A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Feasibility of using genetic linkage analysis to identify the genes encoding T cell-defined minor histocompatibility antigens.

We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA-8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA-A*0201. EBV-transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA-A*0201 transgene, which led to high level expression of the MHC restricting allele HLA-A*0201 on the cell surface. HA-8 expression in the vaccinia-infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA-8 expression in these pedigrees demonstrated that the HA-8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA-8 gene localization further. The data collectively indicate that the HA-8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.

Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice.

The adoptive transfer of tumor-reactive CD8(+) T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8(+) T cell populations that recognize either tyrosinase(369-376) or gp100(209-217) from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-gamma accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8(+) T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

Immune responses to the HLA-A*0201-restricted epitopes of tyrosinase and glycoprotein 100 enable control of melanoma outgrowth in HLA-A*0201-transgenic mice.

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein.

Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1G(V) and MYO1G(M). The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1G(M) endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.

Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors.

We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC-associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor-resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Furthermore, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed.

Tyrosinase degradation via two pathways during reverse translocation to the cytosol.

Previous studies established that after inhibition of proteasome activity, tyrosinase could be detected in the cytosol after initial translation in the endoplasmic reticulum (ER), with a molecular weight consistent with that of a full-length, deglycosylated polypeptide. Here we show that most of these molecules are glycosylated, but have been proteolyzed at the carboxyl terminus by a protease that is insensitive to proteasome inhibitors. We also demonstrate the inhibitor-dependent accumulation of a membrane species that appears structurally homologous to the glycosylated and partially proteolyzed cytosolic form. Under some circumstances, cytosolic tyrosinase that had been deglycosylated and not proteolyzed prior to proteasomal degradation could also be detected. The presence of cytosolic tyrosinase was dependent upon glycosylation of the molecule during synthesis in the ER. These results suggest the existence of at least two alternative pathways for degradation of tyrosinase in the cytosol.

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients.

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients.

Identification of endogenous peptides recognized by in vivo or in vitro generated alloreactive cytotoxic T lymphocytes: distinct characteristics correlated with CD8 dependence.

We studied the molecular basis for CD8 independence of in vivo generated (BM3.3) versus CD8 dependence of in vitro sensitized (KB5.C20/Des) alloreactive H-2K(b)-specific cytotoxic T lymphocytes (CTL). Using microcapillary high-performance liquid chromatography fractionation of H-2K(b) eluates, mass spectrometry and CTL reconstitution assays, we determined that BM3.3 and KB5.C20 recognize, respectively, a single peptide (pBM1) expressed on 8,000 H-2K(b) molecules per allogeneic cell, and three distinct peptides (pKB1, 2, 3), each expressed on around 200 H-2K(b) molecules per allogeneic cell. CD8 (in)dependence was intrinsic to the respective TCR/H-2K(b)-peptide interactions. KB5.C20 and BM3.3 TCR illustrate the correlation that appears to exist between CD8 dependence/low affinity and in vitro sensitization as opposed to low dependency on CD8 and high TCR affinity observed after in vivo sensitization. The results suggest that CD8-dependent alloreactive CTL obtained in vitro with high frequency correspond to low-affinity TCR from the MHC-biased TCR repertoire unpurged by negative selection and have implications for cellular immunotherapeutic approaches.

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65).

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4(+) T cell response in IDDM.

The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.

Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo.

Posttranslational modification of peptide antigens has been shown to alter the ability of T cells to recognize major histocompatibility complex (MHC) class I-restricted peptides. However, the existence and origin of naturally processed phosphorylated peptides presented by MHC class I molecules have not been explored. By using mass spectrometry, significant numbers of naturally processed phosphorylated peptides were detected in association with several human MHC class I molecules. In addition, CD8(+) T cells could be generated that specifically recognized a phosphorylated epitope. Thus, phosphorylated peptides are part of the repertoire of antigens available for recognition by T cells in vivo.

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways.

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines.

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

Direct identification of human tumor-associated peptide antigens and a preclinical model to evaluate their use.

Although the arsenal of a healthy immune system includes both circulating antibodies and cellular components such as T cells, the latter seem to be particularly important in tumor immunology. Under normal conditions, the immune system does not react to the body's cells, which may be described as expressing "self" antigens on the cell surface. When a cell becomes cancerous, however, novel antigens are expressed on the cell surface. These novel "tumor" antigens are recognized as foreign by the body's immune system, and the cells that express them are destroyed or incapacitated. Whereas antibodies may react directly with protein antigens, T cells instead recognize peptide antigens presented by class I and class II molecules of the major histocompatibility complex (MHC). All cells normally break down proteins that they have made. The class I antigen-processing pathway has evolved to display peptides produced by this breakdown process as a way to provide information to cytotoxic T cells about what the cell is making. The display of new peptides as a result of infection or transformation can stimulate cytotoxic T cells to kill the cell. In addition, antigen-processing cells such as dendritic cells engulf dead or dying cells and degradeproteins into peptide fragments. These peptides are then displayed by the MHC class II molecules and presented to T helper cells, which augment the activity of the cytotoxic T cells. Cytotoxic T lymphocytes have recently been isolated from human tumors (especially melanoma) and are critical to the development of promising immunotherapeutic agents. As we shall discuss, these cells can recognize antigens that are common to tumors from different patients. We shall also explore how advances in instrumentation and the use of transgenic mice have increased our understanding of tumor-associated peptides to the point where we can begin to strive for a peptide-based therapeutic vaccine. The caveats for such therapy will also be addressed.

Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.

The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice.

Several HLA-A*0201-restricted peptide epitopes that can be used as targets for active immunotherapy have been identified within melanocyte differentiation proteins. However, uncertainty exists as to the most effective way to elicit CD8+ T cells with these epitopes in vivo. We report the use of transgenic mice expressing a derivative of HLA-A*0201, and dendritic cells, to enhance the activation of CD8+ T cells that recognize peptide epitopes derived from human tyrosinase and glycoprotein 100. We find that by altering the cell surface density of the immunizing peptide on the dendritic cells, either by pulsing with higher concentrations of peptide, or by changing the MHC-peptide-binding affinity by generating variants of the parent peptides, the size of the activated CD8+ T cell populations can be modulated in vivo. Significantly, the density of peptide that produced the largest response was less than the maximum density achievable through short-term peptide pulsing. We have also found, however, that while some variant peptides are effective at eliciting both primary and recall CD8+ T cell responses that can recognize the parental epitope, other variant epitopes lead to the outgrowth of CD8+ T cells that only recognize the variant. HLA-A*0201 transgenic mice provide an important model to define which peptide variants are most likely to stimulate CD8+ T cell populations that recognize the parental, melanoma-specific peptide.