emerge as significant controllers of bacterial growth. These macrophages display a unique chromatin organization pre-infection, indicative of epigenetic priming for pro-inflammatory responses. Moreover, intranasal BCG immunization increases the numbers of CD38+ macrophages, enhancing their capability to restrict Mycobacterium tuberculosis growth. Our findings highlight the dynamic roles of alveolar macrophages in tuberculosis and open pathways for improved vaccines and therapies.

Metabolically active neutrophils represent a permissive niche for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-γ promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB.

Activated B cells in the granulomas of nonhuman primates infected with Mycobacterium tuberculosis.

In an attempt to contain Mycobacterium tuberculosis, host immune cells form a granuloma as a physical and immunological barrier. To date, the contribution of humoral immunity, including antibodies and specific functions of B cells, to M. tuberculosis infection in humans remains largely unknown. Recent studies in mice show that humoral immunity can alter M. tuberculosis infection outcomes. M. tuberculosis infection in cynomolgus macaques recapitulates essentially all aspects of human tuberculosis. As a first step toward understanding the importance of humoral immunity to control of M. tuberculosis infection in primates, we characterized the B-cell and plasma-cell populations in infected animals and found that B cells are present primarily in clusters within the granuloma. The B-cell clusters are in close proximity to peripheral node addressin-positive cells and contain cells positive for Ki-67, a proliferation marker. Granuloma B cells also express CXCR5 and have elevated HLA-DR expression. Tissues containing M. tuberculosis bacilli had higher levels of M. tuberculosis-specific IgG, compared with uninvolved tissue from the same monkeys. Plasma cells detected within the granuloma produced mycobacteria-specific antibodies. Together, these data demonstrate that B cells are present and actively secreting antibodies specific for M. tuberculosis antigens at the site of infection, including lung granulomas and thoracic lymph nodes. These antibodies likely have the capacity to modulate local control of infection in tissues.

Predicting lymph node output efficiency using systems biology.

Dendritic cells (DCs) capture pathogens and foreign antigen (Ag) in peripheral tissues and migrate to secondary lymphoid tissues, such as lymph nodes (LNs), where they present processed Ag as MHC-bound peptide (pMHC) to naïve T cells. Interactions between DCs and T cells result, over periods of hours, in activation, clonal expansion and differentiation of antigen-specific T cells, leading to primed cells that can now participate in immune responses. Two-photon microscopy (2PM) has been widely adopted to analyze lymphocyte dynamics and can serve as a powerful in vivo assay for cell trafficking and activation over short length and time scales. Linking biological phenomena between vastly different spatiotemporal scales can be achieved using a systems biology approach. We developed a 3D agent-based cellular model of a LN that allows for the simultaneous in silico simulation of T cell trafficking, activation and production of effector cells under different antigen (Ag) conditions. The model anatomy is based on in situ analysis of LN sections (from primates and mice) and cell dynamics based on quantitative measurements from 2PM imaging of mice. Our simulations make three important predictions. First, T cell encounters by DCs and T cell receptor (TCR) repertoire scanning are more efficient in a 3D model compared with 2D, suggesting that a 3D model is needed to analyze LN function. Second, LNs are able to produce primed CD4+T cells at the same efficiency over broad ranges of cognate frequencies (from 10(-5) to 10(-2)). Third, reducing the time that naïve T cells are required to bind DCs before becoming activated will increase the rate at which effector cells are produced. This 3D model provides a robust platform to study how T cell trafficking and activation dynamics relate to the efficiency of T cell priming and clonal expansion. We envision that this systems biology approach will provide novel insights for guiding vaccine development and understanding immune responses to infection.

Simian immunodeficiency virus-induced changes in T cell cytokine responses in cynomolgus macaques with latent Mycobacterium tuberculosis infection are associated with timing of reactivation.

Understanding the early immunologic events accompanying reactivated tuberculosis (TB) in HIV-infected individuals may yield insight into causes of reactivation and improve treatment modalities. We used the cynomolgus macaque (Macaca fascicularis) model of HIV-Mycobacterium tuberculosis coinfection to investigate the dynamics of multifunctional T cell responses and granuloma T cell phenotypes in reactivated TB. CD4(+) and CD8(+) T cells expressing Th1 cytokines (IFN-γ, IL-2, TNF) and Th2 cytokines (IL-4 and IL-10) were followed from latent M. tuberculosis infection to reactivation after coinfection with a pathogenic SIV. Coinfected animals experienced increased Th1 cytokine responses to M. tuberculosis Ags above the latent-response baseline 3-5 wk post-SIV infection that corresponded with peak plasma viremia. Th2 cytokine expression was not Ag specific, but strong, transient IL-4 expression was noted 4-7 wk post-SIV infection. Animals reactivating <17 wk post-SIV infection had significantly more multifunctional CD4(+) T cells 3-5 wk post-SIV infection and more Th2-polarized and fewer Th0-, Th1-polarized CD8(+) T cells during weeks 1-10 post-SIV infection than animals reactivating >26 wk post-SIV infection. Granuloma T cells included Th0-, Th1-, and Th2-polarized phenotypes but were particularly rich in cytolytic (CD107(+)) T cells. When combined with the changes in peripheral blood T cells, these factors indicate that events during acute HIV infection are likely to include distortions in proinflammatory and anti-inflammatory T cell responses within the granuloma that have significant effects on reactivation of latent TB. Moreover, it appears that mycobacteria-specific multifunctional T cells are better correlates of Ag load (i.e., disease status) than of protection.