Uninfected Target Cells Research Articles

Synthesis of human immunodeficiency virus DNA in a cell-to-cell transmission model.

Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was modelled by coculturing virus-infected cells with uninfected target cells at a ratio of 1:4. While H9 cells persistently infected with HTLV-IIIB did not contain unintegrated viral DNA detectable by Southern blotting, when cocultured with uninfected HUT-78 cells the mixed culture effectively underwent a new round one-step virus replication which began de novo synthesis of free viral DNA within 4 hours. Linear DNA was synthesized before the accumulation of circular DNA, and two seemingly distinct phases of viral DNA synthesis were involved. When both virus donor cells and recipient cells were arrested in the G0/G1 phase of the cell cycle, accumulation of circular viral DNA was inhibited. In contrast to cell-free virus infection of resting human peripheral blood mononuclear cells (PBMC), where no free viral DNA of discrete sizes could be detected by Southern blot, cell-to-cell transmission infection of resting PBMC resulted in the synthesis of full-length linear as well as circular viral DNA. The efficiency with which cell-to-cell transmission of HIV initiates virus replication underlines the importance of this mode of transmission in virus dissemination in vivo.

Effect of Interleukin-4 on Cytolytic T Cells

Recent studies indicate that interleukin-4 (IL-4) is a potent T-cell growth factor which may have a significant stimulatory effect on alloreactive cytolytic T lymphocytes (CTL). In this study, it was found that exposure of a recombinant retrovirus (RVpC)-specific, MHC-restricted, polyclonal CTL line to IL-4 resulted in the generation of CTL which efficiently lysed RVpC-infected as well as uninfected autologous target cells. A number of CTL cloned lines was generated from the parental line grown for 2 weeks with autologous stimulator cells in IL-4-containing media which again lysed RVpC-expressing as well as uninfected autologous target cells but not their allogeneic HLA-mismatched counterparts. These results extend previous observations in allogeneic systems and suggest that, in a syngeneic system, IL-4 may stimulate the recruitment of autoreactive CTL in vitro. This is a previously unrecognised property of IL-4, the in vivo significance of which remains to be established.

HIV-1 Neutralizing Monoclonal Antibodies Induced by a Synthetic Peptide

We have developed a series of murine monoclonal antibodies to a region of the 120 kD envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1). This region has previously been implicated as a site for virus neutralization by antisera raised to recombinant proteins and by antibodies made to full-length gp120 purified from virus. The antigen employed was a synthetic peptide containing 15 amino acids, representing amino acid residues 308-322, RIQRGPGRAFVTIGK, of env gp120 (HTLV-IIIB isolate). Five of the monoclonal antibodies raised to this antigen have reactivity with gp120 from divergent strains of HIV-1 in Western blot assays. The two of these five which were tested with live cells infected with the divergent HIV-1 isolates IIIB, MN, and RF were specifically reactive by fluorescence analyses with cells infected with the MN and IIIB isolates. Four of the five monoclonal antibodies blocked the fusion of IIIB-infected cells with uninfected MOLT-4 target cells. The monoclonal antibody most reactive with MN-infected cells by fluorescence, #5025A, blocked the fusion of MN-infected cells with uninfected MOLT-4 cells. Four of the five monoclonal antibodies neutralized the IIIB isolate of HIV-1 in vitro, but none neutralized the MN or RF isolates at the levels of antibody tested (less than or equal to 50 micrograms/ml). Taken together these data indicate that monoclonal antibodies to the immunodominant neutralizing domain of HIV-1 gp120 display different levels of group reactivity depending on the assay system being examined.

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.

Virus-specific and autoreactive T cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis

Using a recently described technique for expanding of human T lymphocyte populations from cerebrospinal fluid (CSF), we investigated the local cellular immune response in a patient with chronic rubella panencephalitis. A total of 328 T cell lines (TCLs) was established by seeding CSF cells at limitin dilution into histoplates in the presence of irradiated feeder cells and phytohemagglutinin (PHA)-containing conditioned medium. 80% of TCLs expressed the CD4 + CD8 −, 5% the CD4 − CD8 + phenotype and 15% of TCLs contained different proportions of CD4 + and CD8 + cells. Of 191 TCLs analyzed, 85 were cytotoxic, as shown by their lectin-dependent cytotoxicity against allogeneic uninfected target cells. Eight of them demonstrated specificity for the autologous, rubella virus-infected target cells. When testes for antigen-specific proliferative activity, 26 TCLs responded to rubella antigen, 16 TCLs reacted to myelin basic protein (MBP), four TCLs to proteolipid protein (PLP), four to galactocerebrosides and two to actin. Fourteen out of 16 MBP-specific TCLs also responded, to a minor degree, to rubella antigen and/or actin. The results showed that the persisting rubella infection had given rise to autoreactive T cells. Virus-induced autoreactivity to brain antigens may be an important pathogenic mechanism in other chronic inflammatory disorders of the CNS.

