Repeated determinants within the retinal interphotoreceptor retinoid-binding protein (IRBP): immunological properties of the repeats of an immunodominant determinant.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes in the retina and pineal gland, induces inflammatory changes in these organs (EAU and EAP, respectively) when injected into various mammals. We have previously identified a determinant (residues 1169-1191) in bovine IRBP which is immunodominant and highly immunogenic and immunopathogenic in Lewis rats. IRBP exhibits a fourfold repeat structure and we report here on the comparison between the active sequence 1179-1191 and its three repeat peptides. Only one of the repeats, 271-283, cross-reacted with 1179-1191 and exhibited immunodominance, albeit of a low level. Peptide 271-283 was also immunogenic and immunopathogenic in Lewis rats, but with a minimal dose approximately 100 times higher than that of 1179-1191. Peptide 880-892, a nondominant determinant, resembled 271-283 in its immunogenicity, but was markedly less immunopathogenic. No immunological activity was detected in the fourth repeat peptide, 579-591. Peptide 1179-1191 was superior to the other repeats also in its antigenicity, i.e., the capacity to stimulate presensitized lymphocytes in culture: the minimal stimulatory concentrations of 1179-1191 was greater than 1000 times lower than those of 271-283 or 880-892. Furthermore, 1179-1191 was stimulatory at concentrations lower than those of 271-283 even when tested with lymphocytes sensitized against 271-283. A correlation was also found between the immunological activities of the repeat peptides and their amphipathicity. This study thus identifies two new immunopathogenic determinants of IRBP and provides additional data to show the association between immunodominance of peptides and their various immunological activities.     

Analysis of the pivotal residues of the immunodominant and highly uveitogenic determinant of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), a retinal-specific Ag, induces experimental autoimmune uveitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP is the immunodominant epitope of this protein in Lewis rats and is highly immunogenic and uveitogenic in these rats. The active site of peptide 1169-1191 was determined by testing its truncated forms. The shortest peptide to be immunologically active was found to be 1182-1190 (WEGVGVVPD). To determine the role of individual residues of this sequence, we have tested the immunologic activities of nine analogs of peptide 1181-1191, in which each of residues 1182-1190 was substituted with alanine (A). The tested activities included the capacity to induce experimental autoimmune uveitis and cellular responses in immunized rats, as well as the capability to stimulate lymphocytes sensitized against IRBP or the parent peptide 1181-1191. Analogs that did not stimulate these lymphocytes were also tested for their capacity to competitively inhibit the proliferative response to 1181-1191. Analogs A(1184), A(1186), and A(1187) resembled 1181-1191 in their activities, whereas the other analogs exhibited remarkably reduced activities, with several patterns being noticed. Analog A(1182) was inactive in all tests. Analog A(1190) was very weakly uveitogenic and non-immunogenic, but it stimulated lymphocytes sensitized against IRBP or 1181-1191 when added at exceedingly high concentrations. Analogs A(1183) and A(1185) resembled A(1190) in being weakly uveitogenic and A(1185) was also found to be poorly immunogenic. In addition, relatively high concentrations of A(1183) and A(1185) were needed to stimulate lymphocytes sensitized against IRBP or 1181-1191. However, a different pattern of activities was exhibited by analogs A(1188) and A(1189). These peptides were uveitogenic and immunogenic, but failed to stimulate lymphocytes sensitized to IRBP or 1181-1191. Furthermore, A(1188) and A(1189), but not A(1182), also inhibited the response to 1181-1191 of a cell line specific toward this parent peptide. The data are interpreted to show that residues 1188 and 1189 are involved in the interaction of the peptide with the TCR, whereas residues 1182 and 1190 and, perhaps, 1183 and 1185, are pivotal for the binding of peptide 1181-1190 to the MHC molecules on APC.     

Recognition of peptides that are immunopathogenic but cryptic. Mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes.

