Characterization of Mycobacterium leprae cell wall-associated proteins with the use of T lymphocyte clones

Development of a vaccine against leprosy depends on the identification of Ag that stimulate cell-mediated immune responses. We have previously demonstrated that cell wall proteins of Mycobacterium leprae are highly immunogenic. By using human cell wall-specific T cell clones we have begun to characterize soluble proteins that integrate into the cell wall skeleton. T cells from leprosy lesions were expanded with IL-2 in vitro yet retained specificity to Ag of the insoluble cell wall core (CWC) in vitro, indicating that T cells had been activated by CWC Ag in vivo. A cell wall protein-peptidoglycan complex and cell wall protein preparations lacking carbohydrates and lipids from CWC retained T cell reactivity. To identify immunogenic protein component(s) of cell wall protein, T cell lines were established to cell walls and tested against M. leprae proteins separated by SDS-PAGE and transferred to nitrocellulose. Greatest T cell reactivity was observed to proteins of Mr 7 kDa, 16 kDa, and 28 kDa. T cell clones reactive with 7-kDa and 16-kDa Ag from gels failed to respond to proteins of other Mr separated under either reducing or nonreducing conditions, indicating that these molecules are not subunits of larger proteins and may represent monomeric units polymerized into cell walls. The approaches described herein for characterization of immunodominant T cell Ag of M. leprae may be useful for study of T cell Ag in cell walls of bacterial pathogens of man.     

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.     

A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.     

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.     

T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.     

Regulation of cell wall synthesis and growth

The replication and growth of Mycobacterium tuberculosis are fundamentally linked to the synthesis and extension of its complex cell wall. Incorporation of new wall material must be tightly regulated so that its deposition does not compromise the extant structure. M. tuberculosis also produces an impressive array of complex bioactive lipids that are intimately involved in pathogenesis and protective immunity. The profiles of these lipids are regulated appropriately to allow the bacterium to respond to the prevailing conditions it faces in vivo. A number of regulatory strategies employed by M. tuberculosis to control cell wall biosynthesis and cell division have now been elucidated. The review highlights the role of alternative sigma factors with extracytoplasmic function in the activation of genes for biosynthesis of complex lipids involved in pathogenicity. Rel(Mtb) and CRP(Mt) play roles in cell wall responses to general nutrient deprivation by synthesis and sensing of starvation second messengers, respectively. Recently, the importance of protein phosphorylation networks in cell wall biosynthesis has attracted considerable interest. A plethora of two-component and eukaryotic-like serine/threonine protein kinases systems have been discovered and several are implicated in cell-division, morphogenesis and regulation of the profile of complex bioactive lipids elaborated by the pathogen.     

Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy household contacts of patients with active tuberculosis and healthy controls from the community

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-gamma activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-gamma-producing cells, IFN-gamma concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-gamma-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-gamma-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0. 05). IFN-gamma concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.     

A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia

A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 microg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.     

Innate immunity together with duration of antigen persistence regulate effector T cell induction

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.     

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.     

Identification of mycobacterial HSP70 reactive human T cell clones discriminating between M. tuberculosis and M. leprae

M. tuberculosis reactive CD4⁺ T cell clones were established from a BCG vaccinated donor and tested for proliferative responses against complex mycobacterial antigens like M. tuberculosis , M. leprae , and PPD, as well as the recombinant M. tuberculosis HSP70 and HSP65 antigens from both M. tuberculosis and M. leprae . This screening permitted the identification of T cell clones specifically recognizing the mycobacterial HSP70 or HSP65 antigen. All HSP65 reactive T cell clones were cross-reactive for M. tuberculosis and M. leprae , whereas three HSP70 reactive T cell clones only recognized M. tuberculosis . In addition, HLA typing and blocking experiments with anti-HLA antibodies revealed that antigen presentation to all M. tuberculosis reactive T cell clones was restricted by HLA-DR3 molecules. We have thereby demonstrated the presence of human T cell specificities directed against the mycobacterial HSP70 antigen that are able to discriminate between M. tuberculosis and M. leprae .     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Mycobacterial proteins--immune targets for antituberculous subunit vaccine

Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.     

Human T cell clones recognize two abundant Mycobacterium tuberculosis protein antigens expressed in Escherichia coli

Human T cells reactive to Mycobacterium tuberculosis were cloned from peripheral blood mononuclear cells (PBMC) of four tuberculosis patients by using whole irradiated bacilli as the in vitro stimulatory agent. Twenty-two T cell clones (CD4+) were tested for their reactivity to 12 different mycobacterial species and showed a distribution from limited to broad cross-reactivity. These T cell clones were also tested for their reactivity to three abundant M. tuberculosis proteins of 65, 19, and 14 kD, each expressed from recombinant DNA in Escherichia coli. The three proteins were expressed from DNA clones that were previously isolated from a lambda gt11 genomic DNA library of M. tuberculosis by using monoclonal antibodies directed against this pathogen. A T cell clone from one patient was stimulated by an E. coli lysate containing the 65 kD antigen, and a T cell clone from a second patient was stimulated by an E. coli lysate containing the 19 kD antigen in an in vitro proliferation assay. Both T cell clones showed very limited cross-reactivity when tested against other mycobacteria. We conclude that some patients with tuberculosis exhibit a T cell response to the 65 and 19 kD M. tuberculosis proteins defined by these isolated genes.     

Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement.     

T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens.

Recent evidence suggests the existence of at least two pathways of acquired specific resistance to Mycobacterium tuberculosis infection; the first consisting of cytokine-mediated activation of parasitized host cells by protective T cells, and the second involving the lysis of these cells by cytolytic T cells. Evidence presented in this report shows that both of the above mechanisms are operative in experimentally infected mice, but that they differ markedly in terms of their kinetics of emergence and loss. It was found that protective T cell activity was acquired very early during the course of the infection, and was temporally associated with the onset of bacterial elimination; however, cytolytic activity did not peak until 10 to 20 days later. This report shows further that the target Ag of these effector T cell populations were apparently numerous with no evidence for preferential recognition of a few immunodominant Ag. In view of the preponderance of target proteins in the bacterial filtrate, we present the hypothesis that such proteins secreted or otherwise leaked from the dividing mycobacterium are pinocytosed from the phagosome and used by the infected macrophage as the key protective Ag leading to T cell sensitization. This hypothesis thus explains the preferential requirement for the viable bacterium in the generation of specific resistance, and further explains why protective immunity is generated even while the organism is still multiplying in an apparently unrestrained manner.     

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.     

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.     

CTLA-4 differentially regulates T cell responses to endogenous tissue protein versus exogenous immunogen

CTLA-4 is critical to the regulation of CD4 T cell homeostasis in vivo. However, whether CTLA-4 regulates responses to both self and foreign proteins is not clear. We have directly compared the role of CTLA-4 in controlling T cell responses to the same protein presented as an endogenous tissue Ag vs a foreign immunizing Ag. We show that CTLA-4 only modestly reduces responses to Ag administered with adjuvant, but dramatically inhibits responses to the same Ag expressed transgenically as a tissue self protein. The critical consequence of CTLA-4 engagement is to inhibit T cell accumulation in the local lymph node draining the Ag-bearing tissue, and failure of this control leads to the onset of autoimmune tissue destruction. Thus, CTLA-4 may preferentially dampen pathologic immune responses to self proteins while permitting protective immunity to foreign agents.