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.     

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.     

Probing the structure of processed antigen by using biotin and avidin. MHC-dependent inhibition of responses to selected biotinyl-insulin derivatives

Current models suggest that Ag undergoes proteolytic cleavage in APC and that resultant peptide fragments associate with class II histocompatibility glycoproteins before recognition by helper T cells. Little direct information is available concerning the physical structure and membrane association of Ag processed under physiologic conditions. A model system, employing a series of biotinylated insulin derivatives, was used to examine the domains of Ag that are presented by APC. We reasoned that avidin should block the response of T cells to a given derivative only if biotin is retained on the functionally relevant form of Ag after processing. By utilizing derivatives modified at selected sites one should be able to determine whether specific sites remain after processing. By using F1 APC pulsed with biotinyl-insulin derivatives modified through the free amino groups of the A1, B1, or B29 amino acids, and T cell hybridomas restricted to I-Ad or I-Ab, we found that avidin inhibited the I-Ad-restricted response to A1, but not B1 or B29 derivatives. By contrast, specific inhibition of the I-Ab-restricted response was observed by using all three derivatives. These results suggest that the processed form of insulin recognized in association with I-Ab is largely intact and includes residues from both chains (A1, B1, and B29). The differential inhibition observed by using T cells restricted to different class II alleles demonstrates that processed Ag associated with I-Ab differs in conformation or structure from that associated with I-Ad. This experimental approach should prove valuable in characterizing the actual structure of processed Ag recognized by T cells.     

Time dependence of B cell processing and presentation of peptide and native protein antigens

Th cell recognition of globular proteins requires the uptake and intracellular processing of the native Ag by an APC to produce a peptide fragment containing the T cell antigenic determinant, which is recognized in conjunction with Ia. This report describes the time course of the processing and presentation of a soluble globular protein Ag, pigeon cytochrome c (Pc), and of the presentation of a C-terminal peptide fragment of Pc, residues 81 to 104 (Pc 81-104), which does not require processing. Splenic B cells, acting as APC, require 6 to 8 h incubation with native Pc to process and present it to an I-Ek-restricted Pc-specific T cell hybrid, resulting in the secretion of IL-2. Moreover, the time required for B cells to process Pc is the same whether the Ag is taken up by nonspecific fluid phase pinocytosis or by binding to surface Ig. Once processed, Ag is lost from the B cell surface by 8 to 12 h, although when provided with fresh Pc, the same B cells are still capable of processing and presenting. In contrast to native Pc, only 1 to 2 h are required for the peptide fragment Pc 81-104 to become associated with B cells in a stimulatory fashion, and this time is similar for live and paraformaldehyde-fixed B cells, which cannot internalize or process the peptide. Washed free of excess peptide after 2 h, B cells lose their ability to stimulate T cells by 8 to 12 h, with a time course indistinguishable from that for the loss of processed native Pc. Prolonged incubation of B cells with the peptide for 18 to 24 h results in a dramatic loss of the ability to present Pc 81-104. Even when provided with fresh Pc or Pc 81-104, these cells have diminished ability to present these Ag. This loss is selective, inasmuch as these B cells remain equivalent to untreated B cells in the presentation of an unrelated Ag, OVA, to an I-Ak-restricted specific T cell. However, the ability to present another I-Ek-restricted antigenic peptide of the D glycoprotein of HSV to its specific T cell is also diminished. Loss of activity is observed after incubation only with the peptide and not with the native protein and is not due to a depletion of the antigenic peptide from the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

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.     

Constitutive competition by self proteins for antigen presentation can be overcome by receptor-enhanced uptake