Interphotoreceptor retinoid binding protein (IRBP) is a glycoprotein that localizes in the retina and induces inflammatory changes in this tissue in immunized animals. Certain IRBP-derived peptide determinants are also immunopathogenic, and we have previously shown that these determinants could be either immunodominant or cryptic. Lymphocytes sensitized against the cryptic peptides do not recognize whole IRBP in vitro, and yet these lymphocytes must recognize the protein in vivo to initiate the autoimmune pathogenic process. We have examined here two hypothetical explanations for this dissociation: 1) It is possible that when IRBP is processed in vitro, immunodominant peptide determinants compete with the cryptic ones and inhibit their interaction with the MHC molecules on the APC. This explanation was ruled out here by the finding that the immunodominant peptide 1179-1191 ("W10") did not inhibit the response to a cryptic one, 1158-1180 ("R4"), when added at equivalent and even moderately higher concentrations. 2) The second hypothesis proposes that the cryptic antigenic sites are not generated from IRBP by the APC in vitro, whereas enzymes in the retina digest the protein to yield fragments that generate these antigenic sites upon processing by the APC. In line with this hypothesis, we have found that cleavage of IRBP by certain endoproteinases (Asp-N, Glu-C, or V-8) produced molecules that were recognized in culture by lymphocytes sensitized to the immunopathogenic but cryptic peptide R4. This study, therefore, describes a putative Ag processing mechanism that results in IRBP recognition and, consequently, the initiation of an autoimmune process by lymphocytes sensitized against a cryptic peptide. Furthermore, experiments with R4 and other cryptic peptides have shown that cleavage fragments of up to 38 residues in length can be presented by APC, to stimulate lymphocytes sensitized against these peptides. No responses were stimulated, however, by fragments of 75 or more residues. The data thus provide new insights into the processing and presentation of cryptic peptide determinants by APC.     

The existence of two completely distinct antigenic sites within a decapeptide.

A decapeptide (1182-1191) derived from the bovine interphotoreceptor retinoid-binding protein (IRBP) was found to contain two completely distinct antigenic sites when tested in Lewis rats. One site, localized in sequence 1182-1191, is the core of the immunodominant and highly uveitogenic determinant of IRBP. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at position 1182 is deleted. Lymphocytes sensitized against the first, larger site recognized all longer peptides within sequence 1169-1191, as well as whole IRBP. In contrast, lymphocytes sensitized against the second, short epitope recognized only two peptides, 1184-1191 and (to a lesser degree) 1183-1191. The responses to both sites were restricted by the same major histocompatibility complex (MHC) product (I-A), as shown by monoclonal antibody blocking and by the finding that the lymphocyte response to 1184-1191 was competitively inhibited by peptide 1181-1191. The unique finding of two completely distinct antigenic sites within a decapeptide could be explained by the hypothesis that peptides of the two sites combine with the MHC molecule on antigen-presenting cells by different configurations, thus forming two distinct antigenicities.     

A shared epitope of the interphotoreceptor retinoid-binding protein recognized by the CD4+ and CD8+ autoreactive T cells

We previously demonstrated that cultures of rat uveitogenic T cells rapidly become dominated by CD4+ cells, but activation of CD8+ autoreactive T cells also occurred during the in vitro culture of in vivo-primed T cells. In the present study, we show that the commonly used uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP) 1-20, generated both CD4+ and CD8+ autoreactive T cells in the C57BL/6 (B6) mouse and that this 20-mer contains at least two distinct antigenic epitopes. To determine whether the CD8 response was Ag-specific and whether CD4+ and CD8+ IRBP1-20-specific T cells recognize distinct antigenic epitopes, we prepared highly purified CD4+ and CD8+ T cells from IRBP1-20-primed mice and tested their proliferative response to a large panel of truncated peptides derived from IRBP1-20. The results showed that both CD4+ and CD8+ T cells recognized the same spectrum of peptides. In addition, peptides P10-18 were found to bind effectively to CD8+ IRBP1-20-specific T cells when complexed with recombinant H-2K(b) and also stimulate the proliferation and cytokine production of CD4+ IRBP1-20-specific T cells. Our results document for the first time that CD8+ and CD4+ autoreactive T cells display characteristic epitope recognition and they both recognize the same core epitope.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

Cryptic MBP epitope 1-20 is inducing autoimmune anterior uveitis without EAE in Lewis rats

