Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reflects infection with or exposure to M. leprae.

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-gamma) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-gamma secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-gamma secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.

HLA-class II-associated control of antigen recognition by T cells in leprosy: a prominent role for the 30/31-kDa antigens.

The recognition of 16 mycobacterial Ags by a panel of T cell lines from leprosy patients and healthy exposed individuals from an endemic population was examined within the context of expressed HLA-DR molecules. Although overall no significant differences were found between the frequencies of Ag recognition in the different subject groups, when Ag-specific T cell responses were examined within the context of HLA-DR, a highly significant difference was found in the recognition of the 30/31-kDa Ag. HLA-DR3 appeared to be associated with high T cell responsiveness to the 30/31-kDa Ag in healthy contacts (p = 0.01), but, conversely, with low T cell responsiveness to this Ag in tuberculoid patients (p = 0.005). Within the group of HLA-DR3-positive individuals, differences in 30/31-kDa directed T cell responsiveness were highly significant not only between healthy individuals and tuberculoid patients (p < 0. 0001), but also between healthy individuals and lepromatous patients (p = 0.009), and consequently between healthy individuals compared with leprosy patients as a group (p < 0.0001). A dominant HLA-DR3-restricted epitope was recognized by healthy contacts in this population. It has been proposed that secreted Ags may dominate acquired immunity early in infection. The low T cell response to the secreted, immunodominant 30/31-kDa Ag in HLA-DR3-positive leprosy patients in this population may result in retarded macrophage activation and delayed bacillary clearance, which in turn may lead to enhanced Ag load followed by T cell-mediated immunopathology.

Increased intracellular survival of Mycobacterium smegmatis containing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene.

The thioredoxin (Trx) system of Mycobacterium leprae is expressed as a single hybrid protein containing thioredoxin reductase (TR) at its N terminus and Trx at its C terminus. This hybrid Trx system is unique to M. leprae, since in all other organisms studied to date, including other mycobacteria, both TR and Trx are expressed as two separate proteins. Because Trx has been shown to scavenge reactive oxygen species, we have investigated whether the TR-Trx gene product can inhibit oxygen-dependent killing of mycobacteria by human mononuclear phagocytes and as such could contribute to mycobacterial virulence. The gene encoding M. leprae TR-Trx was cloned into the apathogenic, fast-growing bacterium Mycobacterium smegmatis. Recombinant M. smegmatis containing the gene encoding TR-Trx was killed to a significantly lesser extent than M. smegmatis containing the identical vector with either no insert or a control M. leprae construct unrelated to TR-Trx. Upon phagocytosis, M. smegmatis was shown to be killed predominantly by oxygen-dependent macrophage-killing mechanisms. Coinfection of M. smegmatis expressing the gene encoding TR-Trx together with Staphylococcus aureus, which is known to be killed via oxygen-dependent microbicidal mechanisms, revealed that the TR-Trx gene product interferes with the intracellular killing of this bacterium. A similar coinfection with Streptococcus pyogenes, known to be killed by oxygen-independent mechanisms, showed that the TR-Trx gene product did not influence the oxygen-independent killing pathway. The data obtained in this study suggest that the Trx system of M. leprae can inhibit oxygen-dependent killing of intracellular bacteria and thus may represent one of the mechanisms by which M. leprae can deal with oxidative stress within human mononuclear phagocytes.

Associations between HLA-DRB1 alleles and leprosy in an Indonesian population.

To investigate whether the susceptibility to leprosy (type), subclinical infection with Mycobacterium leprae and the antibody response against M. leprae-specific antigens are associated with HLA-DR phenotypes sequence-specific oligonucleotide HLA-DRB1 and DQA1 typing and antibody assays have been performed in 79 leprosy patients (41 TT/BT and 38 LL/BL) and 50 healthy controls from a Javanese population in Yogyakarta, Indonesia. DRB1*02 was associated with LL/BL [odds ratio (OR) 2.54, 95% confidence interval (CI) 0.97-9.78, p = 0.037 and attributable risk (AR) 41.5%] but not with TT/BT leprosy (p > 0.05). HLA-DRB1*12 was negatively associated with leprosy (either LL/BL or TT/BT [OR 0.33-0.35, p < 0.05, prevented fraction (PF) 58.8%-65.3%]. No significant association was found between HLA-DRB1 or DQA1 type, anti-M. leprae antibody level and subclinical infection with M. leprae. These data indicate that in this population susceptibility to lepromatous leprosy is associated with HLA-DRB1*02, while resistance to leprosy is associated with HLA-DRB1*12. These associations are not paralleled with associations of the same HLA types with anti-M. leprae antibody level. Finally, the results of this study also support the notion that infection with M. leprae per se is not associated with HLA-DRB1 or DQA1 alleles.

