Analysis of human T-cell epitopes in the 19,000 MW antigen of Mycobacterium tuberculosis: influence of HLA-DR.

The potential number of T-cell epitopes in the 19,000 molecular weight (MW) antigen has been investigated using overlapping peptides which comprise the complete sequence. Sixteen potential epitopes could be deduced from the responses to these peptides by polyclonal T cells derived from 22 antigen-responsive donors. The majority of epitopes were not predicted by either of the major paradigms, the Rothbard motif and the amphipathic helix. A hierarchy of epitopes was indicated by the responses, which ranged from strong and frequent in the N-terminal region, to moderate or weak elsewhere. Some epitopes were restricted by single HLA-DR determinants, or families of determinants sharing structural features in common, whilst the two N-terminal peptides were recognized by donors with a diversity of DR types. The high degree of T-cell recognition of the N-terminal region may be of relevance to the design of a sub-unit vaccine capable of priming T cells against Mycobacterium tuberculosis.     

T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae.

The 18-kilodalton (kDa) antigen of Mycobacterium leprae was expressed as a fusion protein with a 2-kDa leader peptide and used in proliferation assays with peripheral blood cells. Fifty percent of untreated tuberculoid leprosy patients and 93% of long-term leprosy contacts responded to the recombinant protein in lymphocyte transformation tests. Comparison of the stimulation indices in the two groups showed that the contacts responded more strongly than the tuberculoid leprosy patients. Seventy percent of Mycobacterium bovis BCG-vaccinated European donors responded, although with low stimulation indices. The isolation of 18-kDa antigen-responsive T-cell lines from a BCG-vaccinated British donor confirmed that the 18-kDa antigen contains at least one cross-reactive epitope. These results indicate that the 18-kDa protein is an important antigen in the immune response to leprosy.     

Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis.

The T-cell response of human donors to secreted antigen fractions of Mycobacterium tuberculosis was investigated. The donors were divided into five groups: active pulmonary tuberculosis (TB) patients with minimal and with advanced disease, Mycobacterium bovis BCG-vaccinated donors with and without contact with TB patients, and nonvaccinated individuals. We found that patients with active minimal TB responded powerfully to secreted antigens contained in a short-term culture filtrate. The response to secreted antigens was mediated by CD4+ Th-1-like lymphocytes, and the gamma interferon release by these cells was markedly higher in patients with active minimal TB than in healthy BCG-vaccinated donors. Patients with active advanced disease exhibited depressed responses to all preparations tested. The specificity of the response to secreted antigens was investigated by stimulating lymphocytes with narrow-molecular-mass fractions of short-term culture filtrate obtained by the multielution technique. Considerable heterogeneity was found within the donor groups. Patients with active minimal TB recognized multiple secreted targets, but interestingly, six of eight patients demonstrated a predominant recognition of a low-mass (< 10-kDa) protein fraction which induced high levels of gamma interferon release in vitro. Only a few of 12 previously characterized secreted antigens were recognized by T cells isolated from TB patients, suggesting the existence of a number of as yet undefined antigenic targets among secreted antigens.     

Vaccination for Mycobacterium tuberculosis infection: reprogramming CD4 T-cell homing into the lung

Development of effective tuberculosis vaccines is hampered by insufficient understanding of protective immunity. Here, Woodworth et al.¹ show secondary effector CD4 T cells generated after Mtb challenge of H56/CAF01 vaccinated mice display superior lung homing compared with primary effectors. Vaccination generates large populations of parenchymal lung effector cells by inducing CXCR3⁺KLRG1⁻ cells that continuously migrate from lymph nodes to lung, and limiting the generation of non-protective CX3CR1⁺KLRG1⁺ intravascular effectors, providing insight vaccine-mediated protection against tuberculosis.     

Human Mycobacterium tuberculosis-reactive CD4+ T-cell clones: heterogeneity in antigen recognition, cytokine production, and cytotoxicity for mononuclear phagocytes.

CD4+ T cells regulate the protective immune response which follows exposure to Mycobacterium tuberculosis by activating macrophages through the cytokines the CD4+ T cells secrete. In addition CD4+ T cells have been shown to be directly cytotoxic for antigen-pulsed mononuclear phagocytes (monocytes-macrophages). To explore the functional interaction between mycobacterial antigen-specific CD4+ T cells and mononuclear phagocytes further, CD4+ T-cell clones were derived from healthy purified protein derivative-positive individuals. Five T-cell clones were selected for detailed analysis. None responded to the purified recombinant or native mycobacterial antigens of 14, 19, 65, 71, and 30 (alpha-antigen/Ag6) kDa. However, the T-cell clones demonstrated heterogeneity in antigen recognition as measured by their Western blot (immunoblot) responses. Some T-cell clones made only interleukin 2, while others made only interleukin 4; all produced gamma interferon, although in differing amounts. Four of five T-cells clones were cytotoxic for purified protein derivative-pulsed monocytes at 1:1 and 10:1 effector-target cell ratios. When monocytes infected with live M. tuberculosis were used as targets, comparable levels of cytotoxicity were observed. The cytotoxicity was major histocompatibility complex class II restricted and inhibited by antibodies to ICAM-1 and LFA-1 and not by antibodies to tumor necrosis factor alpha, lymphotoxin, and gamma interferon. Cytotoxicity by CD4+ T cells for monocytes pulsed with mycobacterial antigens or infected with live M. tuberculosis is a common property of these cells and appears to be independent of the repertoire of lymphokines produced and not limited to recognition of defined mycobacterial heat shock proteins. Lysis of heavily infected mononuclear phagocytes may be one manner in which CD4+ T cells regulate host immune response to M. tuberculosis.     

