Characterization of MHC class II-presented peptides generated from an antigen targeted to different endocytic compartments.

We evaluated the capacity of the secretory pathway or of different endocytic compartments in B cell lines to generate MHC class II-presented peptides from the antigen ovalbumin (OVA). Sorting signals from the transferrin receptor (TFR), targeted a chimeric OVA fusion protein to early endosomes and led to the generation of 8 of 12 presented peptides. Sorting signals from the lysosome-associated membrane protein 1 (LAMP-1), targeted an OVA fusion protein to lysosomes, and led to the generation of 9 of 12 peptides. In contrast, OVA with only a signal sequence led to the generation of only 2 presented peptides. There were both qualitative and quantitative differences in the generation of peptides from the different fusion proteins, suggesting that multiple distinct compartments are involved in generating different epitopes. One peptide was presented better from the TFR fusion protein, while all others were presented better from the LAMP-1 construct. Twelve peptides were generated from exogenously supplied OVA, including 3 peptides that were not generated from any of the fusion proteins. Since most endogenously synthesized foreign antigens are rarely presented on class II molecules, these studies further suggest a strategy whereby antigens in DNA-based vaccines could be targeted to endocytic compartments to enhance immunogenicity.

Functional differences in the specific B-cell compartment in high or low antibody responder mice.

The role of antigen-presenting cells (APC) in quantitative antibody synthesis regulation was studied in mice genetically selected for high (HI) or low (LI) antibody response. Irradiated spleen cells and enriched specific B cells from HI and LI mice co-isogenic at H-2s locus, were compared for their capacity to present chicken ovalbumin (OVA) to specific T-cell hybridomas. Minor differences were observed between HI and LI mice when three distinct hybridomas were stimulated in the presence of OVA and splenic macrophages APC. These differences were totally abolished when APC were pulsed with OVAxAb complexes. Looking at the B-cell compartment, hybridoma IL-2 responses were similar when TNP primed B cells were pulsed with OVA. However, when OVA was targeted on TNP-specific enriched B cells by pulsing with TNP-OVA, the IL-2 production by the T-cell hybridomas was stronger in the presence of HI B cells than in the presence of LI B cells. These results strongly suggest that an efficient Ag handling/processing by specific B cells is a major component of the high Ab responder status in Biozzi mice.

Analysis of MHC class II presentation of particulate antigens of B lymphocytes.

To generate Ab responses to most protein Ags, B cells must first degrade proteins in endocytic compartments and then display antigenic peptides bound to MHC class II molecules. T helper lymphocytes recognize these complexes and stimulate the B cell to synthesize Ab. Although Ab play a key role in host defense against bacteria, it is believed that B cells are incapable of internalizing particulate Ags. However, we find that B lymphoblastoid cell lines and LPS-activated B lymphocytes can present particulate Ag up to 10(5)-fold more efficiently compared with soluble Ag. Moreover, particulate Ags are presented efficiently by unstimulated B cells when they bind to surface Ig. In comparison to B cells, macrophages in general presented particulate Ags 10- to 1000-fold more efficiently and could also present Ag from particles of a much wider range of sizes. We document by ultrastructural and immunofluorescence analysis that B lymphoblastoid cell lines bind and internalize these particles. The internalization and presentation of the particulate Ag is inhibited by cytochalasin B. In contrast, a similar morphologic analysis of normal lymphocytes demonstrated that while Ag beads are bound to the cell surface, they are internalized only rarely. These results suggest there may be both surface and intracellular pathways for the presentation of particulate Ags by B cells. Interestingly, for both macrophages and B cells, the epitopes generated from particulate and soluble Ags were not identical quantitatively or qualitatively, indicating that there are differences in how these forms of Ag are processed and presented.

Specific T-cell tolerance may reflect selective activation of lymphokine synthesis.