Lewis rats immunized with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Although several cryptic epitopes of MBP have strong encephalitogenic and uveitogenic properties, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. In this study, we showed that acetylation of the N-terminal amino acid did not produce encephalitogenicity, did not enhance uveitogenicity, and did not improve T cell proliferation in Lewis rats. The cytokine production profile induced by MBP(1-20) immunization was consistent with a Th1 response. In MBP-injected rats and in peptide-injected rats, the frequency of the IFN-gamma-secreting cells in MBP(69-89)-stimulated T cells was significantly higher than the frequency of IFN-gamma-secreting cells in MBP(1-20)-stimulated T cells. However, similar numbers of IFN-gamma-producing specific cells were found in the eyes of MBP(69-89) and MBP(1-20) immunized rats. In these rats, the iris-infiltrating cells consisted of a much higher percentage of CD4(+) T cells expressing L-selectin (CD62L) than did those cells found in the spinal cord. The results demonstrate that MBP(1-20) is immunogenic and uveitogenic, although it induced only weak proliferation and weak Th1 reaction. The fact that T cells with the same specificity have different effects on target organs suggested that, in the eye and spinal cord, a distinct mechanism might mediate the recruitment of cells to these organs.     

Identification of T cell recognition sites in S-antigen: dissociation of proliferative and pathogenic sites

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.     

IL-22-induced regulatory CD11b+ APCs suppress experimental autoimmune uveitis

We have previously reported that IL-17(+) interphotoreceptor retinoid-binding protein (IRBP) 161-180-specific T cells have a strong pathogenic effect in experimental autoimmune uveitis (EAU) induced in B10RIII mice; however, this pathogenic activity is not solely attributable to the major cytokine, IL-17, produced by these cells. To determine whether other cytokines produced by Th17 cells show a stronger association with their pathogenic activity, we studied the role of IL-22 in EAU. IL-22 is one of the major cytokines produced by these cells. Our results showed that administration of small doses of IL-22 to EAU-susceptible mice significantly reduced the severity of EAU. In addition, mice treated with IL-22 generated decreased numbers of IFN-γ(+) and IL-17(+) uveitogenic T cells, but increased numbers of Foxp3(+) regulatory T cells. Mechanistic studies showed that the effect of the injected IL-22 was on CD11b(+) APCs, which expressed increased levels of IL-22R during induction of disease following immunization with uveitogenic Ag. In vitro IL-22 treatment of CD11b(+) APCs collected from Ag-primed mice resulted in increased expression of programmed death ligand-1 and the production of increased amounts of IL-10 and TGF-β. Moreover, IL-22-treated CD11b(+) APCs caused IRBP161-180-specific T cells to lose their uveitogenic activity and acquire immunosuppressive activity, which suppressed the induction of EAU by additional pathogenic IRBP161-180-specific effector T cells.     

Characterization of a suppressor cell line which downgrades experimental autoimmune uveoretinitis in the rat

A Ts lymphocyte line was isolated from spleens of rats primed with the retinal soluble Ag (SAg) in the anterior chamber of the eye. This line could inhibit in vitro SAg-driven proliferation of uveitogenic Th lymphocytes, in a radioresistant, Ag-independent manner. Adoptively transferred Ts line cells were found to downgrade experimental autoimmune uveoretinitis in actively immunized syngeneic recipients. The initial surface phenotype (OX8+) of the Ts line was unstable in vitro, however, the cells expressed suppressor function irrespective of phenotype. The mechanism of suppression did not appear to involve consumption of IL-2 or direct cytolysis of uveitogenic Th lymphocytes, but rather the production of a soluble suppressor factor. These findings may suggest an in vivo role for suppressor lymphocytes, capable of inhibiting primed Th cells, in the regulation of experimental autoimmune uveoretinitis.     

Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein.

Immunization with bovine interphotoreceptor retinoid-binding protein induces autoimmune uveitis in B10.A mice. We have examined whether this soluble retina-specific Ag can induce anterior chamber-associated immune deviation when injected into the anterior chamber (AC) of the eye, and whether this deviant immune response has any effect on uveitis is susceptible mice. The results of these experiments indicate that interphotoreceptor retinoid-binding protein (IRBP) injected intracamerally altered the subsequent immune response of B10.A mice such that a) they were not able to develop IRBP-specific delayed hypersensitivity, nor (b) were they able to express significant autoimmune uveitis following a uveitogenic regimen. Moreover, spleen cells from mice that received IRBP in the AC suppressed uveitis when adoptively transferred into naive recipients. The splenic suppressor cells were able to prevent autoimmune uveitis in recipient mice when administered after the uveitogenic regimen. Most important, IRBP-specific splenic cells from mice treated with IRBP in the AC when injected into mice with established uveitis caused an abrupt cessation of the intraocular inflammation. The ability of intracamerally-injected soluble Ag to induce suppressor T cells that act on the efferent limb of the immune response suggests that the anterior-chamber-associated immune deviation phenomenon may have physiologic relevance in terms of preservation of the integrity of ocular tissue and renders this approach particularly suitable for treating already established experimental autoimmune diseases of this type. These results are discussed in terms of other methods that have been devised experimentally to suppress and prevent autoimmune uveitis and encephalomyelitis.     