Humoral and cellular immune reactivity to recombinant M. leprae antigens in HLA-typed leprosy patients and healthy controls.

In our search for Mycobacterium leprae antigens that might specifically induce immunity or immunopathology, we have tested both humoral and cellular immune reactivity against purified recombinant M. leprae antigens in 29 paucibacillary (PB), 26 multibacillary (MB) leprosy patients, and 47 matched healthy contacts. The following M. leprae antigens were tested: 2L-1 (65L-1, GroEl-1), 2L-2 (65L-2, GroEl-2), 4L (SoDA), 43L, 10L (B) and 25L (Sra). The individuals were also typed for HLAD-RB1 and DQB1 in order to see whether leprosy status and/or immune reactivity to these antigens might be associated with certain HLA types. We also tested sera from another 48 patients before, during and after multidrug therapy (MDT) to study the relationship between antibody reactivity to recombinant M. leprae antigens and MDT. Antibody titers to the four recombinant M. leprae antigens tested and to D-BSA were higher in MB patients compared to PB patients and healthy controls, and declined with treatment. From a diagnostic or monitoring point of view none of the recombinant antigens seemed to be an improvement over D-BSA, mainly due to the lower sensitivity. IgG subclasses were measured in positive sera of untreated patients. These were mainly of the IgG1 and IgG3 subclasses, but subclass diversity was also observed and antigen dependent: all four subclasses could be detected against 10L (B), only IgG1 and IgG3 against 43L and only IgG1 against 25L and 2L-1. Cellular immune reactivity against the purified recombinant M. leprae antigens was measured in a lymphocyte stimulation test (LST). As for M. leprae, there was an inverse correlation between antibody and T-cell reactivity. However, the number of LST responders to recombinant antigens was much lower than to M. leprae. The 43L antigen was recognized most often (19%-24% of individuals tested) and more often than the 10L (B) antigen (10%-12%). No clear correlation was observed with leprosy type or protection and, in general, M. leprae nonresponders were also negative with recombinant antigens. Finally, we confirmed that HLA-DRB1*02 is associated with leprosy in this population, and we observed an association between DQB1*0601 and lepromatous leprosy. The number of positive individuals was too small to allow a meaningful analysis of the relationship between HLA type and immune reactivity. Although these data do not allow a conclusion as to one of these purified recombinant antigens being either protection or disease related, the antigen-dependent IgG subclass diversity warrants further study on antigen-specific qualitative differences in immune reactivity that may be relevant for the outcome of an infection with M. leprae.

A DR17-restricted T cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH.

The assembly of peptide-major histocompatability class II complexes in vitro is accelerated at low pH, comparable to that found in the intracellular compartments of metabolically active antigen-presenting cells (APC). Mycobacteria such as Mycobacterium tuberculosis reside in phagosomes with only mildly acidic pH. Therefore, we investigated the pH dependency of peptide-HLA-DR binding for several T cell epitopes of mycobacterial proteins, focussing particularly on well-defined, immunodominant HLA-DR17(3)-restricted T cell epitopes: peptide (p) 3-13 from the cytoplasmic 65-kDa heat shock protein of M. tuberculosis/M. leprae, and peptide 56-65 from the secreted 30/31-kDa protein from M. tuberculosis/M. leprae. p3-13 bound to purified, cell-free DR17 under both acidic and neutral conditions. Four other, unrelated DR17-binding peptides showed the same pH-dependent binding characteristics as p3-13. p56-65, however, only bound to purified DR17 at pH 7 but not at all at pH 4.5. These DR17 peptide binding data were confirmed in cell-bound DR17, in T cell stimulation assays in which fixed APC were peptide-pulsed at acidic or neutral pH before addition of peptide-specific DR17-restricted T cells. As far as we are aware, p56-65 is the only human T cell epitope binding to HLA exclusively at neutral pH. The binding characteristics of p56-65 may reflect dominant processing in alternative, less acidic vacuolar compartments specifically related to the generation of epitopes from (secreted) mycobacterial proteins. The observation that p56-65 is an immunodominant epitope for anti-mycobacterial T cells suggests the relevance of such novel processing compartments in T cell-mediated immunity.

