Major histocompatibility complex class I-binding peptides are recycled to the cell surface after internalization

Cytotoxic T lymphocytes (CTL) recognize target antigens as short, processed peptides bound to major histocompatibility complex class I (MHC-I) heavy and light chains (β₂-microglobuhn; β₂ m).The heavy chain, which comprise the actual peptide binding α-1 and α-2 domains, can exist at the cell surface in different forms, either free, bound to β₂m or as a ternary complex with β₂m and peptides. MHC-I chains are also known to internalize, and recycle to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-I-bound peptides also can recycle is not known. We have investigated this by using both peptide transporter mutant RMA-S cells and EL4 cells loaded with D^b-binding peptides, by two different approaches. First, peptides were covalently linked with galabiose (Galα4Gal) at a position which did not interfere with D^b binding or immunogenicity, and peptide recycling tested with Gal₂-specific monoclonal antibodies. By flow cytometry, a return of Gal₂ epitopes to the cell surface was found, after cellular internalization and cell surface clearance by pronase treatment. This peptide recycling could be discriminated from free fluid-phase uptake and was inhibited by methylamine, chloroquine and low temperature (18°C) but not by leupeptin. Second, specific CTL were reacted with peptide-loaded target cells after complete removal of surface D^b molecules by pronase, and after different times of incubation at 37C to allow reexpression. By this procedure, reappearance of target cell susceptibility was confirmed. The results are in agreement with a model for optimizing peptide presentation by recycling through an intracellular compartment similar to early endosomes in certain antigen-presenting cells.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the L^d -binding 12-mer S28 – 39 peptide IPQSLDSWWTSL in H-2 ^d mice, and the K^b -binding 8-mer S208 – 215 peptide ILSPFLPL in H-2^b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the L^d -binding peptide S28 – 39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the K^b -binding peptide S208 – 215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: Implications on immunogenicity and immunodominance

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/K^b (A2/K^b) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/K^b animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/K^b animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.     

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2K^b-bound octapeptides. CTL recognition of such determinants is always sequencedependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on K^b-associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.     

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8 T cells independent of IFN-γ and CD107a responses

CD8⁺ T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8⁺ T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4⁺ T cells. We used a set of SIV_mac239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I^high or MHC class I^intermediate. However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I^low) despite the observations that all CTL clones showed similar IFN-γ responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.     

TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides

The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an αβ TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2D^b MHC molecule. Both peptides were shown to antagonize specifically the T cell cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2D^b using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind D^b. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to D^b. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the D^b pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited D^b-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all D^b-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, D^b-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to D^b, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.     

Induction of CTL in vivo by major histocompatibility complex class I-peptide complexes covalently associated on the cell surface

The identification of endogenously produced antigenic peptides presented by MHC class I molecules has opened the way to peptide-based strategies for CTL induction in vivo. Here we demonstrate that the induction in vivo of CTL directed against naturally processed antigens can be triggered by injection of syngeneic cells expressing covalent major histocompatibility complex class I-peptide complexes. In the model system used, the induction of HLA-Cw3 specific cytotoxic T lymphocytes (CTL) in mice by cell surface-associated, covalent H-2K^d (K^d)-Cw3 peptide complexes was investigated. The K^d-restricted Cw3 peptide 170–179 (RYLKNGKETL), which mimics the major natural epitope recognized by Cw3-specific CTL in H-2^d mice, was converted to a photoreactive derivative by replacing Arg-170 with N-β-(4-azidosalicyloyl)-L-2,3-diaminopropionic acid. This peptide derivative was equivalent to the parental Cw3 peptide in terms of binding to K^d molecules and recognition by Cw3-specific CTL clones and could be cross-linked efficiently and selectively to K^d molecules on the surface of Con A-stimulated spleen cells from H-2^d mice. Photocross-linking prevented the rapid dissociation of K^d-peptide derivative complexes that takes place under physiological conditions. Cultures of spleen cells or peritoneal exudate cells from mice inoculated i.p. with peptide-pulsed and photocross-linked cells developed a strong CTL response following antigenic stimulation in vitro. The cultured cells efficiently lysed not only target cells sensitized with the Cw3 170–179 peptide but also target cells transfected with the Cw3 gene. Moreover, their TCR preferentially expressed Vβ10 and JαpHDS58 segments as well as conserved junctional sequences, as has been observed previously in Cw3-specific CTL responses. In contrast, no Cw3-specific CTL response could be obtained in cultures derived from mice injected with Con A-stimulated spleen cells pulsed with the peptide derivative without photocross-linking.     

Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12

Immunodominance (ID) of T cell epitopes is a well-documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H-2K^d, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half-life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co-injected CTL epitopes can be determined both at the peptide level (binding affinity to H-2K^d) and at the T cell level. This hypothesis is supported by our finding that interleukin-12 strongly modulates ID when it is not correlated with MHC binding.     

Calnexin expression does not enhance the generation of MHC class I-peptide complexes

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K ^d , K^b , K^k , D ^d , D^b , and L^d . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For K^b and K ^d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of K^b molecules bearing the immunodominant peptide from ovalbumin (OVA_{257 – 264} ). This was detected with a recently described monoclonal antibody specific for the K^b/peptide complex. Calnexin expression had no significant effect on the formation of K^b /peptide complexes generated from full-length OVA, cytosolic OVA_{257 – 264} , or endoplasmic reticulum-targeted OVA_{257 – 264} , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K ^d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.     