The cells recognize a bimolecular ligand composed of a self Ia molecule and a fragment of foreign Ag that has been processed by an APC. The effect of self proteins on the processing and presentation of foreign Ag was examined in order to ascertain the mechanisms for competition between foreign and self Ag. How this competition can be overcome to allow an efficient immune response was also examined. Normal mouse serum proteins (NMS) compete for the processing and presentation of the foreign Ag bovine RNase by APC. This competition could have occurred at any of three levels in the APC: 1) Ag uptake, 2) Ag processing, or 3) the binding of Ag to an Ia molecule. No competition for either the uptake or the processing of RNase by self proteins could be demonstrated. However, self peptides do compete with foreign Ag by binding directly to Ia molecules, as has been shown previously. Thus, the observed inhibition by NMS of Ag presentation occurred because of competition for binding to the Ia molecule. We hypothesized that during the generation of an immune response this competition is overcome by enhanced uptake of foreign Ag. To test this, we compared the ability of NMS to compete for the presentation of RNase when it entered the APC via fluid-phase pinocytosis or through receptor-mediated uptake via the mannose receptor. When the RNase entered the APC through the mannose receptor, the ability of NMS to compete was dramatically reduced. Thus, self proteins constitutively compete for the presentation of foreign Ag at the level of binding to an Ia molecule, and this competition can be overcome by receptor-mediated uptake of the Ag.     

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.     

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.     

Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen.

Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.     

Kinetics of MHC-antigen complex formation on antigen-presenting cells

With the use of flow cytometry, we recorded changes in intracellular ionized calcium [Ca2+]i of Indo-1 loaded T cells that were triggered by contact with APC. This rapid readout of TCR perturbation enabled us to monitor the formation of stimulatory Ag-MHC complexes on EBV-transformed B cells that were either pulsed with native tetanus toxoid (TT) or with a 12-amino-acid fragment of this protein. Neither unpulsed APC nor Ag-specific APC that were pulsed with native Ag and kept at +4 degrees C were able to induce changes in basal T cell [Ca2+]i in TT-specific T cell clones. After 1 h at 37 degrees C, however, the Ag-pulsed APC were able to induce a three-to-fourfold increase in [Ca2+]i. This length of time appeared to be almost independent of the concentration of Ag with which the APC were pulsed, suggesting that the lag time was due more to intracellular transit than to association of the processed Ag with the MHC molecule. Furthermore, the same lag time and independence of Ag concentration were found when the EBV-transformed B cells were pulsed with a mouse-anti-transferrin receptor mAb and tested for their capacity to trigger a T cell clone specific for processed mouse Ig. This indicates that, in addition to surface Ig, other receptors that are internalized can function in the same fashion in the uptake and processing of a soluble Ag. In contrast to what was found with intact native Ag, no lag time was observed when the APC were pulsed with high concentrations of a 12-amino-acid peptide, containing the amino acid sequence recognized by a TT-specific T cell clone, suggesting that the formation of MHC-peptide complexes occurs instantly. Pulsing with a lower peptide concentration, however, caused the appearance of a time-dependent increase in efficacy of Ag presentation, suggesting a slow accumulation of MHC-peptide complexes on the B cell membrane.     

T cell-mediated recognition of foreign antigen and the Ia molecule observed by stopped-flow fluorometry

Th cell-mediated rapid recognition of foreign Ag and the Ia molecule was studied using azobenzenearsonate-L-tyrosine (ABA-L-tyrosine)-specific Th cells (I-Ak restricted), foreign Ag (ABA-L-tyrosine), and APC (H-2k). Initial transmembrane signals in Th cell hybridomas (2-45-12) and in Th cell lines (A24-17 or A33-7) were monitored by stopped-flow fluorometry with fluorescent probes. It was found that Th cells recognized foreign Ag within 1 s at 25 degrees C on the APC (B10.BR spleen cells or L cells into which I-Ak genes were transferred). Recognition of foreign Ag and the Ia molecule was shown to deliver the initial signals to Th cell hybridomas and T cell lines. First, Th cells had membrane fluidity increased and then calcium was transported from the external medium into the T cells. The initial transmembrane signals to Th cell hybridomas were inhibited by the addition of an anti-I-Ak mAb. None of the initial signals were observed in the absence of either specific foreign Ag or APC.     

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.     

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.     

