Binding of major histocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II.

Major histocompatibility complex class II-associated invariant chain (Ii) provides several important functions that regulate class II expression and function. One of these is the ability to inhibit class II peptide loading early in biosynthesis. This allows for efficient class II folding and egress from the endoplasmic reticulum, and protects the class II peptide binding site from loading with peptides before entry into endosomal compartments. The ability of Ii to interact with class II and interfere with peptide loading has been mapped to Ii exon 3, which encodes amino acids 82-107. This same region of Ii has been described as a nested set of class II-associated Ii peptides (CLIPs) that are transiently associated with class II in normal cells and accumulate in human histocompatibility leukocyte antigen-DM-negative cell lines. Currently it is not clear how CLIP and the CLIP region of Ii blocks peptide binding. CLIP may bind directly to the class II peptide binding site, or may bind elsewhere on class II and modulate class II peptide binding allosterically. In this report, we show that CLIP can interact with many different murine and human class II molecules, but that the affinity of this interaction is controlled by polymorphic residues in the class II chains. Likewise, structural changes in CLIP also modulate class II binding in an allele-dependent manner. Finally, the specificity and kinetics of CLIP binding to class II molecule is similar to antigenic peptide binding to class II. These data indicate that CLIP binds to class II in an analogous fashion as conventional antigenic peptides, suggesting that the CLIP segment of Ii may actually occupy the class II peptide binding site.

Mechanisms influencing the immunodominance of T cell determinants.

The preferential recognition of certain amino acid sequences from foreign protein antigens by T cells is referred to as T cell epitope immunodominance. To determine the mechanisms underlying this phenomenon, we have studied the correlation between the interaction of a series of synthetic peptides encompassing the entire hen egg-white lysozyme (HEL) sequence with class II molecules of the H-2k haplotype, and T cell responsiveness to these peptides. After HEL priming, three immunodominant T cell epitopes were found: two, included in the HEL sequences 51-61 and 112-129, were recognized in association with I-Ak molecules, and one, included in sequence 1-18, in association with I-Ek molecules. Accordingly, these peptides bound to the appropriate class II molecule, as demonstrated by competition for antigen presentation. Several other HEL peptides, although capable of associating with class II molecules, were not immunodominant. The absence of immunodominance has been shown to arise by three different mechanisms: (a) competition by an immunodominant peptide for presentation in vivo, (b) failure to generate the peptide during antigen processing, and (c) an inherently poor capacity of the T cell repertoire to respond to a particular peptide-MHC complex.

Invariant chain and DM edit self-peptide presentation by major histocompatibility complex (MHC) class II molecules.

We have studied the consequences of invariant chain (Ii) and DM expression on major histocompatibility complex (MHC) class II function. Ii has a number of discrete functions in the biology of class II, including competitive blocking of peptide binding in the endoplasmic reticulum and enhancing localization in the endocytic compartments. DM is thought to act primarily in endosomes to promote dissociation of the Ii-derived (CLIP) peptide from the class II antigen-binding pocket and subsequent peptide loading. In this study, we have evaluated the functional role of Ii and DM by examining their impact on surface expression of epitopes recognized by a large panel of alloreactive T cells. We find most epitopes studied are influenced by both Ii and DM. Most strikingly, we find that surface expression of a significant fraction of peptide-class II complexes is extinguished, rather than enhanced, by DM expression within the APC. The epitopes antagonized by DM do not appear to be specific for CLIP. Finally, we found that DM was also able to extinguish recognition of a defined peptide derived from the internally synthesized H-2Ld protein. Thus, rather than primarily serving in the removal of CLIP, DM may have a more generalized function of editing the array of peptides that are presented by class II. This editing can be either positive or negative, suggesting that DM plays a specifying role in the display of peptides presented to CD4 T cells.

Major histocompatibility complex-controlled, antigen-presenting cell-expressed specificity of T cell antigen recognition. Identification of a site of interaction and its relationship to Ir genes.

