Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.     

Active immunization against virus infections due to antigenic drift by induction of crossreactive cytotoxic T lymphocytes

We have examined whether active immunization with c13 protein, a hybrid protein of the first 81 amino acids of the viral NS1 nonstructural protein and the HA2 subunit of A/PR/8 (H1N1) hemagglutinin, could protect BALB/c mice from challenge with A/PR/8 H1 subtype virus. Mice immunized with the c13 protein had a significant reduction of pulmonary virus titers with A/PR/8 (H1) virus, but failed to limit the replication of A/PC (H3) virus, which reflects the in vitro CTL activity of c13 immune spleen cells. We observed that the epitope recognized by HA2 specific CTL, which are induced by a derivative of c13 protein, is highly conserved among H1 and H2 subtype virus strains. This led us to test whether active immunization with c13 protein would also limit pulmonary virus replication in mice infected with the A/TW virus, a virus of the H1 subtype, which was isolated in 1986, and with a virus of the H2 subtype, A/Japan/305/57. Immunized mice had significantly lower lung virus titers than did control mice, and did not possess any neutralizing antibodies to the challenger viruses. These results indicate that active immunization with a fusion protein containing the cross-reactive CTL epitope protects mice from influenza infection by inducing CTL against influenza A H1 and H2 subtype virus strains, which markedly vary in their antibody binding sites on the HA1. The ability to induce active cross-reactive immunization with a fusion protein which contains a highly conserved CTL epitope offers a model for vaccine approaches against viruses which undergo significant variations in their antibody binding sites.     

Differences in antigen presentation to MHC class I-and class II-restricted influenza virus-specific cytolytic T lymphocyte clones

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA-specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II-restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets.     

Synthetic peptides as antigens and competitors in recognition by H-2-restricted cytolytic T cells specific for HLA

The specificity of peptide recognition by a number of Kd-restricted CTL clones specific for HLA-CW3 or HLA-A24 was investigated. The CTL clones were derived from DBA/2 (H-2d) mice immunized with syngeneic P815 mouse cells transfected with genes encoding HLA-CW3 or HLA-A24 class I molecules. We had previously shown that CTL clones that lysed P815-CW3 transfectant target cells could lyse P815 (HLA-) target cells incubated with synthetic CW3 peptides corresponding to the COOH-terminal end of the alpha 2 domain. In the present study, we found that Kd-restricted CTL clones that lysed P815-A24 transfectant target cells recognized a synthetic peptide from the same region (residues 170-182) of the A24 molecule. CW3 and A24 differ by only one amino acid within this region. Recognition of CW3 or A24 peptides corresponded exactly with lysis of P815-HLA transfectants both for clones that mutually exclusively lysed CW3 or A24 transfectant target cells and for CW3/A24 crossreactive CTL clones. The latter CTL clones that lysed both CW3 and A24 transfectant target cells showed a clear preference for the peptide corresponding to the immunizing HLA allele. The homologous CW3 and A24 peptides could compete with each other for recognition, in contrast to a peptide from the same region of HLA-B7. Peptides from the corresponding region of the endogenous Kd and Dd/Ld molecules could also inhibit recognition of CW3 and A24 peptides. Competition with peptides apparently occurred at the level of the target cell. These results are consistent with a model whereby MHC class I molecules position protein fragments or peptides for specific recognition by T cells.     

The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.     

Use of DNA-mediated gene transfer to analyze the role of H-2Ld in controlling the specificity of anti-vesicular stomatitis virus cytotoxic T cells