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.     

A novel peptide vaccination augments cytotoxic CD8+ T-cell responses against Mycobacterium tuberculosis HspX antigen

Cellular immunity is a critical factor determining the safety and efficacy of newly developed vaccines against Mycobacterium tuberculosis infection. Crosstalk between CD4⁺ and CD8⁺ T-lymphocytes plays central roles in perpetuating the cytotoxic killing to the infected cells for host clearance. Our study proposed a novel alternating MHC-class II restricted peptide vaccination strategy to enhance the antigen-specific CD8⁺ T-cell activity against alpha-crystalline heat-shock protein (HspX) in C57BL/6 mice. Alternating peptide vaccination significantly stimulated a prominent HspX-specific CD8⁺ T-cell response with elevated Th1 and Th17 responses, without interference from Tregs suppression. Heightened central and effector CD8 memory were apparent in mice receiving alternating peptide vaccine, indicating a persisting recall immunity against HspX antigen. It was unlikely for alternating peptide vaccine to cause dysregulation in CD8⁺ T-cells as shown by minimal expression of KLRG1, PD1, LAG3, and CTLA-4 markers. Strong cytotoxic T-lymphocyte (CTL) responses were demonstrated in mice administrated with alternating peptide vaccines, suggesting its capacity in executing killing effector function against targeted cells. In conclusion, our novel vaccination strategy delineated potential benefits of alternating MHC-II peptides to invigorate efficient cytotoxic CD8⁺ T-cell responses against HspX antigen. Such approach might be applicable to serve as alternative immunotherapy for latent tuberculosis infection in future.     

Generation of CD8(+) T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis

Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.     

Function and antigen recognition pattern of L3T4+ T-cell clones from Mycobacterium tuberculosis-immune mice.

T-cell clones were established from Mycobacterium tuberculosis-immunized mice. These clones had the phenotype Thy-1+ L3T4+ Lyt-2- and were restricted by the H-2I-A locus. After antigen stimulation, the T-cell clones secreted interleukin-2 and gamma interferon. Factors produced by these T-cell clones activated normal bone marrow macrophages for antimycobacterial activity in vitro. Furthermore, the T-cell clones could adoptively confer delayed-type hypersensitivity on normal recipient mice. These findings indicate that the T-cell clones clones expressed relevant functions of antimycobacterial immunity. The antigen reactivity of the T-cell clones to different mycobacterial species ranged from broad cross-reactivity to stringent specificity, and none of the clones distinguished between M. tuberculosis and M. bovis. Thus, M. tuberculosis-immune helper/inducer T cells of identical phenotype, genetic restriction, and function varied in their antigen specificity. T-cell clones of the type described will facilitate functional characterization of mycobacterial antigens on the T-cell level.     

Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis.

We report here that during the course of an experimental infection of mice with Mycobacterium tuberculosis, the differential expression of the cell surface antigens CD44 and CD45RB could be used to delineate CD4+ T cells into four phenotypically distinct subsets. The major subset present was designated CD44lo/CD45RBhi and is associated with naive or resting T cells. The three remaining subsets expressed increased levels of the CD44 antigen as the infection progressed and could therefore be considered to be in an activated state. These activated populations could be further divided on the basis of their variable expression of the CD45RB antigen. These populations were designated CD44hi/CD45RBhi, CD44hi/CD45RBlo, and CD44hi/CD45RBneg. Kinetic studies of the emergence of these populations indicated that these subsets arose sequentially from the naive population at times associated with the peak expression of acquired specific resistance. In further studies, in an attempt to associate either the CD44hi/CD45RBlo or the CD44hi/CD45RBneg population with acquired immunologic memory of tuberculosis infection, draining lymph nodes of challenged memory immune animals were analyzed for the accumulation of the CD4+ subsets. The accumulation of both the CD44hi/CD45RBlo and the CD44hi/CD45RBneg populations was observed, but the CD44hi/CD45RBlo population was enriched in a manner consistent with the rapid accumulation of memory T cells during the anamnestic response. While functional roles for each of these subsets remain to be determined, these data provide the first evidence for the evolution of multiple, phenotypically distinct CD4+ T-cell subsets during the in vivo response to an experimental mycobacterial infection.     

