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.     

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.     

Induction of class I MHC-restricted, peptide-specific cytolytic T lymphocytes by peptide priming in vivo

The present study investigated the possibility that protein Ag fragments in the form of peptides could serve as the priming Ag in the generation of a MHC class I-restricted immune response. Trypsin-digested chicken ovalbumin (OVA-TD) fragments were used as the model Ag. The results demonstrate the peptides within OVA-TD, when injected into C57BL/6 mice, could prime T cells which lysed H-2b Ia-EL4 target cells in an OVA-TD-specific manner. In contrast to priming with OVA-TD, immunization of mice with intact OVA did not lead to generation of CTL against OVA-TD or OVA. Furthermore, target cells sensitized with intact OVA failed to be recognized by OVA-peptide-specific CTL indicating that the target cells serving as APC were unable to generate the relevant peptide determinants recognized by the T cells. These results support the idea that the processing pathway within APC for class I-restricted T cells may differ from that used for class II-restricted T cells. Using OVA-TD-specific CTL clones (phenotypically Thy 1+, CD8+, CD4-, Pgp-1+) isolated from primed animals to screen OVA-TD fractions separated by HPLC, two T cell peptide determinants were identified corresponding to OVA sequences 111-122 and 370-381. Both determinants were recognized by CTL clones in the context of the H-2Db molecule.     

Weak base amines can inhibit class I MHC-restricted antigen presentation

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.     

Generation of class I MHC-restricted T-T hybridomas

In this report we describe a system for the generation of functional, class I MHC-restricted, T-T hybridomas. The BW5147 cell line was transfected with the CD8 gene. BW5147 transfectants were obtained that stably expressed CD8 and this expression was maintained after somatic cell hybridization with activated T lymphocytes. To determine whether the stable expression of CD8 would facilitate the generation of class I MHC-specific T-T hybridomas, the transfected cells were fused with alloreactive T cells and the resultant hybrids were screened for their ability to produce lymphokines in response to antigenic stimulation. Somatic cell hybridizations with BW5147-CD8 transfectants give rise to a much higher frequency of class I MHC-specific T-T hybridomas relative to parallel fusions with BW5147. To determine whether the BW5147-CD8 transfectants would also support the generation of Ag-specific, class I MHC-restricted T-T hybridomas, they were fused with OVA-specific CTL. Several T-T hybrid clones were identified that produced lymphokines after stimulation with a transfected APC that was synthesizing OVA, or with a tryptic digest of OVA in the presence of syngeneic APC. The stimulation by Ag was MHC-restricted and mapped to the Kb molecule. An anti-CD8 mAb inhibited the stimulation of these hybridomas by Ag plus APC, whereas their stimulation by mitogen was unaffected. Cytolytic activity was not detected when several of the OVA-specific or alloreactive hybridomas were tested for their ability to kill target cells bearing the appropriate Ag. These results demonstrate that the BW5147-CD8 transfectants allow the generation of class I MHC-restricted T-T hybridomas. The potential utility of this system is discussed.     

Class I-restricted presentation occurs without internalization or processing of exogenous antigenic peptides

Previous studies have shown that glutaraldehyde-fixed cells can present fragmented, but not native, Ag to class II-restricted T cells. This presumably occurs via direct binding of peptides to class II molecules at the cell surface. More recently, it has been shown that viable target cells can present peptides and endogenous, but not exogenous, protein Ag in association with class I MHC molecules to CTL. We have derived CTL specific for a chicken OVA peptide (OVA258-276) recognized in association with H-2Kb. These CTL recognize target cells that endogenously synthesize OVA and cells "loaded" with native OVA but fail to recognize target cells in the presence of exogenous native OVA. Thus, OVA must be intracellularly located to be processed and presented for CTL recognition. It remains unclear, however, whether exogenous peptides require internalization and further processing by target cells or are able to associate directly with class I molecules at the cell surface for CTL recognition. We provide evidence that glutaraldehyde-fixed cells can present synthetic peptides to H-2Kb- and H-2Db-restricted CTL and that such presentation does not require internalization or processing. The peptides used range in size from 16 to 48 amino acids in length. In contrast, glutaraldehyde-fixed cells are incapable of presenting Ag to CTL specific for influenza nucleoprotein and OVA if the cells are fixed within 1 h of viral influenza infection or loading with OVA. Thus, CTL recognition of antigenic peptides appears to occur via direct binding of peptides to class I molecules at the cell surface and does not require any intracellular processing events.     

