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Background The complexity of candidate HIV-1 vaccine formulations is increasing due to extreme challenges faced when trying to prevent or control HIV-1 infection. Methods Immunogen HIVconsv based on the most conserved regions of the HIV-1 proteome was used to explore combinations of seven distinct vaccines modalities in heterologous prime-boost regimens delivered to rhesus macaques to optimize induction of T cell and antibody responses. These include plasmid DNA (P), Semliky Forest virus replicons delivered as DNA (DREP; D) or virus particles (VREP; V), modified vaccinia virus Ankara (MVA; M), adenoviruses of human (HAdV-5; A) and chimpanzee origin (ChAdV-63; C) and adjuvanted synthetic long peptides (SLP; S). Results A number of observations were made. Thus, a very potent combination for induction of HIV-1-specific T cells was an adenovirus vector (A or C) followed by poxvirus M. S boost broadened T cell responses, but did not prime T cells efficiently. D was a stronger prime than P. PPP was the best prime for T cells, while PSS was best for induction of antibodies. Even very complex regimen PPPAMSSCMV continued to recruit new T cell clones into the response to a single epitope, although a ceiling for immunodominant responses was reached; subdominant responses could be boosted up to the last V delivery. Finally, PPSS, but not SSSS could protect 2/6 animals from SIVmac251 acquisition. Conclusion These results will guide initial design of human trials. So far, human studies in Oxford testing CM, PPPCM and PPPMC regimen concur with observations made in rhesus macaques.

Bispecific Abs (BsAb) represent a novel format of immunotherapy, recognizing immune effector cells (e.g., T cells), on the one hand, and target cells (e.g., tumor cells), on the other hand. To be successful, cross-linking of the two cell types is necessary for effector cell activation and subsequent killing of the malignant target cells. We asked the question, whether CTL that were incubated with the BsAb aCD3 x aCD19 and malignant B cells and activated to kill the malignant B cells were still able to eliminate their natural target cells (e.g., virus-infected autologous body cells). To test this, HLA-A*0201-restricted, influenza-specific CTL were incubated with BsAb- and HLA-A*0201-positive B lymphoid tumor cells in combination with HLA-A*0201-positive, virus-infected non-B lymphoid cells as natural target cells. The results showed that even in the presence of BsAb and high amounts of tumor B cells, CTL were still capable of eliminating the virus-infected non-B lymphoid target cells; actually, CTL recognized and eliminated the homologous original target cells preferentially.

The application of molecularly defined vaccines composed of a limited number of tumor-specific T cell epitopes has resulted in protective antitumor T cell immunity in several mouse tumor models. The first encouraging results with such vaccines have been obtained in human beings. The development of the next generation of rationally designed vaccines that are both effective and safe for application in a clinical setting requires comparison of different modes of delivery of tumor-associated T cell epitopes in multiple epitope constructs.

CTL can play an important role in the defense against tumors. Protective CTL-mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases.

The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

CTL that lyse melanoma cells were previously isolated from several melanoma patients. Such CTL recognize autologous proteins, indicating the occurrence of autoreactive T cells in melanoma patients. We have now raised CTL, using responding T lymphocytes from healthy donor blood, that lysed not only cells incubated with an HLA-A*0201-binding tyrosinase peptide but also melanoma cells endogenously processing and presenting the epitope. Our results suggest that autoreactive CTL precursors are present in healthy donor blood and can be activated in vitro with synthetic peptides presented on appropriate APCs. Therefore, tissue-specific, autoantigen-derived peptides might be useful in immunotherapy of both poorly and nonimmunogenic tumors.

Mouse embryo cells (C57BL/6, H-2b) transformed by the E1A and E1B genes of adenovirus type 5 (Ad5E1 MEC) are highly immunogenic. Previously, CTL were cloned from mice immunized with Ad5E1 MEC. These CTL clones were capable of tumor eradication in nude mice, and were directed against the Ad5E1A-encoded decapeptide SGPSNTPPEI, presented by the H-2Db MHC molecule. We have now generated Ad5E1 MEC containing a mutated Ad5E1A-encoded epitope. The mutant Ad5E1 MEC induce a strong CTL response when injected into immunocompetent mice. CTL clones generated against mutant Ad5E1-transformed tumor cells recognize an Ad5E1B-encoded epitope (VNIRNCCYI) in the context of H-2Db. Because this epitope is also present on wild-type Ad5E1 MEC, it is concluded that Ad5E1-transformed tumor cells express at least two CTL epitopes. Interestingly, the lysis of Ad5E1 MEC by the Ad5E1B-specific, but not by the Ad5E1A-specific, CTL clones was strongly diminished by the action of the activated ras oncogene. CTL directed against the Ad5E1B-encoded epitope were, like Ad5E1A-specific CTL, able to eradicate large established Ad5E1-induced tumors in B6 nude mice, demonstrating that CTL activity directed against different CTL epitopes expressed by the same tumor can be exploited for immunotherapy of cancer.

