Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.     

Tumor antigen presentation by murine epidermal cells

The ability of epidermal Langerhans cells to present Ag for CD4-dependent immunity is well documented, and it has been hypothesized that Langerhans cells participate in the generation of immunity against incipient epidermal neoplasms by presentation of tumor-associated Ag in situ. This study examined the ability of murine epidermal cells (EC) to present tumor-associated Ag for the induction of in vivo antitumor immunity. Murine epidermal cells were deleted of Thy-1-bearing cells, cultured in 50 U/ml granulocyte-macrophage-CSF for 14 to 18 h, and pulsed with tumor fragments (TF) derived from S1509a-fibrosarcoma cells. These TF-pulsed EC were injected s.c. into syngeneic recipients at weekly intervals for a total of three immunizations and challenged with viable S1509a tumor cells 1 wk after the last immunization. Control animals received TF-pulsed allogeneic EC or EC treated identically but not pulsed with TF. EC that were pulsed with tumor cell fragments were able to induce protective immunity to tumor growth in vivo and to immunize for a significant delayed-type hypersensitivity response to injected tumor cells. The induction of antitumor immunity with TF-pulsed EC was genetically restricted, and culture of EC in granulocyte-macrophage-CSF was required for development of significant immunity. Furthermore, deletion of I-A+ cells by antibody and complement-mediated lysis eliminated the generation of immunity. Thus, I-A+ epidermal cells are capable of presenting S1509a tumor Ag for the generation of protective antitumor immunity in vivo.     

Biochemistry and biology of antigen presentation by macrophages

The presentation of antigen by macrophages has been studied. We have shown that globular proteins must be processed in endocytic vesicles of low pH prior to presentation. Some of the structural requirements of one such processed peptide have been determined, as has the affinity for Ia of that peptide. Finally, we have shown that a membrane-associated form of interleukin-1 is also required for presentation of processed antigen to T cells.     

Inability of bm14 mice to respond to Theiler's murine encephalomyelitis virus is caused by defective antigen presentation, not repertoire selection

Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.     

Intracellular Salmonella inhibit antigen presentation by dendritic cells

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4(+) as well as CD8(+) T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.     

The mannose receptor fails to enhance processing and presentation of a glycoprotein antigen in transfected fibroblasts

One function proposed for the mannose receptor found on dendritic cells as well as on macrophages and hepatic endothelial cells is in enhancing uptake and processing of glycoprotein antigens for presentation by major histocompatibility complex (MHC) class II molecules. In this study, a direct assessment of the possible role of the mannose receptor in this process was made in the absence of other endocytic receptors that can internalize glycoproteins. Presentation of RNase A and B peptides was compared in transfected fibroblasts coexpressing the mannose receptor and MHC class II molecules. RNase B bears a high-mannose oligosaccharide and is a ligand for the mannose receptor, whereas RNase A is not glycosylated and is taken up by pinocytosis. Incubation of RNase A or B with the transfected cells resulted in identical stimulation of ribonuclease-specific T cells, indicating that endocytosis of the glycosylated protein by the mannose receptor does not enhance presentation of this antigen. The postulated role of the mannose receptor in presentation of glycoprotein-derived antigen is reevaluated in light of these results.     

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

BACKGROUND: Transplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC). MATERIALS AND METHODS: We used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC. RESULTS: The majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed. CONCLUSION: In summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.     

Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes

T lymphocytes and macrophages from Listeria-infected mice were used to evaluate the processing and presentation of live Listeria monocytogenes in vitro. Antigen presentation to T cells was quantitated by interleukin-2 production. In contrast to inert antigens such as heat-killed Listeria, live bacteria were processed and presented poorly. To evaluate the role of hemolysin (Hly), we used isogenic pairs of Hly+ and Hly- Listeria as antigens. In contrast to live Hly- bacteria, which were presented as well as heat-killed Listeria, live Hly+ bacteria were presented poorly. Hly+ bacteria also inhibited the presentation of heat-killed Listeria. This effect was apparent with as few as 10 bacteria/macrophage and was not due to loss of macrophage viability or decreased Ia expression after exposure to the live bacteria. With respect to murine listeriosis, the LD50 values for the Hly- strains were at least 1000 times higher than those for the Hly+ strains. These results suggest that the ability of Hly+ bacteria to inhibit antigen processing and presentation may be an important determining factor in Listeria infection and immunity.     

