H2-O expression in primary dendritic cells

H2-O is a nonpolymorphic class II molecule whose biological role remains to be determined. H2-O modulates H2-M function, and it has been generally believed to be expressed only in B lymphocytes and thymic medullary epithelial cells, but not in dendritic cells (DCs). In this study, we report identification of H2-O expression in primary murine DCs. Similar to B cells, H2-O is associated with H2-M in DCs, and its expression is differentially regulated in DC subsets as well as during cell maturation and activation. Primary bone marrow DCs and plasmacytoid DCs in the spleen and lymph nodes express MHC class II and H2-M, but not the inhibitor H2-O. In contrast, myeloid DCs in secondary lymphoid organs express both H2-M and H2-O. In CD8alphaalpha(+) DCs, the ratio of H2-O to H2-M is higher than in CD8alphaalpha(-) DCs. In DCs generated from GM-CSF- and IL-4-conditioned bone marrow cultures, H2-O expression is not detected regardless of the maturation status of the cells. Administration of LPS induces in vivo activation of myeloid DCs, and this activation is associated with down-regulation of H2-O expression. Primary splenic DCs from H2-O(-/-) and H2-O(+/+) mice present exogenous protein Ags to T cell hybridomas similarly well, but H2-O(-/-) DCs induce stronger allogeneic CD4 T cell response than the H2-O(+/+) DCs in mixed leukocyte reactions. Our results suggest that H2-O has a broader role than previously appreciated in regulating Ag presentation.     

Expression patterns of H2-O in mouse B cells and dendritic cells correlate with cell function

In the endosomes of APCs, the MHC class II-like molecule H2-M catalyzes the exchange of class II-associated invariant chain peptides (CLIP) for antigenic peptides. H2-O is another class II-like molecule that modulates the peptide exchange activity of H2-M. Although the expression pattern of H2-O in mice has not been fully evaluated, H2-O is expressed by thymic epithelial cells, B cells, and dendritic cells (DCs). In this study, we investigated H2-O, H2-M, and I-A(b)-CLIP expression patterns in B cell subsets during B cell development and activation. H2-O was first detected in the transitional 1 B cell subset and high levels were maintained in marginal zone and follicular B cells. H2-O levels were down-regulated specifically in germinal center B cells. Unexpectedly, we found that mouse B cells may have a pool of H2-O that is not associated with H2-M. Additionally, we further evaluate H2-O and H2-M interactions in mouse DCs, as well as H2-O expression in bone marrow-derived DCs. We also evaluated H2-O, H2-M, I-A(b), and I-A(b)-CLIP expression in splenic DC subsets, in which H2-O expression levels varied among the splenic DC subsets. Although it has previously been shown that H2-O modifies the peptide repertoire, H2-O expression did not alter DC presentation of a number of endogenous and exogenous Ags. Our further characterization of H2-O expression in DCs, as well as the identification of a potential free pool of H2-O in mouse splenic B cells, suggest that H2-O may have a yet to be elucidated role in immune responses.     

Facilitated antigen presentation by B cells expressing IgD when responding T cells express IgD-receptors

In vitro studies have confirmed that cognate interactions between T and B cells are required to demonstrate enhanced helper activity using T cells with upregulated IgD-receptors (IgD-Rs). We studied the mechanism by which IgD-R+ T cells facilitate antibody responses by examining whether T cells also benefit from their expression of IgD-R. Experiments were designed to determine whether upregulation of IgD-R on T cells facilitates antigen presentation by IgD+ B cells. Goat Ig-primed splenic T cells from BALB/c mice were tested for their ability to respond to antigen-presenting B cells treated with goat anti-mouse (GAM) IgM or GAM IgD. T cell responses to GAM IgM and GAM IgD presented by B cells were significantly higher when goat Ig-primed cells were induced to express IgD-R by exposure to oligomeric IgD compared with goat Ig-primed control T cells. This effect was inhibited when monomeric IgD was added to the cultures. No differences in T and IgD-R+ T cell responses were seen using adherent cells as APCs. B cells from IgD-/- mice were also tested. Such B cells present antigen to IgD-R+ T cells without promoting enhanced responses compared with B cells from heterozygous IgD+/- mice. These studies suggest that IgD may play a costimulatory role during antigen presentation. We conclude that when T cells are induced to express IgD-R, these lectin-like receptors can ligate B cell membrane IgD during antigen presentation to facilitate responses of each of the cells engaged in cognate interaction, yielding enhanced antigen-specific T cell and B cell responses.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.     

