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)     

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.     

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.     

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-gamma in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-gamma in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-gamma production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-gamma production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.     

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.     

Molecular mapping of interactions between a Mycobacterium leprae-specific T cell epitope, the restricting HLA-DR2 molecule, and two specific T cell receptors.

A systematic series of 89 single residue substitution analogs of the Mycobacterium leprae 65-kDa protein-derived peptide LQAAPALDKL were tested for stimulation of two HLA-DR2 restricted 65 kDa-reactive T cell clones from a tuberculoid leprosy patient. Some analogs with substitutions outside a "core" region showed enhanced stimulation of the T cell clones. This core region of seven or eight residues was essential for recognition, whereas substitution of amino acids outside this region did not affect T cell recognition although these residues could not be omitted. Thus these core residues interact directly with the presenting HLA-DR2 molecule and/or the TCR. Except for analogs of position 419 for clone 2B6, the majority of the nonstimulatory substitution analogs did not inhibit the presentation of LQAAPALDKL and thus probably failed to bind to the HLA-DR2 molecule. Unless all of the core residues are physically involved in binding to DR2, substitution at a position not directly involved in binding appears to have an influence on other residues that do bind to the DR2 molecule. Active peptide analogs with two or more internal prolines suggest that not all analogs need be helical for activity with clone 2F10.     

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 .     

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).     

T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein

The immune response to mycobacterial pathogens comprises a significant percentage of T cells with specificity for a 65-kDa heat shock protein (hsp) which is highly conserved in bacteria and man. PBMC were activated in vitro with killed Mycobacterium tuberculosis and afterward tested for CTL activity on autologous target cells primed with 1) killed M. tuberculosis, 2) intact recombinant 65-kDa hsp of Mycobacterium bovis/M. tuberculosis; or 3) tryptic fragments of the recombinant 65-kDa hsp. Strong CTL activity was observed on targets primed with killed M. tuberculosis or with tryptic fragments of the 65-kDa hsp, but not on those primed with the intact 65-kDa hsp. M. tuberculosis activated T cells from 2/13 donors tested exerted killer activity against unprimed targets. To assess whether T cell responses were directed against self-epitopes shared by the mycobacterial and human 65-kDa hsp, four peptides of at least 10 amino acids length were synthesized corresponding to fully or almost identical regions of these molecules. Peripheral blood T cells from 8/9 individuals tested, after activation with killed M. tuberculosis, expressed strong CTL activity toward autologous targets primed with one or more of these synthetic peptides. By using HLA-DR transfected murine L cells we found that the epitopes were recognized in the context of histocompatible HLA-DR (class II) molecules. We conclude that the demonstration of T cells with specificity to self-epitopes in vitro is not indicative for autoimmune disease. However, if at certain stages of infection such T cells are activated by crossreactive microbial epitopes they could cause autoimmune responses.     

M. leprae and PPD-triggered T cell lines in tuberculoid and lepromatous leprosy

Proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and bacillus Calmette Guerin-derived purified protein derivative (PPD) were studied in the presence or absence of interleukin 2 (IL 2) in high M. leprae responders (tuberculoid leprosy patients and healthy subjects) and low M. leprae responders (lepromatous leprosy patients). High responders in most cases developed a strong proliferative response to both antigens in the absence of IL 2. Additional IL 2 and restimulation with antigen plus autologous antigen-presenting cells (APC) allowed the derivation of antigen-specific T cell lines. The lines were assayed for proliferative responses to several mycobacterial antigens. Both PPD and M. leprae-triggered T cell lines exhibited a good proliferative response to either antigen and showed in addition a broad cross-reactivity with other mycobacteria, suggesting a preferential T cell response to epitopes shared by several mycobacterial species. Within the lepromatous group, 50% of the patients studied could mount a proliferative response to PPD antigen in the absence of IL 2, but none of them was able to do so with M. leprae antigen. The addition of IL 2 increased the number of positive responders to PPD in this group, and in some patients IL 2 was able to restore M. leprae reactivity as well, suggesting that IL 2 had overcome a suppressor mechanism. PPD and M. leprae-triggered T cell lines were obtained from these subjects (with IL 2 added from the beginning of the culture when required). M. leprae lines exhibited variable and unstable pattern of specificity, most lines exhibiting, at least transiently, a cross-reactive response to other mycobacteria, but some displaying only M. leprae-specific response. In contrast, PPD lines from these subjects consistently exhibited a good response to PPD, a lesser response to various other mycobacteria and no response to M. leprae, a pattern differing from that obtained with PPD lines of high M. leprae responders. Co-cultures of irradiated lepromatous PPD triggered T cell lines with fresh autologous PBMC non-specifically reduced the proliferative response of the latter to PPD, as well as to unrelated antigens. A similar suppression was also observed when PPD lines from one of the tuberculoid patients were assayed. PPD and M. leprae T cell lines from both high and low responders initially exhibited the same CD4+ CD8- phenotype. In all cases, antigenic specificity declined and could not be maintained after 5 to 8 wk of continuous culture, a change associated with the progressive appearance of CD8+ and Leu8+ cells.     

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.     

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.     

Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.     

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

HLA-DR/DQ Transgenic, class II deficient mice as a novel model to select for HSP T cell epitopes with immunotherapeutic or preventative vaccine potential

Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4⁺ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific mannerin vitro. In order to develop a preclinical model in which p1-20 APL can be testedin vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab⁰) mice, in which all CD4⁺ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab⁰ and DQ8.Ab⁰ mice both responded well to hsp65. Furthermore, DR3.Ab⁰ mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab⁰ mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab⁰ mice and DR3⁺ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab⁰ mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein

Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.     

Natural killer cell clones can efficiently process and present protein antigens.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.