Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells.

In earlier studies, primary proliferative and cytotoxic T-cell (CTL) responses to influenza virus were produced in vitro by using mouse dendritic cells (DC) pulsed with virus or viral peptide as the stimulus for syngeneic T cells in 20-microliters hanging-drop cultures. We have now adapted this system for producing primary responses with cells from non-immune donors to produce primary proliferative and CTL responses to human immunodeficiency virus I (HIV) and to HIV peptides in vitro using cells from normal human peripheral blood. All donors in this study were laboratory personnel with no history of HIV infection. DC enriched from peripheral blood were exposed to HIV in vitro and small numbers were added to T lymphocytes in 20-microliters hanging drops. Proliferative responses to virus-infected DC were obtained after 3 days in culture. After 6 days, CTL were obtained that killed virus-infected autologous--but not allogeneic--phytohaemagglutinin (PHA)-stimulated blast cells. Proliferative and CTL responses were obtained using cells from 14 random donors expressing a spectrum of major histocompatibility complex (MHC) types but the CTL, once produced, showed killing restricted by the MHC class I type. Treatment of cultures with monoclonal antibody (mAb) to CD4-positive cells at the beginning of culture blocked the development of both proliferative and CTL responses, but treatment after 5 days had no effect on the CTL activity. Treatment with MCA to CD8-positive cells at the beginning of culture did not block proliferation significantly, but treatment either before or after the 5-day culture period blocked CTL responses. Collaboration between proliferating CD4-positive cells and CD8-positive cells may thus be required to produce CTL of the CD8 phenotype. DC exposed to HIV also produced CTL that killed autologous blast cells pulsed with gp120 envelope glycoprotein. However, DC infected with whole virus did not produce CTL that lysed target cells pulsed with a synthetic peptide, which included a known T-cell epitope of gp120 (representing amino acids 111-126). DC pulsed with gp120 were a poor stimulus for the development of CTL. In contrast, DC pulsed with the peptide (111-126) stimulated both proliferative and CTL responses. The latter killed not only target cells pulsed with the peptide itself or with gp120 but also killed virus-infected autologous blast cells. CTL were again obtained reproducibly with this peptide using donors expressing a spectrum of MHC types.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain

The cytotoxic T-lymphocyte (CTL) response to rabies virus glycoprotein has been studied. A primary in vivo CTL response was obtained following inoculation of A/J mice with 10 micrograms of glycoprotein, but only when in the form of reconstituted glycoprotein-lipid vesicles. These glycoprotein-lipid vesicles were prepared with lipids from BHK-21 cells, and did not incorporate mouse major histocompatibility complex (MHC) antigens. Secondary in vitro stimulation of rabies virus-specific CTL was obtained with inactivated virus and with larger quantities of glycoprotein. This response, but not that induced by rabies virus-infected stimulator cells, was dependent on the presence of radiation-resistant accessory cells which could not be replaced with T-cell growth factors. An analysis of the molecular requirements for stimulation of CTL by glycoprotein revealed that cleavage by cyanogen bromide (CNBr) or limited tryptic digestion actually enhanced stimulation of CTL. In contrast, reduction and alkylation destroyed activity. Following separation of CNBr or tryptic fragments by gel electrophoresis or high-pressure liquid chromatography (HPLC) (under nonreducing conditions), a nominal determinant of glycoprotein was identified. One CNBr peptide (residues 103-178) and one peak of tryptic peptides were found to stimulate rabies virus-specific CTL. The tryptic peak was further analyzed by Edman degradation-sequencing, and found to consist of three peptides with amino terminals at residues 130, 251 and 279. This evidence suggests that a nominal determinant of glycoprotein responsible for stimulating rabies virus-specific CTL is located between residues 130-178 of the glycoprotein, and incorporates a single disulfide loop (159-169) which is necessary for biologic activity.     

Superior induction of anti-tumor CTL immunity by extended peptide vaccines involves prolonged, DC-focused antigen presentation

Anti-tumor vaccines consisting of extended CTL peptides in combination with CpG-ODN were shown to be superior to those comprising minimal CTL epitopes and CpG-ODN, in that they elicit stronger effector CTL responses with greater tumoricidal potential. We now demonstrate that this improved performance is primarily due to the focusing of CTL epitope presentation onto activated DC in the inflamed lymph nodes draining the vaccination site. In the case of vaccination with minimal peptides, additional APC including T and B cells are also loaded with CTL epitopes. Our data suggest that circulation of these peptide-loaded lymphocytes leads to epitope presentation in non-inflamed lymphoid organs distal from the vaccination site, in the absence of potent costimulatory signals required for efficient CTL priming. The resulting blend of pro-immunogenic and tolerogenic signals, which results in suboptimal activation of the CTL response, is avoided by vaccinating with extended CTL peptides. An additional advantage of extended CTL peptide vaccines is an increased duration of in vivo epitope presentation.     

Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.     

Influenza basic polymerase 2 peptides are recognized by influenza nucleoprotein-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.     

Cytotoxic T cell response against lymphoblasts infected with Moloney (Abelson) murine leukemia virus. Methodological aspects and H-2 requirements

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2^b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2^b. In analogy with previous studies on Moloney virus-specific CTL. it was observed that C57BL/6 (H-2^b) effector cells predominantly lysed D^b-compatible, virus-infected target cells; B10.A(5R), (K^bD^d) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.     

Short peptides sensitize target cells to CTL specific for the MHC class Ib molecule,H2-M3

The MHC class Ib molecule H2-M3 presents N-formylated peptides from the N terminus of proteins encoded by the mitochondrial genome to CTL. A panel of CTL specific for a peptide derived from a mitochondrial protein, either COI or ND1, was used to determine the optimal peptide length for sensitizing antigen-deficient target cells. All long-term CTL lines and most CTL clones lysed target cells sensitized with either a COI hexamer or an ND1 heptamer. Only 3 out of 12 anti-ND1 clones preferred an octamer or nonamer peptide and no CTL required to longer peptides. The CTL preference for short peptides matches a shortened groove in M3. The CTL all lysed lymphoblasts encoding the appropriate mitochondrial antigen, suggesting that these target cells express naturally processed, endogenous, formylated peptides, ranging from six to nine amino acids in length.     

In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.     

In vitro human cytotoxic T cell responses against influenza A virus can be induced and selected by synthetic peptides

Studies on human anti-influenza cytolytic activities have demonstrated that cytotoxic T lymphocytes (CTL) from HLA-B37 individuals react preferentially with the peptide corresponding to residues 335-349 of the nucleoprotein, whereas CTL from HLA-A2 donors recognize peptide 57-68 from the viral matrix as a dominant epitope. We studied the secondary CTL response, obtained from peripheral blood mononuclear cells, of an HLA-A2+,B37+ individual stimulated either by infectious virus or by synthetic peptides. Only an HLA-B37-restricted response was detected after stimulation by the whole virus, showing an immunodominance of this activity over that restricted by HLA-A2. Moreover, human cytotoxic cell lines were successfully obtained after stimulation of peripheral blood mononuclear cells with synthetic peptides. Under these conditions, it was possible to selectively reveal the existence of an HLA-A2-restricted activity directed against the matrix peptide. These results demonstrate that, at least in vitro, it is possible to stimulate a latent repertoire by using synthetic peptides. Nevertheless, we could not induce a response against the matrix or the nucleoprotein peptides in HLA-A2- or B37- individuals, suggesting that a finer selection of synthetic peptides would be necessary for their possible utilization to induce CTL during vaccination.     

Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16-residue nucleoprotein peptide (NPP; 365-380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H-2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP-conjugated to ovalbumin as a carrier. However, in contrast to NPP-specific CTL lines raised from virus-primed animals none of the peptide-induced CTL lines were able to lyse virus-infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100-fold lower than that of CTL from virus-primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide-induced CTL was very sensitive to blocking by anti-CD8 antibodies as compared to virus-specific CTL. Furthermore, all peptide-induced CTL showed a high second reactivity for allogeneic H-2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.     

Vaccination with dendritic cells pulsed in vitro with tumor antigen conjugated to cholera toxin efficiently induces specific tumoricidal CD8+ cytotoxic lymphocytes dependent on cyclic AMP activation of dendritic cells

We investigated the development of CD8+ tumor-specific cytotoxic lymphocytes (CTL) and protection against tumor growth after vaccination with bone marrow-derived dendritic cells (DC) pulsed with a model protein ovalbumin conjugated to cholera toxin (OVA-CT) in B6 mice using E.G7 tumor cells expressing OVA(257-264) peptide (SIINFEKL) as target cells in vitro and in vivo. Vaccination with OVA-CT-pulsed DC concurrently induced strong CTL in vitro activity and anti-E.G7 tumor protection in vivo in WT, NK-depleted and CD4-deficient mice as well as in IL-12-/- and IFN-gamma-/- mice but not in CD8-deficient mice. Importantly, activation of CTL by OVA-CT-pulsed DC was dependent on CT-induced activation of adenylate cyclase and increased cAMP production by DC associated with increased expression of MHC class I and co-stimulatory molecules (CD80, CD86 and CD40). These results show that vaccination with DC pulsed with antigens (Ag) conjugated to CT induces a strong CTL response and suggest that conjugation of tumor Ag to CT for DC vaccination represents a promising approach for tumor vaccination and immunotherapy.     