Autophagy and antigen presentation

CD4(+) T cells co-ordinate adaptive immunity and are required for immunological memory establishment and maintenance. They are thought to primarily recognize extracellular antigens, which are endocytosed, processed by lysosomal proteases and then presented on major histocompatibility complex (MHC) class II. However, recent studies have demonstrated that viral, tumour and autoantigens can gain access to this antigen presentation pathway from within cells by autophagy. This review will discuss the autophagic pathways that contribute to endogenous MHC class II antigen processing. Furthermore, potential characteristics of autophagy substrates, qualifying them to access these pathways, and regulation of autophagy will be considered. Finally, I will suggest how antigen presentation after autophagy might contribute to immune surveillance of infected and transformed cells.     

Murine gammaherpesvirus-68 inhibits antigen presentation by dendritic cells

Dendritic cells (DCs) play a central role in initiating adaptive immunity. Murine gammaherpesvirus-68 (MHV-68), like many persistent viruses, infects DCs during normal host colonization. It therefore provides a means to understanding what host and viral genes contribute to this aspect of pathogenesis. The infected DC phenotype is likely to depend on whether viral gene expression is lytic or latent and whether antigen presentation is maintained. For MHV-68, neither parameter has been well defined. Here we show that MHV-68 infects immature but not mature bone marrow-derived DCs. Infection was predominantly latent and these DCs showed no obvious defect in antigen presentation. Lytically infected DCs were very different. These down-regulated CD86 and MHC class I expression and presented a viral epitope poorly to CD8(+) T cells. Antigen presentation improved markedly when the MHV-68 K3 gene was disrupted, indicating that K3 fulfils an important function in infected DCs. MHV-68 infects only a small fraction of the DCs present in lymphoid tissue, so K3 expression is unlikely to compromise significantly global CD8(+) T cell priming. Instead it probably helps to maintain lytic gene expression in DCs once CD8(+) T cell priming has occurred.     

Amplification mediated by polyomavirus large T antigen defective in replication.

The polyomavirus large T antigen promotes homologous recombination at high rates when expressed in rat cells carrying the viral replication origin and two repeats of viral DNA sequences stably integrated into the cellular genome. Recombination consists of both reciprocal and nonreciprocal events and is promoted by mutants defective in the initiation of viral DNA synthesis (L. St-Onge, L. Bouchard, and M. Bastin, J. Virol. 67:1788-1795, 1993). We have extended our studies to a rat cell line undergoing amplification of the viral insert. We show that large T antigen promotes amplification independently of its replicative function but that its origin-specific DNA binding activity is not sufficient to promote homologous recombination.     

Differential antigen presentation by cloned populations of mouse splenic macrophages

Soft agar colonies of mouse splenic macrophages differ in their ability to process and present complex Ag to T cell hybridomas. To determine if the basis for this differential activity was the synthesis of molecules that might interfere with the activity of either the hybridoma or the indicator cells used for the bioassay of IL-2, culture supernatants were compared from Ag-presenting and nonpresenting cultures for their content of suppressor activity, using mitogen-treated mouse SC. No correlation was found between a colony's Ag-presenting activity and its secretion of suppressor factors, nor did colonies unable to present Ag release factors that interfered with the detection of IL-2. In a second approach, paired subcultures from individual colonies were tested for their ability to present, to the same hybridoma, both native Ag and the "preprocessed" peptide of the Ag. The presentation of native Ag was restricted to the progeny of a minority of the cloned macrophage progenitors, but all of the progeny cultures presented the peptide. Together, these results suggest that the basis for differential Ag presentation may be in the manner in which the cloned macrophages degrade and process ingested Ag.     

Antigen stability controls antigen presentation

We investigated whether protein stability controls antigen presentation using a four disulfide-containing snake toxin and three derivatives carrying one or two mutations (L1A, L1A/H4Y, and H4Y). These mutations were anticipated to increase (H4Y) or decrease (L1A) the antigen non-covalent stabilizing interactions, H4Y being naturally and frequently observed in neurotoxins. The chemically synthesized derivatives shared similar three-dimensional structure, biological activity, and T epitope pattern. However, they displayed differential thermal unfolding capacities, ranging from 65 to 98 degrees C. Using these differentially stable derivatives, we demonstrated that antigen stability controls antigen proteolysis, antigen processing in antigen-presenting cells, T cell stimulation, and kinetics of expression of T cell determinants. Therefore, non-covalent interactions that control the unfolding capacity of an antigen are key parameters in the efficacy of antigen presentation. By affecting the stabilizing interaction network of proteins, some natural mutations may modulate the subsequent T-cell stimulation and might help microorganisms to escape the immune response.     

Host glycans and antigen presentation

The cell-mediated adaptive immune response depends upon the activation of T cells via recognition of antigen in the context of a major histocompatibility complex (MHC) molecule. Although studies have shown that alterations in T cell receptor glycosylation reduces the activation threshold, the data on MHC is far less definitive. Here, we discuss the data on MHC glycosylation and the role the glycans might play during the adaptive host response.