H-2b mice are immunologic responders to the tumorigenic MCF1233 murine leukemia virus (MuLV), an AKV-related virus derived from endogenous C57BL MuLV. We have identified an immunodominant CTL epitope that is expressed on MCF1233 MuLV-induced lymphomas of H-2b mice. C57BL/10 (B10) mice were immunized with an MCF1233-induced B10 B cell lymphoma, and tumor-specific CTL cultures were generated in vitro. These were tested for recognition of synthetic class I-binding MuLV peptides, selected for class I allele-specific motifs. One of 28 candidate peptides sensitized target cells for CTL recognition. This peptide seems to be an immuno-dominant epitope, because it was recognized by all independent CTL clones, isolated from the tumor-specific bulk culture. The epitope (KSPWFTTL) is derived from the MCF1233 MuLV envelope (env)-p15E region and is shared by all endogenous AKV types of MuLV. It has an optimal length of eight amino acids and is presented by the Kb H-2 class I molecule. Interestingly, Friend, Moloney, and Rauscher (FMR) types of MuLV are not recognized by MCF MuLV-directed CTL. The FMR env-p15E proteins have a single amino acid difference at the first position of the MCF1233 MuLV epitope (RSPWFTTL instead of KSPWFTTL). The corresponding FMR-encoded peptide bound class I H-2 Kb equally well as the MCF peptide, but it was poorly recognized by MCF1233 MuLV-specific CTL. Moreover, in the Rauscher MuLV-induced cell line RMA the FMr peptide seems not to be processed for recognition by CTL, which was illustrated by experiments with CTL elicited against this peptide. Altered TCR interaction as well as lack of processing thus may explain the type specificity of MCF1233 MuLV-directed CTL.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.

C57BL/6 mice are protected from a lethal pneumonia caused by Sendai virus when treated with low doses of mAb directed to the CD3 Ag. The protective mechanism is not due to an accelerated Sendai virus-specific Th cell, CTL, or antibody response but to a strong NK cell response via the in vivo induction of lymphokines. Antibodies directed against the NK1.1 and asialo GM1 marker totally reversed the protective effect of anti-CD3 treatment. In vivo treatment with rIL-2 also induced NK activity and induced antiviral protection. Treatment with anti-CD3 protects when given in a narrow time window (1 day before until 1 day after Sendai virus inoculation), indicating that NK activity is protective in the early phase of virus infection.

We have previously shown that small B16 melanomas can be successfully treated using a combination of anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibody with a granulocyte/macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cell vaccine. Regression of tumors results in long-lasting immunity and is frequently accompanied by autoimmune depigmentation. Here we examine the cellular and molecular mechanisms of this combined treatment. Histological examination of depigmented lesions revealed infiltration of polymorphonuclear cells and deposition of antibody. The combination therapy also induced tumor rejection and skin depigmentation in B cell-deficient and in CD4(+) T cell-depleted mice. Both effects of the treatment absolutely required CD8(+) T cells. Analysis of the response in successfully treated mice revealed elevated levels of CD8(+) T cells specific for a nonameric peptide consisting of residues 180-188 of the melanocyte differentiation antigen tyrosinase-related protein (TRP)2. There was no evidence of reactivity to the melanocyte antigens gp100, tyrosinase, Mart1/MelanA, or TRP1. Fas-FasL interactions and perforin played a role in mounting the effector response, whereas the tumor necrosis factor pathway was not required. The cellular requirements for tumor rejection in this therapeutic setting were strikingly different from those in a prophylactic setting. In particular, if mice received a prophylactic vaccine consisting of anti-CTLA-4 and B16-GM-CSF before tumor challenge, full protection was obtained even in the absence of CD8(+) T cells. Our data demonstrate that therapeutic autoreactive CD8(+) T cell responses can effectively be generated in tumor-bearing mice and stresses the value of studying tumor immunity in a therapeutic rather than a prophylactic setting.