In previous work (5,6), we have reported studies on a T lymphocyte hybridoma clone and the peritoneal exudate T cells (PETLES) from B10.A(5R) mice primed with the cytochrome c carboxyl terminal peptide (residues 81-103) of the tobacco horn worm moth (Manducca sextus). As expected, since B10.A(5R) is a low responder to pigeon fragment 81-104, it was found that the B10.A(5R) lymphocytes were unable to respond to the pigeon cytochrome c 81-104 fragment presented on syngeneic B10.A(5R) antigen-presenting cells (APC). However, these same T lymphocytes did respond to the pigeon fragment when presented on B10.A APC. Thus, some structural difference between the pigeon and moth peptides had prevented B10.A(5R) APC from effectively presenting the pigeon fragment to moth-primed B10.A(5R) lymphocytes. This structural difference was found to be the deletion of an alanine at position -103 (Ala103) from the pigeon sequence in the moth peptide. Two additional T cell specificities were created by changing residue-99. These T cell populations from the B10.A(5R) showed an identical dependence on the Ala103 deletion when B10.A and B10.A(5R) APC were compared. The relationship of APC-expressed antigen specificity and MHC-linked immune responsiveness differences was also examined. The B10.A(5R) was found to be a high responder to each of three peptides that lack Ala103 but not to the Ala103-containing analogues. B10.A mice, in contrast, respond to both types of peptides. Utilizing allogeneic antigen-presentation to B10.A PETLES by pulsed APC, it was shown that the poor response of the B10.A(5R) to the Ala103-containing peptides was, in two of three cases, not associated with any differences in T cell repertoires but due to two different APC capabilities of B10.A and B10.A(5R). The exception apparently represents a case of T cell repertoire polymorphism between B10.A and B10.A(5R) that can also affect immune responsiveness.

Structural evidence of T cell xeno-reactivity in the absence of molecular mimicry.

The T cell receptor (TCR), from a xeno-reactive murine cytotoxic T lymphocyte clone AHIII12.2, recognizes murine H-2Db complexed with peptide p1027 (FAPGVFPYM), as well as human HLA-A2.1 complexed with peptide p1049 (ALWGFFPVL). A commonly proposed model (the molecular mimicry model) used to explain TCR cross-reactivity suggests that the molecular surfaces of the recognized complexes are similar in shape, charge, or both, in spite of the primary sequence differences. To examine the mechanism of xeno-reactivity of AHIII12.2, we have determined the crystal structures of A2/p1049 and Db/p1027 to 2.5 A and 2.8 A resolution, respectively. The crystal structures show that the TCR footprint regions of the two class I complexes are significantly different in shape and charge. We propose that rather than simple molecular mimicry, unpredictable arrays of common and differential contacts on the two class I complexes are used for their recognition by the same TCR.

Expression and function of a nonglycosylated major histocompatibility class I antigen.

The major histocompatibility class I antigens, expressed in most somatic cells, have carbohydrate moieties. We constructed mutant mouse MHC class I genes in which codons for the N-linked glycosylation sites were replaced by those of other amino acids. L cell transformants expressing the nonglycosylated class I antigens allowed us to investigate biological roles of carbohydrates with the highest specificity possible. The nonglycosylated antigen was unchanged in its overall serological specificities, and was recognized by alloreactive cytotoxic T cells. Further, the antigen was capable of mediating cytotoxic activity of vesicular stomatitis virus-specific T cells. These studies indicate that carbohydrates are not essential for immunological function of the MHC class I antigens. Cell surface expression of the nonglycosylated antigen was markedly reduced as compared with the native antigen, which was not attributable to accelerated degradation or rapid shedding. We conclude that the primary role of carbohydrates of the class I antigens is to facilitate the intracellular transport of the nascent proteins to the plasma membrane. The possible involvement of carbohydrate-receptor interactions in this process is discussed.

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved.

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains.

Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis.

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.

Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.

The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity.

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.

Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants.

Two chemoattractants for human monocytes were purified to apparent homogeneity from the culture supernatant of a glioma cell line (U-105MG) by sequential chromatography on Orange A-Sepharose, an HPLC cation exchanger, and a reverse phase HPLC column. On SDS-PAGE gels under reducing or nonreducing conditions, the molecular masses of the two peptides glioma-derived chemotactic factor 1 and 2 were 15 and 13 kD, respectively. Amino acid composition of these molecules was almost identical, and differed from other cytokines that have been reported. The NH2 terminus of each peptide was apparently blocked. When tested for chemotactic efficacy, the peptides attracted approximately 30% of the monocytes added to chemotaxis chambers, at the optimal concentration of 10(-9) M. Potency and efficacy were comparable with that of FMLP, which is often used as a reference attractant. The activity was chemotactic rather than chemokinetic. In contrast to their interaction with human monocytes, the pure peptides did not attract neutrophils. These pure tumor-derived chemoattractants can now be compared with attractants produced by normal cells and evaluated for their biological significance in human neoplastic disease.

Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.

Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART-1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.

Contribution of antigen-presenting cell major histocompatibility complex gene products to the specificity of antigen-induced T cell activation.

