Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host''s immune response. It has been suggested that mycobacteria may contain genes that reduce the host''s ability to elicit CD8⁺ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8⁺ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8⁺ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8⁺ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.     

Human Memory CD4+ T Cell Immune Responses against Giardia lamblia

The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, T_H17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4⁺ T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4⁺ effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4⁺ EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4⁺ T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4⁺ EM T cell response of which IL-17A production seems to be an important component.     

Downmodulation of Tumor Suppressor p53 by T Cell Receptor Signaling Is Critical for Antigen-Specific CD4+ T Cell Responses

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Gamma interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis

The human immune system efficiently limits the replication of Mycobacterium tuberculosis in most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response against M. tuberculosis and to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+ and CD8+ lymphocytes that produced gamma interferon (IFN-gamma). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-gamma. These results suggest an important role for the early appearance of IFN-gamma-producing lymphocytes in the lung in resistance to infection with M. tuberculosis.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

Biological functions of Mycobacterium tuberculosis-specific CD4+T cells were impaired by tuberculosis pleural fluid

Cell surface molecules are present on several lymphocyte subsets and are differentially expressed during lymphocyte development and activation. Human Leukocyte Differentiation Antigen (HLDA) Workshops have played an essential role in the identification and characterization of the molecules found in the membrane of hematopoietic cells. In the present study, the reactivities of sixty-five monoclonal antibodies (mAbs) submitted to the HLDA9 Workshop were tested. A multicolor flow cytometric analysis was performed in order to determine the expression profiles of these proteins on peripheral blood lymphocytes, hematopoietic cell lines, and tonsil B-cells. The following B-cell subsets were assessed: mature na?ve, pre-germinal center, germinal center, unswitched and switched memory, plasmablasts, and plasma cells. Immunohistochemical analysis on formalin-fixed paraffin-embedded tonsils was also carried out. Remarkably, a large group of immunoglobulin family inhibitory cell surface molecules were observed on several distinct B-cell subsets including: CD152 (CTLA4), CD170 (Siglec-5), CD272 (BTLA), CD305 (LAIR1), CD307d (FCRL4), and CD329 (Siglec-9). The following molecules were also found to be differentially expressed on B-cell subsets (CD80, CD185 (CXCR5), CD196 (CCR6), CD270 (TNFRSF14), CD307a-c (FCRL1-3), CD319 (SLAMF7) and CD362 (SDC2)) or delineated B-cell subpopulations (CD126 (IL-6R), CD255 (TNFSF12), CD264 (TNFRSF10D), CD267 (TNFRSF13B) and CD329 (Siglec-9)). Of these, only CD307a, CD307b, and CD307d presented a B-cell-specific expression pattern. Our results show that several of these molecules are capable of further subdividing the known B-cell subsets and, in fact, may represent new markers for research, diagnosis, and eventually targets for the treatment of B-cell malignancies and autoimmune diseases.     

CD69 expression on CD4+ T lymphocytes after in vitro stimulation with tuberculin is an indicator of immune sensitization against Mycobacterium tuberculosis antigens

The expression of the CD69 antigen on CD4 T lymphocytes after in vitro stimulation with purified protein derivative (2 tuberculin units) was used to evaluate the tuberculin reactivities of 52 individuals from four experimental groups: Mycobacterium bovis BCG-vaccinated healthy individuals with a negative tuberculin skin test (TST) result (group A), BCG-vaccinated healthy individuals with a positive TST result (group B), patients with active tuberculosis (TB) before treatment (group C), and individuals with clinically inactive TB who had previously completed a prescribed course of chemotherapy (group D). The expression of CD69 on CD4 T lymphocytes was significantly higher in patients with active TB (16.2%+/-7.3%), individuals with clinically inactive TB (10.5%+/-7.4%), and healthy individuals with a positive TST result (15.5%+/-7.2%) than in healthy individuals with a negative TST result (3.8%+/-4.3%) (P<0.005). We confirmed the correlation between CD69 antigen expression on T lymphocytes after stimulation with tuberculin and the TST induration diameter (Spearman rho=0.783; P<0.001), an assay for gamma interferon (the Quantiferon-TB assay; Spearman rho=0.613; P<0.001), and the lymphocyte BLAST transformation test (Spearman rho=0.537; P<0.001). Our results demonstrate the usefulness of the determination of CD69 on CD4 T lymphocytes after in vitro stimulation with tuberculin as a rapid indicator of immune sensitization against Mycobacterium tuberculosis.     

Functionally Diverse Subsets in CD4 T Cell Responses Against Influenza

Background  Antibody alone cannot provide optimal protection against many infectious diseases impacting global heath. In these cases, our challenge is to develop innovative vaccines that generate protective populations of memory T cells. However, our studies suggest that current paradigms explaining how memory CD4 T cells provide protection are inadequate. This is likely due to both the paucity of and heterogeneity of memory CD4 T cells observed in vivo, which make analysis extremely difficult. Summary  Here, we discuss new findings that indicate there is extensive functional heterogeneity within effector and memory CD4 T cell populations both in vivo and in vitro. Using influenza as an example, we also discuss the merits of employing reductionist approaches to explore how unique subsets of CD4 T cells are generated, what mechanisms of protection they use, and where they stand on the axes of differentiation that define T cell subsets.     

Intracellular expression of interleukin-4 and interferon-gamma by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients

In some chronic pathological conditions, antigen persistence activates and expands the CD4+ CD57+ T-cell subset. The host immune response against tuberculosis infection is maintained through the continuous presence of antigen-stimulated effector/memory helper T cells. To determine whether CD4+ CD57+ T cells were also expanded in human tuberculosis, we analysed (by flow cytometry) the phenotype of peripheral blood CD4+ T cells from 30 tuberculosis patients and 30 healthy controls. We observed a significant increase in the CD4+ CD57+ T-cell subset in tuberculosis patients in comparison to healthy controls (P < 0.001). Most CD4+ CD57+ T cells exhibited a CD28- CD45RO+ CD62L- phenotype, which is associated with memory cells. In vitro, a higher number of antigen-stimulated CD4+ CD57+ T cells produced intracellular interferon-gamma and interleukin-4 compared with antigen-stimulated CD4+ CD57- T cells (P < 0.001). These findings suggest that the majority of CD4+ CD57+ T cells correspond to a phenotype of activated memory T cells.     

Interleukin-2 is essential for CD4+CD25+ regulatory T cell function

Constitutive expression of CD25, the IL-2 receptor alpha-chain, defines a distinct population of CD4+ T cells (Treg) with suppressive activity in vitro and in vivo. IL-2 has been implicated in the generation and maintenance of Treg, however, a functional contribution of the IL-2 receptor during suppression is thus far unknown. We show that IL-2 is required for Treg function in vitro, since suppression is completely abrogated by selective blocking of the IL-2 receptor on Treg during co-culture with responder T cells. We demonstrate that Treg, which do not produce IL-2, compete for IL-2 secreted by responder T cells. In accordance with the idea of competition being part of the suppressive mechanism, in vitro neutralization of IL-2 mimics all effects of Treg. Conversely, recombinant IL-2 abrogates inhibition of IL-2 production in responder T cells, the hallmark of Treg suppression. Finally, activation in the presence of IL-2 primes Treg to produce IL-10 upon secondary stimulation, indicating that IL-2 uptake is also required to induce additional suppressive factors that might be more relevant for suppression in vivo. We propose the parakrine uptake of soluble mediators as a flexible mechanism to adapt Treg activity to the strength of the responder T cell reaction.     

Rv2468c, a novel Mycobacterium tuberculosis protein that costimulates human CD4+ T cells through VLA-5

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5β1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.     

Suppression of CD4+ Effector Responses by Naturally Occurring CD4+ CD25+ Foxp3+ Regulatory T Cells Contributes to Experimental Cerebral Malaria

The role of naturally occurring CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (nTreg) in the pathogenesis of cerebral malaria (CM), which involves both pathogenic T cell responses and parasite sequestration in the brain, is still unclear. To assess the contribution and dynamics of nTreg during the neuropathogenesis, we unbalanced the ratio between nTreg and naive CD4⁺ T cells in an attenuated model of Plasmodium berghei ANKA-induced experimental CM (ECM) by using a selective cell enrichment strategy. We found that nTreg adoptive transfer accelerated the onset and increased the severity of CM in syngeneic C57BL/6 (B6) P. berghei ANKA-infected mice without affecting the level of parasitemia. In contrast, naive CD4⁺ T cell enrichment prevented CM and promoted parasite clearance. Furthermore, early during the infection nTreg expanded in the spleen but did not efficiently migrate to the site of neuroinflammation, suggesting that nTreg exert their pathogenic action early in the spleen by suppressing the protective naive CD4⁺ T cell response to P. berghei ANKA infection in vivo in both CM-susceptible (B6) and CM-resistant (B6-CD4^−/−) mice. However, their sole transfer was not sufficient to restore CM susceptibility in two CM-resistant congenic strains tested. Altogether, these results demonstrate that nTreg are activated and functional during P. berghei ANKA infection and that they contribute to the pathogenesis of CM. They further suggest that nTreg may represent an early target for the modulation of the immune response to malaria.     

Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10

Mycobacterium tuberculosis infection elicits antigen-specific CD8(+) T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted by M. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion by M. tuberculosis is required for the priming of CD8(+) T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8(+) T cells in vivo. By using well-defined M. tuberculosis mutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8(+) T cells specific for CFP10. CD4(+) and CD8(+) T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8(+) T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8(+) T-cell response during M. tuberculosis infection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation during M. tuberculosis infection and in vaccine development.     

