Molecular requirements for trinitrophenyl recognition by antihapten cytotoxic T lymphocytes

The requirement of direct covalent association of trinitrophenyl (Tnp) groups with cell surface components for functional interactions with anti-Tnp cytotoxic T lymphocytes (CTLs) was analyzed. This question was approached by comparing the ability of two methods of trinitrophenylation to render cells susceptible to lysis by anti-Tnp CTLs. As previously shown, cells modified with trinitrobenzene sulfonic acid were susceptible to H-2-restricted lysis by anti-Tnp CTLs. However, cells incubated with Sendai virus covalently associated with Tnp groups, were not rendered susceptible to lysis by anti-Tnp CTLs. These same target cells, however, were susceptible to H-2-restricted lysis by anti-Sendai virus CTLs. Direct analysis of the number of Tnp groups on cells modified by either method indicates no significant difference in the number of Tnp molecules associated with the different target cells. The results suggest that direct covalent association of Tnp groups with cell surface specific components is a minimal molecular requirement for recognition and lysis by anti-Tnp CTLs.     

Introduction of soluble protein into the class I pathway of antigen processing and presentation

In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphocytes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2Kb-restricted CTL specific for the OVA258-276 peptide. This synthetic peptide and CNBr fragments of OVA (242-285 and 242-273) were able to target H-2b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the Kb restricted determinant formed readily.     

Lysis of target cells infected with vesicular stomatitis virus (VSV) in the presence of tunicamycin by anti-VSV cytotoxic T lymphocytes

We have analyzed the requirement for the expression of the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) on target cells for recognition and lysis by anti-VSV cytotoxic T lymphocytes (CTL). In addition, we have attempted to determine if the carbohydrate moieties on the G protein are required for recognition and lysis by anti-VSV CTL. When VSV (Orsay) is grown at 30 degrees C in the presence of tunicamycin (TM), glycosylation of G protein is inhibited; however, nonglycosylated G protein is found on the surface of the cell and active virus particles are produced. In contrast, VSV (Orsay) grown at 39 degrees C in the presence of TM produces low titers of virus and the presence of G protein on the surface of cells is not detectable. The susceptibility of these target cells to lysis by anti-VSV CTL was analyzed. The results suggest that expression of the G protein is required for target cell lysis by anti-VSV CTL. However, the presence of the carbohydrate moieties on the G protein are nt an absolute requirement for recognition by anti-VSV CTL. VSV-infected target cells incubated in the presence of TM were lysed by anti-VSV CTL up to 50 to 80% of the infected target cell control. This result suggests either that some clones of anti-VSV CTL recognize carbohydrate moieties or that carbohydrate moieties play some as yet undefined nonantigenic role in the recognition of the target antigen by the CTL receptor.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Possible mechanisms by which the H-2Kbm3 mutation may decrease cytotoxic T-lymphocyte recognition of vesicular stomatitis virus nucleoprotein antigen.

Spleen cells from C57BL/6 (B6) mice generate a strong in vitro cytotoxic T-lymphocyte (CTL) response specific for vesicular stomatitis virus (VSV). Spleen cells from VSV-primed B6-H-2bm3 (bm3) mice, which have a mutation in H-2Kb, require approximately 10-fold more UV-inactivated VSV to generate in vitro secondary anti-VSV CTL, compared with spleen cells from primed B6 mice. Anti-VSV CTL elicited in both bm3 and B6 mice are primarily specific for the viral nucleocapsid protein (N protein), as demonstrated by using recombinant vaccinia viruses that express the VSV N protein. bm3 CTL were found to exhibit only a very low level of lytic activity when tested against autologous VSV-infected concanavalin A spleen cell blasts as well as several H-2b tumor cell lines. The weak anti-VSV response of bm3 CTL was found to be the result of a combination of inefficient recognition of VSV-infected target cells and decreased elicitation of secondary effector cells. VSV-infected bm3 target cells were not killed as well as B6 targets by either bm3 or B6 effectors. This is because of the inefficient recognition of targets, as demonstrated by the fact that VSV-infected bm3 cells were unable to competitively inhibit the lysis of VSV-infected B6 target cells by either bm3 or B6 effectors. By using cells from recombinant mice, it was shown that the CTL response restricted by H-2Kb was low in the bm3 mice, compared with that of the B6 mice. However, the H-2Db-restricted CTL activity was similarly low in both the B6 and bm3 mice. The possibility that the low response to VSV-infected bm3 cells is caused by differences between the bm3 and B6 cells in expression of either viral antigens or H-2K was investigated by radiolabeling and immunoprecipitation. VSV-infected B6 and bm3 cells were found to express equivalent levels of both viral antigens and H-2K. These results indicate that the bm3 mutation alters a functional site on the H-2Kb molecule that is involved in the recognition of VSV-infected cells. The observation that elicitation of bm3 CTL can occur at high antigen doses further suggests that the bm3 mutation results in a lower affinity of H-2K either for viral antigen or for receptor sites on the CTL.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

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.     