Identification of a potent new pathogenic site in human retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of the eye which can be induced in LEW rats by immunization with either human or bovine S-antigen (S-Ag). In previous reports, two nonimmunodominant pathogenic sites were found using synthetic peptides corresponding to conserved sequences at amino acid residues 303-314 and 286-297 of the bovine sequence. In this report, a 20-residue synthetic peptide encompassing amino acids 343-362 located near the C-terminus was found to be highly immunopathogenic in LEW rats. The onset of EAU was observed at as early as 8 days when high doses of a peptide-encompassing residues 343-362 were used. EAU was elicited with as little as 0.5 microgram of peptide per animal. Smaller peptides from this region were also tested for uveitogenicity, further refining the site to 13 amino acids. Uveitogenic T cell lines were made to this site in two ways; first, by the in vitro selection of a bulk T cell line raised to human S-Ag with peptide 343-362. Second, by the in vitro selection of a peptide-specific line from an animal immunized with peptide 352-364, which corresponds to the minimal uveitogenic site. Both of these lines adoptively transferred EAU to LEW rats, further establishing the pathogenicity of this site. A proliferative site distinct from, but overlapping, the uveitogenic site was also found. The potent uveitopathogenicity of peptides from this region indicates that it is a major pathogenic site responsible for EAU induced in LEW rats by immunization with human S-Ag.     

Hydrodynamic vaccination with DNA encoding an immunologically privileged retinal antigen protects from autoimmunity through induction of regulatory T cells

The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 microg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8(+) cells, IL-10, or TGF-beta. In contrast, depletion of CD25(+) cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4(+)CD25(high) T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3(+)CD4(+)CD25(+) regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.     

Elucidation and role of critical residues of immunodominant peptide associated with T cell-mediated parasitic disease.

Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234-246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234-246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.     

Rat T-cell lines specific to a nonimmunodominant determinant of a retinal protein (IRBP) produce uveoretinitis and pinealitis

Rat lymphocyte lines were established, with specificity toward two synthetic peptides derived from the interphotoreceptor retinoid-binding protein (IRBP), which specifically localizes in the retina and pineal gland. One of the peptides, R4, is immunopathogenic, producing experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in immunized rats, while the other peptide, R3, exhibits no detectable immunopathogenicity in rats. The cell lines carry surface markers specific for the helper/inducer subset of T-lymphocytes. When tested by the proliferation assay, the line cells demonstrated major histocompatibility-restricted vigorous responses against the immunizing (homologous) peptide, but failed to recognize the intact IRBP molecule. This finding is in line with other data indicating that peptides R3 and R4 are nonimmunodominant determinants of IRBP for the Lewis rat. Yet, the cell lines specific for R4 were highly immunopathogenic, producing EAU and EAP in naive rats at numbers as low as 0.25 x 10(6), with histopathological changes similar to those induced by active immunization with this peptide. The immunological capacity of the cell lines was further demonstrated by the finding that spleen cells from recipient rats of these lines responded well against the homologous peptides. The uniqueness of this system, in which lymphocytes specific toward a nondominant determinant are immunopathogenic, is underscored and the possible mechanisms of disease induction are discussed.     

In vivo priming of HIV-specific CTLs determines selective cross-reactive immune responses against poorly immunogenic HIV-natural variants

Degeneracy of the TCR repertoire might allow for cross-recognition of epitope variants. However, it is unclear how the first encounter with HIV Ags determines recognition of emerging epitope variants. This question remains crucial in the choice of HIV vaccine sequences given the virus variability. In this study, we individualized nine natural mutations within an HIV-Nef(180-189) epitope selected from several HIV-infected individuals. These variants of Nef(180-189) sequence display slightly different HLA-A2 binding capacities and stabilities and we have shown that only two induced a strong CTL response in vivo in HLA-A2 transgenic mice after a single injection. We demonstrated that priming with these two immunogenic variants generated a specific pattern of cross-reactive CTL repertoire directed against poorly immunogenic peptides. Thus, the range of peptide variants recognized by HIV-specific CTL depends upon the Ag encountered during primary immunization of CD8 lymphocytes. These data have practical implications in the development of cross-reactive vaccines against HIV.     