Peptide-specific CTL induction in HBV-seropositive PBMC by stimulation with peptides in vitro: novel epitopes identified from chronic carriers

Cytotoxic T lymphocytes (CTL) recognize and destroy virus-infected cells, and it has been established that epitope-based peptides could induce such CTL in vivo as well as in vitro. In this study attempts were made to define the epitopes that are recognized by the CTL, and thus a series of 9- to 10-mer peptides derived from the amino acid sequences of hepatitis B virus (HBV) proteins were synthesized on the basis of the previously described HLA-A2 peptide binding motif. The binding assay of the synthetic peptides using transporter-associated with antigen processing (TAP)-deficient human cell line, T2, showed that eight out of 11 peptides tested enhanced the expression of HLA-A2 molecules on the T2 cell surface. Some of these peptides triggered activation of CTL in peripheral blood mononuclear cells of HBV-seropositive chronic carriers. The activated CTL in turn recognized and killed the T2 cells pulsed with the same peptides. This study shows that novel HLA-A2-restricted epitopes exist in the natural repertoire of immunity against HBV. These findings can be useful in developing peptide-based therapeutics against viral infections.     

Interaction of In Vitro- and In Vivo-Generated Cytotoxic T Cells with SV40 T Antigen: Analysis with Synthetic Peptides

Virus-specific cytotoxic T cells recognize antigens in the form of peptides (8 or 9 amino acids long) bound to MHC class-I molecules. Exposure of unprimed murine splenocytes to synthetic peptides of viral antigens elicits primary CTL in vitro. The fme specificity of such CTL as well as the correlation between binding affinity of peptides to class-I molecules and CTL induction was analysed using synthetic peptides corresponding to overlapping and distinct amino-acid residues in SV40 T antigen (Tag) D_b-restricted T-cell epitopes I, II-III, and V. The peptides induced cross-reactive CD8₊ primary CTL in spienocytes of naive C57 BL/6 mice. This reactivity was seen regardless of the peptides allelic anchor motifs or their abilities to stabilize empty class-I molecules. However, none of the primary CTL and CTL lines lysed Tag-expressing cells. In contrast, CTL generated in vivo by immunizing mice with Tag-expressing cells recognized endogenously processed Tag as well as synthetic peptides. The peptides recognized by these CTL depended on the intracellular concentration of Tag antigen in the immunizing cells. The reactivity of these CTL was peptide specific as shown by a functional peptide competition assay. Moreover, three peptides bound to and were recognized in the context of both K^b and D^b molecules. These results have revealed a flexible disposition of MHC class-I molecules with regard to peptide binding and also reflected lack of correlation between binding affinity to class-1 molecules and the capacity of peptides to induce primary CTL or to serve as potential targets. The significance of these findings in relation to identifying major T-cell epitopes using allele specific peptide motif and in vitro maintained CTL clones is discussed.     

Identification of H-2Kb Binding and Immunogenic Peptides from Human Papilloma Virus Tumour Antigens E6 and E7

Peptides can be used to induce MHC class I restricted cytotoxic T cells (CTL) tbrough in vivo immunization. This approach may enable the development of peptide vaccination schemes for immunization against viral infection in humans. Human papillomavirus (HPV) is one of a few viruses associated with human cancer and the development of an anti-cancer vaccine seems possible. As a model approach, we searched the E6 and E7 proteins of the human papillomavirus type 16 for possible murine MHC class I restricted peptide epitopes. We utilized the mouse H2-K^b peptide binding motif which consists of phenylalanine or tyrosine at position five and leucine at the carboxy-terminus with the modification that leucine could be replaced by other aliphatic but non-aromatic amino acids. Four peptide sequences from E6 and two from E7 were selected. These peptides were tested for their ability to bind and stabilize K^b and for their immunogenicity in vivo . It was shown tbat one peptide from E6, E6.1 (50–57), bound K^b, but was not able to prime mice in vivo . In contrast, the two selected E7 peptides E7.1 (21–28) and E7.2 (48–55) bound K^b and were immunogenic in vivo . The peptide induced CTL lysed syngeneic EL-4 cells transfected with the open reading frame of E7 but not vector only transfectants. This implies tbat both peptides were naturally processed and presented by K^b on the surface of target cells. MHC class I peptide binding motifs therefore appear to be an effective and useful tool to predict peptide epitopes of proteins associated with cancer.     