Selective T-cell unresponsiveness, as measured by interleukin 2 (IL-2) synthesis upon challenge with antigen, was induced in SJL mice by ovalbumin (OVA) in incomplete or complete Freund's adjuvant administered i.p. or s.c. Ten days later, the mice were given booster injections of 100 micrograms of OVA/complete Freund's adjuvant. On day 20, lymph node and spleen cells were challenged in vitro with serial dilutions of OVA. There was an antigen-specific dose-dependent down regulation of IL-2 production and T-cell proliferation in lymph node T cells. Concomitantly, 100 micrograms of OVA up regulated IL-4 and, to a lesser extent, interferon gamma (IFN-gamma) production, particularly by spleen T cells. Altogether, these data indicate that the drop of IL-2 production and T-cell proliferation, as well as the up regulation of IL-4 and IFN-gamma production, are complex manifestations of an evolving T-cell response. The maturation of the T-cell response leads to the production of different patterns of lymphokines, which may be significantly affected, as desired, by dosage, timing, and route of immunization, as well as by the choice of adjuvants.

Specific T-cell tolerance may be preceded by a primary response.

We have evaluated the ability of ovalbumin to induce T-cell-specific tolerance in SJL mice. A significant decrease of interleukin 2 in lymph-node culture supernatants from tolerant mice upon antigen stimulation was seen. Oral tolerization was less effective than i.p.- or s.c.-tolerization protocols. Transfer experiments of either splenic or lymph-node T cells from tolerant mice to naive mice definitely ruled out suppression as a mechanism involved in tolerant mice. Surprisingly, we found that, before the establishment of specific T-cell tolerance to ovalbumin, T cells from mice that will display tolerance were responsive and synthesized interleukin 2 upon antigen challenge in vitro. Thus, we concluded that anergy cannot account solely for the T-cell unresponsiveness in tolerant mice. Furthermore, although we cannot rule out the hypothesis that the T-cell unresponsiveness in tolerant mice can be explained by programmed cell death of ovalbumin-specific T cells, these data led us to speculate that T-cell "refractoriness" could explain the drop of interleukin 2 production in lymph-node T-cell culture supernatant from tolerant mice.

Heterogeneity in antigen processing by different types of antigen-presenting cells. Effect of cell culture on antigen processing ability.

The ability of normal B cells, peritoneal macrophages, and splenic APC to process and present OVA to a panel of T-T hybridomas with different specificities was investigated. In all cases, B cells were less efficient than unfractionated splenocytes in presenting OVA or its peptides. However, when the presentation of native Ag was compared to the presentation of peptides, it was obvious that there were marked differences in the ability of these two APC populations to generate different epitopes from OVA. Leupeptin inhibits the processing of selected epitopes from native OVA differently when it was presented by spleen cells or B cells, suggesting that these two APC populations differ in their protease content. The effect of in vitro culture on the ability of splenic and peritoneal APC to process OVA was also investigated. Native OVA presentation by macrophages and spleen cells was affected by in vitro culture, more for some epitopes than for other epitopes. In contrast, presentation of exogenous peptides by paraformaldehyde-fixed APC was either not affected by previous culturing for 3 days, or very much improved. Altogether, these data demonstrate that different epitopes on the same protein may be independently and differentially processed by B cells and spleen cells. Furthermore, the precise peptides that are produced may vary with the physiologic state of the APC.

Processing and presentation of ovalbumin in mice genetically selected for antibody response.

Lines of mice selected for high or low antibody production to sheep red blood cells (H-I and L-I) were studied for their ability to process and present ovalbumin to a panel of 12 T-T hybridomas in two different H-2 haplotypes. When H-I and L-I spleen cells were used as antigen-presenting cells, no difference could be observed in the peptide generation by these mice compared to H-2-compatible B.10.Q and B.10.S spleen cells, respectively. Neither normal splenic L-I B-cells nor L-I thioglycolate-induced peritoneal macrophages were defective at presenting native ovalbumin, to six and eight different I-As-restricted T-T hybrids, respectively. Altogether, these results differ from previous findings which had indicated a deficiency in the processing and presentation of antigen by the low line, L-I.

Diversity in MHC class II ovalbumin T cell epitopes generated by distinct proteases.