Anti-MIF activity of serum from a rabbit immunized with human B-lymphoid cell line migration inhibitory factor.

Sensitized parental strain lymphocytes can specifically inhibit proliferation of semiallogeneic erythroid cells grafted to irradiated syngeneic F1 hybrid mice. In H-2-incompatible strain combinations, inhibition is mainly caused by a direct attack of the lymphocytes against the grafted bone marrow. However, some indirect effect is also caused by a reaction of the lymphocytes against cells of the recipient. In this investigation we have shown that lymphocytes which have been sensitized against Mls-antigen-incompatible cells inhibit erythroid proliferation indirectly by reacting against host cells. The sensitized lymphocytes exhibit very little direct effect on the grafted bone marrow cells.     

Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis

Mycobacterium tuberculosis cell walls are likely to contain critical T cell Ag capable of inducing protective immunity against the development of tuberculosis in animal models. Therefore, we characterized cell wall-associated Ag that stimulate T lymphocytes in tuberculosis patients and clinically well tuberculin-positive individuals. A protein-peptidoglycan complex isolated from the M. tuberculosis cell wall had potent immunologic activity, evoking PBMC proliferative responses similar to those induced by sonicated whole M. tuberculosis. In order to characterize the immunoreactive protein determinants associated with the protein-peptidoglycan complex, T cell lines were established to cell wall Ag and used to probe M. tuberculosis proteins separated by SDS-PAGE. These T cell lines proliferated primarily to protein Ag of 10, 19, 23, 28, 30, 40 to 50, and 65 kDa. Cell wall-reactive T cell clones that recognized the 10-, 23-, 28-, and 30-kDa proteins as single bands on SDS-PAGE did so under reducing and nonreducing conditions, suggesting that these are not proteolytic fragments or subunits of larger protein aggregates. We propose that these protein monomers, when post-translationally complexed with peptidoglycan, are the key ingredients of the immunogenic protein-peptidoglycan complex. In order to assess the relationship of the cell wall-associated Ag to those secreted proteins from "early culture filtrates" of actively growing M. tuberculosis recently implicated in eliciting protective immunity, cell wall-reactive T cell clones were tested for their ability to recognize early culture filtrates. Results revealed that at least three proteins shared with the cell wall complex are contained within early culture filtrates. Our data indicate that antigenic determinants associated with the protein-peptidoglycan complex of the M. tuberculosis cell wall may be involved in protective immunity and hence are potential candidates for inclusion in an effective antituberculosis vaccine.     

Rejection of mouse sarcoma cells after transfection of MHC class II genes

Th cells are stimulated by peptide Ag presented in the context of MHC class II molecules. We have reasoned that immune responses against tumors may be more efficient if tumor cells were class II Ag positive, and thereby able to directly function as APC to stimulate tumor-specific Th cell proliferation. We have tested this hypothesis by using DNA-mediated gene transfer to generate syngeneic MHC class II Ag-expressing mouse Sal sarcoma cells (Sal/Ak transfectants). Autologous A/J mice challenged i.p. or s.c. with Sal/Ak transfectants do not develop tumors, whereas A/J mice challenged with the class II negative parental Sal tumor have a high tumor incidence. Furthermore, immunization of the autologous host with Sal/Ak transfectants completely protects against subsequent challenge with wild-type Sal cells. MHC class II-expressing tumor cells, therefore, stimulate an improved tumor-specific immune response, and the immunity is cross-reactive with the class II negative tumor. Inasmuch as the transfected MHC class II gene product is not functioning as a target molecule for autologous tumor rejection, the improved immunogenicity of the Sal/Ak cells is probably due to stimulation of a tumor-specific Th cell population. The increased immunogenicity of Sal/Ak cells is, therefore, probably the result of direct presentation of Sal tumor-associated Ag in the context of tumor cell MHC class II molecules to Th lymphocytes. These studies demonstrate that induction of tumor cell MHC class II Ag expression is a potential strategy for tumor-specific immunotherapy, and suggest that tumor immunity may be enhanced by improved Th cell generation.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.