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We previously reported that cells contain endogenous adjuvants in their cytoplasm that when released markedly augment the generation of CD8 T cell responses. In the present study we found that these cytosolic adjuvants similarly augmented the generation of CD4 T cell responses, and therefore must be affecting a step that is common to the generation of helper and cytotoxic T cell responses. The endogenous adjuvants work differently than a classical bacterial adjuvant, Freund's complete adjuvant. Remarkably, they stimulate the immune response to particulate and cell-associated antigens but not to the same antigens in soluble form. To gain insight into the underlying mechanisms for these effects, we studied the effect of the cytosolic adjuvants on the fate of particulate antigens and antigen-presenting cells (APC) in vivo. Injection of cytosol by itself causes no detectable change in the number or phenotype of APC in draining lymph nodes. However, co-injection of cytosol and fluorescent particles leads to the increased accumulation in the draining lymph node of dendritic cells and macrophages containing phagocytosed particles and expressing high levels of costimulatory molecules. Therefore, cell injury releases cytosolic factors that selectively enhance immune surveillance of particulate antigens released from dying cells by stimulating APC in tissues to acquire these antigens, mature and migrate to lymph nodes. This process will allow the immune system to rapidly detect and respond to viral infections and tumors.

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.

The molecular nature of the defect in two mouse antigen processing-defective cell lines was examined. Both mutants were derived from the A20 (BALB/c, H2d) B cell line, and both were found to have defects in the H2-DMa gene. Mutant 3A5 exhibits severely reduced amounts of H2-DMa message, and no detectable DMalpha protein. cDNA sequence revealed a C-->T transition at nucleotide 118, introducing a premature stop codon in exon 2 of the H2-DMa gene. In contrast, mutant 2A2 exhibits reduced but detectable levels of H2-DMa message and DMalpha protein only after treatment with IL-4, which induces the expression of both the H2-DMa and the H2-DMb genes in B cells. In this mutant the cDNA sequence revealed a missense mutation in exon 3 resulting in the conversion of a conserved proline residue in the Ig-like domain to serine. Stable transfection with full-length H2-DMa cDNA reconstitutes the antigen processing capacity of both mutants, as demonstrated by the ability to present native antigen to T cell clones, and by restored class II SDS stability.

There is an antigen presenting cell (APC) in the lymphoid organs capable of presenting exogenous antigen (Ag) with major histocompatibility complex (MHC) class I molecules. This study was initiated to isolate clones of these APC to definitively establish their phenotype and to further study their properties. Murine bone marrow macrophages (BM M psi) were immortalized by overexpression myc and raf oncogenes. Five BM M psi cell lines were generated that are phagocytic and expressed at their surface M psi differentiation Ag. All five cell lines processed and presented exogenous ovalbumin (OVA) with MHC class I molecules. They all presented OVA-linked to a phagocytic substrate 10(2)-10(4)-fold more efficiently than soluble Ag. Clonal isolates of two of the M psi cell lines had an identical phenotype and functional properties as the uncloned lines. These results definitively establish that M psi are APC with the capacity of presenting exogenous Ag with MHC class I molecules. Interferon (IFN)-gamma interleukin-4, granulocyte-macrophage colony stimulating factor and lipopolysaccharide either alone or in combination induced little or no augmentation and in some cases decreased presentation of exogenous OVA with MHC class I. In contrast, all of M psi activating factors increased MHC class I expression. Moreover, IFN-gamma increased the presentation of cytosolic OVA, demonstrating differences between the presentation of cytosolic Ag versus exogenous Ag with MHC class I. Finally, some lines constitutively processed and presented exogenous OVA with MHC class II while others only presented after stimulation with IFN-gamma. These results demonstrate that the pathways involved in the presentation of exogenous Ag with MHC class I and class II are independently regulated and that a cloned cell is capable of presenting exogenous Ag through both pathways.

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.

Ly-6A.2 (T cell-activating protein, TAP) and Thy-1 are glycosyl-phosphatidyl-inositol (GPI)-anchored proteins expressed on the surface of murine T lymphocytes. We have found that Ly-6A.2 (TAP) and Thy-1 are internalized by T cells. In the present study we have investigated whether these GPI-anchored proteins enter cells by endocytosis through coated pits. Two lines of evidence argue against the involvement of coated pits in the internalization of Ly-6A.2 (TAP) and Thy-1. First, drugs that effectively blocked the endocytosis of transferrin receptor and H-2 class I molecules, (which are known to be internalized via coated pits) did not inhibit the internalization of the GPI-anchored proteins. Second, in ultrastructural analyses, Ly-6A2 (TAP) and Thy-1, in contrast to the transferrin receptor, were rarely found in coated pits or vesicles. These observations suggest that the GPI-anchored proteins on T lymphocytes are internalized by a distinct pathway that does not involve endocytosis through coated pits.

The ability of antigen-specific murine B lymphocytes to present antigen to a normal (nontransformed) antigen-specific, interleukin (IL)1-dependent T cell clone and to heterogeneous T lymphocytes was investigated. Hapten-specific lymphocytes present protein antigens to both the D10G.4.1 T cell clone and heterogeneous immune T cells. When the antigen bears an epitope recognized by the specific B cell, the subsequent presentation of this antigen is 10(2)-10(4)-fold more efficient, as compared to the same, but nonhaptenated protein. The requisite B lymphocyte binds hapten, is radiosensitive and is nonadherent to plastic. The hapten-specific antigen presentation is blocked by antibodies to the surface Ig receptor, while the presentation of unmodified protein is unaffected. These observations are identical to our findings in studies of B cell antigen presentation to T-T hybridomas and therefore demonstrate the generality of the immunoglobulin-dependent pathway of presentation. However, in contrast to the results with T-T hybridomas, the specific B lymphocytes are necessary, but not sufficient, to activate IL 1-dependent T cells. A third cell is required for both B lymphocyte immunoglobulin-dependent and nonspecific antigen-presentation. This cell is radioresistant, plastic adherent and of low density. The requirement for this third cell can be circumvented by supplementing cultures with highly purified IL 1. These results demonstrate that the remarkably efficient ability of B lymphocytes to present antigen is operative with factor-dependent T lymphocytes. Furthermore, conventional accessory cells also appear to play a role in this process, which is consistant with their known requirement in antigen-specific T-B interactions in the generation of antibody responses.