Identification and design of p53-derived HLA-A2-binding peptides with increased CTL immunogenicity

The replacement of a suboptimal amino acid in a primary anchor position with an optimal residue improves human leucocyte antigen (HLA) binding and immunogenicity, while maintaining cytotoxic T lymphocyte (CTL) specificity. Using a neural network capable of performing quantitative predictions of peptide binding to HLA-A2 molecules, we identified three p53 protein-derived nonamer peptides with intermediate binding owing to suboptimal amino acids in the P2 anchor position. These peptides were synthesized along with the corresponding analogs, where the natural P2 residue had been replaced with the optimal leucine residue. All three modified peptides bound to and more efficiently stabilized HLA-A2 molecules than the corresponding nonmodified peptides. The HLA-A2 transgenic mice were used for immunization. Two of the epitopes were more immunogenic in their modified than in their natural versions. The CTLs raised against the modified peptides efficiently killed the target cells pulsed with the corresponding native peptide. In terms of sensitizing the targets cells for the CTL killing, the modified peptides were more efficient than native peptides. Finally, the CTLs induced by modified peptide killed HLA-A2 transgenic mouse fibrosarcoma cells transfected with human p53 DNA. The data suggest that modified self-peptides derived from overexpressed tumour-associated proteins can be used in vaccine development against cancer, and that quantitative predictions of HLA binding is of value in the rational selection and improvement of target epitopes recognized by CTLs.     

Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro

Cytolysis and interferon(IFN)-gamma production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc-cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 coreceptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFNgamma-production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFN-gamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for the IFN-gamma production.     

Specificity of CD8+ T cells from subunit-vaccinated and infected H-2b mice recognizing the 38 kDa antigen of Mycobacterium tuberculosis

CD8+ T cells have been implicated in protective anti-tuberculous immune responses, but little is known about the identity of mycobacterial antigens recognized by CD8+ T cells. In this study we identified the Mycobacterium tuberculosis 38 kDa protein as a target for murine CD8+ cytotoxic T lymphocytes (CTL) which were induced by vaccination of C57BL/6 mice with DNA delivered with a plasmid, with transfected tumour cells or by infection with tubercle bacilli. Using overlapping synthetic peptides covering the whole protein sequence, peptides predicted to contain H-2Kb or H-2Db motifs, as well as naturally processed peptides, we were able to identify CTL epitopes. Differences were demonstrated in peptide specificity between CTL from immunized or M. tuberculosis-infected mice. The identified CTL epitopes could be important for future analysis of the involvement of CD8+ T cells in M. tuberculosis infections and for vaccine development.      

Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma

A role of CD4(+) cells in the regulation of immune responses has steadily gained renewed recognition. The understanding of these T-regulatory (T-reg) cells in the generation of antitumor cytolytic T lymphocyte (CTL) response is therefore important. It has been shown that immunization with specific peptides, DNA, or tumor lysate-based vaccines can induce CTL responses in vivo. We have immunized melanoma patients with major histocompatibility complex (MHC) class I restricted peptide- or melanoma tumor lysate-loaded antigen-presenting cell (APC)-based vaccines and have monitored the generation of CTL responses and T-reg cell responses, if any. Using tetramer staining and limiting dilution analyses as monitors of CTL responses, we found significant increases in the number of antigen-specific CTL in circulation after vaccination with the MART-1(27-35) peptide (AAGIGILTV)-pulsed autologous APC, the MAGE-1(161-169) peptide (EADPTGHSY)-pulsed APC, or with autologous tumor lysate-pulsed APC. The antigen-specific CTL reached the peak expansion by day 7 and then declined to the prevaccine levels by day 28. The decline in the CTL response was associated by a concomitant expansion of CD4(+) CD25(+)T cells. Analysis of postvaccine peripheral blood lymphocytes (PBL) from patients showed an increased amount of interleukin (IL)-10 secretion on in vitro stimulation with IL-2 after successive vaccination. Triple color flow cytometric analyses revealed cytoplasmic IL-10 in the CD4(+)CD25(+) T-cell fraction and the number of CD4(+)CD25(+) IL-10(+) T cells were found to increase significantly in postvaccine PBL. These observations have implications in tumor antigen and APC/dendritic cell (DC)-based cancer vaccine strategies.     

HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not processed and presented

Human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies and a high percentage of cervical carcinoma cells express HPV-16 E6 and E7 oncoproteins. These proteins are attractive targets for cytolytic T lymphocyte (CTL) mediated immunotherapy. We screened peptides derived from the HPV-16 E7 protein for binding to HLA-A2 and tested their potential to induce specific CTL responses in chimeric HLA-A2/H2-Kb transgenic mice. From eight potential binding peptides four displayed binding and were tested for immunogenicity. CTL activity was tested using target cells pulsed with peptide or expressing E7 protein. While there was no CTL induction observed with the peptides 7-15, 66-74 and 82-90, CTL from mice immunized with 86-93 lysed targets presenting the peptide in the context of the HLA-A2/H2-Kb molecule or wild-type HLA-A2. In contrast, 86-93 induced CTL showed no cytolytic activity against cells expressing the protein E7 and vaccination with the E7 protein did not lead to cytotoxicity against targets pulsed with the 86-93 peptide. Therefore the peptide 86-93, which binds to HLA-A2, is able to induce CTL responses in context of HLA-A2, but the peptide appears not to be processed or presented by HPV type 16 infected cells.