Richard Wang

A Critical Role for CD8 T Cells in a Nonhuman Primate Model of Tuberculosis

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell–mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.

Definition of APC Presentation of Phosphoantigen (E)-4-Hydroxy-3-methyl-but-2-enyl Pyrophosphate to Vγ2Vδ2 TCR

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vγ2Vδ2 T cells, molecular mechanisms by which HMBPP interacts with Vγ2Vδ2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vγ2Vδ2 TCR of rhesus macaques to define HMBPP/APC interaction with Vγ2Vδ2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vγ2Vδ2 TCR tetramer. The Vγ2Vδ2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vγ2Vδ2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vγ2Vδ2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vγ2Vδ2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vγ2Vδ2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vγ2Vδ2 TCR.

Severe Tuberculosis Induces Unbalanced Up-Regulation of Gene Networks and Overexpression of IL-22, MIP-1α, CCL27, IP-10, CCR4, CCR5, CXCR3, PD1, PDL2, IL-3, IFN-β, TIM1, and TLR2 but Low Antigen-Specific Cellular Responses

The immune mechanisms by which early host-mycobacterium interaction leads to the development of severe tuberculosis (TB) remain poorly characterized in humans. Here, we demonstrate that severe TB in juvenile rhesus monkeys down-regulated many genes in the blood but up-regulated selected genes constituting gene networks of Th17 and Th1 responses, T cell activation and migration, and inflammation and chemoattractants in the pulmonary and lymphoid compartments. Overexpression (450-2740-fold) of 13 genes encoding inflammatory cytokines and receptors (IL-22, CCL27, MIP-1alpha, IP-10, CCR4, CCR5, and CXCR3), immune dysfunctional receptors and ligands (PD1 and PDL2), and immune activation elements (IL-3, IFN-beta, TIM1, and TLR2) was seen in tissues, with low antigen-specific cellular responses. Thus, severe TB in macaques features unbalanced up-regulation of immune-gene networks without proportional increases in antigen-specific cellular responses.

Differentiation, Distribution and γδ T Cell-Driven Regulation of IL-22-Producing T Cells in Tuberculosis

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vγ2Vδ2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNγ-producing Vγ2Vδ2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNγ neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vγ2Vδ2 T-cell-driven IFNγ-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vγ2Vδ2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB.

IL-2 Simultaneously Expands Foxp3+ T Regulatory and T Effector Cells and Confers Resistance to Severe Tuberculosis (TB): Implicative Treg–T Effector Cooperation in Immunity to TB

The possibility that simultaneous expansion of T regulatory cells (Treg) and T effector cells early postinfection can confer some immunological benefits has not been studied. In this study, we tested the hypothesis that early, simultaneous cytokine expansion of Treg and T effector cells in a tissue infection site can allow these T cell populations to act in concert to control tissue inflammation/damage while containing infection. IL-2 treatments early after Mycobacterium tuberculosis infection of macaques induced simultaneous expansion of CD4+CD25+Foxp3+ Treg, CD8+CD25+Foxp3+ T cells, and CD4+ T effector/CD8+ T effector/Vγ2Vδ2 T effector populations producing anti-M. tuberculosis cytokines IFN-γ and perforin, and conferred resistance to severe TB inflammation and lesions. IL-2–expanded Foxp3+ Treg readily accumulated in pulmonary compartment, but despite this, rapid pulmonary trafficking/accumulation of IL-2–activated T effector populations still occurred. Such simultaneous recruitments of IL-2–expanded Treg and T effector populations to pulmonary compartment during M. tuberculosis infection correlated with IL-2–induced resistance to TB lesions without causing Treg-associated increases in M. tuberculosis burdens. In vivo depletion of IL-2–expanded CD4+Foxp3+ Treg and CD4+ T effectors during IL-2 treatment of M. tuberculosis-infected macaques significantly reduced IL-2–induced resistance to TB lesions, suggesting that IL-2–expanded CD4+ T effector cells and Treg contributed to anti-TB immunity. Thus, IL-2 can simultaneously activate and expand T effector cells and Foxp3+ Treg populations and confer resistance to severe TB without enhancing M. tuberculosis infection.

Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.

Membrane-Bound IL-22 after De Novo Production in Tuberculosis and Anti-Mycobacterium tuberculosis Effector Function of IL-22+ CD4+ T Cells

The role of IL-22–producing CD4+ T cells in intracellular pathogen infections is poorly characterized. IL-22–producing CD4+ T cells may express some effector molecules on the membrane, and therefore synergize or contribute to antimicrobial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22+ T cells cannot be purified for evaluation due to secretion nature of cytokines. In this study, we surprisingly found that upon activation, CD4+ T cells in Mycobacterium tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22+ CD4+ T effector cells appeared to mature in vivo and sustain membrane distribution in highly inflammatory environments during active M. tuberculosis infection. Near-field scanning optical microscopy/quantum dot-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ∼100–200 nm nanoclusters or ∼300–600 nm nanodomains for potential interaction with IL-22R. Importantly, purified membrane-bound IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22–producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 t1/2 and to exert efficient cell–cell interaction for anti-M. tuberculosis effector function.

NSOM/QD-Based Direct Visualization of CD3-Induced and CD28-Enhanced Nanospatial Coclustering of TCR and Coreceptor in Nanodomains in T Cell Activation

Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2–4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and ∼6–10% of CD3 were co-clustering with CD4 or CD8 as 70–110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200–500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3–CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.

Antigen-specific Vγ2Vδ2 T effector cells confer homeostatic protection against pneumonic plaque lesions

The possibility that Vγ2Vδ2 T effector cells can confer protection against pulmonary infectious diseases has not been tested. We have recently demonstrated that single-dose (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment can induce prolonged accumulation of Vγ2Vδ2 T effector cells in lungs. Here, we show that a delayed HMBPP/IL-2 administration after inhalational Yersinia pestis infection induced marked expansion of Vγ2Vδ2 T cells but failed to control extracellular plague bacterial replication/infection. Surprisingly, despite the absence of infection control, expansion of Vγ2Vδ2 T cells after HMBPP/IL-2 treatment led to the attenuation of inhalation plague lesions in lungs. Consistently, HMBPP-activated Vγ2Vδ2 T cells accumulated and localized in pulmonary interstitials surrounding small blood vessels and airway mucosa in the lung tissues with no or mild plague lesions. These infiltrating Vγ2Vδ2 T cells produced FGF-7, a homeostatic mediator against tissue damages. In contrast, control macaques treated with glucose plus IL-2 or glucose alone exhibited severe hemorrhages and necrosis in most lung lobes, with no or very few Vγ2Vδ2 T cells detectable in lung tissues. The findings are consist with the paradigm that circulating Vγ2Vδ2 T cells can traffic to lungs for homeostatic protection against tissue damages in infection.

CD4+ T Cells Contain Early Extrapulmonary Tuberculosis (TB) Dissemination and Rapid TB Progression and Sustain Multieffector Functions of CD8+ T and CD3− Lymphocytes: Mechanisms of CD4+ T Cell Immunity

The possibility that CD4+ T cells can act as “innate-like” cells to contain very early Mycobacterium tuberculosis dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3− lymphocytes during development of adaptive immunity against primary tuberculosis (TB) has not been demonstrated. We showed that pulmonary M. tuberculosis infection of CD4-depleted macaques surprisingly led to very early extrapulmonary M. tuberculosis dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during M. tuberculosis infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. Whereas CD3− non–T lymphocytes in the presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to M. tuberculosis infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3− lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNF-α, IL-17, IL-22, and perforin at the endpoint of more severe TB, but they presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite the presence of IL-17+ and IL-4+ cells. These results implicate a previously unknown innate-like ability of CD4+ T cells to contain extrapulmonary M. tuberculosis dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3− lymphocytes and to prevent rapid TB progression during M. tuberculosis infection of nonhuman primates.