Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2K^d to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2K^d. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2K^d-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2K^d; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2K^d. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2K^d to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2K^d class I molecule, and only a fraction of these serve as CTL epitopes.     

In vitro induction of naive cytotoxic T lymphocytes with complexes of peptide and recombinant MHC class I molecules coated onto beads: role of TCR/ligand density

We previously reported that complexes of peptide with soluble single-chain recombinant MHC (SC-MHC) class I molecules are able to induce cytotoxic T lymphocytes (CTL) in vitro in a murine system with an efficiency comparable to that observed with peptide-pulsed dendritic cells as antigen-presenting cells. In this report, we have assessed the capacity of preformed peptide/SC-Kd complexes in monomeric or dimeric form as well as of peptide/SC-Kd-loaded beads to generate in vitro specific CTL responses from naive DBA/2 spleen cells. Peptide/SC-Kd-coated beads were consistently more efficient. We evaluated the role of co-stimulatory molecules, using monoclonal antibodies anti-CD80 or anti-CD86. In addition, the capacity of peptide/SC-Kd-coated beads to generate a CTL response from purified naive CD8⁺ T cells was ascertained. Taken together, the results indicate that, under our conditions, CTL priming does not require the participation of co-stimulatory molecules and is the consequence of a direct interaction between the cognate TCR on peptide-specific CTL precursors and the peptide/SC-Kd-loaded beads. Titration of the amount of preformed complexes of SC-Kd and peptide 170 –179 of HLA-CW3 that need to be coated onto the beads to prime CTL precursors shows an activation threshold which can be calculated to be between 25 000 and 50 000 complexes. In effect, in cultures stimulated with specific peptide CW3/SC-Kd complexes representing less than 50 % occupancy of the total (10⁵ ) complexes on the beads, no peptide-specific cytolytic activity was observed. These results suggest that the efficiency of the primary CTL induction depends on the density of specific peptide/SC-Kd complexes present on the beads.     

Cytotoxic T lymphocyte responses to wild-type and mutant mouse p53 peptides

Cytotoxic T lymphocytes (CTL) recognize peptides presented at the cell surface in association with major histocompatibility complex (MHC) class I molecules. The finding that peptides binding to MHC class I molecules share common amino acid motifs renders feasible the selection of antigenic peptides by simply scanning protein sequences, and thus, provides the possibility of inducing CTL to pre-defined specificities. Tumor cells possess antigens known to generate MHC class I-restricted CD8⁺ CTL responses. Thus, these antigens represent good targets to induce tumor-specific immunity. Among these antigens, the p53 tumor suppressor gene product is an attractive candidate for cancer immunotherapy. Mutations in the p53 gene have been found to be very frequently associated with a malignant transformation and often lead to p53 protein overexpression. Thus, we investigated the possibility of inducing CTL to wild-type or mutant p53 peptides in a BALB/c (H-2^d) mouse model. Peptides possessing the H2-K^d binding motif were selected and tested for binding to the H-2K^d molecules in vitro. Synthetic peptides p53_122–130 wild-type or “mutant” (Lys → Glu substitution at position 129) were shown to be the best binder peptides and were tested for their immunogenicity in mice. H-2K^d-restricted p53-specific CD8⁺ CTL were generated following immunization of mice with either wild-type (wt) p53_122–130 or mutant (mut) p53_122–130 (E129) peptides. Only low-affinity CTL can be obtained by immunization with the wt sequence. In contrast, CTL elicited with the mut peptide recognized the mut sequence at a 10–100-fold lower concentration. This indicates that CTL elicited with the mut peptide recognized the mut sequence very efficiently, whereas the wt sequence is poorly recognized, if at all. Taken together, these results thus suggest that p53-specific tumor immunotherapy may be successful only if the mutated protein is taken into consideration.     

Generation of hen egg lysozyme-specific and major histocompatibility complex class I-restricted cytolytic T lymphocytes: recognition of cytosolic and secreted antigen expressed by transfected cells

Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K^b. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8⁺ clones were found to be H-2K^k restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived ?) presented by the MHC class I (H-2K^b or H-2K^k) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.     

Original H-2d and alien H-2k-like antigens of a BALB/c fibrosarcoma as defined by in vitro and in vivo studies of cell-mediated immunity