Clonal dominance and selection for similar complementarity determining region 3 motifs among T lymphocytes responding to the HLA-DR3-associated Mycobacterium leprae heat shock protein 65-kd peptide 3-13.

In order to establish whether specific MHC class II-peptide complexes are capable of selecting TCR V regions, we investigated in detail the TCR beta chain used in the recognition of HLA-DR3 restricted hsp65 peptide 3-13 in a tuberculoid leprosy patient. Using RT-PCR, a clear dominance of the TCRBV5 gene family was observed in a hsp65 peptide 3-13-specific T-cell line; however, not in fresh, unstimulated PBMCs, PHA-stimulated PBMCs, or a T-cell line specific for tetanus toxoid. DNA sequence analysis of the TCR V regions, comprising TCRBV5 genes, derived from the hsp65 peptide 3-13-specific T-cell line revealed the exclusive usage of the TCRBV55S1 gene segment and a predominance of one V-D-J gene rearrangement, which is indicative of clonal expansion of these T lymphocytes. Additional highly similar V-D-J gene rearrangements were detected at a low level in this hsp65 peptide 3-13 specific T-cell line. These conserved junctional regions (CDR3 regions) could not be detected within the TCRBV5 gene family of fresh PBMCs, PHA-stimulated PBMCs, hsp65, and tetanus-toxoid-specific T-cell lines from this patient. The observations in this tuberculoid leprosy patient reveal that an HLA class-II-restricted T-cell response results in selection of TCRBV regions which are highly similar in amino acid composition to the CDR3 region within the expanding TCRBV regions.

Definition of a human suppressor T-cell epitope.

The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.

Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B, which is specifically recognized by antibodies from patients with leprosy.

Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions (AA) 206-230, 251-280, and 291-325. Sera of patients with active tuberculosis who responded to the native 30-kDa antigen did not recognize these peptides. The antibody-binding specificity to the defined B cell regions was evaluated in a blind study with 71 serum samples from patients and household contacts living in Ethiopia where leprosy is endemic. The peptide of AA 256-280 was recognized by 88% of LL patients, 15% of patients with tuberculoid leprosy, and none of the contacts. These findings suggest that there are major linear B cell epitopes on the M. leprae 30-kDa protein that are recognized by lepromin-negative LL patients, whereas lepromin-positive patients respond preferentially to conformational epitopes.

A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders.

It is now well established that cultured human melanocytes are capable of expressing immunologically important cell surface molecules and that they can produce cytokines. Not all cells with the ability to express MHC class II molecules are capable of effective Ag presentation. However, the dendritic nature of melanocytes, their strategic position within the skin, and their phagocytic capacity seem to suggest a role for these cells in processing and presenting Ag. This study demonstrates that cultured normal human skin melanocytes can present peptide Ag, and process and present the mycobacterial HSP65 kDa protein and whole Mycobacterium leprae sonicate to CD4+ cytotoxic proliferative T cell clones in an Ag-specific and HLA-class II-restricted manner. T cell stimulation was dependent on costimulatory signals, i.e., LFA-3/CD2 and LFA-1/ICAM-1. Besides eliciting a T cell proliferative response, our studies further demonstrate that melanocytes can function as target cells for T cell-mediated cytotoxicity. The described Ag-processing and -presenting functions of melanocytes, taken together with in vivo behavior of melanocytes in hypopigmentation, provide new clues for the etiopathogenesis of melanin pigmentary disorders.

The biologic importance of conserved major histocompatibility complex class II motifs in primates.

