Lack of Ir gene control in the immune response to malaria. I. A thymus-independent antibody response to the repetitive surface protein of sporozoites

The anamnestic antibody response to synthetic peptide antimalarial vaccines is under Ir gene control. It has therefore been inferred that the development of antibody responses to the native repetitive Ag of malaria parasites also requires linkage of T and B cell epitopes, presentation of Ag in the context of MHC class II components, and cognate T cell help for antibody production. In this study, we sought to test this assumption, by utilizing classical protocols to determine whether the antibody response to the repetitive surface Ag of malaria sporozoites, the circumsporozoite (CS) protein, is under Ir gene control. In contrast to vaccine constructs, such as recombinant proteins or synthetic peptides, secondary responses to the repetitive oligomeric domains of the native CS protein of intact malaria sporozoites do not require the presence of Ag-specific Th cells. Conferral of CS-specific Th cells does not appear to influence the magnitude of this thymus-independent response to sporozoites. In further contrast to synthetic CS analogs, exposure to the parasite appears to be associated with low levels of Ag-specific Th cell sensitization. These observations suggest a functional role in immune evasion for the immunodominant repetitive domains found within protein Ag of malaria and other parasites.     

Identification of proteases that process distinct epitopes on the same protein

Proteolytic degradation of protein antigens is thought to be a major step in the processing of Ag for presentation to T cells, but the range of proteases involved is unknown. Here we used a large panel of protease inhibitors to determine the role of each of the four classes of proteases in antigen processing. Moreover, we asked whether different proteases were necessary for presentation of different known epitopes, defined by three Th cell clones. For all three epitopes of myoglobin, intracellular thiol proteases such as cathepsins B or L were the only proteases necessary. Furthermore, myoglobin pre-digested with cathepsin B could be presented to all three clones without further processing. Thus, a single protease may be both necessary and sufficient for Ag processing to present the majority of epitopes, at least for myoglobin. This finding provides an explanation of earlier data on the fragments produced from processed myoglobin, and so may contribute to a much needed solution to the long standing problem of predicting where a protein will be cleaved during processing.     

Immunodominance: intermolecular competition between MHC class II molecules by covalently linked T cell epitopes.

The T cell response to complex protein Ag typically focuses on a few, and frequently a single, immunodominant epitope. Several groups have proposed that the mechanism of immunodominance is determined by the steps of Ag processing and presentation including protein unfolding, the sites of proteolytic cleavage, and the affinity of binding to MHC molecules. Also, the failure of the TCR repertoire to recognize MHC-bound peptides, termed a hole in the repertoire, can prevent recognition of a potentially dominant processed peptide. In the present study, we demonstrate that immunodominance can be determined by intermolecular competition for binding to MHC class II molecules between covalently linked T cell epitopes. In addition, we have analyzed the factors controlling T cell recognition of the covalently linked epitopes. In our system, T cell recognition of the dominant epitope is not altered by Ag processing, and is not simply a function of MHC-binding affinity. We propose that adjacent sequences can subtly alter the conformation of an epitope, creating significant changes in T cell recognition. These observations are discussed in terms of the mechanisms of immunodominance and in terms of the development of synthetic peptide vaccines.     

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.     

Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by Ia-restricted Th2 helper T cell lines

The mechanism of help for resting B cell growth in MHC-restricted T-B collaboration was investigated using an in vitro polyclonal model for these T cell-B cell interactions. In the presence of rabbit anti-mouse Ig, small, size-selected B cells elicit help from syngeneic Ia-restricted Th2 cell lines specific for F(ab')2 rabbit globulin. Both Ag-presenting and bystander B cells receive signals from stimulated Th cells that lead to B cell proliferation. The results suggest that the direct activation of resting Ag-presenting and bystander B cells by Th2 cells is mediated by a similar effector mechanism. Although proliferative responses by Ag-presenting B cells are of greater magnitude, help for both Ag-presenting and bystander B cell populations is characterized by the lack of a requirement for membrane Ig cross-linking, by identical kinetics, and by the necessity for direct cell contact or close proximity with Th cells. B cell proliferation is not induced by exposure to the sequence of diffusable mediators released from a synchronized Ag-specific T-B interaction. The T cell-dependent proliferation by both B cell populations can be inhibited by excess mitomycin C-treated syngeneic "cold target" B cells, demonstrating a requirement for a short-range T cell-B cell interaction. mAb inhibition experiments fail to identify a role for class II, LFA-1, or CD4 membrane molecules in the delivery of help to bystander B cells. Antibody against H2d bystander class II molecules has no effect on bystander B cell proliferation at concentrations that completely block Ag presentation by H2d B cells to an H2d-restricted Th cell line. Antibodies against the cell adhesion molecule LFA-1 or the Th cell molecule CD4 do inhibit bystander B cell proliferation, but only to the extent that they block T cell activation and the induction of help. The inductive stimulus leading to resting B cell growth results from an early, short-range interaction with Th cells. B cell proliferation is supported by T cell soluble mediators as a consequence of this interaction, which is required for at least 8 hr after T cell recognition of Ag/Ia on the surface of Ag-presenting B cells.     