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.     

Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.     

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.     

Autoimmune myocarditis does not require B cells for antigen presentation.

T cells constitute the pathogenic effector cell population in autoimmune myocarditis in BALB/c mice. Using mice rendered deficient for B cells by a targeted disruption to the IgM transmembrane domain or by treatment with anti-IgM Ab from birth, we asked whether B cells are a critical APC in the induction of autoimmune myocarditis. B cell-deficient mice immunized with cardiac myosin develop myocarditis comparable in incidence and severity to that in wild-type mice, suggesting that autoreactive T cells that cause myocarditis in BALB/c mice are activated by macrophages or dendritic cells. Since it does not appear that presentation of cryptic epitopes is critical for the breakdown of self tolerance, potentially pathogenic T cells recognizing dominant myosin epitopes must have escaped tolerization. Either anatomic sequestration of cardiac myosin peptide-MHC complexes or subthreshold presentation of cardiac myosin peptides by conventional APC can explain the survival of these autoreactive T cells.     

Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells

Vaccination with dendritic cells (DCs) pulsed with antigenic peptides derived from various tumor antigens has great, but as yet significantly unrealized, potential in cancer treatment. Here, we describe a strategy for prolonged presentation of an MHC class I-restricted self-peptide on DCs through linkage of it to a cell penetrating peptide (CPP). DCs loaded with a peptide derived from tyrosinase-related protein 2 (TRP2) covalently linked to a CPP1 sequence retained full capacity to stimulate T cells for at least 24 h, completely protected immunized mice from subsequent tumor challenge, and significantly inhibited lung metastases in a 3-day tumor model. DCs pulsed with TRP2 alone failed to provide any of these protections. In addition, we demonstrate that both CD4+ and CD8+ T cells were required for potent antitumor immunity. This CPP-based approach may be generally applicable to enhance the efficacy of DC-based peptide vaccines against cancer and other diseases.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

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.     

TLR agonists downregulate H2-O in CD8alpha- dendritic cells

Peptide loading of MHC class II (MHCII) molecules is catalyzed by the nonclassical MHCII-related molecule H2-M. H2-O, another MHCII-like molecule, associates with H2-M and modulates H2-M function. The MHCII presentation pathway is tightly regulated in dendritic cells (DCs), yet how the key modulators of MHCII presentation, H2-M and H2-O, are affected in different DC subsets in response to maturation is unknown. In this study, we show that H2-O is markedly downregulated in vivo in mouse CD8α(-) DCs in response to a broad array of TLR agonists. In contrast, CD8α(+) DCs only modestly downregulated H2-O in response to TLR agonists. H2-M levels were slightly downmodulated in both CD8α(-) and CD8α(+) DCs. As a consequence, H2-M/H2-O ratios significantly increased for CD8α(-) but not for CD8α(+) DCs. The TLR-mediated downregulation was DC specific, as B cells did not show significant H2-O and H2-M downregulation. TLR4 signaling was required to mediate DC H2-O downregulation in response to LPS. Finally, our studies showed that the mechanism of H2-O downregulation was likely due to direct protein degradation of H2-O as well as downregulation of H2-O mRNA levels. The differential H2-O and H2-M modulation after DC maturation supports the proposed roles of CD8α(-) DCs in initiating CD4-restricted immune responses by optimal MHCII presentation and of CD8α(+) DCs in promoting immune tolerance via presentation of low levels of MHCII-peptide.     