Phylogenetic comparisons of polymorphic second-exon sequences of MHC class II DRB genes showed that equivalents of the HLA-DRB1*03 alleles are present in various nonhuman primate species such as chimpanzees, gorillas, and rhesus macaques. These alleles must root from ancestral structure(s) that were once present in a progenitor species that lived about 35 million years ago. Due to accumulation of genetic variation, however, sequences that cluster into a lineage are generally unique to a species. To investigate the biologic importance of such conservation and variation, the peptide-binding capacity of various Mhc-DRB1*03 lineage members was studied. Primate Mhc-DRB1*03 lineage members successfully binding the p3-13 peptide of the 65-kD heat-shock protein of Mycobacterium tuberculosis/leprae share a motif that maps to the floor of the peptide-binding site. Apart from that, some rhesus macaque MHC class-II-positive cells were able to present the p3-13 peptide to HLA-DR17-restricted T cells whereas cells obtained from great ape species failed to do so. Therefore, these studies open ways to understand which MHC polymorphisms have been maintained in evolution and which MHC residues are essential for peptide binding and T-cell recognition.

Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates.

Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.

T-cell receptor beta-chain gene usage in the T-cell recognition of Mycobacterium leprae antigens in one tuberculoid leprosy patient.

The beta chain of the T-cell antigen receptor present on 20 T-cell clones isolated from a tuberculoid leprosy patient was studied by gene rearrangement and PCR analysis. These T-cell clones all responded to Mycobacterium leprae-encoded protein antigens, and 8 of them specifically recognized peptides of the mycobacterial 65-kDa heat shock polypeptide (65hsp). All T-cell clones studied were HLA-DR-restricted (DR2 or -3). In the DR3-restricted group, 7 of 10 used a beta-chain variable region V beta 5 gene family member, whereas in the DR2-restricted group, 2 of 10 T-cell clones used a V beta 5 gene segment and 5 used the V beta 18 gene segment. The deduced amino acid sequences of the beta chain from 8 T-cell clones have revealed that 3 of 4 DR3-restricted T-cell clones expressed the V beta 5.1 gene segment whereas the fourth DR3-restricted T-cell clone employed a V beta 5 family member not previously described. The V beta 5.1-positive T-cell clones all recognized the same 65hsp peptide from residues 2 to 12. The N-D-N segment (where D is diversity) of the junctional region of these T-cell clones was very similar, despite different beta-chain joining gene segments. Of the 4 DR2-restricted T-cell clones investigated, 3 used the V beta 18 gene segment and recognized the 65hsp peptide from residues 418 to 427. In conclusion, within this panel of M. leprae-reactive T-cell clones, the DR3-restricted T-cell clones mainly used a V beta 5 gene segment, whereas the DR2-restricted clones employed preferentially the V beta 18 gene segment.

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.

Molecular and immunological analysis of a fibronectin-binding protein antigen secreted by Mycobacterium leprae.

By screening a Mycobacterium leprae lambda gt11 genomic DNA library with leprosy-patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin-binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327-amino-acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75-85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55-266 and 266-327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55-266 and 265-327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin-binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

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)

An immunogenetic view of delayed type hypersensitivity.

This review, the third in the series on cellular immune reactivity to tubercle bacilli in the centenary year of Koch's classical paper describing this phenomenon and its possible implications, represents an immunogenetic point of view. In fact this will be quite a broad point of view by an immunogeneticist who is not hampered by specific knowledge on therapy or prevention of tuberculosis. In this respect I probably do not differ very much from Robert Koch 100 years ago! An important difference, however, is that we think we now understand a great deal of the cellular and molecular basis of the immunological phenomena observed by Koch. Immunogenetics has contributed considerably to our current understanding and I will try to review that contribution here. Because thus far my main research interest has been in another mycobacterium, namely Mycobacterium leprae, I will use M. leprae and leprosy as an example to illustrate some ideas. The message of this review is that there is a reason for optimism: the knowledge recently gained by cellular and molecular immunologists as well as immunogeneticists has straightforward implications for the rational development of subunit vaccines and immunotherapeutic strategies.