Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Whole blood production of thromboxane, prostacyclin and leukotriene B4 after dietary fish oil supplementation in man: effect of vitamin E

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.     

Efficient tumor eradication by adoptively transferred cytotoxic T-cell clones in allogeneic hosts

Tumor-specific cytotoxic T cells (CTLs) can play an important role against cancer as illustrated by the observation that adoptive transfer of tumor-specific CTLs can mediate potent anti-tumor effects. Although such CTLs can be detected at the tumor site, relatively little is known about the mechanisms by which they enter the tumor. In this study, the role of major histocompatibility complex (MHC) class 1 molecules on vascular endothelium in the tumor in entry of, and tumor eradication by, tumor-specific CTL was investigated. Two H-2Db-restricted CTL clones recognizing peptide VNIRNCCYI on human adenovirus type 5 early region 1-(Ad5E1)-induced tumors were used to test whether CTLs were able to cross the vascular endothelium lacking the restricting MHC molecule. One CTL clone recognizes peptide VNIRNCCYI in the context of both H-2Db and H-2Dbm14 molecules. The other CTL clone recognizes this peptide only in the context of H-2Db. Adoptive transfer of these CTLs leads to eradication of Ad5E 1-induced, H-2Db-expressing tumors in B6(H-2Db+) and Bm14(H-2Db-) nude mice. Our data show that presentation of tumor-derived peptides by MHC molecules on endothelial cells of blood vessels in a tumor do not play a major role in eradication of tumors by adoptively transferred CTL in combination with interleukin-2. Moreover, our data show that successful adoptive CTL immunotherapy is possible across allogeneic barriers, without inducing severe side effects, provided the tumor expresses the MHC class 1-peptide complex recognized by the CTLs.     

Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.     

Efficient direct priming of tumor-specific cytotoxic T lymphocyte in vivo by an engineered APC

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.     

A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle

Alloreactive T cells are often specific for individual peptides that are bound to allogeneic major histocompatibility complex (MHC) molecules. Other alloreactive T cells are reported to be peptide-independent or to recognize MHC conformational changes that are induced by multiple peptides. We tested 12 anti-HLA-B7 alloreactive cytotoxic T lymphocyte (CTL) clones that bind a restricted region of HLA-B7, including three CTL clones that were generated in a protocol designed to stimulate peptide-independent T cells. All 12 CTLs recognized multiple point mutations in the HLA-B7 peptide-binding groove. Eleven of the 12 CTLs recognized specific peptides that eluted in one or two fractions on high-performance liquid chromatography (HPLC). None of the CTLs promiscuously recognized 16 HLA-B7-binding synthetic peptides, although one CTL recognized minor by-products in one synthetic peptide preparation. CTL clone KID-9 cross-reacted with allogeneic HLA-B7 and HLA-B27 molecules and recognized a distinct peptide bound to each MHC molecule. CTL clone KD-11 recognized peptides that eluted in two HPLC fractions and recognized HLA-B7-transfected peptide antigen processing defective T2 cells. These results indicate that CTL allorecognition is peptide-specific whether the allogeneic MHC molecules are expressed on normal cells or antigen processing-deficient cells.     

Differential antigen recognition by T cells from the spleen and central nervous system of coronavirus-infected mice

CD8+ cytotoxic T lymphocytes (CTLs) isolated from the central nervous system (CNS) of C57BI/6 mice acutely infected with mouse hepatitis virus, strain JHM (MHV-JHM), and analyzed in a direct ex vivo cytotoxicity assay recognize two epitopes (H-2Db- and H-2Kb-restricted encompassing amino acids 510-518 and 598-605, respectively) within the surface (S) glycoprotein. In contrast, CD8+ T cells isolated from the spleens of mice inoculated intraperitoneally with MHV-JHM and restimulated in vitro only respond to the H-2Db-restricted epitope. In this report, the preferential recognition of the H-2Db-restricted epitope is confirmed using splenocytes stimulated in vitro with either MHV-JHM-infected MC57 cells or with a cell line expressing the S protein and analyzed in secondary CTL assays. To determine whether these results represent a difference in epitope recognition between the spleen and CNS, secondary CTL assays were performed using spleen cells coated with peptides encompassing the CTL epitopes as stimulators. Under these conditions, both epitopes sensitized cells for lysis by spleen-derived CTLs, suggesting that both epitopes were recognized by splenic CD8+ T cells after infection in vivo. Furthermore, limiting dilution analysis indicated that the precursor frequency of splenic CD8+ T cells specific for both the H-2Kb- and H-2Db-restricted epitopes were not significantly different. Thus, the results suggest that in vitro stimulation of splenocytes specific for the H-2Kb-restricted epitope is inefficient after endogenous processing but that this inefficiency can be corrected if peptide is provided exogenously at sufficiently high concentrations. As a consequence, the results also show that cells responsive to both of the previously identified CNS-derived CD8+ T cell epitopes are present in the infected spleen at nearly the same frequency.     