Mouse thymidine kinase (tk-) C3H L (H-2k) cells transformed by the technique of DNA-mediated gene transfer with the herpes simplex virus tk gene together with the BALB/c H-2Ld gene express H-2Ld molecules indistinguishable from their counterparts on spleen cells. An established cloned cell line (8-5) was used to assess the function of the H-2Ld antigen in determining the specificity of alloreactive as well as anti-vesicular stomatitis virus (VSV) cytotoxic T cells (CTL). Both anti-H-2d and anti-H-2Ld CTL displayed a cytotoxic effect against 8-5 cells but not a control cell line transformed with the tk gene only (tk+ cells). Further evidence that 8-5 cells express H-2Ld was provided by the finding that monoclonal anti-H-2Ld but not H-2Dd antibodies blocked target cell lysis by the effector cells. Both BALB/c (H-2d) and DBA/2 (H-2d) animals generated anti-VSV CTL that lysed infected 8-5 but not tk+ cells. To further establish that H-2Ld controlled the specificity of the effector cells, a monoclonal antibody directed against H-2Ld was shown to inhibit lysis of infected 8-5 target cells. To determine whether other H-2d-encoded gene products could serve as restricting antigens for anti-VSV CTL in BALB/c animals, unlabeled VSV infected 8-5 cells were tested for their ability to block lysis of 51chromium-labeled P815 (H-2d)-infected target cells. The 8-5-VSV inhibitor cells inhibited lysis to a slightly lesser extent than unlabeled P815-VSV cells, indicating that H-2Ld plays a major if not exclusive role in restricting anti-VSV CTL in H-2d animals.     

Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants

This paper shows that most murine (C57BL/6) influenza A virus-specific memory cytotoxic T lymphocyte (CTL) clones tested in limiting dilution did not react with the influenza A virus surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). This lysis of syngeneic target cells infected with the influenza A virus strains, Aichi (H3N2), PR8 (H1N1), or recombinant strain X31 (H3N2) indicates that most antigenic epitopes recognized are associated with internal virus determinants. X31 and PR8 share the internal, and X31 and Aichi the external, viral determinants. Extensive CTL cross-reactivity was observed in experiments with target cells infected with virus carrying internal determinants homologous with the priming virus. In contrast, when the internal viral determinants differed between the priming virus and the virus used to infect the target cells, and although HA and NA were homologous, we found almost complete CTL-specificity for the priming virus. Thus, the predominant reactivity of influenza A virus-specific CTL differs from that of anti-influenza A antibodies, which are primarily directed towards epitopes on the virus surface glycoproteins. This finding may be relevant for the role of influenza A virus-specific CTL in recurrent infections with different influenza A viruses.     

Fine specificity of cytotoxic T lymphocytes primed in vivo either with virus or synthetic lipopeptide vaccine or primed in vitro with peptide

Standard synthetic peptide preparations contain numerous peptidic byproducts in small amounts, which may be efficiently recognized by cytotoxic T lymphocytes (CTL). Recognition patterns of such peptide mixtures by CTL may serve as a kind of fingerprint for CTL fine specificity. Three types of H-2Db-restricted CTL were compared in this way. CTL primed in vivo either with A/PR/8/34 influenza virus or with a synthetic lipopeptide vaccine prepared from influenza nucleoprotein (NP) peptide 365-380 showed identical fine specificity. Both recognize virus-infected cells. In contrast, CTL primed in vitro with NP 365-380 had a different fine specificity and they did not recognize virus-infected cells. Most significantly, the two in vivo primed CTL types efficiently recognized the natural viral nonapeptide NP 366-374 presented by virus-infected H-2b cells, whereas the in vitro primed CTL failed to do so.     

Antigen recognition by cloned cytotoxic T lymphocytes follows rules predicted by the altered-self hypothesis

Radiation chimeras prepared by injecting H-2 heterozygous F1 stem cells into lethally irradiated parental hosts show a marked, but not absolute, preference for host-type H-2 antigens in the H-2-restricted cytotoxic T lymphocyte (CTL) response to minor histocompatibility (minor H) antigens. We have selected for the anti-minor HCTL that are restricted to the parental H-2 type absent from the chimeric host and found that in two out of eight cases, such CTL lysed target cells of either parental H-2 type. From one of these CTL populations that lysed H-2d and H-2k target cells expressing BALB minor H antigens, clones were derived and further analyzed. The results showed that: (a) lysis of both H-2d and H-2k target cells was H-2 restricted; (b) H-2d restriction mapped to Dd, and H-2k restriction mapped to Kk; (c) testing against various H-2d and H-2k strains of different and partially overlapping minor H backgrounds as well as against the appropriate F1 crosses revealed that in Dd- and Kk-restricted killing, different minor H antigens were recognized. In a second system, a CTL population was selected from normal (H-2d x H-2k)F1 mice that was specific for H-2d plus minor H antigens and for H-2k plus trinitrophenylated bovine serum albumin. We interpret these findings in terms of the altered-self hypothesis: The association of one H-2 antigen with one conventional antigen X may be recognized by the same T cell receptor specific for the complex formed by a different H-2 antigen in association with a second conventional antigen Y. The implications of these observations for the influence of self H-2 on the generation of the T cell receptor repertoire are discussed.     

