Induction Of Cytotoxic T-lymphocyte Responses Research Articles

Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning

AbstractLentiviral vectors are increasingly being used for transferring genes into hematopoietic stem cells (HSCs) due to their ability to transduce nondividing cells. Whereas results in in vitro studies and the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) model have been highly encourgaging, studies in large animals have not confirmed the superior transduction of HSCs using lentiviral vectors versus oncoretroviral vectors. In contrast to the stable gene marking we have consistently achieved with oncoretroviral vectors in animals that received myeloablative conditioning, we observed the complete disappearance of genetically modified enhanced green or yellow fluorescent protein–expressing cells in 5 baboons that received transplants of HSCs transduced with lentiviral vectors alone or in combination with oncoretroviral vectors. Immune responses to transgene products have been found to be involved in the disappearance of gene-modified cells after nonmyeloablative conditioning. Thus, we examined whether the disappearance of genemodified cells after ablative conditioning may be due to an immune response. In 4 of 5 animals, cytotoxic T lymphocytes specific for the transgene protein were readily detected, demonstrating that immune reactions were responsible for the disappearance of the gene-marked cells in the animals. In summary, we report the induction of transgene-specific immune responses after transplantation of lentivirally transduced repopulating cells in a myeloablative setting.

Induction of cytotoxic T-cell responses against the oncofetal antigen-immature laminin receptor for the treatment of hematologic malignancies

AbstractThe oncofetal antigen immature laminin receptor protein (OFA-iLRP) is a highly conserved protein that is preferentially expressed in fetal tissues and in many types of cancer, including hematopoietic malignancies, whereas OFA-iLRP is not detectable on healthy differentiated adult cells. To investigate whether OFA-iLRP-specific cytotoxic T lymphocytes (CTLs) are capable of killing OFA-iLRP-expressing hematologic targets, CTLs were generated from healthy HLA-A*0201-positive volunteers by incubating T cells with autologous dendritic cells (DCs) transfected with OFA-iLRP RNA. OFA-iLRP-specific CTLs lysed HLA-A2+OFA-iLRP+tumor cells, including several lymphoma and leukemia cell lines, as well as fresh leukemic targets from patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), indicating that OFA-iLRP-derived peptides are naturally processed and presented by hematologic tumors. Healthy OFA-iLRP-negative target cells (CD14+monocytes, activated B cells, DCs, bone marrow cells) were not attacked by OFA-iLRP-specific CTLs. Furthermore, in an established murine B-cell lymphoma model (A20), treatment with syngeneic DCs transfected with OFA-iLRP-coding RNA resulted in powerful antitumor effects in a significant portion of mice. For the first time, these data show that OFA-iLRP can be used as a target for T-cell-based immunotherapeutic strategies against hematologic malignancies. (Blood. 2003;102:4416-4423)

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model.

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Intranasal immunization with formalin-inactivated virus vaccine induces a broad spectrum of heterosubtypic immunity against influenza A virus infection in mice

It has been known that influenza A virus infection induces a cross-protective immunity against infection by viruses with different subtypes of viral envelope proteins, hemagglutinin (HA) and neuraminidase (NA). This heterosubtypic immunity is generally mediated by cytotoxic T lymphocytes (CTL) reactive to specific epitopes in the viral internal proteins, such as nucleoprotein and matrix protein. By contrast, immunization with inactivated virus antigens has been thought to be unable to generate heterosubtypic immunity, since inactivated antigens do not usually induce CTL responses. However, we show that intranasal immunization with formalin-inactivated intact virus, but not ether-split vaccines, induced a broad spectrum of heterosubtypic protective immunity in mice. The protection may be mediated by the mucosal immune response, most likely secretory IgA antibodies to the viral proteins. This approach may overcome limitations in the efficacy of inactivated influenza vaccines and confer potent immunity to humans against viruses with new pandemic potential.

Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes.

Recent studies have shown that tumor necrosis factor alpha (TNF-alpha) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-alpha knockout (TNF-alpha(-/-)) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-alpha(-/-) mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-alpha(-/-) mice after two sessions of immunization with the HBV plasmid; however, TNF-alpha was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-alpha(-/-) mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-alpha enhanced the proliferation of a TNF-alpha(-/-) clone but suppressed that of a TNF-alpha(+/+) clone in vitro, TNF-alpha also has a direct effect on the proliferation of CTLs. In conclusion, TNF-alpha is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.

Estimating cumulative probabilities from incomplete longitudinal binary responses with application to HIV vaccine trials.

When describing longitudinal binary response data, it may be desirable to estimate the cumulative probability of at least one positive response by some time point. For example, in phase I and II human immunodeficiency virus (HIV) vaccine trials, investigators are often interested in the probability of at least one vaccine-induced CD8+ cytotoxic T-lymphocyte (CTL) response to HIV proteins at different times over the course of the trial. In this setting, traditional estimates of the cumulative probabilities have been based on observed proportions. We show that if the missing data mechanism is ignorable, the traditional estimator of the cumulative success probabilities is biased and tends to underestimate a candidate vaccine's ability to induce CTL responses. As an alternative, we propose applying standard optimization techniques to obtain maximum likelihood estimates of the response profiles and, in turn, the cumulative probabilities of interest. Comparisons of the empirical and maximum likelihood estimates are investigated using data from simulations and HIV vaccine trials. We conclude that maximum likelihood offers a more accurate method of estimation, which is especially important in the HIV vaccine setting as cumulative CTL responses will likely be used as a key criterion for large scale efficacy trial qualification.

CD4-Independent Protective Cytotoxic T Cells Induced in Early Life by a Non-Replicative Delivery System Based on Virus-like Particles

The relative immaturity of the neonatal immune system limits CD4+ Th1 and cytotoxic T lymphocyte (CTL) responses, and represents a significant challenge for the development of vaccines against intracellular pathogens. In this report, we demonstrate the ability of a non-replicative delivery system based on parvovirus-like particles (VLP) to induce CTL responses in the neonatal period. A single immunization of 1-week-old BALB/c mice with recombinant VLP carrying a CD8+ T cell determinant from lymphocytic choriomeningitis virus (VLP-LCMV) induced antigen-specific CD8+ cytotoxic T cells that were similar to those elicited by adult immunization, as assessed by cytotoxic activity, interferon (IFN)-γ secretion, cytotoxic precursor cell frequencies, in vitro avidity for antigen and protective activity against viral challenge. These CTL responses are elicited within 2 weeks of a single immunization, in the absence of adjuvant and independently of the presence and help of CD4+ T cells, highlighting the potential of VLP as candidate vaccine vectors in early life.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8.

The human herpesvirus 8 (HHV-8) is a human gamma2-herpesvirus that is implicated in the development of Kaposi's sarcoma (KS), primary effusion lymphoma and Castelman's disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV-8 viral antigens. In this study, using peptide-binding motifs, we selected potential human leucocyte antigen (HLA)-A2-binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV-8 antigens, respectively. HLA-A2-binding peptides were tested for their capacity to induce CTL responses in HHV-8-negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV-8-derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16-25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59-68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease-free individuals infected by HHV-8 demonstrating that the two epitopes are relevant targets of CTL-mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV-8-associated malignancies.

CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules.

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8α− DCs, whereas 15 hours later they are presented mainly by CD8α+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8α and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8α in CD11b+ DCs suggests that CD8α expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag−/−), CD11b+ DCs did not express CD8α and PPV-VLPs presentation by CD8α+ DCs was severely diminished. These results indicate that both CD8α− and CD8α+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

Identification of the HLA-A24 peptide epitope within cytomegalovirus protein pp65 recognized by CMV-specific cytotoxic T lymphocytes.

Among cytomegalovirus (CMV) tegument proteins, phosphoprotein 65 (pp65) has been identified as the important target antigen of the cytotoxic T lymphocyte (CTL) response against the virus. We synthesized seven CMV-pp65-derived peptides carrying an HLA-A24-binding motif, and investigated the ability of these peptides to induce CMV-specific CTL. We identified one nonamer peptide (pp65113-121; VYALPLKML) able to bind HLA-A24 and induce CTL responses in vitro in peripheral blood mononuclear cells (PBMC) from CMV-seropositive individuals. The peptide-specific CTLs generated were capable of recognizing pp65 expressed on CMV-infected fibroblasts as well as pp65113-121 peptide bound to the surface of C1R-A*2402 cells in an HLA-A24-restricted manner. The pp65113-121 peptide thus might be considered a synthetic peptide vaccine in HLA-A24-positive individuals.

Rapid induction of cytotoxic T-cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.

We have shown that the pulsing of dendritic cells (DCs) with human papillomavirus type 16 (HPV-16) antigen proteins by lipofection stimulates class I-restricted cytotoxic T lymphocyte (CTL) response against primary cervical cancer cells. Also, we have shown that adeno-associated virus (AAV) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T-cell priming by DCs. Here, DCs are pulsed (infected) with an AAV vector containing the HPV-16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV/E6 vector was used. Most importantly, AAV/E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti-MHC class I antibodies. Fluorescence-activated cell sorter (FACS) analysis of resulting primed cell populations revealed higher levels of CD8+ T cells by AAV-based pulsing, with little evidence of CD56 (NK). FACS analysis of the DC populations revealed that AAV/E6 vector-pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein-pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques.

Induction of cytotoxic T-cell responses against immunoglobulin V region–derived peptides modified at human leukocyte antigen–A2 binding residues

AbstractCytotoxic T-lymphocyte (CTL) responses can be generated against peptides derived from the immunoglobulin (Ig) V region in some but not all patients. The main reason for this appears to be the low peptide-binding affinity of Ig-derived peptides to major histocompatibility complex (MHC) class I molecules and their resulting low immunogenicity. This might be improved by conservative amino acid modifications at the MHC-binding residues of the peptides (heteroclitic peptides). In this study, it was found that in 18 Ig-derived peptides, that heteroclitic peptides from the Ig gene with improved binding to human leukocyte antigen (HLA)-A*0201 can be used to improve CTL responses. Amino acid substitution substantially increased predicted binding affinity, and there was a strong correlation between predicted and actual binding to HLA-A*0201. CTLs generated against the heteroclitic peptide had not only enhanced cytotoxicity against the heteroclitic peptide but also increased killing of antigen-presenting cells pulsed with the native peptide. Surprisingly, no difference was observed in the frequency of T cells detected by MHC class I peptide tetramers after stimulation with the heteroclitic peptide compared with the native peptide. CTLs generated against heteroclitic peptides could kill patients' tumor cells, showing that Ig-derived peptides can be presented by the tumor cell and that the failure to mount an immune response (among other reasons) likely results from the low immunogenicity of the native Ig-derived peptide. These results suggest that heteroclitic Ig-derived peptides can enhance immunogenicity, thereby eliciting immune responses, and that they might be useful tools for enhancing immunotherapy approaches to treating B-cell malignant diseases.

Induction of CTL responses and identification of a novel epitope of hepatitis B virus surface antigens in C57BL/6 mice immunized with recombinant vaccinia viruses

MHC class I-restricted cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV) surface antigens (HBsAg) has been suggested to play essential roles in viral clearance and pathogenesis of HBV-induced hepatitis. In the present study, we analyzed CTL responses to endogenously synthesized or exogenously introduced HBsAg in C57BL/6 mice (H-2 b). We show that the endogenously synthesized surface antigens of adr-type HBV encoded by recombinant vaccinia virus efficiently elicit CTL responses in C57BL/6 mice previously defined as non-responders to vaccinia-HBV immunization. We also show that two peptides, S 179–186 (FVQWFVGL) and S 208–216 (ILSPFLPLL), serve as effective motifs for CTL response in H-2 b system after in vitro restimulation of the primed T cells with either of the two synthetic peptides. S 208–215 has recently been identified as a CTL epitope which could be produced by exogenous pathway only, in contrast to the current result, while S 179–186 appeared a novel epitope for CTL response. In addition, we show that soluble HBsAg also elicits CTL responses in H-2 b mice upon in vitro restimulation with the two peptides, although less efficiently compared with the recombinant vaccinia viruses. These findings may provide an efficient experimental system for studying H-2 b-restricted immune responses against endogenously synthesized and exogenously introduced HBsAg.

Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells

Vaccination of patients with cancer using dendritic cells (DCs) was shown to be effective for B-cell lymphoma and malignant melanoma. Here we provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DCs pulsed with HER-2/neu– or MUC1-derived peptides. Ten patients were included in this pilot study. The DC vaccinations were well tolerated with no side effects. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTLs) could be detected in the peripheral blood using both intracellular IFN-γ staining and 51Cr-release assays. The major CTL response in vivo was induced with the HER-2/neu–derived E75 and the MUC1-derived M1.2 peptide, which lasted for more than 6 months, suggesting that these peptides might be immunodominant. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after 7 immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Our results show that vaccination of DCs pulsed with a single tumor antigen may induce immunologic responses in patients with breast and ovarian cancer. This study may be relevant to the design of future clinical trials of other peptide-based vaccines.

Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells

AbstractVaccination of patients with cancer using dendritic cells (DCs) was shown to be effective for B-cell lymphoma and malignant melanoma. Here we provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DCs pulsed with HER-2/neu– or MUC1-derived peptides. Ten patients were included in this pilot study. The DC vaccinations were well tolerated with no side effects. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTLs) could be detected in the peripheral blood using both intracellular IFN-γ staining and 51Cr-release assays. The major CTL response in vivo was induced with the HER-2/neu–derived E75 and the MUC1-derived M1.2 peptide, which lasted for more than 6 months, suggesting that these peptides might be immunodominant. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after 7 immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Our results show that vaccination of DCs pulsed with a single tumor antigen may induce immunologic responses in patients with breast and ovarian cancer. This study may be relevant to the design of future clinical trials of other peptide-based vaccines.

Genetically detoxified mutants of heat-labile enterotoxin from Escherichia coli are effective adjuvants for induction of cytotoxic T-cell responses against HIV-1 gag-p55.

There is an urgent need for prophylactic and therapeutic vaccines against human immunodeficiency virus (HIV). Mucosal immunization strategies have great potential to elicit both mucosal and systemic cellular immunity required to protect against HIV-induced acquired immune deficiency syndrome (AIDS). However, mucosal immunizations with soluble protein antigens generally require adjuvants. In this study, we tested two mutants of the heat-labile enterotoxin (LT) from Escherichia coli, LTK63: with no measurable ADP-ribosyltransferase activity, and LTR72: with residual ADP-ribosyltransferase activity, as mucosal adjuvants for induction of cytotoxic T lymphocyte (CTL) responses to coadministered HIV gag p55 protein. We found that intranasal (i.n.) immunizations with HIV gag p55 protein coadministered with LTK63 or LTR72 induced systemic CTL responses comparable to that obtained following intramuscular (i. m.) immunizations with the same adjuvants. Moreover, oral coadministration of LTR72, but not LTK63, resulted in local as well as systemic p55-specific CTL responses in mesenteric lymph nodes (MLN) and spleens (SP) of the immunized mice. These data have important implications for current efforts to develop a safe vaccine against HIV.

Creating HIV-1 reverse transcriptase cytotoxic T lymphocyte target structures by HLA-A2 heavy chain modifications.

Vigorous HIV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHC-viral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8(+) CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes.

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D b and H-2L d). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D b MHC-I motif was replaced by alanine. Three out of five peptides with H-2D b or H-2L d binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D b molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D b MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D b molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2 b) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D b molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.

Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine-serine-glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70. In vivo immunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4(+) T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8(+) cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.

Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70.

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine-serine-glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70. In vivo immunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4(+) T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8(+) cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.