Tumor necrosis factor alpha is a cytotoxin induced by murine Chlamydia trachomatis infection.

A mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent) was used to demonstrate that whole spleen cells from both nude athymic mice (nu/nu) and heterozygous mice (nu/+) produced tumor necrosis factor alpha in vitro in response to mouse pneumonitis agent antigen. The tumor necrosis factor alpha measured in these supernatants by immunoassay was shown to have bioactivity in a cytotoxic assay in which uninfected target cells were used. This cytotoxicity was distinct from the gamma interferon-related cytotoxicity against C. trachomatis-infected targets that we described previously.

Polymorphism of cytotoxic T-lymphocyte clones that recognize a defined nine-amino-acid immunodominant domain of lymphocytic choriomeningitis virus glycoprotein

To assess the heterogeneity of cytotoxic T lymphocytes (CTLs) directed against viral epitopes, we studied six class I major histocompatibility complex-restricted (H-2Db) CTL clones that recognize the same 9-amino-acid immunodominant epitope, amino acids 278 to 286 from envelope glycoprotein 2 (GP2) of lymphocytic choriomeningitis virus (LCMV). Using Southern blot analysis of beta-chain rearrangements, we found that each clone has a unique restriction pattern, providing evidence of the independent derivation of the clones and suggesting that the clones express different beta-chain sequences for their T-cell receptor. All these clones killed syngeneic target cells infected with strain Armstrong or WE of LCMV; however, two of the six clones failed to recognize target cells infected with the Pasteur strain of LCMV. Sequence analysis of LCMV Armstrong, WE, and Pasteur GP in the region of amino acids 272 to 293 demonstrated a single-amino-acid substitution at amino acid 278 in the region of the defined epitope in the Pasteur strain. Interestingly, one of the two CTL clones that failed to lyse LCMV Pasteur-infected target cells nevertheless efficiently and specifically killed uninfected target cells coated with the appropriate LCMV Pasteur peptide, while the other clone failed to do so. This indicated a dichotomy between processing of the synthesized protein initiated by infection and a peptide exogenously applied. Dose-response studies utilizing several peptides with substitutions in GP amino acid 278 indicate that CTL recognition occurs at the level of a single amino acid and suggest that this difference is likely recognized at the level of the T-cell receptor.

Gamma interferon-mediated cytotoxicity related to murine Chlamydia trachomatis infection.

After infection with the mouse pneumonitis agent (MoPn; murine Chlamydia trachomatis), heterozygous (nu/+) but not nude athymic (nu/nu) mice produced enhanced amounts of gamma interferon (IFN-gamma) in vitro in response to MoPn antigen that exhibited cytotoxic activity when added to host cells already infected with chlamydiae. Antibody-complement lysis showed the cytotoxic activity to be dependent, at least in part, on L3T4+ T cells for production. The cytotoxic responses were directed primarily against Chlamydia-infected target cells, but a second type of toxicity was demonstrable against uninfected target cells after treatment of the generating cell population with anti-Lyt-2 antibody plus complement at certain time points after infection. This additional nonspecific cytotoxic activity was presumably due to a second factor (factor X) acting in concert with IFN-gamma. Lyt-2+ cells, however, also were shown to play a role in IFN-gamma production and cytotoxicity directed against infected targets at later time points after infection. Neutralization of IFN-gamma in the samples containing cytotoxic activity abrogated the cytotoxicity against both infected and uninfected targets, but cloned murine IFN-gamma exhibited toxicity in a dose-dependent manner only against infected target cells. The data provides evidence that cytotoxicity against infected targets is due to antigen-specific induction of IFN-gamma, but other cytokine activity, most demonstrable after removal of Lyt-2.2+ cells and cytotoxic to uninfected targets, also is present.

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.

Antibody-dependent cell-mediated cytotoxicity against cells infected with the human immunodeficiency virus.

Human immunodeficiency virus (HIV) elicits the production of virus-specific antibodies in infected individuals. We investigated the ability of serum from HIV-infected individuals to mediate antibody-dependent cellular cytotoxicity in an in vitro 51Cr release assay system. Fresh peripheral blood mononuclear cells from healthy donors seronegative for HIV were used as cellular effectors against HIV-infected and uninfected H9 target cells in the presence of serum from HIV-infected or uninfected donors. Serum from HIV-infected, but not uninfected, donors significantly augmented cytolysis of virus-infected targets (P less than .005). There was no augmented killing of uninfected H9 cells with sera from either group. Studies using serum from mice that had been immunized with synthetic peptides from the HIV envelope region suggested that this response is directed, at least in part, at several determinants of the transmembrane portion of the HIV envelope glycoprotein.