Specificity studies on cytolytic T lymphocytes directed against murine leukemia virus-induced tumors. Analysis of monoclonal cytolytic T lymphocytes

The specificities of cloned cytolytic T lymphocytes (CTL) were studied for the analysis of CTL populations generated against murine leukemia viruses (MuLV) in H-2 congenic BALB/c (H-2d) and BALB.B (H-2b) mice. In particular, CTL generated in response to tumors induced by Gross MuLV and Friend MuLV were studied; these tumors expressed virus-induced antigens that do not cross-react and that can be distinguished from each other. The systematic study of 92 CTL clones clearly indicated that MuLV-immune CTL were highly heterogeneous with respect to both the intensities of target cell lysis that they mediated and to their specificity of recognition of MuLV-induced tumor target cells. Various categories of CTL clones were identified, ranging from CTL clones tht were tightly H-2 restricted and specific for the immunizing tumor to CTL clones that displayed no discernible patterns of specificity and that attacked a large number of different target cells. In addition, the surface markers of these cloned CTL were defined, and the best conditions for their prolonged maintenance in culture were determined. The present data indicate that future efforts in the definition of target antigens recognized by tumor-specific CTL should be performed with monoclonal lymphocytes.     

H-2L-restricted recognition of viral antigens In the H-2d haplotype, anti-vesicular stomatitis virus cytotoxic T cells are restricted solely by H-2L

H-2d-encoded gene products were analyzed as restriction antigens for anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). Cold target competition experiments revealed that VSV recognition was H-2D region-restricted; H-2K-end-restricted recognition of VSV could not be demonstrated. That VSV is not recognized in the context of K-region-encoded gene products is also supported by the observation that H-2dm1 and H-2dm2 mice, strains that contain H-2Kd but have an alteration in H-2L and/or H-2D/L, are nonresponders in the CTL assay. Two different lines of evidence eliminated H-2Dd, H-2Md, and H-2Rd as the restriction antigens: (a) H-2dm2-VSV inhibitors that express H-2Dd and H-2Md did not block the lysis of P815-VSV targets by Balb/c anti-VSV killer cells, and (b) a hybridoma specific for H-2Dd failed to inhibit killer cell activity in this same effector/target combination. However, two monoclonal antibodies specific for H-2Ld but not H-2Rd completely blocked anti-VSV cytotoxic activity. Taken together, in the H-2d haplotype, anti-VSV CTL recognize VSV solely in the context of the H-2Ld molecule. This is the first demonstration of the exclusive use by a mouse stain of the H-2L molecule only for H-2-restricted recognition, and thus supports the notion that H-2L plays a major role in restricting antigen specific recognition. Finally, the fact that an anti-H-2Ld monoclone completely blocked an H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 but not BALB/c, does not sensitize H-2 alloreactive CTL.     

In vivo effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific

Cloned lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza virus confer complete protection upon adoptive transfer to syngeneic mice lethally infected by influenza virus. The exquisite specificity exhibited by a subtype-specific cloned CTL in culture is reflected in its capacity to eliminate pulmonary virus and mediate recovery only in those mice infected by the virus subtype recognized by this cloned line in vitro. A cross-reactive CTL cloned line protects mice infected by either of two influenza virus subtypes. In mice dually infected with two virus subtypes, the subtype-specific CTL clone only reduces lung virus levels of the recognized virus subtype and cannot prevent these mice from dying. In contrast, adoptive transfer of the cross-reactive CTL clone into mice simultaneously infected with two virus subtypes results in reduction of pulmonary titers of both subtypes and promotes complete recovery. These results directly implicate CTL as an important antiviral defense mechanism in experimental influenza infection. In addition, these results indicate that both the induction and expression of antiviral effector activity by CTL in vivo is highly specific and therefore favor the concept that CTL express their antiviral effect in vivo by direct cytolysis of infected cells.     

