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We have previously reported that during mycobacterial infection, naïve CD4 + T-cell activation is enhanced in the lungs. We investigated the role of chemokine receptor CCR7 and its ligands in the ability of CD11c + lung dendritic cells (DCs) to activate naïve CD4 + T cells during pulmonary infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection resulted in the accumulation and maturation in the lungs of DCs that persisted as the mycobacterial burden declined. Lung DCs from infected mice expressed more major histocompatibility complex class II (MHC-II) than those from uninfected mice. CCR7 expression levels on lung DCs were comparable among uninfected and infected mice. The gene expression of the CCR7 ligand CCL19 progressively increased throughout BCG infection, and its expression was MyD88 dependent. CD11c + lung cells from BCG-infected mice activated ovalbumin (OVA)-specific naïve CD4 + T cells more than CD11c + lung cells from uninfected mice. Interestingly, during peak mycobacterial infection, CD11c hi MHC hi lung DCs had slightly decreased chemotaxis toward the CCR7 ligand CCL21 and less efficiency in activating naive CD4 + T cells than DCs from mice during late-stage infection, when few bacilli are found in the lung. These findings suggest that during BCG infection, the inflammation and sustained expression of CCL19 result in the recruitment, activation, and retention in the lung of DCs that can activate naïve CD4 + T cells in situ.

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4 + T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c + cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4 + T cells in the lungs in response to airway antigen challenge.

Mycobacterium tuberculosisresides in phagosomes inside macrophages. In this study, we analyzed the kinetics and location ofM. tuberculosispeptide-major histocompatibility complex class II (MHC-II) complexes inM. tuberculosis-infected human macrophages.M. tuberculosispeptide-MHC-II complexes were detected with polyclonal autologousM. tuberculosis-specific CD4+T cells or F9A6 T hybridoma cells specific forM. tuberculosisantigen (Ag) 85B (96-111). Macrophages processed heat-killedM. tuberculosismore rapidly and efficiently than liveM. tuberculosis. To determine whereM. tuberculosispeptide-MHC-II complexes were formed intracellularly, macrophages incubated with heat-killedM. tuberculosiswere homogenized, and subcellular compartments were separated on Percoll density gradients analyzed with T cells. In THP-1 cells,M. tuberculosisAg 85B (96- 111)-DR1 complexes appeared initially in phagosomes, followed by MHC class II compartment (MIIC) and the plasma membrane fractions. In monocyte-derived macrophages,M. tuberculosispeptide-MHC-II complexes appeared only in MIIC fractions and subsequently on the plasma membrane. Although phagosomes from both cell types acquired lysosome-associated membrane protein 1 (LAMP-1) and MHC-II, THP-1 phagosomes that support formation ofM. tuberculosispeptide-MHC-II complexes had increased levels of both LAMP-1 and MHC-II. Thus,M. tuberculosisphagosomes with high levels of MHC-II and LAMP-1 and MIIC both have the potential to form peptide-MHC-II complexes fromM. tuberculosisantigens in human macrophages.

Control ofMycobacterium tuberculosisinfection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing ofM. tuberculosisantigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK)M. tuberculosismore efficiently than liveM. tuberculosis. These observations suggested that liveM. tuberculosismay inhibit Ag processing by inhibiting phagosome maturation or that HKM. tuberculosismay be less resistant to Ag processing. In the present study we examined the correlation betweenM. tuberculosisviability and phagosome maturation and efficiency of Ag processing. Since heat treatment could renderM. tuberculosisAgs more accessible to proteolysis,M. tuberculosiswas additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK)M. tuberculosisin activated murine bone marrow macrophages was examined by using an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HKM. tuberculosismore rapidly and efficiently than they processed live, RadK, or RifKM. tuberculosis. Live, RadK, and RifKM. tuberculosiscells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadKM. tuberculosisexpressed fewerM. tuberculosispeptide-MHC-II complexes than phagosomes containing HKM. tuberculosisexpressed. Since only liveM. tuberculosiswas able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms ofM. tuberculosis. These findings suggest that the mechanisms used byM. tuberculosisto inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.

The pneumococcal (Pn) conjugate vaccine includes seven different polysaccharides (PS) conjugated to CRM197. Utilizing antigen-processing cells and a CRM197-specific mouse T-cell hybridoma, we found that the serotype of conjugated PnPS dramatically affected antigen processing of CRM197. Unconjugated CRM197and serotype conjugates 14 and 18C were processed more efficiently.

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses againstStreptococcus pneumoniaepolysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM197. BALB/c ByJ mice were injected with 19F-CRM197or 6B-CRM197with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM197alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM197alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM197IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.