Two distinct mechanisms account for the immune response (Ir) gene control of the T cell response to pigeon cytochrome c

Previous experiments have demonstrated that the immune response of MHC congenic mice to pigeon cytochrome c is under Ir gene control. Expression of I-E-encoded gene products influences both the magnitude and fine specificity of the Th cell response to pigeon cytochrome c and phylogenetic derivatives. Results of those experiments implicate both determinant selection and repertoire selection as mechanisms of Ir gene control in this system. In this report we have compared the TCR expressed in pigeon cytochrome c-reactive Th cells from B10.A(I-Ek), B10.A(5R) (I-Eb), and B10.S(9R) (I-Es) mice. The B10.A(5R) strain is a low responder to pigeon cytochrome c, but in response to moth cytochrome c this strain produces T cells which respond to pigeon or moth cytochrome c on B10.A APC. These cells are phenotypically identical to the predominant clonal phenotype seen in the B10.A response to pigeon cytochrome c. In this report, we show that the B10.A and B10.A(5R) pigeon cytochrome c-reactive T cells express essentially identical T cell receptors. These results, coupled with recent studies reporting a relatively low affinity for I-Eb molecules by pigeon cytochrome c peptides compared with moth cytochrome c peptides, strongly argue that the immune response defect in the B10.A(5R) strain is due to a defect in Ag presentation (determinant selection). In contrast, B10.A and B10.S(9R) strains are high responders to pigeon cytochrome c. Both strains produce T cell clones which are capable of responding to cytochrome c presented by either B10.A or B10.S(9R) APC in vitro. We show that, even in T cells with this MHC restriction degeneracy, the TCR expressed in the two strains are different. Because the APC of both strains can clearly present the cytochrome c Ag, we conclude that the differential expression of the TCR in the responses is due to a T cell repertoire selection difference in the two strains. Thus, for the response to one Ag in three MHC congenic strains, there exists evidence that both determinant selection and repertoire selection can be mechanisms of Ir gene control of an immune response.     

Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.     

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.     

HIV-1 tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.     

Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.     

Inducible cell contact signals regulate early activation gene expression during B-T lymphocyte collaboration.

B cells get help in the antibody response by presenting processed Ag to Th cells. We asked whether the Ag-presenting B cell must induce Th functions before receiving help, or whether B cell activation is a direct consequence of T cell recognition of Ag on the B cell surface. To obtain a prompt and sensitive indication of the receipt of growth signals, we measured mRNA levels of the immediate early genes, c-myc and egr-1, in T and B cells separated from Ag-specific B-T conjugates of normal, resting murine B cells and a Th line. Although Ag-dependent increases in B cell c-myc expression occur as early as 2 h after conjugation, early c-myc expression in the B cell was also seen when the Th cells were activated with immobilized anti-CD3 in the absence of Ag recognition. Therefore, T cell activation rather than Ag recognition per se appears to be responsible for the early c-myc signal in the B cells. The c-myc response in the B cell depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A acting on the T cell. Delivery of contact help is not blocked by anti-class II MHC antibody. Contact with activated Th cells induces a different pattern of immediate early gene expression from that induced by cross-linking the B cell Ag receptor.     

Analysis of H2-O influence on antigen presentation by B cells

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.     

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.     

Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4(+) Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.     

Cellular interactions involved in Th cell memory

The cellular interactions involved in maintaining CD4+ T cell memory have hitherto not been identified. In this report, we have investigated the roles played by B cells and dendritic cells (DCs) in this process. We show that long-lasting Th cell memory depends on the presence of B cells, but that direct Ag presentation by B cells is not required. Instead, Ag presentation by DCs is critical for the survival of memory Th cells. DCs presenting specific Ag can be detected in animals long after immunization. These findings support a model in which B cells provide an environment in which Ags may be trapped and retained. This Ag is periodically presented to memory CD4+ T cells by DCs, providing an essential survival signal.     

Flexibility in MHC and TCR recognition: degenerate specificity at the T cell level in the recognition of promiscuous Th epitopes exhibiting no primary sequence homology

Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3(378-395) and TT(830-844))-specific CD4+ T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3(378-395) peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3(378-395)-specific CD4+ T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host's response to a pathogen may be modified by its previous experience with other unrelated pathogens.     

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.     

Efficient in vivo presentation of Listeria monocytogenes- derived CD4 and CD8 T cell epitopes in the absence of IFN-gamma

IFN-gamma is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-gamma on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-gamma-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-gamma on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-gamma is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.     

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.     

Epstein-Barr nuclear antigen 1-specific CD4(+) Th1 cells kill Burkitt's lymphoma cells

The gamma-herpesvirus, EBV, is reliably found in a latent state in endemic Burkitt's lymphoma. A single EBV gene product, Epstein-Barr nuclear Ag 1 (EBNA1), is expressed at the protein level. Several mechanisms prevent immune recognition of these tumor cells, including a block in EBNA1 presentation to CD8(+) killer T cells. Therefore, no EBV-specific immune response has yet been found to target Burkitt's lymphoma. We now find that EBNA1-specific, Th1 CD4(+) cytotoxic T cells recognize Burkitt's lymphoma lines. CD4(+) T cell epitopes of EBNA1 are predominantly found in the C-terminal, episome-binding domain of EBNA1, and approximately 0.5% of peripheral blood CD4(+) T cells are specific for EBNA1. Therefore, adaptive immunity can be directed against Burkitt's lymphoma, and perhaps this role for CD4(+) Th1 cells extends to other tumors that escape MHC class I presentation.