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.     

High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.     

Persistent infection with lymphocytic choriomeningitis virus enhances expression of MHC class I glycoprotein on cultured mouse brain endothelial cells

Brain endothelial cells (EC) represent a major component of the blood/brain barrier, which activated CTL cross to enter the central nervous system. Several viruses also penetrate the central nervous system through the blood stream via the brain EC. The studies reported here focus on understanding the principles and consequences of interactions among viruses, lymphocytes, and EC in the brain. As shown persistent but not acute infection by lymphocytic choriomeningitis virus enhances the expression of MHC class I glycoproteins on the brain EC of mice. This increase in MHC expression during viral infection does not seem to result from the release of cytokines. However, replicative virus is required, because UV inactivated virus fails to enhance MHC expression. Viral determinants appear on EC surfaces after infection and serve as targets for CTL directed lysis. In contrast, neurons (OBL 21 neuronal cell line), which express negligible amounts of MHC class I glycoproteins, show no gain in MHC markers during persistent viral infection and are not targets for virus-specific CTL killing.     

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.     

Mechanism of escape of endogenous murine leukemia virus emv-14 from recognition by anti-AKR/Gross virus cytolytic T lymphocytes.

It was previously shown that spleen cells from endogenous ecotropic murine leukemia virus emv-14+ AKXL-5 mice fail to stimulate an anti-AKR/Gross virus cytolytic T-lymphocyte (CTL) response in a mixed lymphocyte culture with primed C57BL/6 responder spleen cells, whereas spleen cells from AKXL strains carrying the very similar emv-11 provirus do stimulate a response (Green and Graziano, Immunogenetics 23:106-110, 1986). We wished to determine whether the lack of response with AKXL-5 spleen cells was at the level of recognition between effector cell and target cell and whether the relevant mutation was within the emv-14 provirus. It is shown here that EMV-negative SC-1 fibroblast cells transfected with the major histocompatibility complex class I Kb gene and infected with virus isolated from the AKXL-5 strain (SC.Kb/5 cells) were not lysed by H-2b-restricted anti-AKR/Gross virus CTL. SC.Kb cells infected with virus isolated from emv-11+ strains, however, were efficiently lysed by anti-AKR/Gross virus CTL, indicating that there is nothing intrinsic to EMV-infected SC.Kb cells that would prevent them from being recognized and lysed efficiently by anti-AKR/Gross virus CTL. Analysis of virus expression for the infected SC.Kb cells by XC plaque assay and by flow cytometry indicated that emv-14 virus expression for SC.Kb/5 cells was not significantly different from that for emv-11-containing SC.Kb/9 or SC.Kb/21 cells. These data show that the mutation responsible for the lack of CTL recognition and lysis is at the level of recognition between target cell and effector cell. Furthermore, these data strongly suggest that the mutation is within the emv-14 genome. Flow cytometry experiments with monoclonal antibodies against a number of viral determinants indicated that there was no gross mutation detectable in the viral determinants analyzed. The data suggest that the relevant mutation may be a point mutation or a small insertion or deletion within a coding sequence that is critical for CTL recognition.     

Recognition of Nonstructural Protein Peptides by Cytotoxic T Lymphocytes Raised against Japanese Encephalitis Virus

We have previously reported that Lyt2⁺ cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K^d-restricted CTL and to examine their cross-recognition of West Nile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K^d binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.     

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.     

Detection and induction of equine infectious anemia virus-specific cytotoxic T-lymphocyte responses by use of recombinant retroviral vectors

Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. V. Recognition is restricted to gene products encoded by the viral S RNA segment

Lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) strain-specific, H-2d-restricted CTL effectively lyse syngeneic targets infected by LCMV ARM, but show reduced killing of LCMV Pasteur (PAST) strain-infected H-2d cells. We have reassorted the two RNA segments, large (L) and small (S), of LCMV ARM and PAST to generate LCMV with genotypes of L ARM/S PAST and L PAST/S ARM. By using these reassortants and both LCMV primary CTL and CTL clones, we report that the induction, recognition, and lysis of LCMV-specific CTL depend on the S RNA segment and the genes it encodes.     

Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins

PBL from chimpanzees immunized with a recombinant vaccinia virus that expresses HIV envelope glycoproteins ("env"), were found to proliferate after stimulation with HIV or with "env". Furthermore, CTL clones lytic for autologous target cells expressing HIV envelope glycoproteins were generated after stimulation of the chimpanzees' PBL with "env", indicating that immunization of these primates with a recombinant vaccinia virus primes HIV-specific CTL and also that HIV envelope glycoproteins serve as target antigens for CTL.     

Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells

Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens.