A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes

A recombinant vaccinia has been designed to express amino acids 366-379 of influenza nucleoprotein, previously shown to be the minimal epitope recognized by a class I-restricted cytotoxic T cell clone. Target cells infected with the recombinant vaccinia virus expressing this peptide are recognized by CTL as efficiently as target cells expressing the complete nucleoprotein. The results imply the existence of a peptide transport system that constitutively passes the products of degraded proteins from the cytoplasm into a membrane-bound compartment of the cell.     

HLA B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes

Human influenza A virus-specific, cytotoxic T cells have been shown previously to recognize the virus nucleoprotein on infected cells. CTL preparations from four HLA B37-positive donors were shown to recognize a synthetic peptide that corresponded to amino acids 335-349 of the nucleoprotein sequence. Influenza-specific CTL from 10 donors of other HLA types failed to recognize this epitope. CD8+ CTL lines were derived from lymphocytes of two HLA B37-positive donors and used to show that the peptide was represented on virus-infected cells and to determine the probable boundaries of the epitope.     

Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.     

RMA/S cells present endogenously synthesized cytosolic proteins to class I-restricted cytotoxic T lymphocytes.

RMA/S is a mutant cell line with decreased cell surface expression of major histocompatibility complex class I molecules that has been reported to be deficient in presenting endogenously synthesized influenza virus nucleoprotein (NP) to cytotoxic T lymphocytes (CTL). In the present study we show that RMA/S cells can present vesicular stomatitis virus nucleocapsid protein, and, under some conditions, NP, to Kb-and Db-restricted CTL, respectively. Antigen presentation results from processing of cytosolic pools of endogenously synthesized proteins, and not the binding to cell surface class I molecules of antigenic peptides present in the virus inoculum or released from infected cells. Antigen processing of RMA/S differs, however, from processing by wild-type cells in requiring greater amounts of antigen, longer times to assemble or transport class I-peptide complexes, and in being more sensitive to blocking by anti-CD8 antibody. Thus, the antigen processing deficit in RMA/S cells is of a partial rather than absolute nature.     

Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.     

Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes

Human cytotoxic T cells specific for influenza A virus were tested for recognition of each of the ten influenza A virus proteins expressed in target cells using recombinant vaccinia viruses. They recognized the matrix M1, polymerase PB2, and nucleoproteins of influenza virus in association with MHC class I antigens. These internal viral proteins were seen by CTL in conjunction with one or more of the available dependent HLA gene products. There was no detectable recognition of influenza virus surface glycoproteins in target cells.     

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)     

Identical peptides recognized by MHC class I- and II-restricted T cells

Previous data from many groups show that both class I and class II-restricted T cells recognize short synthetic peptides in the context of their respective MHC molecules (9-18), all of the peptides described to date are restricted to only a single class of MHC molecules; however, structural homology between the class I and II MHC molecules and the use of similar TCRs by class I and II-restricted T cells suggest that antigen recognition mechanisms are similar in both systems. To directly compare antigen recognition in the two systems, we analyzed peptides for the ability to function in both a class I and II-restricted system and found that seven of seven individual peptides tested stimulate both class I and II-restricted T cell responses. In addition, two of the peptides can function in different species stimulating both human class I and murine class II T cell responses. Thus, the process of T cell recognition of antigen in the context of MHC molecules was highly conserved in evolution not only between the class I and class II MHC systems, but also between the murine and human species.     

肽刺激培养法体外扩增巨细胞病毒特异细胞毒性T淋巴细胞

目的研制一种扩增特异细胞毒性T淋巴细胞频数的方法。方法用不同浓度的巨细胞病毒（CMV）特异的表位肽pp65刺激人外周血单个核细胞（PBMCs）使CMV特异CTLs在PBMCs中的百分比增高。用四聚体一PE和CD。一FITC双标记PBMCs中的CMV特异CTLs，然后用流式细胞仪分析。结果这个方法能有效扩增CMV特异CTLs，使在外周血中占淋巴细胞百分比不到l％的CMV特异CTLs迅速增加到接近20％，占CDsT淋巴细胞的40％以上，并能从单份血样中获得大量特异CTLs用于流式分析。结论肽刺激培养法简便、易操作，能有效扩增CMV特异CTLs，使不同人的数据更容易对比和连续的评价。该方法也为过继性免疫疗法治疗巨细胞病毒疾病打下了基础。     

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.     

Tumor antigens recognized by T lymphocytes

In the last five years, knowledge of human tumor antigens recognized by autologous cytolytic T lymphocytes (CTL) has increased considerably. So far, genetic and biochemical approaches have led to the molecular identification of three classes of antigens. Most of these antigens consist of peptides that are presented to T cells by HLA molecules. The first class comprises antigens encoded by genes such as MAGE, BAGE, and GAGE, which are expressed in various tumors of different histological origins, but not in normal tissues other than testis. The second class represents differentiation antigens encoded by genes that are only expressed in melanoma and normal melanocytes like tyrosinase, Melan-A/MART-1, gp100 and gp75. The third class includes antigens produced by unique point mutations in genes that are ubiquitously expressed. In most cases, the antigenic peptide is encoded by the mutated region of the gene. A number of these antigens provide promising targets for new protocols of specific cancer immunotherapy.     

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.     

Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.     

Determinants recognized by human cytotoxic T cells on a natural hybrid class I HLA molecule

The major histocompatibility complex class I HLA molecules are the primary determinants recognized by allogeneic cytotoxic T lymphocytes (CTL), and serve as restricting elements for CTL recognition of viral, chemical, or minor histocompatibility antigens. HLA-Aw69 is a naturally occurring hybrid class I molecule that we have used to investigate the regions of class I antigens involved in human CTL recognition. HLA-Aw69 appears to have resulted from an exon shuffle between two closely related class I genes: the alpha 1 domain of HLA-Aw69 is identical to that of HLA-Aw68, while the alpha 2 and alpha 3 domains are identical to HLA-A2. The determinants recognized by human allogeneic CTL clones specific for HLA-A2, -Aw68, and/or -Aw69 fall into three patterns: (a) CTL determinants are located on both the alpha 1 and alpha 2 domains; (b) interaction of the alpha 1 and alpha 2 domains results in new combinatorial determinants; (c) interaction of the alpha 1 and alpha 2 domains in the hybrid molecule results in the loss of CTL determinants that are present on both parental molecules. Thus, using human CTL clones, target cells, and HLA molecules, we show that the interaction of the alpha 1 and alpha 2 domains alters CTL determinants in ways not directly predictable from primary structure.     

Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis.

Autoimmune helper T lymphocytes were selected from the blood of two myasthenic patients of different HLA-DR type, using acetylcholine receptor (AChR) from Torpedo californica. These polyclonal T cell lines were tested for reactivity with three synthetic peptides corresponding to the NH2-terminal region of the human AChR alpha subunit. This segment is a good candidate for T cell epitopes since it has a propensity to form an amphipathic alpha helix. The peptides elicited 10-30% of the response induced by native Torpedo AChR. Different peptides were recognized by the autoreactive T cells of the two patients. These results suggest that the NH2-terminal region of the AChR alpha chain contains T cell-stimulating epitopes, and that the T cell autoimmune response in myasthenia gravis, like the B cell response, is heterogeneous.     

Alloreactive cytotoxic T lymphocytes recognize epitopes determined by both the alpha helices and beta sheets of the class I peptide binding site.

A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha helices and the beta-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the alpha helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (Kb alpha Ld beta) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha helices of Kb can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing Kb alpha Ld beta. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing Kb alpha Ld beta. Thus, alloreactive CTL did not recognize structures determined by the alpha helices alone or by the beta sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with Kb alpha Ld beta. In addition to the Kb alpha helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with Kb alpha Ld beta. Amino acid 24 is located on the beta 2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and Kb alpha Ld beta identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the alpha helices and beta sheets of the ARS. These interactions are best explained by the influence of the alpha helices and beta sheets on the peptide-binding properties of these antigen-presenting molecules.     

Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin

Antigen-specific human T cell clones specific for defined peptides of influenza A hemagglutinin were found to be rendered unresponsive by incubation with moderately high concentrations of antigen. This was the case whether the synthetic peptide antigen was present for the duration of the culture or the cloned T cells were preincubated with antigen for 3-18 h at 37 degrees C, before stimulation with T-depleted irradiated sheep erythrocyte non-rosette-forming lymphocytes (E-) pulsed with the optimal dose of peptide. Tolerance could not be overcome by culture with various numbers of E- cells and antigen. The induction of unresponsiveness was antigen specific, since it depended upon incubation with the appropriate peptide recognized by that clone. In addition, the tolerant T cells remained unresponsive to stimulation with the specific peptide for at least 7 d after induction even though maintained in culture in the presence of T cell growth factor. This state of antigen-specific unresponsiveness is akin to immunological tolerance. Furthermore, the experiments reported here demonstrate that the helper T cell clone can be inhibited by the relevant peptide in the absence of any suppressor cells or their precursors. This suggests that antigen-induced unresponsiveness need not always depend on the presence of suppressor T cells. The induction of tolerance in T cell clones does not result in early T cell death, since cells that no longer proliferate in response to the specific antigen and accessory cells still proliferate in response to T cell growth factor.