The human antimurine xenogeneic cytotoxic response. I. Dependence on responder antigen-presenting cells

We have examined the role of the human responder APC in the generation of CTL responses to xenogeneic antigens. Of six xenogeneic responses evaluated, only the human antimurine response was dependent on human APC for CTL generation. APC requirements for the other five xenogeneic responses more closely resembled those observed in the generation of human or murine alloreactive CTL. Depletion studies identified a defective human CD4+ Th cell-murine stimulator cell interaction that could be bypassed by the addition of exogenous IL-2. The function of the responder APC involved in the human antimurine CTL response was inhibited by chloroquine, suggesting a requirement for Ag processing. Effective presentation of murine stimulator Ag by human APC was completely blocked by anti-human Ia mAb, indicating that the Ag is presented to Th cells via the human class II molecule. These results are consistent with an Ia-dependent recognition of processed murine Ag by human T cells and represents a model for investigating human T cell activation requirements, Th cell function, and MHC restriction.     

High efficiency presentation of TNP-conjugated islet antigen to islet-specific T cells by a hybrid B cell line expressing TNP-specific surface Ig.

There is compelling evidence from animal models that type I diabetes is a consequence of T cell-mediated destruction of islet beta-cells. The recent isolation of islet-specific T cell clones from nonobese diabetic mice provides a means of identification of the Ag on islet cells that are responsible for stimulation of autoreactive T cells. We describe an APC line constructed by fusion of spleen B cells obtained from nonobese diabetic mice to a B lymphoma that was transfected with the H and L chains of an IgM specific to the hapten TNP. Using this hybrid APC we have observed a dramatic increase in the efficiency of presentation of TNP-conjugated islet cell protein preparations compared to that seen with conventional APC. Our results illustrate the potential use of this APC line for isolation and characterization of islet Ag relevant to the T cell response.     

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.     

Human T cell clones present antigen

Two human T cells clones are described which react with influenza virus hemagglutinin type H3 and synthetic peptides of H3 when presented by PBMC APC. Both T cell clones also responded to peptide Ag in the absence of additional APC suggesting that T cells can simultaneously present and respond to Ag. T cell clones could only present peptide Ag and not an appropriate strain of inactivated whole influenza virus thus indicating an inability to process Ag conventionally. Peptide presentation by T cells was dose dependent, restricted by MHC class II Ag and was dependent on the number of Ag presenting T cells per culture. Experiments with nested peptides showed that the same epitope was recognized in the presence and absence of PBMC APC. No Ag or IL-2 from the propagation procedure was carried over into assays and two-color fluorescence-activated cell sorter analysis of each clone detected no contaminating cells with the phenotype of monocytes, macrophages or B cells; in each T cell clone, all cells expressing MHC class II Ag co-expressed CD3. These date therefore provide strong evidence that human T cell clones can simultaneously present and respond to appropriate forms of Ag.     

Functional sites on the A alpha-chain. Polymorphic residues involved in antigen presentation to insulin-specific, Ab alpha:Ak beta-restricted T cells

The interaction between the clonally selected TCR, the processed Ag peptide and the Ia molecule is not fully understood in molecular terms. Our study intended to delineate the residues of Ab alpha molecules that function as contact sites for Ag and for the TCR of a panel of T cells specific for the A chain of insulin in combination with mixed haplotype Ab alpha:Ak beta molecules. Multiple L cell transfectants expressing alpha,beta-heterodimers composed of wild-type A beta- and chimeric or mutant A alpha-chains served as antigen presenting cells. The recombinant A alpha-chains had been generated by an exchange of allelically hypervariable regions (ahv) or amino acids. The results point out a broad spectrum of b sequence requirements for the bovine insulin-specific activation of the various T cell populations. Activation of some T cells seemed quite permissive, requiring b-haplotype amino acids in any one of the three ahv, while others had strict requirements, demanding b-haplotype sequence in all three ahv. Our data stress the role of ahvII and especially ahvIII in T cell activation. Interestingly, single amino-acid substitutions in ahvII or ahvIII of Ak alpha were sufficient to bring up full stimulation potential for two T cell hybridomas. We also found that some ahv permutations influenced the Ag preference (beef insulin versus pig insulin) of some T cells. These data suggest a critical role for the three-dimensional structure of the complex formed by Ia and the processed Ag peptide. The stability of the trimolecular complex essential for T cell activation is envisioned as being the sum of the interactions between Ag/I-A, TCR/Ag, and TCR/I-A, each variable in strength and compensated for by the others.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.