It is generally accepted that a limited number of T cell epitopes are generated by APC from an immunogenic protein. To ascertain the number of determinants on OVA recognized in the context of the H-2s haplotype, we generated 19 T-T hybridomas against OVA and H-2s and we synthesized 46 overlapping peptides spanning the entire protein. Eighteen T-T hybrids were stimulated by eight different peptides. The peptide recognized by one T cell hybrid was not identified. The effect of four protease inhibitors on the processing and presentation of OVA by the LS.102.9 B cell hybridoma seemed to implicate several groups of proteases in the processing of this Ag. Alkylation of cysteine residues with iodoacetic acid showed in a few cases a dramatic decrease in the capacity of OVA to stimulate T-T hybrids recognizing cysteine-free peptides. In contrast, two T-T hybrids recognizing cysteine containing peptides were not affected by the alkylation, suggesting that alkylation inhibited the processing of OVA without affecting peptide interaction with class II MHC molecules. These data demonstrate that the repertoire of peptides generated by APC from OVA is not limited to one or few immunodominant peptides, and results from the activity of several endopeptidases and/or exopeptidases. In addition, the structure of the Ag (native or denatured) was shown to affect the efficiency with which different epitopes are generated.

The generation of immunogenic peptides can be selectively increased or decreased by proteolytic enzyme inhibitors.

The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment.

Resistance to collagen-induced arthritis in Biozzi mice is not associated with T cell receptor V beta gene polymorphism.

H-I and H-II high antibody-responder Biozzi mice, which express the H-2q permissive haplotype, were shown to be sensitive and refractory to collagen-induced arthritis, respectively. To assess a possible role of T cell receptor (TcR) V beta gene deletion or polymorphism in the resistance to arthritis in H-II mice, the germinal structure of TcR V beta genes and their expression at the membrane level were compared in both lines. In contrast to H-2q refractory SWR mice, which exhibit a deletion of about 50% of TcR V beta gene segments. H-I and H-II lines have an identical and complete set of V beta genes and exhibit no difference in the average expression of V beta 6, V beta 8 and V beta 11 gene products on T cell surface. These results indicate that mechanisms other than V beta gene deletion or polymorphism can be involved in the resistance of H-2q-positive mice to experimental arthritis.

Induction of interleukin-3 by interleukin-1 in the absence of other exogenous stimuli.

We have previously reported that lipopolysaccharide (LPS) could induce the production of interleukin-3 (IL-3) by mouse spleen cells. In the present study, we show that recombinant human interleukin-1, in the absence of other stimuli, is able to induce the production of IL-3. IL-3 was detected in the supernatants of adult, although neither in young nor in nude mouse splenocytes and was assessed by its capacity to support the growth of the IL-3-dependent FDC-P2 cell line. The presence of IL-3 was antigenically confirmed with a monoclonal anti-IL-3 antibody. Both recombinant IL-1 alpha and IL-1 beta had similar potential for inducing IL-3 production. IL-3 activity was detected in the supernatants of cells cultured in the presence of 100 pg/ml IL-1; maximal IL-3 levels were obtained with 10-30 ng/ml IL-1. Kinetic studies of IL-1-induced IL-3 production indicated that 4-6 days of culture were required for optimal production, whereas 1-2 days were sufficient in cultures stimulated with concanavalin A. Recombinant IL-6 failed to induce significant amounts of IL-3, and TNF alpha induced only weak IL-3 production. GM-CSF but not M-CSF could lead to the appearance of IL-3 in spleen cell culture supernatants. Removal of macrophages decreased the production of IL-3 induced by LPS and GMF-CSF though did not affect the IL-3 production induced by IL-1. This observation suggests that IL-1 production might be an intermediate event in IL-3 production induced by LPS and GM-CSF through the activation of macrophages. IL-3 was detected in culture supernatants of B-cell-depleted splenocytes indicating that T-cells were the source of IL-3. Surprisingly T-cell-depleted populations could also produce IL-3 upon IL-1 stimulation. Preliminary experiments with an autoreactive CD4- CD8- V beta 8+ clone suggested that these cells might also be involved in the described IL-3 production.

Polymorphism of Tcrb and Tcrg genes in Biozzi mice: segregation analysis of a new Tcrg haplotype with antibody responsiveness.

Tcrb and Tcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriate V and C probes. No polymorphism of the Tcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a new Tcrg haplotype shared by L-I and L-II mice was identified and characterized by C gamma 1, 2, 3, C gamma 4, V gamma 1, 2, 3, V gamma 5, and V gamma 6 restriction fragment length polymorphisms (RFLPs). Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-I g genotype with the low responder status by analyzing g haplotypes among highest and lowest responder (H-I X L-I)F2 hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with the H-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and the Tcrg genotype was consistent with the heterogenous g haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the same Tcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F2 segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.