Human Papillomaviruses (HPVs) are sexually transmitted pathogens, which are implicated in the etiology of cervical cancer. The early proteins E6 and E7 of HPV have transforming capacity and interfere with the cell cycle control of infected host cells and are essential for the maintenance of the transformed state. Identification of MHC class I-restricted, immunogenic peptides derived from either the E6 or the E7 protein is essential for the design of vaccines as well as the monitoring of clinical trials and immunotherapeutic approaches for the treatment of HPV-18-induced carcinomas. We have determined the binding affinities for all possible 9-mer peptides spanning the entire E6 and E7 amino acid sequence for the HLA-A*0101, HLA-A*0201, HLA-A*0302, HLA-A*1102, and HLA-A*2402101 molecules by a competition assay with reference peptides, thereby establishing the binding peptides as potential cytotoxic T-cell epitopes. From the HLA-A*0201 binding peptides, we selected five E6-derived and one E7-derived peptide with high affinities for HLA-A*0201. These six peptides were tested for their immunogenicity by in vitro immunization assays with purified human CD8+ T cells. We identified three HPV-18 E6-derived peptides (ELTEVFEFA, KTVLELTEV, and KLPDLCTEL) and the E7-derived peptide TLQDIVLHL to be highly immunogenic. Overall, these results will help to design vaccines for the prevention or treatment of HPV-18-induced cervical cancer.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.

The human epithelial mucin MUC1 is over-expressed in more than 90% of carcinomas of the breast, ovary, and pancreas as well as in some other tumours, making it a potential target for tumour immunotherapy. We have identified several MUC1-derived peptides mapping outside the variable number tandem repeat region that comply with the peptide-binding motif for HLA-A*0201 and that become processed into stable major histocompatibility complex-peptide complexes as assessed by in vitro assays. In A2/K(b) transgenic mice, 3 peptides, namely MUC(79-87) (TLAPATEPA), MUC(167-175) (ALGSTAPPV) and MUC(264-272) (FLSFHISNL) elicit peptide-specific cytotoxic T lymphocyte (CTL) immunity, which protects these mice against a challenge with MUC1, A2/K(b)-expressing tumour cells. These peptides therefore represent naturally processed MUC1-derived CTL epitopes that could be used as components in peptide-based vaccines and for the analysis of anti-MUC1 CTL responses in A*0201-positive patients with MUC1-expressing tumours.

Colorectal carcinoma is commonly associated with mutation and overexpression of p53, making this antigen a potential target for immune intervention. We analyzed humoral and proliferative immunity against p53 in the blood of patients with resected primary colorectal cancer. The majority of these patients displayed anti-p53 T helper (Th) immunity in the absence of measurable p53 specific antibody levels. The Th responses were long-lasting since they could be detected up to several years after resection of the primary tumor. In a number of cases the Th responses were highly sensitive, reflected by the recognition of naturally processed p53 protein. Our data argue that boosting of these responses in patients with minimal residual disease through p53-specific vaccination, may be employed for improving the chance of disease-free survival of these patients.

CTLs specific to p53 were previously shown to efficiently eradicate p53-overexpressing tumor cells in vitro as well as in vivo. In this report, we demonstrate that these CTLs can also eliminate tumors that display moderate or even low levels of p53. Neither high steady-state levels of p53 nor elevated p53 synthesis is a prerequisite for recognition of tumors by p53-specific CTLs. Instead, our data show that a high p53 turnover rate is an important factor in determining the sensitivity of tumor cells to p53-specific CTLs. Our data suggest that p53 turnover is related to the MHC class I-restricted presentation of p53-derived epitopes at the tumor cell surface and indicate that CTL-mediated immunotherapy that targets p53 can be applied to a wider range of tumors than has thus far been anticipated.