Cytotoxic T lymphocyte responses to wild-type and mutant mouse p53 peptides

Cytotoxic T lymphocytes (CTL) recognize peptides presented at the cell surface in association with major histocompatibility complex (MHC) class I molecules. The finding that peptides binding to MHC class I molecules share common amino acid motifs renders feasible the selection of antigenic peptides by simply scanning protein sequences, and thus, provides the possibility of inducing CTL to pre-defined specificities. Tumor cells possess antigens known to generate MHC class I-restricted CD8⁺ CTL responses. Thus, these antigens represent good targets to induce tumor-specific immunity. Among these antigens, the p53 tumor suppressor gene product is an attractive candidate for cancer immunotherapy. Mutations in the p53 gene have been found to be very frequently associated with a malignant transformation and often lead to p53 protein overexpression. Thus, we investigated the possibility of inducing CTL to wild-type or mutant p53 peptides in a BALB/c (H-2^d) mouse model. Peptides possessing the H2-K^d binding motif were selected and tested for binding to the H-2K^d molecules in vitro. Synthetic peptides p53_122–130 wild-type or “mutant” (Lys → Glu substitution at position 129) were shown to be the best binder peptides and were tested for their immunogenicity in mice. H-2K^d-restricted p53-specific CD8⁺ CTL were generated following immunization of mice with either wild-type (wt) p53_122–130 or mutant (mut) p53_122–130 (E129) peptides. Only low-affinity CTL can be obtained by immunization with the wt sequence. In contrast, CTL elicited with the mut peptide recognized the mut sequence at a 10–100-fold lower concentration. This indicates that CTL elicited with the mut peptide recognized the mut sequence very efficiently, whereas the wt sequence is poorly recognized, if at all. Taken together, these results thus suggest that p53-specific tumor immunotherapy may be successful only if the mutated protein is taken into consideration.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

Selection of T-cell receptors with a recurrent CDR3β peptide-contact motif within the repertoire of alloreactive CD8(+) T cells

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo‐rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H‐2K^b (K^b)‐associated peptides and critically required to induce full activation of H‐2^k monoclonal CD8⁺ TLs expressing the cognate TCR‐BM3.3. Here, we asked whether a pBM1/K^b‐specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K^b‐binding CD8⁺ TLs increased from <0.04% in naïve mice to 3% of activated CD44⁺ CD8⁺ TLs in H‐2^k mice rejecting K^b‐expressing cells. Among these, TCR‐Vβ2 usage was greatly enriched, and 75% of them shared a TCR‐Vβ2 CDR3β motif with the prototype TCR‐BM3.3. Fewer than 5% of K^b‐reactive CD44⁺ CD8⁺ TLs not binding pBM1/K^b displayed this CDR3β motif. We found that the recurrent CDR3β motif of pBM1/K^b‐binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR‐peptide/MHC fit. Thus, a CDR3β motif generated by a process akin to “convergent recombination” accounts for a sizable fraction of the alloreactive anti‐K^b TCR repertoire.     

Glycosylphosphatidylinositol-linked Db does not induce an influenza-specific cytotoxic T lymphocyte response or recycle membrane-bound peptides

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I-bound peptides, are known to recycle between the cell surface and an undefined, endosomal-like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H-2D^b molecule expressed in transgenic mice, either transmembranous (D^b-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (D^b-GPI). The recycling capacity of peptides bound to D^b-tm and D^b-GPI was investigated using glycosylated D^b-binding glycopeptides, which were detected by flow cytometry. Only the tm form of D^b was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366–374, ASNENMETM), onyl D^b-tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I-bound peptides on antigen-presenting cells may be important for the generation of certain CTL responses.     

The influence of allo-class II MHC-specific Th2 cells on the generation of CD4 and CD8 cytotoxic T cells to associated class I and class II MHC alloantigen

There is considerable interest in whether CD4 T cell function can affect the outcome of allogeneic transplants. In mice tolerant to an isolated class II MHC disparity, the normal Th1 activity in vitro associated with graft rejection is switched to Th2 in tolerant animals. Because clinical transplants involve multiple class I and II MHC disparities we tested how the switch to Th2 activity of tolerant mice would affect the generation of CD4 and CD8 cytotoxic T cells (CTL) against MHC alloantigens to which the mice were not tolerant. A.TH mice (K^kI^sD^d) were rendered neonatally tolerant of A.TL (K^kI^kD^d) and the generation of CD4 or CD8 CTL measured in a mixed lymphocyte reaction (MLR) against (A.TLxB6)F₁ stimulators. Normal mice generated CD4 CTL against both A.TL and B6 (K^bI^bD^b), but tolerant mice were unable to generate cytotoxicity against either A.TL or B6. However, tolerant cells were able to generate CD8 CTL against B6. IL-4 inhibited the generation of CD4, but not CD8, CTL by normal cells and anti-IL-4 antibody was shown to increase the generation of CD4 CTL against B6 in F₁ stimulated cultures. Overall the results showed that a Th2 response could inhibit the generation of CD4 CTL against concomitant alloantigen in a process at least partially involving IL-4, but that, conversely, tolerant Th2 cells could help in the generation of CD8 CTL. The results suggest that with whole MHC disparities a simple change of CD4 T cells to Th2 would not be enough to procure graft acceptance.