CD4(+) T-cell subsets that mediate immunological memory to Mycobacterium tuberculosis infection in mice

We have studied CD4(+) T cells that mediate immunological memory to an intravenous infection with Mycobacterium tuberculosis. The studies were conducted with a mouse model of memory immunity in which mice are rendered immune by a primary infection followed by antibiotic treatment and rest. Shortly after reinfection, tuberculosis-specific memory cells were recruited from the recirculating pool, leading to rapidly increasing precursor frequencies in the liver and a simultaneous decrease in the blood. A small subset of the infiltrating T cells was rapidly activated (<20 h) and expressed high levels of intracellular gamma interferon and the T-cell activation markers CD69 and CD25. These memory effector T cells expressed intermediate levels of CD45RB and were heterogeneous with regard to the L-selectin and CD44 markers. By adoptive transfer into nude mice, the highest level of resistance to a challenge with M. tuberculosis was mediated by CD45RB(high), L-selectin(high), CD44(low) cells. Taken together, these two lines of evidence support an important role for memory cells which have reverted to a naive phenotype in the long-term protection against M. tuberculosis.     

结核分枝杆菌Rv0577抗原T细胞多肽预测及实验验证

目的建立一个预测和验证具有T细胞免疫功能的多肽的方法 ,用于耐多药结核病(MDR-TB)和广泛耐药结核病(XDR-TB)治疗或者辅助治疗。方法采用生物信息学方法预测Rv0577的T细胞表位,筛选并化学合成表位集中、亲和力较强、理化性质稳定的长链多肽,应用酶联免疫斑点(ELISPOT)技术检测多肽刺激活动性结核病患者免疫细胞分泌IFN-γ的能力。结果预测并筛选出Rv0577长链多肽2条,ELISPOT结果表明2条多肽可以激活活动性结核病患者的免疫细胞。结论软件预测与初步鉴定结果具有一致性,此方法的建立为寻找更多的T细胞免疫多肽奠定基础。     

Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells.

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection.     

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.     

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.     

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.     

T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls

Background: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. Objective: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 μg Fe/mL) and low iron (0.02 μg Fe / mL) cultures of M. tuberculosis. Materials and Methods: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. Results: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). Conclusions: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.     

T-cell recognition of the HspX protein of Mycobacterium tuberculosis correlates with latent M. tuberculosis infection but not with M. bovis BCG vaccination

During stationary growth or in vitro conditions mimicking relevant aspects of latency, the HspX protein (Rv2031c) is specifically upregulated by Mycobacterium tuberculosis. In this study we compared T-cell responses against HspX and the secreted M. tuberculosis protein Ag85B (Rv1886c) in tuberculosis (TB) patients, tuberculin skin test-positive individuals, M. bovis BCG-vaccinated individuals, and healthy negative controls. Gamma interferon responses to HspX were significantly higher in M. tuberculosis-exposed individuals than in M. tuberculosis-unexposed BCG vaccinees. In contrast, no such differences were found with respect to T-cell responses against Ag85B. Therefore, BCG-based vaccines containing relevant fragments of HspX may induce improved responses against this TB latency antigen. To identify relevant major histocompatibility complex class I- and class II-restricted HspX-specific T-cell epitopes, we immunized HLA-A2/K(b) and HLA-DR3.Ab(0) transgenic (tg) mice with HspX. Two new T-cell epitopes were identified, p91-105 and p31-50, restricted via HLA-A*0201 and HLA-DRB1*0301, respectively. These epitopes were recognized by human T cells as well, underlining the relevance of HspX T-cell recognition both in vivo and in vitro. In line with the data in humans, BCG immunization of both tg strains did not lead to T-cell responses against HspX-derived epitopes, whereas nonlatency antigens were efficiently recognized. These data support the notion that BCG vaccination per se does not induce T-cell responses against the latency antigen, HspX. Thus, we suggest that subunit vaccines incorporating HspX and/or other latency antigens, as well as recombinant BCG strains expressing latency antigens need to be considered as new vaccines against TB.     

Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines

Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.     

Influence of HLA-DR on the phenotype of CD4+ T lymphocytes specific for an epitope of the 16-kDa alpha-crystallin antigen of Mycobacterium tuberculosis.

T helper phenotype may be influenced by cytokine milieu, the differential expression of co-stimulatory molecules, antigen dose, and by differences in affinity at the TCR-peptide-MHC interface. We investigated the latter hypothesis by examining the response of six HLA-DR-restricted CD4+ T cell lines specific for the immunodominant and permissively recognized p91-110 epitope of the 16-kDa alpha-crystallin protein of Mycobacterium tuberculosis. Each line was generated from a sensitized HLA-DR-heterozygous donor and all proliferated when peptide was presented by autologous irradiated peripheral blood mononuclear cells. However, when HLA-DR-matched homozygous Epstein-Barr-virus-transformed B cell lines (L-BCL) were used as peptide-presenting cells there was heterogeneity in the response. The most pronounced proliferative response, and the highest IFN-gamma secretion and cytolytic activity was stimulated by L-BCL expressing molecules (DRB1*0101, *1501 and *0401) with high affinity (IC50 < 10 microM) for the 16p91-110 peptide. By comparison, IL-4 secretion or a lower proliferative response could occur when peptide was presented by alleles of high, or of intermediate (10 microM < IC50 < 100 microM), affinity. These data support the hypothesis that the host MHC can influence CD4+ phenotype and have implications for subunit vaccination against tuberculosis.