The tumor suppressor protein p53 is overexpressed in up to 50% of all human malignancies, both in solid tumors as well as hematological malignancies, and is therefore an attractive target for immunotherapy. We have recently shown that cytotoxic T lymphocytes (CTL), raised in p53 gene deficient (p53 -/-) mice and recognizing a murine wild-type (wt) p53 peptide, were able to eradicate a mutant p53-induced and overexpressing tumor in p53 +/+ nude mice. These CTL also prevented the outgrowth of a more aggressive p53-overexpressing tumor in immunocompetent C57BL/6 mice. Importantly, this occurred in the absence of demonstrable damage to normal tissue. Possibly due to the aggressive nature of the latter tumor, adoptive transfer of wtp53-specific CTL did not result in the eradication of established tumors, either in nude or immunocompetent mice. Therefore, we explored whether the cytotoxic drug cyclophosphamide (CY) could potentiate the therapeutic activity of wtp53-specific CTL. We show here that CY acts synergistically with adoptively transferred wtp53-specific CTL in controlling the growth of an aggressive mutant p53-induced and overexpressing tumor. Previously described mechanisms underlying the synergism between CY and immune T cells were evaluated, but were not found to be operational in this model.

Immune privilege in the eye is considered essential in the protection against local sight-threatening inflammatory responses. However, the deviant immune responses in the eye may also provide an ideal opportunity to uncontrolled growth of viruses or tumors by inhibiting intraocular immunological attack. To establish to what extent immune privilege interferes with T cell-mediated antitumor immunotherapy, we established a new ocular tumor model in the mouse and tested whether well-defined tumor-specific CTLs can eradicate an immunogenic intraocularly growing tumor. Tumor cells, transformed by human adenovirus type 5 early region 1 (Ad5E1), injected s.c. in a dose of 10(7) cells, did not induce s.c. tumor growth in C57BL/6 mice. However, an injection of 0.3 x 10(6) of these cells into the anterior chamber of the eye led to intraocular tumor growth in 95% of mice (n = 20). Tumor growth in the eye did not induce systemic tumor-specific tolerance, because 70% of the mice were able to eradicate the tumor spontaneously after 5 weeks. Mice vaccinated s.c. with irradiated tumor cells were protected against intraocular tumor challenge, indicating that preactivated memory T cells are able to protect against intraocular tumor growth. Moreover, an i.v. injection of an Ad5E1-specific CTL clone was able to eradicate established intraocular Ad5E1-transformed tumors, whereas the anatomy of the eye remained intact. These results demonstrate that tumor-specific, CTL-mediated immunity can be used successfully for the prevention and eradication of tumors growing in the immune-privileged anterior chamber of the eye, without detectable destruction of the eye.

Peptides corresponding to the fusion site in 210 kD BCR-ABL protein b3a2 (p210b3a2) were previously shown to bind to several HLA class I and II alleles. We have found that b3a2 peptide-specific CD4-positive T-helper cells were able to recognize p210b3a2-positive chronic myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until now, there were no reports of b2a2 breakpoint-specific human T-cell responses. Here we show that repetitive stimulation of T lymphocytes with a 17mer peptide covering the fusion region in p210b2a2 also leads to specific T-cell responses. CD4 and CD4/CD8 double-positive clones obtained from a b2a2 peptide-specific cell line were cytotoxic and proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous Epstein-Barr virus (EBV) transformed cells, expressing BCR-ABL(b2a2) on transfection, and allogeneic HLA-DR matched p210b2a2-positive cells from CML patients were, however, not lysed. BCR-ABL peptide-specific T-cell clones did respond to autologous EBV cells transfected with invariant chain (li) cDNA in which the HLA class II-associated invariant chain peptide (CLIP) was replaced by a BCR-ABL b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABL(b2a2) ligand.

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.

Although numerous studies have documented a role for B7-1 (CD80) in the induction of antitumor CTL immunity, it is presently unclear to what extent expression of this costimulatory molecule truly endows tumors with significant in vivo APC (antigen-presenting cell) capacity. Recent studies have, in fact, demonstrated that cross-priming, rather than direct priming, may constitute the major mechanism of CTL induction by B7-1 expressing tumors. We have, therefore, investigated the requirements for antigen density and costimulatory molecules in direct CTL priming with a prototype cell-based vaccine that uses a signal sequence-containing minigene to direct expression of a tumor-specific CTL epitope to the endoplasmic reticulum. This design limits sources of antigen available to professional APC in the host and, thereby, the contribution of cross-priming. Induction of antitumor CTL immunity by our prototype APC was shown to solely involve direct priming, independent of host APC, NKI.1+ cells, and CD4+ T cell help. CTL induction through this mechanism required the engineered APC to express the B7-1 molecule as well as a sufficiently high density of peptide/MHC complexes at its surface. Our data, in contrast to previous studies using modified tumor cells, clearly define the antigenic and costimulatory requirements for a suitably engineered "artificial" APC to directly prime peptide-specific CTL in vivo, and demonstrate that the signal sequence minigene approach allows the engineering of highly effective and well-defined cellular vaccines for activation of CTL against epitopes of choice.

Murine tumor cells obtained through transfection of expression plasmids carrying activated cellular and/or viral oncogenes constitute formidable tools for immunological tumor research. As reported previously, mouse embryo cells of C57BL/6 origin, transformed by mutated p53 or human papilloma virus type 16 (HPV16), present, at their surface, MHC-bound peptides that are derived from the p53 and the HPV16 E7 oncoproteins, respectively, which can serve as a target for a highly effective antitumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide that is presented by the aforementioned HPV16- and p53-transformed cells. This peptide is encoded by a cryptic open reading frame in the backbone sequences of the plasmids that had been used to generate these cells. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were the result of the bidirectional nature of the retroviral long terminal repeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR. Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhibited lower levels of this peptide. LTR-driven expression plasmids were also shown to encode this peptide epitope when used for DNA vaccination, as mice vaccinated with such a plasmid developed a CTL response against this peptide. Our data show that awareness of plasmid backbone-derived epitopes is of crucial importance for the correct interpretation of preclinical experiments and for the design of DNA vaccines.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

Bispecific Abs (BsAb) represent a novel format of immunotherapy, recognizing immune effector cells (e.g., T cells), on the one hand, and target cells (e.g., tumor cells), on the other hand. To be successful, cross-linking of the two cell types is necessary for effector cell activation and subsequent killing of the malignant target cells. We asked the question, whether CTL that were incubated with the BsAb aCD3 x aCD19 and malignant B cells and activated to kill the malignant B cells were still able to eliminate their natural target cells (e.g., virus-infected autologous body cells). To test this, HLA-A*0201-restricted, influenza-specific CTL were incubated with BsAb- and HLA-A*0201-positive B lymphoid tumor cells in combination with HLA-A*0201-positive, virus-infected non-B lymphoid cells as natural target cells. The results showed that even in the presence of BsAb and high amounts of tumor B cells, CTL were still capable of eliminating the virus-infected non-B lymphoid target cells; actually, CTL recognized and eliminated the homologous original target cells preferentially.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.

MHC-class-I-restricted cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens expressed by malignant cells are important components of the immune response against cancer. Recently, tumor-specific CTL could be generated in vitro, with responding lymphocytes from the blood of healthy blood donors. In the present study, we confirm that peptide-specific stimulation in vitro can induce high-affinity CTL capable of recognizing tumor cells expressing the appropriate tumor antigen. These tyrosinase-specific CTL display a restricted usage of TCRAV and TCRBV gene segments but of diverse CDR3 regions, resulting in a distinct fine-specificity for each CTL clone. This suggests that, similar to in vivo priming, peptide-pulsed APC are capable of stimulating a T-cell response in vitro expressing a limited TCR repertoire against autologous tumors. The generated CTL can recognize their target structure with high affinity, and this correlates in part with tumor-cell lysis. This methodology may be used to treat melanoma patients with infusion of ex vivo-induced and -expanded CTL.

MAGE-2 is expressed in many tumors, including melanoma, laryngeal tumors, lung tumors and sarcomas, but not in healthy tissue, with the exception of testis. Thus, MAGE-2-derived peptides that bind to HLA class I molecules and elicit cytotoxic T lymphocyte (CTL) responses could be of significant therapeutic importance. In this study, we show that several MAGE-2-derived peptides bind with high affinity to HLA-A*0201. Three of them form complexes with HLA-A*0201 that are stable at 37 degrees C and are immunogenic in HLA-A*0201Kb transgenic mice. Moreover, CTLs against 2 of them (M2 112-120, and M2 157-166) specifically recognize cells that express both the MAGE-2 protein and HLA-A*0201Kb. These 2 peptides are processed and presented in the context of HLA-A*0201. Therefore, these peptides are candidate components in peptide-based vaccines for the treatment and prevention of several types of MAGE-2-expressing cancers.