The present study examines the immunosensitivity and the immunogenicity of both original H-2^d and alien H-2^k-like antigens of the BALB/c (H-2^d) fibrosarcoma C-1 as detected by in vitro and in vivo cell-mediated cytotoxicity (CMC) assays. It was found that ⁵¹Cr-labeled C-1 cells were lysed in vitro by C 57 BL/6 anti-H-2^d lymphocytes. The specificity of this reaction was shown by cold inhibition experiments in which the anti-H-2^d cytotoxic activity on YC8 (H-2^d) targets was inhibited by unlabeled YC8 or C-1 but not by C3UR11 (H-2^k) tumor cells. Both D^d- and K^d-encoded antigens were recognized by appropriate cytotoxic effectors. The immunogenicity of H-2^d antigens of C-1 was revealed by the ability of C 57 BL/6 anti-C-1 lymphocytes to lyse YC8 targets. The expression of H-2^k-like alien alloantigens on C-1 was indicated by the finding that anti-H-2^k cytotoxic T lymphocytes (CTL), generated by culturing BALB/c spleen cells immune to BALB.K (H-2^k), C3Hf (H-2^k) or A (H-2^a = H-2^k/d) tissues with the cells of the same strain used for immunization, lysed C-1 targets. The cytotoxicity of these anti-H-2^k CTL against C 3 UR11 (H-2^k) targets could be specifically inhibited by cold C 3 UR 11 or C-1 cells but not by two other BALB/c tumors. Using recombinant H-2-congenic mice, it was shown that both D^k and K^k antigens were recognized by CTL on C-1 cells. The immunogenicity of the H-2^k-like antigens, however, could not be detected in vitro. In fact, effector spleen cells from BALB/c mice immune to C-1 did not develop any detectable cytotoxicity against C 3 UR 11 targets as assayed either by a direct in vitro test or after in vitro restimulation with C-1 sarcoma cells. A similar experimental design was adopted in Winn assays carried out by mixing spleen cells of BALB/c immune mice with either C-1 or C3 UR 11 targets and injecting the mixtures in BALB/c or hybrid recipients. These in vivo tests revealed the presence of both H-2^d (K^d and D^d) and H-2^k-like (K^k and D^k) antigens on C-1. At variance with the in vitro CMC assays, however, the Winn assay also detected the immunogenicity of the H-2^k alien antigens, since BALB/c anti-C-1 spleen cells were able to significantly reduce the growth of C 3 UR 11 lymphoma cells in (BALB/c × C 3 Hf)F₁ hosts.     

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2K^b-bound octapeptides. CTL recognition of such determinants is always sequencedependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on K^b-associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.     

Cytotoxic T lymphocyte recognition of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region

Allotype- or idiotype-specific CD4⁺ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8⁺ T cells. We have therefore investigated whether T-depleted spleen cells from Ig x light chain-expressing 129/Sv mice (129x^+/+) could induce, in Cx knockout mice (129 x^?/?), the generation of Ig constant x light chain region (Cx)-specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x^+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H-2K^b class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the L^d -binding 12-mer S28 – 39 peptide IPQSLDSWWTSL in H-2 ^d mice, and the K^b -binding 8-mer S208 – 215 peptide ILSPFLPL in H-2^b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the L^d -binding peptide S28 – 39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the K^b -binding peptide S208 – 215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.     

Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12

Immunodominance (ID) of T cell epitopes is a well-documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H-2K^d, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half-life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co-injected CTL epitopes can be determined both at the peptide level (binding affinity to H-2K^d) and at the T cell level. This hypothesis is supported by our finding that interleukin-12 strongly modulates ID when it is not correlated with MHC binding.     

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8⁺ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4–12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K^d; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8⁺ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4⁺ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2K^d was demonstrated with the mutant ras4–12(V12) peptide, but not the normal ras4–12(G12) peptide, which specifically inhibited an H-2K^d-restricted, anti-nucleoprotein NP147–155 CTL response in a dose-dependent fashion. An anti-ras CD8⁺ T cell line was then established from immune splenocytes of BALB/c (H-2^d) mice injected with ras4–12(V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2K^d and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly → Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras 4–12(V12) peptide was immunogenic for the production of antigen-specific CD8⁺ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.     

An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein

The immunodominant H-2D^d-restricted cytotoxic T lymphocyte (CTL) response to the HIV-1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320-327). However, 9mer and l0mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing D^d-expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10-amino acid determinant (residues 318-327) and a 67-amino acid peptide (residues 281-348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild-type gpl60 in their ability to sensitize target cells for lysis by gpl60-specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160-specific CTL. These data demonstrate that both the V3 loop itself and a 10-residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene-encoded CTL epitopes for recombinant vaccines designed to induce protective T cell-mediated immunity against HIV-1.     

SPECIFICITY OF ANTI-H-2 CLASS I ANTIBODIES INDUCED BY SYNGENEIC IMMUNIZATION WITH SENDAI VIRUS-TREATED CELLS IS REGULATED BY THE MOUSE MHC AND VIRAL ANTIGENS. NO EVIDENCE FOR MHC-RESTRICTED VIRUS- SPECIFIC ANTIBODIES

In a previous study, we searched for Sendai virus (SV)-specific antibodies that were restricted in their binding by self-major histocompatability complex (MHC) antigens. In C57BL/6 (B6; H-2^b) mice, most of the sera obtained after i.p. injections with syngeneic SV-coated (SV⁺) spleen cells contained auto- and alloreactive lymphocytotoxic antibodies directed against H-2 class I molecules, but no viral-specific, MHC-restricted antibodies. Here we report that syngeneic immunization with SV⁺ cells regularly induced H-2-specific antibodies in various mouse strains. From a total of 12 strains tested, only the B10.S (H-2^s) strain appeared to be a low responder. The immune responses are of two types: (i) mice of some strains produce autoreactive antibodies and a broad variety of alloreactive antibodies; and (ii) mice of some strains produce only narrow or widely alloreactive antibodies. Because most of the strains differ only in the H-2 region, the patterns observed are regulated by the MHC. To locate the genes involved in the induction of H-2-specific antibodies more precisely, two B6 mutant strains, bml (K^b mutant) and bm13 (D^b mutant), were immunized with syngeneic SV⁺ cells. The results suggest that the H-2D^b region plays an important role in the induction and specificity of the lymphocytotoxic H-2 class I-specific antibodies present in sera of H-2^b mice after syngeneic immunization with SV⁺ cells.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

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.     