The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.     

On the role of the transmembrane anchor sequence of influenza hemagglutinin in target cell recognition by class I MHC-restricted, hemagglutinin-specific cytolytic T lymphocytes

We have examined the requirement for the transmembrane hydrophobic anchor sequence of the influenza hemagglutinin (HA) in the formation of the antigenic moiety on the surface of target cells recognized by class I MHC-restricted murine CTL. For this analysis we have used a line of CV-1 monkey epithelial cells that express the transfected murine H-2Kd gene product as target cells and have used recombinant SV40-based late replacement vectors to achieve expression of genes encoding wild-type and mutant forms of HA. We have found that the majority of Kd-restricted HA-specific CTL clones recognize target cells that express a secreted HA molecule that lacks the transmembrane and cytoplasmic domains of the parent glycoprotein. Several Kd-restricted CTL clones that recognize subtype-specific and crossreactive epitopes on HA fail to recognize the anchor-negative, secreted HA or chimeric HA molecules containing the transmembrane and cytoplasmic domains of unrelated glycoproteins. These CTL clones appear to be directed to antigenic epitopes located within the transmembrane domain of HA, as defined by their capacity to recognize target cells sensitized with a synthetic 23-amino-acid peptide corresponding to sequences within this domain. The implications of these results for class I MHC-restricted CTL recognition are discussed.     

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.     

Participation of the major histocompatibility complex in antibody recognition of viral antigens expressed on infected cells

The capacity of the antibody repertoire to recognize complex antigens on viral-infected cells was investigated at the level of monoclonal B cell responses. A majority of primary B cells responsive to PR8(H1N1)-infected H-2 syngeneic cells produced antibody that bound viral determinants only in the context of infected cells and not the isolated virion. An examination of the fine specificity of such antibodies revealed that most could be distinguished by a panel of cells infected with closely related heterologous H1 influenza strains. Indeed, most antibodies bound hemagglutinin determinants of PR8 exclusively, and few were broadly cross-reactive. An examination of the same antibodies for their recognition of cell surface antigens revealed that the majority recognized MHC-encoded antigenic determinants. Thus, most BALB.K (H-2k) primary B cells responsive to PR8-L919 (H-2k) cells produced monoclonal antibodies that bound PR8-antigens only in the context of H-2Dk-infected cells. Most C57BL/10 (H-2b) B cells responsive to PR8-EL4 (H-2b) cells produced monoclonal antibodies that bound PR8 antigens only in the context of H-2Kb-infected cells. These latter antibodies were further shown to recognize that H-2Kb molecule by virtue of their capacity to be discriminated by a panel of PR8-infected H-2Kb mutant cells. These findings demonstrate that much of the antibody repertoire is capable of highly specific complex recognition of viral antigenic determinants in the context of the appropriate MHC alloantigen.     

An immunodominant epitope present in multiple class I MHC molecules and recognized by cytotoxic T lymphocytes