A small group of HIV-1-infected subjects who either do not progress to AIDS or progress only slowly have sustained HIV-1-specific CTL responses. It has been suggested that the specificities of these responses differ from the CTL responses of rapid progressors due to recognition of epitopes that are under structural or functional constraints. We have, in this respect, studied the CTL response to reverse transcriptase (RT) in long term survivors (LTS) and in HIV-1-infected individuals who progressed to AIDS within 3 to 6 yr. Both LTS and progressors displayed vigorous RT-specific CTL responses of comparable magnitude during the asymptomatic phase. From each individual at least two CTL lines were obtained from blood samples drawn at different time points during follow-up. A total of 19 CTL lines recognized nine different RT-derived epitopes. CTL obtained from progressors recognized epitopes with a similar degree of amino acid conservation as epitopes targeted by CTL from LTS. Furthermore, five of seven epitopes were recognized by both LTS and progressors. Moreover, one of the epitopes recognized by progressors contained the highly conserved YMDD motif that is essential for RT activity. In conclusion, our data imply that neither the magnitude nor the specificity of HIV-1-specific CTL against RT is a major cause of a more protracted course of disease.

After allogeneic bone marrow transplantation (BMT), the beneficial graft-versus-leukemia (GVL) effect but also the life-threatening graft-versus-host disease (GVHD) are mediated by T cells of the grafted marrow. The identification of leukemia cell-reactive T cells and their ligands are, therefore, crucial for the development of new anti leukemia strategies. Here we describe a leukemia-reactive allo-HLA class II restricted CD4+ T-cell clone, 6.2, isolated from a healthy individual after stimulation with allogeneic leukemic cells. Clone 6.2 recognizes leukemic cells from several AML patients without showing reactivity to unfractioned peripheral blood mononuclear cells, monocytes, B cells, T-cell blasts, and proximal tubulus epithelial cells. Interestingly, clone 6.2 also recognizes BM cells derived from healthy individuals and inhibits the colony formation of myeloid and erythroid cell lineages. In the BM, clone 6.2 recognizes only CD34+ early precursor cells but not CD34-, more differentiated cells. Thus, the target antigen of clone 6.2 is developmentally regulated and expressed only by leukemic cells and CD34+ early progenitor cells in the hematopoietic system. We suggest that targeting the T-cell immune response to leukemia-associated, developmentally regulated antigens of the hematopoietic system can provide a basis for the separation of GVL from GVHD, and may lead to new therapeutic approaches for residual and relapsed leukemia.

The MHC class-I binding affinity of an epitope is an important parameter determining the immunogenicity of the peptide-MHC complex. In order to improve the immunogenicity of an epitope derived from melanocyte lineage-specific antigen gp100, we performed amino-acid substitutions within the epitope and assayed both HLA-A*0201 binding and CTL recognition. Anchor replacements towards the HLA-A*0201 peptide-binding motif gave rise to peptides with higher HLA-A*0201 binding capacity compared to the wild-type epitope. In addition, several of the gp100 154-162 epitope-analogues were more efficient at target-cell sensitization for lysis by anti-gp100 154-162 CTL compared to the wild-type epitope. These altered gp100 154-162 epitopes were subsequently tested for their capacity to induce CTL responses in vivo using HLA-A*0201/Kb transgenic mice, and in vitro using HLA-A*0201 + donor-derived lymphocytes. Interestingly, the peptide-specific CTL obtained, which were raised against the different gp100 154-162 epitope-analogues, displayed cross-reactivity with target cells endogenously processing and presenting the native epitope. These data demonstrate that altered epitopes can be exploited to elicit native epitope-reactive CTL. The use of epitope-analogues with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

The mouse strains C57BL/6 (B6, H2b) and Kbm1 mutant bm1 have a defined difference of three amino acids at position 152, 155, and 156 in the MHC class I K molecule. This causes a change in the side and the bottom of the antigen presenting groove of the K molecule resulting in strong allogeneic responses in vitro and in vivo. Here we report on the peptide specificity of CD4+ T cells of B6 origin directed against the Kbm1 mutant and speculate on the peptide specificity of CD8+ bm1-specific T lymphocytes of B6 origin. Bm1-specific CD4+ T helper cells recognized a peptide derived from the Kbm1 molecule encompassing the three mutations, presented by MHC class II molecules on syngeneic cells. The ability of this peptide to bind to MHC class II resulted from amino acid mutations at positions 155 and 156. Furthermore, the recognition of the natural peptide derived from the Kbm1 molecule presented by MHC class II I-Ab molecules on cells of bml origin could be blocked by addition of an MHC class II I-Ab binding competitor peptide. Thus, due to the mutations in an MHC class I molecule, indirect presentation via MHC class II molecules and MHC class II-restricted recognition of a peptide derived from such a MHC class I molecule is demonstrable.

In chronic myeloid leukemia (CML) the classical 9;22 translocation results in a BCR-ABL fusion gene, which encodes chimeric BCR-ABL fusion 210 kD oncoproteins (p210BCR-ABL). The two main p210BCR-ABL fusion variants in CML, b2a2 and b3a2 are examples of well characterized antigens expressed by malignant cells. The possibility of an immunotherapeutic approach involving the fusion part of p210BCR-ABL in CML has previously been illustrated by observed peptide binding to major histocompatibility complex (MHC) class I alleles and by demonstrating the immunogenicity of p210BCR-ABL breakpoint peptides. In this report we show that in vitro immunization of human T cells with a 17 amino acid (aa) peptide representing the p210BCR-ABL fusion region resulted in peptide specific CD4+ T-cell lines designated P4, P6, and P7. HLA DR4 (DRB1*0401) restricted T-cell line P4 and several subsequently derived clones recognized HLA-DRB1*0401 and p210b3a2-mRNA expressing blasts from an allogeneic patient with CML in blast crisis. Recognition appeared DR expression-dependent. No responses were observed with DR4 positive p210BCR-ABL negative cells or with p210b3a2 leukemic cells with absent or insufficient expression of DR4. These observations indicate that oncoprotein p210b3a2 can be degraded and processed for presentation by MHC class II molecules at the surface of leukemic cells. The BCR-ABL fusion region is in all likelihood presented as peptides by HLA DR and thus capable to act as a distinctive tumor antigen to peptide specific CD4+ T cells.

An impaired immune response is frequently observed in patients and experimental animals with advanced cancer. We and others have shown alterations in CD3-associated signal-transducing zeta molecules in tumor-infiltrating T cells and peripheral blood lymphocytes (PBLs) of patients with advanced cancer. By using flow cytometric analysis of permeabilized cells with a monoclonal antibody (TIA-2) that reacts with the cytoplasmic domain of the zeta chain, here we demonstrate a marked decrease (P0.01) in the expression of the signal-transducing CD3 zeta chain of PBLs in patients with cervical cancer (n = 22) as compared to PBLs from healthy donors (n = 21). In addition, PBLs isolated from patients (n = 23) with cervical intraepithelial neoplasia (CIN), to a lesser but significant (P0. 01) extent, expressed reduced CD3 zeta levels as compared to those from healthy donors. This decreased expression of zeta chains was also observed on CD16(+) natural killer cells in PBLs from patients with cervical cancer. Surface expression of CD3 epsilon on PBLs was also decreased in cervical cancer patients as compared to healthy donors, but not on PBLs from patients with CIN. CD3 zeta chain expression significantly (r = 0.53, P0.01) correlated with the ability of the PBLs to produce tumor necrosis factor in response to anti-CD3 stimulation. These findings suggest that alterations of signal-transducing zeta molecules commonly occur in patients with cervical cancer and to a lesser extent with CIN, and that they are associated with reduced cellular functions such as production of tumor necrosis factor.

CTL can play an important role in the defense against tumors. Protective CTL-mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases.

The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines.

Most cervical carcinoma (Cxca) cells constitutively express human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins. These proteins are, therefore, attractive targets for T cell-based immunotherapy. Previously, we identified HVP16 E7-encoded CTL epitopes. In patients with cervical intraepithelial neoplasia or Cxca, little is known concerning T-cell activity against viruses in general and against HPV16 in particular. Here, we have screened the blood of 10 healthy donor controls and of 22 patients with HPV16+ cervical lesions for the presence of CTLs directed against HPV16 E7- and control influenza virus matrix-derived epitopes presented by HLA-A *0201. We detected influenza virus-specific CTLs in all donors and in the majority of patients, indicating that most patients have functioning T-cell responses despite their lesions or therapeutic interventions. Moreover, we show that patients with HPV16+ lesions occasionally have memory CTLs against a HPV16 E7-encoded epitope (sequence YMLD-LQPETT), providing evidence for natural CTL immunity against HPV16 in patients with cervical lesions. Combined, these findings raise possibilities for vaccination with HPV16 E7-encoded peptides to induce or augment CTL responses for treatment or prevention of Cxca.