Mycobacterium tuberculosis Cell Wall Glycolipids Directly Inhibit CD4+ T-Cell Activation by Interfering with Proximal T-Cell-Receptor Signaling

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4⁺ T-cell responses. We have previously shown that M. tuberculosis can indirectly inhibit CD4⁺ T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine if M. tuberculosis could directly inhibit CD4⁺ T-cell activation. Murine CD4⁺ T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4⁺ T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence of M. tuberculosis and its subcellular fractions. CD4⁺ T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence of M. tuberculosis. Fractionation identified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose-capped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosis glycolipids directly inhibited CD4⁺ T-cell activation by interfering with proximal T-cell-receptor signaling.Aerosolized Mycobacterium tuberculosis infects alveolar and lung parenchymal macrophages, where it replicates unrestrained in the face of innate responses until T-cell immunity controls its growth. Despite robust activation of innate and adaptive immunity, M. tuberculosis survives and persists as a latent infection (15, 18). CD4⁺ T cells have a central role in controlling M. tuberculosis during acute and latent infections (53). Animal studies have shown that depletion or absence of CD4⁺ T cells during primary infection results in unchecked M. tuberculosis growth in the lung and decreased survival (37, 38, 41). Depletion of CD4⁺ T cells during latent infection also worsens disease and survival (46). In humans, loss of CD4⁺ T cells from progressive human immunodeficiency virus infection is directly responsible for the high rates of tuberculosis in human immunodeficiency virus-infected persons (48).Much is known about how M. tuberculosis manipulates macrophages for its survival (19, 29, 44). However, the way in which M. tuberculosis interferes with adaptive T-cell immunity is not well understood. Our recent studies have demonstrated that M. tuberculosis can modulate CD4⁺ T-cell function both indirectly and directly. M. tuberculosis, through Toll-like receptor 2 (TLR-2), inhibits gamma-interferon-regulated genes that result in decreased major histocompatibility complex class II (MHC-II) antigen processing by macrophages for effector and memory CD4⁺ T cells (22, 23, 40, 43). M. tuberculosis can also induce increased adhesion to fibronectin through α₅β₁ integrin on CD4⁺ T cells (45).M. tuberculosis molecules responsible for modulating CD4⁺ T-cell function reside in the mycobacterial cell wall and include the lipoproteins LpqH, LprG, and LprA as well as the glycolipid phosphatidylinositol mannoside (PIM). The lipoproteins bind to TLR-2 on macrophages, and PIM binds to VLA-5 (α₅β₁) on CD4⁺ T cells. The M. tuberculosis cell wall also contains lipoarabinomannan (LAM); complex lipids, such as phthiocerol dimycocerosate, and cord factor/dimycolytrehalose; and sulfolipids that are both targets of the immune response (e.g., glycolipids presented by CD1 to T cells) and agonists of host cell receptors (9, 10, 32). Although M. tuberculosis bacilli largely reside within macrophages, mycobacterial cell wall components, including glycolipids, can traffic outside infected macrophages through the production of exosomes. These exosomes can then deliver M. tuberculosis molecules to T cells and other host cells that are not directly interacting with M. tuberculosis-infected cells and thereby affect host immune responses (3, 4).The purpose of this study was to determine if M. tuberculosis can directly (i.e., independently from its effect on MHC-II antigen processing) interfere with CD4⁺ T-cell activation and, if so, what the M. tuberculosis molecules(s) and mechanism(s) are. Highly purified murine CD4⁺ T cells devoid of antigen-presenting cells (APCs) were activated by CD3 and CD28 monoclonal antibodies (MAbs) in the presence or absence of M. tuberculosis and biochemical fractions. We have found that M. tuberculosis bacilli directly inhibited CD4⁺ T-cell activation. Biochemical fractionation identified cell wall glycolipids as potent inhibitors of signaling through the T-cell receptor (TCR) complex by interfering with ZAP-70 phosphorylation.     

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A

The contribution of CD8+ and CD4+ T cell-mediated effector functions against Mycobacterium tuberculosis infection elicited by i.m. vaccination with plasmid DNA encoding the immunodominant Ag85A antigen of M. tuberculosis was studied. Ag85A DNA-vaccinated beta2-microglobulin gene-deficient (beta2m-/-) mice, which lack CD8+ T cells, produced Ag85-specific antibodies and Th1 type cytokines similar to wild-type mice. Although beta2m-/- mice were more susceptible to M. tuberculosis infection, following vaccination they efficiently controlled bacterial replication in spleen and lungs 4 weeks post-infection. In contrast, mice lacking CD4+ T cells were neither sensitized by the Ag85A DNA vaccine to produce Ag85-specific antibodies or Th1 type cytokines nor did they contain a M. tuberculosis challenge infection. In addition, Ag85A DNA-vaccinated IFN-gamma gene knockout mice produced Ag85-specific antibodies and IL-2 but died rapidly following a M. tuberculosis challenge infection. Collectively, these data support the view that IFN-gamma-producing CD4+ T cells, independently of CD8+ T cells, may mediate the protective effect of the Ag85A DNA vaccine.