H-2K RESTRICTION OF THE T CELL-MEDIATED LYSIS OF A CHEMICALLY-INDUCED BALB/c FIBROSARCOMA

Previous work has shown that a cytotoxic T lymphocyte (CTL) immune response of syngeneic mice immunized with a chemically-induced BALB/c (H-2^d) fibrosarcoma was directed against an individual tumour-associated antigen. To see whether this reaction was restricted by products of the major histocompatibility complex (MHC), anti-H-2 alloantisera to K or D antigens were used to interfere with the CTL-mediated immune response. Antisera to K^d but not to D^d antigens inhibited the lytic activity of CTL against fibrosarcoma cells. In addition, the study of the CTL response in F₁→ P antitumour immunized chimeric mice showed that antitumour cytotoxicity developed only when F₁ and parental host shared the K^d region. Both experiments strongly indicate that recognition of the individual tumour-associated antigen of the BALB/c fibrosarcoma is restricted by the products of H-2K^d genes.     

Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector

This study examines the suitability of replication-defective adenovirus vectors for engineering recombinant vaccines. The immunological abilities and limitations of E1-deleted adenoviruses containing the lac Z gene (Ad-β-gal) were investigated by examining the humoral and cellular immune responses to the β-galactosidase protein. BALB/c mice (H-2^d) were given in a single injection of recombinant adenovirus. The cytotoxic T lymphocyte (CTL) response of spleen cells was evaluated. Recognized target cells were H-2^d-derived tumor cells transfected by the lac Z gene, or incubated with the 876–884 β-galactosidase peptide known to be restricted by the L^d molecule of the major histocompatibility complex. A long-lasting β-galactosidase-specific cytotoxic T cell response was obtained. By contrast, CTL from mice immunized with the L^d-restricted peptide were less specific for the endogenous epitope presented by the transfectants expressing β-galactosidase. Ad-β-gal-immunized mice were also protected against an intra-cerebral challenge with a recombinant vaccinia virus expressing the lac-Z gene. These results suggest that Ad-β-gal-induced CTL have protective abilities in vivo. The induction of β-galactosidase-specific T helper lymphocytes and humoral IgG responses were also examined. A proliferative response occurred only late after immunization and the primed T lymphocytes produced interleukin-2, but no interleukin-4. A humoral IgG response to the β-galactosidase protein was detected 15–30 days after a single immunization and remained stable for 6 months without boosting. Lastly, we followed the evolution of the immune response over the course of successive immunizations. The magnitude and kinetics of the cellular and humoral responses were similar to those obtained after a single immunization. Consistent with these observations, an adenovirus-specific neutralizing antibody response was detected as early as the second immunization. Thus, a single immunization with a replication-defective adenovirus recombinant vector induces long-lasting humoral and cellular immune responses specific to the transgene product.     

Proliferation and MHC-unrestricted bystander lysis by virus-specific cytotoxic T cells following antigen self-presentation

Cytotoxic T cells (CTL) not only act as effector cells, but can also serve as antigen-presenting cells (APC) for other CTL due to their expression of major histocompatibility complex (MHC) class I molecules. In the present study we show that independently derived CTL lines (CTLL) with specificity for an L^d-presented nonapeptide corresponding to amino acids 168–176 of the immediate-early 1 (IE1) protein of murine cytomegalovirus not only lyse syngeneic but also allogeneic target cells, if the peptide is present during the cytolytic assay. Whereas a short peptide pulse is sufficient to render syngeneic cells susceptible to lysis, continued presence of soluble peptide is mandatory for the lysis of allogeneic target cells. This indicates a difference in the mechanisms involved. Syngeneic BALB/c B cell blasts (K^dD^dL^d) and mutant BALB/ c-H-2^dm2 B cell blasts lacking the restricting L^d molecules (K^dD^d0) were lysed to a similar extent in the absence of the IE1 nonapeptide, provided that the IE1-specific CTL had been pre-incubated with the peptide before the cytolytic assay. Since the mutant cells cannot present the IE1 peptide, their lysis indicates an MHC-unrestricted, peptide-independent mode of recognition by the CTLL. In addition, proliferation of the CTLL takes place after incubation with the cognate peptide, even in the absence of professional APC. These data indicate inter-CTL antigen self-presentation, resulting in activation of the lytic machinery leading to peptide-independent bystander lysis of allogeneic as well as syngeneic target cells. Received: 13 February 1998