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A better understanding of all immune components involved in protecting against Mycobacterium tuberculosis infection is urgently needed to inform strategies for novel immunotherapy and tuberculosis (TB) vaccine development. Although cell-mediated immunity is critical, increasing evidence supports that antibodies also have a protective role against TB. Yet knowledge of protective antigens is limited. Analyzing sera from 97 US immigrants at various stages of M. tuberculosis infection, we showed protective in vitro and in vivo efficacy of polyclonal IgG against the M. tuberculosis capsular polysaccharide arabinomannan (AM). Using recently developed glycan arrays, we established that anti-AM IgG induced in natural infection is highly heterogeneous in its binding specificity and differs in both its reactivity to oligosaccharide motifs within AM and its functions in bacillus Calmette-Guérin vaccination and/or in controlled (latent) versus uncontrolled (TB) M. tuberculosis infection. We showed that anti-AM IgG from asymptomatic but not from diseased individuals was protective and provided data suggesting a potential role of IgG2 and specific AM oligosaccharides. Filling a gap in the current knowledge of protective antigens in humans, our data support the key role of the M. tuberculosis surface glycan AM and suggest the importance of targeting specific glycan epitopes within AM in antibody-mediated immunity against TB.

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

Interleukin-3 (IL-3) is produced during infections caused by parasites, bacteria and viruses, but its contribution to immunity in this context remains largely unknown. In mouse models of parasitic infections, in which the effects of IL-3 have been most extensively studied, IL-3 has been variously reported as protective, detrimental or inconsequential. Similarly, mixed results have been reported in viral and bacterial infection models. Here, we investigated the effects of IL-3 in mouse models of Mycobacterium tuberculosis and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections by assessing the pathogen burden, disease manifestations and survival following infection. After infection with M. tuberculosis, IL-3 deficient mice showed higher bacillary burden, increased lung pathology and reduced survival compared to wild type mice. After infection with HSV-1 through cutaneous route and HSV-2 through vaginal route, IL-3 deficient mice showed higher viral burden, increased disease manifestations and reduced survival compared to wild type mice. Our results show that IL-3 makes a subtle but significant contribution to protective immunity in these mouse models of bacterial and viral infections.

Analysis of Ag-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4+ T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4+ T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from bacillus Calmette-Guérin–vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4+ CD154+ cells was distinct from that of CD154− cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

Background Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored. Methods We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis Results Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence. Conclusions The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis. We previously reported that the mycobacterial ribosome is a major target of CD4(+) T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4(+) T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4(+) T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4(+) T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4(+) T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4(+) T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Analysis of antigen-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. While several methods exist for identifying antigen-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC-II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live, antigen-specific CD4+ T cells but this approach remains underexplored, and to our knowledge has not previously been applied in mycobacteria-infected animals. Here we show that CD154 expression identifies adoptively transferred or endogenous antigen-specific CD4+ T cells induced by Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination. We confirmed that antigen-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from BCG-vaccinated mice, and show that high quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of the CD4+ CD154+ cells was distinct from that of CD154− cells, and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells characterized by the production of interleukin-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live, antigen-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Approximately one-third of the human population is latently infected withMycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintainingM. tuberculosisin the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five relatedM. tuberculosisproteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role ofM. tuberculosis rpfgenes in an established mouse model ofM. tuberculosispersistence and reactivation. After an aerosol infection with theM. tuberculosisErdman wild type (Erdman) or single-deletionrpfmutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the fiverpfdeletion mutants tested, one (ΔRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and ΔRv1009-infected mice at advanced stages of reactivation. Analysis ofrpfgene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the fiverpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting thatrpfgene expression may be regulated in a growth phase-dependent manner. To our knowledge, ΔRv1009 is the firstM. tuberculosismutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

The granulomatous reaction is the hallmark of the host response to infection with Mycobacterium tuberculosis. Despite its apparent importance to host defence against the tubercle bacillus, the granulomatous response remains to be completely defined. The present study used histological, immunohistochemical and flow-cytometric analyses to characterize pulmonic granulomatous tissues of tuberculous mice and humans. The kinetics of recruitment of neutrophils, macrophages, dendritic cells, and T and B lymphocytes into the lungs of mice infected aerogenically with the virulent Erdman strain of M. tuberculosis was evaluated in detail in both the acute and persistent phase of infection. A hypoxia-sensing compound based on the 2-nitroimidazole structure (EF5), together with immunohistochemical studies targeting endothelial cells were used to examine the relative oxygen tension in tuberculous granulomatous tissues in mice. The results have provided evidence that: (i) the granulomatous tissues are a highly organized structure whose formation is regulated by orderly recruitment of specific immune cells exhibiting distinct spatial relationship with one another; (ii) the granulomatous reaction, at least in the mouse, may represent an exaggerated response to the tubercle bacillus that can play a role in the development of immunopathology; (iii) B lymphoid aggregates are a prominent feature in both murine and human granulomatous tissues, although the immune cells that are most prominently associated with these clusters vary among the two species; (iv) murine tuberculous granulomatous tissues are relatively aerobic, suggesting that mouse models of persistent tuberculosis may not be suitable for the study of any hypoxic response of M. tuberculosis.

Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis., IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization., Author Summary Mycobacterium tuberculosis poses a serious threat to public health globally. It has been well established that T cells are critical in protection against M. tuberculosis. The role of B cells and humoral immunity in the process is less well understood. We previously showed that B cells and humoral immunity regulate the immune response against M. tuberculosis. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may modulate the development of vaccine-induced protective immunity. The data reveal that B cells can regulate neutrophilia during M. tuberculosis infection and BCG vaccination by modulating the IL-17 response. Vaccination studies show that excess neutrophilia adversely affects the development of BCG-elicited Th1 response. These observations suggest that B cells can optimize the development of protective immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Understanding the mechanisms by which B cells and humoral immunity modulate the immune response during M. tuberculosis infection and BCG immunization, particularly those that regulate IL-17 levels and neutrophilia, may lead to the development of novel strategies for the control of the tubercle bacillus, including efficacious vaccines.

B cell aggregates, which display characteristics of germinal center reactions, are a prominent feature in the lungs of Mycobacterium tuberculosis (Mtb)-infected hosts. Using the B cell-deficient mouse strain μMT, we have previously shown that B cells are required for optimal protection against aerosol Mtb infection. μMT mice display exacerbated lung granulomatous inflammation during acute tuberculosis (TB), and this can be ameliorated by adoptive transfer of B cells or treatment with immune serum, suggesting a role for immunoglobulin in modulating the local lung response to Mtb. To investigate the role of antibody in a protective host response to Mtb, we utilized mice deficient in various immunoglobulins. Mice unable to secrete IgM (μS−/−) were more susceptible to aerosol Mtb infection than those lacking all other immunoglobulin isotypes, and displayed increased mortality during chronic TB. Relative to wild-type (WT) mice, Mtb-infected μS−/− mice had fewer lymphoid aggregates and a delayed granulomatous response in the lungs, despite having greater numbers of activated pulmonary CD4+ T cells. Treatment of μS−/− mice with immune serum (derived from Mtb-infected WT mice) during acute infection, led to increased lymphoid aggregates and plasma cells in the lungs, more pulmonary infiltrate, a concomitant decrease in the lung bacterial burden, and ultimately delayed the onset of death of μS−/− mice during chronic TB. These data suggest IgM plays an important role in controlling Mtb infection by orchestrating local granulomatous responses in the lungs, which leads to improved host survival during the chronic phase of infection.

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell−/−) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell−/− mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell−/− mice show increased production of IL-10 in the lungs, whereas IFN-γ, TNF-α, and IL-10R remain unchanged from wild type. B cell−/− mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell−/− mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.

The chemokine receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis, compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3−/− and wild-type mice were found, the most striking being the increase in the number of CD4+ T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4+ T lymphocytes in the lungs of CXCR3−/− mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-γ-producing CD4+ T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-γ, IL-12, TNF-α, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4+ T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4+ T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.

The role of humoral immunity in the immune response to Mycobacterium tuberculosis (Mtb) is poorly defined. B cells form aggregates with features of germinal centers and are prominent components of the lung granuloma in Mtb-infected humans, mice and macaques. Using muMT B cell-deficient mice, we showed that B cells were required for optimal granulomatous response and protection upon aerosol infection. muMT mice were more susceptible to Mtb with increased mortality and elevated lung bacterial burden. B cell-deficiency also resulted in exacerbated immunopathology with increased neutrophil influx and IL-10 production independent of tissue bacterial load. Transfer of B cells reversed the phenotypes of the muMT mice. This occurred in the presence of immunoglobulins (Igs) in the serum but without the presence of lung B cells. We showed that engagement of distinct Fcγ receptors (FcγR) divergently affected susceptibility to Mtb. Mice lacking activating FcγR showed increased mortality and bacterial load with enhanced lung neutrophil influx and IL-10 expression compared to wildtype, whereas mice lacking inhibitory FcγRIIB exhibited an augmented lung Th1 response. Thus, B cells via their ability to produce Igs may regulate T cell responses by immune complex engagement of FcγR on antigen presenting cells. The mechanisms by which B cells modulate the immune response to Mtb are being characterized by testing the immune response of transgenic mice deficient in B cells or Igs to infection.