Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.     

Recognition of viral antigens by human influenza A virus-specific T lymphocyte clones

The nature of the viral antigens recognized by influenza A virus-immune cytotoxic T lymphocytes (CTL) is still a matter of debate. We have used four human influenza A virus-specific T lymphocyte clones with antigen-specific cytotoxic and proliferative activity to investigate the requirements for recognition of viral antigens on infected cells. One clone recognized a cross-reactive determinant on the viral hemagglutinin, and two clones were specific for different epitopes on the viral nucleoprotein (NP). A fourth clone seemed to be specific for the viral M protein. Target cell recognition was abrogated by the addition, during infection, of the lysosomotropic drug chloroquine, known to inhibit antigen processing. Furthermore, target cells that had been pulsed with soluble purified NP were recognized and were lysed by the NP-specific clone. This reaction could also be abrogated by the addition of chloroquine during pulsing. These results were obtained irrespective of whether EBV-transformed B lymphoblastoid cells or Ia antigen-expressing T cell blasts were used as target cells. It is concluded that CTL can recognize internal viral proteins that are actively presented at the surface of the target cell. These data indicate that probably every viral protein can function as a target molecule for virus-immune CTL.     

Fine specificity and antigen receptor expression among influenza virus-specific cytolytic T lymphocyte clones

Influenza virus stimulates a vigorous cytolytic T lymphocyte (CTL) response in the mouse that is directed to several virion polypeptides. This report examines the fine specificity of a panel of murine influenza-specific CTL clones restricted by MHC class I products of the H-2d haplotype. Ten of 22 A/JAPAN/305/57-specific CTL clones analyzed were directed to the A/JAPAN/305/57 hemagglutinin protein as detected by using target cells infected with a recombinant vaccinia virus containing hemagglutinin gene. Based on their fine specificity of hemagglutinin recognition, these clones defined four functional epitopes on the hemagglutinin. The remaining 12 cytolytic clones exhibited cross-reactivity for type A influenza viruses of the major human subtypes, and approximately 60% of these clones were directed to the nucleocapsid protein. KJ16-133 monoclonal antibody analysis of the utilization of the T cell receptor V beta 8 gene segment subfamily revealed that members of this V beta gene subfamily are expressed by both hemagglutinin- and nucleocapsid-specific MHC class I-restricted CTL (and by influenza-specific MHC class II-restricted T lymphocytes as well). These results suggest that CTL detect several distinct antigenic sites on the hemagglutinin. In addition, these results reveal no direct correlation between viral antigenic specificity and V beta gene expression by these virus-specific CLT clones.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.     

Induction of class I MHC-restricted, peptide-specific cytolytic T lymphocytes by peptide priming in vivo

The present study investigated the possibility that protein Ag fragments in the form of peptides could serve as the priming Ag in the generation of a MHC class I-restricted immune response. Trypsin-digested chicken ovalbumin (OVA-TD) fragments were used as the model Ag. The results demonstrate the peptides within OVA-TD, when injected into C57BL/6 mice, could prime T cells which lysed H-2b Ia-EL4 target cells in an OVA-TD-specific manner. In contrast to priming with OVA-TD, immunization of mice with intact OVA did not lead to generation of CTL against OVA-TD or OVA. Furthermore, target cells sensitized with intact OVA failed to be recognized by OVA-peptide-specific CTL indicating that the target cells serving as APC were unable to generate the relevant peptide determinants recognized by the T cells. These results support the idea that the processing pathway within APC for class I-restricted T cells may differ from that used for class II-restricted T cells. Using OVA-TD-specific CTL clones (phenotypically Thy 1+, CD8+, CD4-, Pgp-1+) isolated from primed animals to screen OVA-TD fractions separated by HPLC, two T cell peptide determinants were identified corresponding to OVA sequences 111-122 and 370-381. Both determinants were recognized by CTL clones in the context of the H-2Db molecule.     

Anti-influenza virus cytotoxic T lymphocytes recognize the three viral polymerases and a nonstructural protein: responsiveness to individual viral antigens is major histocompatibility complex controlled

It has recently been shown that antiviral major histocompatibility complex class I-restricted cytotoxic T lymphocytes can recognize proteins that serve as internal viral structural components (influenza A virus nucleoprotein, vesicular stomatitis virus nucleocapsid protein). To further examine the role of internal viral proteins in cytotoxic T-lymphocyte recognition, we constructed recombinant vaccinia viruses containing individual influenza A virus genes encoding three viral polymerases (PB1, PB2, PA) and a protein not incorporated into virions (NS1). We found that cells infected with each of these recombinant vaccinia viruses could be lysed by anti-influenza cytotoxic T lymphocytes. Cytotoxic T-lymphocyte responsiveness to the individual viral antigens varied greatly between mouse strains. By using congenic mouse strains, responsiveness to PB1 and PB2 was found to cosegregate with major histocompatibility complex haplotype. These findings provide further evidence that internal antigens play a critical role in cytotoxic T-lymphocyte recognition of virus-infected cells. Additionally, they suggest that the cytotoxic T-lymphocyte response to viral antigens may often be restricted to only a fraction of the major histocompatibility complex class I repertoire.     

HA2 subunit of influenza A H1 and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response

Influenza H1 subtype-specific CTL can be induced by secondary stimulation of a hybrid protein of the first 81 amino acids of the viral NS1 non-structural protein and the HA2 subunit of A/Puerto Rico/8/34(H1N1) hemagglutinin. In addition, a derivative of this protein with 65 amino acids deleted from the N-terminal end of HA2 can also generate H1 subtype-specific CTL in bulk cultures. CTL clones established by stimulation with the derivative protein demonstrated cross-reactive lysis of target cells infected with virus strains of the H1 and H2 subtypes. Cold target competition experiments with CTL clones as effectors demonstrated that the Ag specificity between these two hybrid proteins is identical. Adoptive transfer of the CTL clone significantly reduced virus titers in the lungs of mice infected with the virus strains of the H1 or H2 subtype but not those infected with the H3 subtype virus in vivo, which reflects the in vitro CTL clone activity. These experiments demonstrate that an epitope on the hemagglutinin that is conserved on virus strains of the H1 and H2 subtypes induces a protective CTL response. These results suggest an alternative approach for developing influenza vaccines by using conserved antigenic sites on the hemagglutinin HA2 subunit to avoid the problem of frequent antigenic mutations of the HA1 subunit antibody binding sites.     

Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.     

Influenza A virus—a model for viral antigen presentation to cytotoxic T lymphocytes

Human influenza A virus is characterized by its high degree of variability and by its ability to cause frequent epidemics of disease. Most of the variation occurs in the two surface glycoproteins of the virus, against which protective antibodies are directed. In contrast, the strong MHC class I-restricted CTL response to infection with virus is predominantly specific for internal viral proteins which are relatively well conserved, and is cross-reactive between different strains of influenza A virus. However, the natural evolution of influenza viruses is largely driven by selection with antibody, with no firm evidence of selection by CTL. In normal individuals influenza virus produces an acute, localized infection, and this in part may reflect an inability to escape the CTL response.     

The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.     

Ultrastructural changes during lysis of L929 target cells by class II-restricted influenza virus-specific murine cytotoxic T-lymphocyte clones

Lysis of virus-infected L929 target cells transfected with the H-2 class II IAk gene by class II-restricted influenza virus-specific murine cytotoxic T lymphocyte (CTL) clones was studied by electron microscopy and compared with lysis of L929 cells by class I-restricted CTL clones. T lymphocytes predominantly approached the basal surface of target cells grown on a plastic dish and also approached uninfected L929 target cells, although virus maturation exhibited no polarity with respect to the cell surface site. After incubation for 30 min, the target cell nuclei began to change: chromatin became irregularly redistributed and aggregated, and the nuclei appeared swollen. Later, electron-dense and -light areas of nuclei became segregated, and the cytoplasm became disorganized with many vacuoles. The ultrastructural changes of target cells during lysis by class I- and class II-restricted CTL clones appeared to be similar. These findings and other cytotoxicity data of class I and class II CTLs are discussed.     

Class I MHC-restricted recognition of cells expressing a gene encoding a 41 amino acid product of the influenza hemagglutinin

Class I H-2Kd-restricted influenza hemagglutinin (HA)-specific CTL recognize two immuno-dominant sites as represented by synthetic peptides spanning epitopes located in the HA1 and hydrophobic transmembrane domains of the influenza HA. Using a vaccinia virus recombinant expression system, we have examined CTL recognition of HA deletion mutants expressed in target cells. We have demonstrated that a truncated influenza HA gene encoding only the transmembrane anchor region containing a class I recognition site and a short segment of the cytoplasmic tail of the HA can be efficiently presented to class I CTL. These results set the stage for detailed analyses of the intracellular events associated with Ag presentation to class I CTL and offer novel possibilities for future vaccine design.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.     

Role of de novo protein synthesis in target cells recognized by cytotoxic T lymphocytes specific for vesicular stomatitis virus.

The requirements for viral and host protein synthesis in the generation of target antigens for cytotoxic T lymphocytes (CTL) was evaluated by using vesicular stomatitis virus (VSV) inactivated by UV irradiation (UV-VSV). EL4 target cells incubated with UV-VSV were recognized and lysed by anti-VSV CTL, indicating that de novo synthesis of viral proteins was not required for the generation of antigens recognized by antiviral CTL. Anti-VSV CTL from H-2b mice primarily recognize determinants derived from the VSV N protein bound to the class I major histocompatibility complex (MHC) antigen H-2Kb. Comparison of a cloned CTL line representing this specificity and a heterogeneous population of anti-VSV CTL showed that determinants other than that recognized by the cloned CTL were generated more efficiently from UV-VSV. By using vaccinia virus recombinants that express deletion fragments of the N protein, it was shown that these additional determinants were probably derived from VSV proteins other than the N protein. The protein synthesis inhibitor emetine was used to determine whether newly synthesized host proteins were required for antigen generation. The addition of emetine to target cells prior to or at the time of the addition of UV-VSV inhibited lysis by anti-VSV CTL. This inhibition could be due to depletion of newly synthesized MHC molecules from intracellular membranes. This hypothesis was supported by using brefeldin A to delay membrane protein transport in target cells during the time of incubation with emetine and UV-VSV, which resulted in partial reversal of the effect of emetine. These results suggest that newly synthesized class I MHC molecules are required for the generation of antigens recognized by anti-VSV CTL.     

Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression.

The human pathogen CMV, is a major cause of morbidity and mortality in immunocompromised hosts. The CD8+ class I-restricted CTL response to CMV assists in preventing progression of CMV infection to life-threatening disease; however, the viral Ag recognized by CD8+ CTL are not well characterized. In general, virus-specific CTL recognize endogenously synthesized viral proteins processed and presented associated with class I MHC molecules. Although proteins or inactivated virions have been experimentally delivered to the cytoplasm to result in class I MHC presentation, this mode of Ag delivery to the class I processing pathway after natural viral entry has not been documented in humans. Our data demonstrate that the CMV-specific class I-restricted CTL response in individuals latently infected with CMV is predominantly specific for selected structural virion proteins introduced into the cell after viral penetration and efficient recognition occurs in the absence of de novo viral gene expression. This CTL response may provide a biological advantage for limiting the spread of infection after CMV reactivation because infected cells are lysed before viral assembly.     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis.

Hepatitis C virus (HCV) is a major cause of post-transfusion and sporadic hepatitis worldwide, leading to chronic liver disease in at least 50% of infected individuals. The pathogenic mechanisms that result in chronic hepatitis are unknown. Lymphocytes are typically observed within the hepatic parenchyma, but the functional characteristics of these cells have not been defined. In this study, liver-infiltrating lymphocytes from two subjects with chronic HCV hepatitis were cloned at limiting dilution and tested for HCV-specific cytolytic activity using autologous target cells infected with vaccinia viruses expressing recombinant HCV Ag or sensitized with synthetic HCV peptides. In both subjects, HCV-specific, HLA class I-restricted CTL were identified that recognized epitopes in variable regions of either the envelope or nonstructural proteins. These results demonstrate the presence of HCV-specific CTL at the site of tissue damage in persons with chronic HCV hepatitis, and provide a means to evaluate the possible pathogenic role of these cells in HCV infection.