Identification of new epitopes from four different tumor-associated antigens: recognition of naturally processed epitopes correlates with HLA-A*0201-binding affinity.

Forty-two wild-type and analogue peptides derived from p53, carcinoembryonic Ag, Her2/neu, and MAGE2/3 were screened for their capacity to induce CTLs, in vitro, capable of recognizing tumor target lines. All the peptides bound HLA-A*0201 and two or more additional A2 supertype alleles with an IC(50) of 500 nM or less. A total of 20 of 22 wild-type and 9 of 12 single amino acid substitution analogues were found to be immunogenic in primary in vitro CTL induction assays, using normal PBMCs and GM-CSF/IL-4-induced dendritic cells. These results suggest that peripheral T cell tolerance does not prevent, in this system, induction of CTL responses against tumor-associated Ag peptides, and confirm that an HLA class I affinity of 500 nM or less is associated with CTL epitope immunogenicity. CTLs generated by 13 of 20 of the wild-type epitopes, 6 of 9 of the single, and 2 of 5 of the double substitution analogues tested recognized epitopes generated by endogenous processing of tumor-associated Ags and expressed by HLA-matched cancer cell lines. Further analysis revealed that recognition of naturally processed Ag was correlated with high HLA-A2.1-binding affinity (IC(50) = 200 nM or less; p = 0.008), suggesting that high binding affinity epitopes are frequently generated and can be recognized as a result of natural Ag processing. These results have implications for the development of cancer vaccines, in particular, and for the process of epitope selection in general.     

Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.     

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.     

The naive repertoire of human T helper cells specific for gp120, the envelope glycoprotein of HIV.

The envelope glycoprotein of HIV gp120 is a T cell Ag in experimental animals and in humans infected with HIV or deliberately immunized with gp120 in various forms. Inasmuch as T cell responses result from the interaction of Ag processed and presented by APC with the unprimed T cell repertoire, we have investigated the human T cell repertoire specific for gp120 in seronegative, normal individuals. T cell lines and clones specific for HIV gp120 were generated by repeated in vitro stimulation of peripheral blood T lymphocytes with gp120-pulsed APC, followed by IL-2 expansion. We observed that the T cell response to whole gp120 involved single restricted immunodominant epitopes in gp120 that differ between responding individuals. Focusing of the response to limited regions of gp120 when the whole Ag is used for priming suggests that one or more adjacent epitopes are immunodominant and mask responses to "immunorecessive" epitopes. We have been able to generate primary in vitro responses to recessive epitopes by stimulation in vitro with synthetic peptides of gp120. The results indicate that a much broader T repertoire can be detected when individual peptides are used for priming in vitro rather than gp120. This information has important implications for the development of vaccination protocols aimed at eliciting diverse immune responses to "immunorecessive" regions of envelope glycoprotein.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.     

DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323-339 epitope

The OVA323-339 epitope recognized by DO11.10 (H-2d) and OT-II (H-2b) T cells was investigated using amino- and carboxy-terminal truncations to locate the approximate ends of the epitopes and single amino acid substitutions of OVA323-339 to identify critical TCR contact residues of the OVA323-339 peptide. DO11.10 and OT-II T cells are both specific for a C-terminal epitope whose core encompasses amino acids 329-337. Amino acid 333 was identified as the primary TCR contact residue for both cells, and amino acid 331 was found to be an important secondary TCR contact residue; however, the importance of other secondary TCR contact residues and peptide flanking residues differ between the cells. Additional OVA323-339-specific clones were generated that recognized epitopes found in the N-terminal end or in the center of the peptide. These findings indicate that OVA323-339 can be presented by I-Ad in at least three binding registers. This study highlights some of the complexities of peptide Ags such as OVA323-339, which contain a nested set of overlapping T cell epitopes and MHC binding registers.     

Processing of exogenous liposome-encapsulated antigens in vivo generates class I MHC-restricted T cell responses.