Early postoperative gadolinium-DTPA-enhanced MR imaging after successful lumbar discectomy

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.     

Association between sleep apnea syndrome and coronary artery disease

This study compared ligand densities on antigen-presenting cells (APCs) needed for in vitro restimulation of in vivo primed T cells and for in vitro assessed T cell effector function. Spleen cells of lymphocytic choriomeningitis virus (LCMV)-primed mice were restimulated in vitro with graded amounts of virus-derived peptides using macrophages or a cloned dendritic cell line as APCs. To test for effector function of these cytotoxic T cells, the same APCs pulsed with graded amounts of the peptides were used as target cells in an in vitro 51Cr release assay. The same peptide concentration that rendered an APC restimulatory for primed cytotoxic T lymphocytes (CTLs) also rendered it susceptible for lysis by the same CTLs. In addition, the same peptide concentrations that made macrophages susceptible for CTL-mediated lysis induced proliferative responses in vitro of in vivo primed memory CTLs. Thus, restimulation of in vivo primed T cells--measured by either proliferation or cytotoxic effector function--or sensibilization of target cells for lysis requires similar ligand densities on APCs and is therefore, contrary to expectations, governed by similar overall avidity thresholds. These results have implications for CTL memory.     

The preferential cytolytic T lymphocyte response to immunodominant minor histocompatibility antigen peptides

C57BL/6 mice preferentially generate cytolytic T lymphocytes (CTL) to a limited number of immunodominant minor antigens and associated immunogenic peptides when primed with H2-matched Balb.B spleen cells despite multiple minor histocompatibility (H) antigen differences. We have examined the complexity of dominant H antigens recognized by these CTLs to estimate the number of peptides associated with single antigens. Peptides eluted from Kb molecules of lymphoblasts from Balb.B and CXB recombinant inbred (RI) strains were tested for sensitization of RMA-S cells for lysis by short-term C57BL/6 CTL lines specific for Balb.B and CXB strains. Anti-Balb.B CTLs recognized four Kb-bound peptides; subsets of these peptides were recognized by anti-CXB CTLs when tested with peptides from the respective CXB strains. Single peptides segregated independently among the CXB strains, confirming that single peptides were encoded by independently segregating alleles. These peptides were expressed in diverse inbred mouse strains and were recognized preferentially by C57BL/6 CTLs stimulated by different inbred mouse strains. This set of peptides was subclassified by their capacity to sensitize targets when presented in unfractionated mixtures of Kb-bound peptides. The peptide associated with the previously classified dominant CTT-2 antigen was the only peptide to strongly sensitize RMA-S cells for lysis under these conditions. These results suggest that dominant peptides have a wide strain distribution and may have a distinct advantage over dominated peptides in binding to class I molecules and/or in presentation to CTLs.     

Heteroclitic immunization induces tumor immunity

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.     

Intraductal papillary mucinous tumors of the pancreas: imaging studies and treatment strategies

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.     

Effect of ischemia--reperfusion on heat shock protein 70 and 90 gene expression in rat liver: relation to nutritional status

p53 gene mutations occur in most human cancers and result in an altered protein product that accumulates within the cell. Although the observed endogenous human CTL response to p53 is weak, high-affinity, human p53-specific CTLs have been generated from HLA A2.1 transgenic mice immunized with human CTL epitope peptides. In this study, we examine the ability of HLA A2.1-restricted and human p53-specific CTLs from HLA A2.1 transgenic mice to suppress the growth of p53-overexpressing human tumors in severe combined immunodeficient (SCID) mice. In vitro, murine p53(149-157)-specific CTLs selectively lysed the p53-overexpressing pancreatic carcinoma cell line Panc-1 but did not recognize HLA A2.1- tumor cells or HLA A2.1+ normal human fibroblasts. Furthermore, in vivo, the growth of established human tumor xenografts in SCID mice was significantly reduced and survival was prolonged after the administration of p53-specific CTLs but not after the administration of control CTLs or PBS alone. Following treatment with p53(149-157)-specific CTLs, regressing Panc-1 tumors were infiltrated by the CD8+ CTLs, as demonstrated by immunohistochemistry. These findings suggest that p53(149-157)-specific and HLA A2.1-restricted murine CTLs suppress the growth of established Panc-1 tumors following adoptive transfer into SCID hosts and prolong their survival.     