Modulation of antigen presentation and B cell receptor signaling in B cells of beige mice

Binding of Ag by B cells leads to signal transduction downstream of the BCR and to delivery of the internalized Ag-BCR complex to lysosomes where the Ag is processed and presented on MHC class II molecules. T cells that recognize the peptide-MHC complexes provide cognate help to B cells in the form of costimulatory signals and cytokines. Recruitment of T cell help shapes the Ab response by facilitating isotype switching and somatic hypermutation, and promoting the generation of memory cells and long-lived plasma cells. We have used the beige (Bg) mouse, which is deficient in endosome biogenesis, to evaluate the effect of potentially altered Ag presentation in shaping the humoral response. We show that movement of the endocytosed Ag-BCR complex to lysosomes is delayed in Bg B cells and leads to relatively poorer stimulation of Ag-specific T cells. Nevertheless, this does not affect Bg B cell activation or proliferation when competing with wild-type B cells for limiting T cell help in vitro. Interestingly, Bg B cells show more prolonged phosphorylation of signaling intermediates after BCR ligation and proliferate better to low levels of BCR cross-linking. Primary Ab responses are similar in both strains, but memory responses and plasma cell frequencies in bone marrow are higher in Bg mice. Further, Bg B cells mount a higher primary Ab response when competing with wild-type cells in vivo. Thus, the intensity and duration of BCR signaling may play a more important part in shaping B cell responses than early Ag presentation for T cell help.     

Regulation of antigen presentation. II. Anti-Ig and IL-2 induce IL-1 production by murine splenic B cells

Murine splenic B cells did not constitutively express IL-1 activity. After culture with anti-Ig and T cell-conditioned media and then fixation, B cells expressed membrane IL-1 and were able to stimulate growth of the IL-1-dependent T cell clone D10. Expression of membrane IL-1 required stimulation of B cells for 2 days before fixation. Significant IL-1 activity was detectable in freeze-thaw lysates of identical B cell preparations by 12 h. B cells also released IL-1 into the culture media. In situ hybridization studies by using probes to murine IL-1 alpha and IL-1 beta genes supported these observations. Thus, messenger RNA for IL-1 alpha and IL-1 beta rose in parallel, were detected between 6 and 24 h of culture, and declined to low levels by 30 h. Despite the presence of mRNA for IL-1 alpha and IL-1 beta, only IL-1 alpha had functional activity as determined by the use of a mAb to IL-1 alpha. IL-2 was found to be an essential component of the T cell-derived supernatant. Although IL-4 or TNF did not induce significant B cell IL-1 expression, they both caused a modest, but reproducible enhancement when added in combination with IL-2. IFN-gamma, by contrast, partially inhibited IL-1 induction.     

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.     

MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A

The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment. However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process. The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules. An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins. To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A. Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas. No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells. Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins.     

Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures

Summary The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3–8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3–8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.     

Characterization of the signaling function of MHC class II molecules during antigen presentation by B cells.

In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.     

IL-2 production by B cells stimulated with a specific antigen

The ability of a specific antigen (Ag) to stimulate B cells to produce IL-2 was examined with a murine B lymphoma line, A20-HL, which expressed surface IgM specific for trinitrophenyl (TNP). The culture supernatant of A20-HL cells stimulated with TNP3.9-ovalbumin (-OVA) or anti-IgM goat IgG contained an activity which supported the proliferation of an IL-2-dependent T cell line, CTLL-2. Neither TNP3.9-OVA nor anti-IgM antibody stimulated the parent line, A20.2J, which did not bear TNP-specific sIg, whereas anti-mouse Ig rabbit IgG F(ab)2 did stimulate both A20-HL cells and A20.2J cells. The active material in the culture supernatant was identified as IL-2 based on the experiments in which the activity was inhibited by anti-IL-2 mAb, and IL-2 mRNA was expressed in A20-HL cells stimulated with TNP3.9-OVA or anti-IgM antibody. These results support the conclusion that a specific Ag can stimulate A20-HL cells to produce IL-2. For IL-2 production, TNP receptors on A20-HL cells have to be appropriately cross-linked, inasmuch as either TNP3.9-OVA or TNP6.7-OVA was much more effective than TNP1.2-OVA and TNP22.9-OVA in the induction of IL-2 production by A20-HL cells.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.