Conjugation of various serotypes of pneumococcal polysaccharide (PnPS) to carrier protein enhances the magnitude of the polysaccharide-specific antibody response, presumably by eliciting T-cell help. However, variability in PnPS serotype-specific immunogenicity has been observed. CBA/J mice immunized with either 6B or 19F PnPS conjugated to the protein carrier Cross Reactive Material 197 (CRM 197 ) produce a strong anti-PnPS antibody response; however, when mice are immunized with 23F PnPS conjugated to CRM 197 , they fail to produce a significant anti-PnPS response. In order to determine whether this difference was related to alterations in antigen processing of the carrier protein and the subsequent T-cell responses, we studied proliferation of lymphocytes from CBA/J mice immunized with CRM 197 alone or conjugated to 6B, 19F, or 23F PnPS. T-cell proliferative responses to synthetic peptides demonstrated that lymph node cells elicited by the poorly immunogenic conjugate 23F-CRM 197 recognized many, but not all, of the epitopes recognized by lymph node cells elicited by 6B- and 19F-CRM 197 as well as additional epitopes. Despite marked differences in PnPS-specific immunogenicity, all mice made high titers of CRM 197 antibodies of the immunoglobulin G 1 isotype. Cells from mice immunized with any of the conjugates yielded vigorous T-cell responses to whole antigen. We conclude that the serotype of PnPS can alter the peptide specificities of T-cell responses, but even a poorly immunogenic PnPS conjugate can elicit a significant T-cell response. Thus, conjugation of PnPS to a carrier protein that elicits carrier-specific T- and B-cell responses does not necessarily enhance PnPS immunogenicity.

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.

We investigated parameters that affect the efficiency with which antigenic epitopes from Salmonella typhimurium are processed for presentation to T lymphocytes. As a model system, the hen egg white lysozyme 52-61 [HEL(52-61)] epitope, which binds the murine major histocompatibility complex class II (MHC-II) molecule I-Ak, was expressed in soluble fusion proteins in S. typhimurium. Murine peritoneal macrophages mediated phagocytic processing of viable S. typhimurium expressing fusion proteins of the HEL epitope for presentation via I-Ak regardless of the bacterial compartment in which the epitope was contained (i.e., surface exposed, facing the periplasmic space, or in the cytoplasm). Minor differences in processing efficiency observed with different epitope compartmentalizations could be overcome by altering the relative expression level, indicating that epitope abundance is an important factor for efficient processing of epitopes from S. typhimurium. This processing pathway required phagocytosis of bacteria followed by passage through an acidic compartment, suggesting a pathway involving phagolysosomal degradation of the bacteria to liberate epitopes that bind MHC-II. HEL(52-61) was processed more efficiently from heat-killed S. typhimurium than from viable bacteria, and in addition, the HEL epitope was processed more efficiently from a rough lipopolysaccharide (LPS) strain than from its isogenic smooth LPS counterpart, most likely because of enhanced phagocytosis of the rough LPS strain. These data suggest that the efficiency of epitope processing from S. typhimurium for presentation via MHC-II is affected by bacterial viability, epitope abundance, and LPS phenotype, factors which may be important to consider in development of recombinant S. typhimurium vaccine strains.

The effect of abundance and compartmentalization of antigenic epitopes expressed in Escherichia coli on phagocytic processing was studied by expressing fusion proteins containing the epitope from positions 52 to 61 of hen egg white lysozyme [HEL(52-61)], which binds the I-Ak murine major histocompatibility complex class II (MHC-II) molecule or the epitope from positions 257 to 264 of chicken egg ovalbumin [OVA(257-264]), which binds the Kb murine MHC-I molecule. Epitopes expressed as fusion proteins in the outer membrane protein LamB allowed exposure of the epitopes either at the bacterial surface, in the periplasmic space, or in the cytoplasm. Regardless of epitope compartmentalization within the bacterium, MHC-II-restricted or MHC-I-restricted presentation to T hybridoma cells occurred after macrophages phagocytosed bacteria producing the HEL(52-61) epitope or the OVA(257-264) epitope, respectively. Increased epitope abundance within a given microbial compartment resulted in increased processing and presentation to epitope-specific T hybridoma cells. Minor differences in the efficiency of epitope processing between the constructs was observed, and the HEL or OVA epitope exposed in the periplasmic space was processed most efficiently compared with the surface- or cytoplasm-localized epitopes. These differences could be overcome by increasing the amount of epitope per bacterium as little as two to five times. The minor differences in processing efficiency may be due to differing protein contexts of the epitope as well as differing epitope compartmentalizations within the bacteria. Thus, production of abundant epitope is the important parameter influencing processing of epitopes expressed in E. coli to induce T-cell responses rather than targeting of an epitope to a specific bacterial compartment.

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4 + T cells. In the absence of APCs, activation of memory CD4 + T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4 + T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4 + T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.

Although the 38-kDa glycolipoprotein ofMycobacterium tuberculosisH37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-α and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-α and IL-6 expression during mycobacterial infection.