Acid-sensitive liposomes have been developed for cytosolic delivery of encapsulated substances. We now demonstrate delivery of liposome-encapsulated Ag into the class I MHC Ag processing pathway in peritoneal macrophages in vitro using several types of acid-sensitive liposomes, including those composed of dioleoylphosphatidylethanolamine (DOPE)/palmitoylhomocysteine, DOPE/cholesterol hemisuccinate, DOPE/dioleoylsuccinylglycerol, and DOPE/dipalmitoylsuccinylglycerol. Our previous studies showed that acid-resistant liposomes (dioleoylphosphatidylcholine/dioleoylphosphatidylserine) did not engender class I-mediated presentation in vitro. However, in vivo immunization with OVA encapsulated in acid-resistant as well as acid-sensitive liposomes generated class I MHC-restricted T cell responses, as determined by subsequent in vitro cytotoxicity assays using OVA-transfected target cells. Target lysis by these cells was OVA- and class I MHC (Kb)-specific. This response was not generated by immunization with equivalent amounts of soluble OVA. Thus, a pathway for in vivo class I processing of Ag encapsulated in acid-resistant liposomes has been missed in vitro, perhaps because it is dependent on specific populations of APC or interactions between cells that have not been reconstituted in vitro. This pathway may explain the ability of many exogenous particulate Ag (liposomes, bacteria, parasites, and mammalian cells) to generate class I MHC-restricted T cell responses.     

Processing of tetanus toxin by human antigen-presenting cells. Evidence for donor and epitope-specific processing pathways

Human T cell clones specific for epitopes 830-843 and 947-967 of tetanus toxin can be differentially activated in vitro when APC (PBL or LCL) from different donors are pulsed with tetanus toxin. Although PBL tested do not seem to exhibit substantial differences in the number of precursor T cells specific for these epitopes, APC from the same donors activate clone KT-2 specific for peptide 830-843 but not clone KT-30 specific for peptide 947-967. These APC express the proper restriction element because they can present the corresponding synthetic peptides. The failure to present a particular epitope might, however, be explained by the absence or presence of a protease(s) required for Ag presentation that may vary for different epitopes. Indeed, the protease inhibitor leupeptin was found to inhibit activation of KT-2 but not KT-30 T cell clone by the KK.35 B cell line normally capable of presenting either epitope. In summary, these data suggest that tetanus toxin processing and epitope formation by APC is distinct in different donors and for different epitopes.     

MHC class I-restricted presentation of exogenous antigen by thymic antigen-presenting cells in vitro and in vivo.

We have used a T-T hybridoma, RF33.70, to detect the MHC class I-restricted presentation of exogenous native OVA by thymic APC in vitro. Presentation of OVA with class I molecules by thymic APC requires intracellular processing. Phenotypic analyses indicated that low bouyant density, MHC class II+, FcR+ cells are capable of using this presentation pathway. In order to determine whether thymic APC have this function in vivo, thymic APC were isolated from mice after i.v. injection of native OVA. We find that OVA is presented in association with MHC class I, but not class II, molecules in the thymus. This is in contrast to splenic APC, which present exogenous OVA with both class I and class II molecules under these conditions. Our findings have implications for the repertoire of self-peptides that might be presented by thymic APC to developing T lymphocytes.     

Antigen processing and the human T cell receptor repertoire for insulin

Three human T cell lines specific for the A loop of beef insulin were studied to determine the requirements for Ag processing. The data show that the conformation of the A loop of insulin is required for recognition and that the B chain of insulin per se is not necessary for this response. Processing of native insulin was required for responses of all three T cell lines; however, each displayed a different pattern of sensitivity to inhibition of processing and aldehyde fixation of APC. A peptide comprised of two disulfide-linked A chains was partially stimulatory when presented by fixed APC whereas A chain monomers and disulfide-linked A and B chain peptides were not. The response to native insulin, peptides, and A chain dimers was sensitive to chloroquine suggesting that none of these moieties is the terminal processed peptide recognized by insulin immune T cells. The unique patterns of fine specificity, processing requirements, and recognition of aldehyde-fixed antigen-MHC for each T cell line suggest the hypothesis that Ag processing leads to heterogeneity of the T cell repertoire for a single epitope of insulin.     

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.     

The role of endoplasmic reticulum-associated aminopeptidase 1 in immunity to infection and in cross-presentation

Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.