CTL derived from (C3H x B6.K1)F1 animals were sensitized against L cells that express the transfected gene product Q10d/Ld. These CTL were highly crossreactive against three other class I molecules, H-2Kbm1, H-2Ld, and H-2Kd. In an attempt to define this crossreactive epitope it was noted that between 25 and 39% of amino acids in the alpha helices and central beta strands of these three molecules vary from Q10d. These amino acids represent residues that have been proposed to potentially interact with a peptide antigen or TCR (21). However, all four molecules share the amino acid tyrosine at positions 155 and 156. Additionally, Q10d, H-2Kbm1, and H-2Ld share alanine at position 152, while H-2Kd has an aspartic acid. We showed that these residues were important in controlling this epitope by the finding that anti-Q10d CTL did not recognize H-2Kbm1 revertant molecules that had either the position 152 alanine changed back to the wild-type H-2Kb residue (glutamic acid) or position 155 and 156 tyrosines changed back to wild-type residues arginine and leucine. Further evidence that these molecules share a crossreactive epitope was noted by the failure of (C3H x H-2Kbm1)F1 animals to generate CTL that recognized H-2Ld or H-2Kd, and the inability of (C3H x BALB/c)F1 animals to generate CTL reactive against H-2Kbm1. CTL from these mice were still able to recognize Q10d/Ld indicating that other epitopes could be detected if natural tolerance prevented recognition of the crossreactive epitope. To further define the epitope, CTL clones were generated against Q10d/Ld and maintained on either H-2Kbm1 or BALB/c feeder cells. In addition to testing these clones on the target cells described above, mutant molecules derived from H-2Ld, which have amino acid substitutions in their alpha 1 domain, were analyzed. It was noted that some anti-Q10 clones that did not crossreact on H-2Ld did react against H-2Ld mutant antigens that had H-2Dd amino acid substitutions in the alpha 1 domain at positions 63, 65, 66, and 70. Other clones had differential reactivities on these H-2Ld mutants further substantiating that alpha 1 domain amino acids play a role in controlling the expression of the crossreactive epitope. Thus, four class I molecules with multiple amino acid differences in their alpha 1 and alpha 2 domains share a crossreactive epitope readily recognized by alloreactive CTL.(ABSTRACT TRUNCATED AT 250 WORDS)     

The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice

We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice.     

小鼠肝素酶H-2Kb限制性CTL表位的实验鉴定

目的实验鉴定H-2Kb限制性小鼠肝素酶(mHpa)CTL表位。方法5条mHpa表位分别负载C57BL/6小鼠骨髓来源树突状细胞,并进一步诱导产生表位特异性的效应细胞,采用标准4 h51Cr释放试验检测效应细胞对不同靶细胞的免疫杀伤效应;采用ELISPOT检测效应细胞分泌IFN-γ的能力。结果5条mHpa CTL表位中,仅mHpa398-405和mHpa519-526可以诱导小鼠产生特异性CTL,对同来源的B16黑色素瘤细胞、Lewis肺癌细胞和EL-4淋巴瘤细胞具有明显的免疫杀伤效应,而对H-2Kb阴性的P815肥大细胞瘤细胞不具有免疫杀伤效应,进一步研究发现,上述多肽特异性CTL对自体淋巴细胞和DC细胞不具有杀伤效应,另一方面,mHpa398-405和mHpa519-526还可促进效应细胞分泌IFN-γ的能力。结论mH-pa398-405、mHpa519-526为小鼠肝素酶来源且受H-2Kb限制性CTL表位,小鼠体内可以诱导产生表位特异性的CTL反应。     

Recognition of viral glycoproteins by influenza A-specific cross- reactive cytolytic T lymphocytes

Two populations of cytolytic T lymphocytes (CTL) generated after influenza A virus infection can be distinguished into one with specificity for the sensitizing hemagglutinin type and a second with cross-reactivity for antigens induced by other type-A influenza viruses. The molecules carrying the antigenic determinants recognized by the cross-reactive CTL were studied. In L-929 cells abortively infected with fowl plague virus, matrix (M) protein synthesis is specifically inhibited, whereas the envelope glycoproteins, hemagglutinin and neuraminidase, are synthesized and incorporated into the plasma membrane. These target cells were lysed by cross-reactive CTL. The envelope proteins of type A/Victoria virus were separated from the other virion components and reconstituted into lipid vesicles that lacked M protein that subsequently were used to prepare artificial target cells. Target-cell formation with vesicles was achieved by addition of fusion-active Sendai virus. These artificial target cells were also susceptible to lysis by cross-reactive CTL. In contrast to previous observations that suggested that the M protein of influenza viruses is recognized by these effector cells, we present evidence that the antigencic determinants induced by the viral glycoproteins are recognized.