A peptide-binding assay employing the HLA class I molecules on intact human B cells is described. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment, after which the cells are incubated with a FL-labeled reference peptide together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACscan analysis. The assay is easy to perform because there is no need to purify HLA class I molecules, or to transfect cells with HLA class I molecules, and no radioactive label is used. Moreover, large panels of HLA-typed human B-cell lines are available as tools for peptide binding to a vast array of HLA molecules. The binding assay was optimized and validated with peptides of known binding capacity to either HLA-A*0201 or HLA-A*0301. The kinetics of peptide binding in this assay were shown to be comparable to that in assays employing soluble HLA class I molecules. Application of the assay in the search for potential HLA-A*0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five high-affinity binding peptides.

In chronic myeloid leukemia (CML) the proto-oncogene c-abl from chromosome 9 q34 is translocated to the breakpoint cluster region (bcr) gene on chromosome 22 q11. This translocation results in a BCR-ABL fusion gene, which encodes chimeric fusion oncoproteins p210BCR-ABL. Here we demonstrate that a peptide with joining region sequence ATGFKQSSKALQRPVAS (eight amino acids (aa) encoded by BCR exon 3; one novel lysine, encoded by the fusion codon; eight aa encoded by ABL exon 2) is immunogenic to human T cells. Primary immune response induction with this peptide resulted in a HLA DR2(DRB1*1501) restricted CD4+ BCR-ABL peptide specific T cell line P1. Responses of P1 were negatively affected by individual aa replacement by alanine at eight aa positions within the 17mer peptide (F4, K5, Q6, K9, L11, Q12, R13, P14). These findings were supported by experiments with a panel of overlapping 11mer b3a2 peptides. Only two of these peptides with an aa sequence encompassing all residues which could not be replaced by alanine induced P1 proliferation. Since presentation of cytosolic oncoproteins as peptides by DR molecules has been observed, the present findings provide a possible explanation for post interferon-alpha persisting remissions in spite of the presence of BCR-ABL PCR positive progenitors.

MHC class II (MHC-II) molecules bind fragments of exogenous Ags in an intracellular endocytotic compartment. In view of divergent data on the MHC-II distribution in different cell lines, it was of interest to localize MHC-II molecules in a natural and the most potent APC type, the dendritic cell (DC). By using immunogold labeling of ultrathin cryosections of cultured mouse spleen DC, we found that MHC-II molecules were present abundantly at the plasma membrane and in intracellular compartments containing internal membrane vesicles and/or membrane sheets. The majority of these compartments was situated late in the endocytotic route, as demonstrated by the late appearance (after a lag of 30 min) of internalized exogenous tracer. These compartments contained the lysosomal enzymes cathepsin D and beta-hexosaminidase, but lacked the late endosomal marker cation-dependent mannose-6-phosphate receptor. We conclude that most of the intracellular MHC-II molecules in cultured spleen DC reside in a compartment with (pre)lysosomal characteristics, resembling the so-called MHC-II-enriched compartments (MIIC), originally described in B cells. We also investigated whether the presence of MHC-II molecules in endocytotic compartments was related to the kinetics of Ag processing and presentation by these cells. Pulse-chase endocytosis experiments with hen egg lysozyme (HEL) as a model Ag showed that activated spleen DC were able to efficiently process and present this Ag to an HEL-specific T hybridoma cell line. However, presentation started only after a lag of 2 h and was maximal after 6 h. The difference in time between the arrival of Ag in proteolytic endocytotic compartments, in particular MIIC, and effective Ag presentation is discussed in the context of DC maturation.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

CTL that lyse melanoma cells were previously isolated from several melanoma patients. Such CTL recognize autologous proteins, indicating the occurrence of autoreactive T cells in melanoma patients. We have now raised CTL, using responding T lymphocytes from healthy donor blood, that lysed not only cells incubated with an HLA-A*0201-binding tyrosinase peptide but also melanoma cells endogenously processing and presenting the epitope. Our results suggest that autoreactive CTL precursors are present in healthy donor blood and can be activated in vitro with synthetic peptides presented on appropriate APCs. Therefore, tissue-specific, autoantigen-derived peptides might be useful in immunotherapy of both poorly and nonimmunogenic tumors.