Peptides derived from self-proteins as partial agonists and antagonists of human CD8+ T-cell clones reactive to melanoma/melanocyte epitope MART1(27-35)

The self-peptide MART1(27-35) derives from the melanocyte/melanoma protein Melan A/MART1 and is a target epitope of CD8+ T cells, commonly recovered from tumor-infiltrating lymphocytes of HLA-A2.1+ melanoma patients. Despite their prevalence in such patients, these CTLs generally appear to be ineffective in mediating tumor regression in vivo. We have noted previously that numerous peptides from both endogenous and foreign proteins are similar to MART1(27-35) and, potentially, are capable of productively engaging the T-cell receptors of patient-derived CTLs. This observation raised the question of whether CTLs in vivo might encounter self-peptide analogues of MART1(27-35) that lack full agonist activity, perhaps to the detriment of the antitumor CTL response. This possibility was evaluated using cloned, patient-derived CTLs with a panel of self-derived natural analogues of MART1(27-35) in assays for cytolysis, cytokine release, and phosphorylation of T-cell receptor signaling constituents. Several peptides were identified as partial agonists, capable of eliciting cytolysis and/or release of cytokines tumor necrosis factor-alpha and IFN-gamma but not interleukin 2. Several other peptides showed antagonist behavior, effectively inhibiting cytolysis of MART1(27-35)-pulsed targets, but did not inhibit killing of cells prepulsed with a synthetic, heteroclitic variant of MART1(27-35). Some of these antagonists also had lasting effects on interleukin 2 secretion by CTLs under experimental conditions involving sequential exposure to ligands. Together, these observations suggest that encounters with self-peptide analogues of MART1(27-35) may contribute to the peripheral maintenance of these CTLs, while ultimately impairing the efficacy of this antitumor T-cell response.     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth, Galleria mellonella L

The aim of the current study was to determine whether immunization with synthetic peptides corresponding to the joining region segment of p210 bcr-abl chimeric protein can elicit CD8+ cytotoxic T lymphocytes (CTLs) capable of specifically lysing leukemia cells. BALB/c mice were immunized with peptides identical to the joining region segment of p210 bcr-abl protein. Class I major histocompatibility complex (MHC)-restricted bcr-abl peptide-specific CD8+ CTLs were elicited. The CTL clones were H-2 Kd restricted and specifically recognized a nonamer peptide of the combined sequence of bcr-abl amino acids but neither bcr nor abl amino acid sequence alone. Despite specificity and substantial lytic potential against syngeneic cell line incubated with exogenously supplied peptides, the bcr-abl peptide-specific CTLs failed to lyse syngeneic murine leukemia cells expressing human p210 bcr-abl protein containing the same bcr-abl joining region peptide sequence. Similarly, the bcr-abl peptide-specific CTLs did not lyse human bcr-abl-positive chronic myelogenous leukemia cells expressing murine class I MHC antigen (i.e., K562 cells infected with vaccinia virus expressing H-2 Kd). The appropriateness of the joining region segment of bcr-abl protein to serve as a T cell target depends upon whether that segment is presented by class I MHC in a concentration high enough to stimulate CTLs. The current experiments using murine peptide-specific CTLs could not establish that the joining region of bcr-abl protein is processed and presented by class I MHC antigen-processing pathway, but the possibility was not ruled out. Alternative models and/or strategies are necessary.     

Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors

This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.     

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells

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.     

Frequency of ras mutations in liver neoplasms from B6C3F1 mice exposed to tetrafluoroethylene for two years

BACKGROUND: Cytotoxic T lymphocytes (CTLs) contribute to the rejection of transplanted tissues through two pathways: first, by direct recognition of foreign graft major histocompatibility complex (MHC) class I molecules; and second, by recognition of foreign graft-derived peptides presented by classical MHC class Ia molecules that are matched between graft and donor. However, a number of observations suggest that additional categories of CTL recognition patterns may exist, but they remain to be defined molecularly. METHODS: Previous studies showed that the murine nonclassical MHC molecule H2 M3 may be involved in allorecognition. We investigated whether other members of nonclassical MHC class Ib, namely Qa1 and Qa2, may be recognized. Alloreactive CTLs were generated from mice mismatched for non-MHC and/or MHC genetic backgrounds and tested using various target cells, including cells transfected with Qa1 or Qa2. Furthermore, candidate peptides were synthesized and used to generate CTLs specific for peptide presented by Qa1 or Qa2. RESULTS: The experiments demonstrate that allogeneic and xenogeneic peptides were recognized by CTLs when presented on shared nonclassical MHC class Ib Qa1 and Qa2 molecules. CONCLUSIONS: The results confirm that MHC class Ib molecules present peptides to CTLs. This potentially important alloreactivity pathway may be functional between most individuals because sharing of MHC class Ib alleles is frequent.