Previous studies from our laboratory showed that B 10.A mice are high responders to pigeon cytochrome c fragment 81-104, whereas'B 10.A(5R) mice are low responders. In the present studies, the C-terminal cyanogen bromide cleavage fragment and homologous synthetic peptides of tobacco horn worm moth cytochrome c were shown to be immunogenic in both B10.A and B10.A(5R) mice. These strains, however, showed different patterns of cross-reactivity when immune lymph node T cells were stimulated with cytochrome c fragments from other species. To examine the two patterns of responsiveness at a clonal level, cytochrome c fragment-specific T cell hybridomas were made and found to secrete interleukin 2 in response to antigen. The patterns of cross- reactivity of these B 10.A and B 10.A(5R) clones were similar to that seen in the whole lymph node population. Surprisingly, when these clones were tested for major histocompatibility complex (MHC)-restricted antigen recognition, they were all found to respond to antigen with both B10.A and B10.A(5R) antigen-presenting cells (APC). Furthermore, the cross-reactivity pattern appeared to be largely determined by the genotype of the APC, not the genotype of the T cell clone. That is, a given T cell clone displayed a different fine specificity when assayed with B10.A or B10.A(5R) APC. This observation indicates that the APC MHC gene product and antigen interact during the stimulation of the T cell response and that as a consequence the specificity of antigen-induced T cell activation is influenced by these MHC gene products. (During the preparation of this manuscript it has come to our attention that results similar to our own, concerning the fine specificity of cytotoxic T cell clones, have been obtained by Dr. T. R. Hunig and Dr. M. J. Bevan, Massachusetts Institute of Technology, Boston, MA. T. R. Hunig and M. J. Bevan. 1981. Specificity of T-cell clones illustrates altered self hypothesis. Nature. 294:460.)

A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes.

A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent the products of mutated genes. In contrast, most of the antigens recognized by human tumor-reactive T cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation of a cDNA clone encoding beta-catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. The cDNA clone, which was isolated from the autologous melanoma cDNA library, differed by a single base pair from the published beta-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The beta-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype.

Properties of monocyte chemotactic and activating factor (MCAF) purified from a human fibrosarcoma cell line.

A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.

Developmental and tissue-specific expression of nuclear proteins that bind the regulatory element of the major histocompatibility complex class I gene.

Expression of MHC class I genes varies according to developmental stage and type of tissues. To study the basis of class I gene regulation in tissues in vivo, we examined binding of nuclear proteins to the conserved cis sequence of the murine H-2 gene, class I regulatory element (CRE), which contains two independent factor-binding sites, region I and region II. In gel mobility shift analyses we found that extracts from adult tissues that express class I genes, such as spleen and liver, had binding activity to region I. In contrast, extracts from brain, which does not express class I genes, did not show region I binding activity. In addition, fetal tissues that express class I gene at very low levels, also did not reveal region I binding activity. Binding activity to region I became detectable during the neonatal period when class I gene expression sharply increases. Most of these tissues showed binding activity to region II, irrespective of class I gene expression. Although region II contained a sequence similar to the AP-1 recognition site, AP-1 was not responsible for the region II binding activity detected in this work. These results illustrate a correlation between region I binding activity and developmental and tissue-specific expression of MHC class I genes. The CRE exerts an enhancer-like activity in cultured fibroblasts. We evaluated the significance of each factor binding to CRE. Single 2-bp mutations were introduced into the CRE by site-directed mutagenesis and the ability of each mutant to elicit the enhancer activity was tested in transient CAT assays. A mutation that eliminated region I protein binding greatly impaired enhancer activity. A mutation that eliminated region II binding also caused a lesser but measurable effect. We conclude that region I and region II are both capable of enhancing transcription of the class I gene. These results indicate that in vivo regulation of MHC class I gene expression is mediated by binding of trans-acting factors to the CRE.

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor.

The cDNA coding for human monocyte-derived neutrophil-specific chemotactic factor (MDNCF) was cloned from LPS-stimulated human monocyte mRNA. The cDNA sequence codes for a polypeptide consisting of 99 amino acids, including a putative signal sequence. Comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of natural MDNCF shows that the mature functional protein comprises 72 amino acids, beginning with serine at residue 28. The deduced amino acid sequence shows striking similarity to several platelet-derived factors, a v-src-induced protein, a growth-regulated gene product (gro), and an IFN-gamma inducible protein. The availability of the MDNCF cDNA enabled us to use it as a probe to identify inducers of MDNCF mRNA expression in human PBMC. MDNCF mRNA was increased greater than 10-fold within 1 h after stimulation with LPS, IL-1, or TNF, but not by IFN-gamma, IFN-alpha, or IL-2. Furthermore, we also determined that LPS, IL-1, and TNF stimulated the mononuclear cells to produce biologically active MDNCF. This observation may account for the in vivo capacity of IL-1 and TNF to induce netrophil infiltrates.

Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus-immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.