Detection of antibodies which mediate human immunodeficiency virus-specific cellular cytotoxicity (ADCC) in vitro

A method to detect antibodies which mediate antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected target cells was developed. Normal lymphocytes were used as effector cells and different HIV-infected cell lines as target cells. The level of ADCC varied depending on both the effector cell function and the target cell susceptibility. For evaluation of HIV-specific ADCC, the effector function was simultaneously tested against a standard antigen ( β 2 microglobulin), expressed both on infected and uninfected target cells. The HIV-infected monocytoid cell line U937, clone 2, was found to be the most useful target cell in the present system. The detection of antibodies which induced HIV-specific ADCC killing will permit clinical and experimental analysis of the possible importance of such antibodies in protection against HIV-associated disease symptoms.

Augmentation of natural immune defence mechanisms and therapeutic potential of a mismatched double-stranded polynucleotide in cutaneous herpes simplex virus type 2 infection.

We studied the effect of an analogue of polyinosinic acid:polycytidylic acid, the mismatched poly(rI).poly(rC12U), on herpes simplex virus type 2 (HSV-2)-induced cutaneous disease in the guinea-pig. Recurrence patterns and HSV-2-induced immune responses were also defined. Intranasal administration (1.5 micrograms/g body weight, five doses at 48 h intervals) of poly(rI).poly(rC12U) during initial HSV-2 infection caused a significant (P less than 0.05) reduction in virus titres in the skin and decreased (P less than 0.01) the duration and severity of the primary cutaneous lesions. The incidence and frequency of subsequent recurrent episodes were also significantly (P less than 0.01) reduced. Titres of serum neutralizing antibody were identical in treated and untreated animals. Interferon (IFN) activity was detectable in the sera from poly(rI).poly(rC12U)-treated animals. Peripheral blood mononuclear (PBL) and spleen cells from treated animals had enhanced cytotoxic activity for HSV-2-infected and uninfected target cells. The cytotoxic activity of the PBL was enhanced by treatment in vitro with poly(rI).poly(rC12U) or IFN.

Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and alloreactive cytotoxic T cells.

Cross-reactive recognition of alloantigen by "self + X"-reactive cytotoxic T lymphocytes (CTL) has been documented in a variety of systems. It has been shown previously that the H-2Kb-restricted CTL response of C57BL/6 (B6) mice to vesicular stomatitis virus (VSV) infection is partially cross-reactive on uninfected target cells expressing the H-2Kbm8 mutation. In this report, we describe the isolation and detailed characterization of such dual-reactive CTL. By employing EL4 tumor lines transfected with genes encoding various VSV proteins, we demonstrated that the majority of dual-reactive CTL recognize the internal N protein of VSV and are also reactive against uninfected bm8 targets. Although the response of normal B6 mice to bm8 stimulators shows no measurable cross-reactivity on VSV-infected targets, the response of VSV-primed B6 mice to bm8 stimulation is almost entirely cross-reactive, lysing VSV-B6 targets and uninfected bm8 targets roughly equally. Furthermore, about 70% of CTL clones isolated from such mice by bm8 stimulation are dual-reactive with respect to effector function. Analysis at the population and clonal levels with cold target competition and antibody blocking suggests that the bulk of dual-reactive CTL have a higher avidity for VSV-B6 targets than for bm8 targets. The extreme case of this is illustrated by a fraction of CTL clones, isolated and maintained on bm8 stimulators, which lyse VSV-B6 targets but do not lyse bm8 targets. One such CTL clone is shown to be specific for the bm8 antigen in proliferation assays. These results demonstrate that: the specificity of an alloreactive CTL response may be dramatically altered by previous antigenic encounters; and dual-reactive CTL display a significant difference in affinity of the CTL receptor-determinant interaction, depending on the target which is recognized.

Activation of human natural killer cells by herpes simplex virus type 1-infected cells.

Human natural killer (NK) cells were shown to be much more cytolytic for WISH cells infected with herpes simplex virus type 1 (HSV-1) than for uninfected cells during 18-hour 51Cr release assays. Cold-target competition experiments involving high cold target to radiolabeled target cell ratios demonstrated that infected cells specifically competed for lytic activity against infected cell targets, and, therefore, the infected cells were not inherently more sensitive to lysis than uninfected cells. In contrast to these findings, depletion experiments involving low ratios of cold HSV-1-infected targets to labeled infected target cells resulted in increased lytic activity against uninfected cell targets. This finding suggested that NK effectors with specificity for WISH cell surface determinants had become highly activated during the depletion incubation. The addition of interferon alpha and particularly interleukin 2 to NK cytotoxicity assays enhanced NK cytolytic activity against both infected and uninfected target cells in a dose-dependent manner. Interleukin 1 did not give this effect. However, the enhanced lytic activity of NK cells following exposure to low doses of infected cell cold targets cannot be explained solely on the basis of release of the three lymphokines tested, since interleukin 1 was not effective, interleukin 2 was not detected in culture supernatants derived from the competition experiments, and NK cells preferentially lyse HSV-1-infected target cells independent of the enhancing effects of interferon. Together, the results indicated that NK cells recognize and bind to specific target cell surface structures which may, in turn, enhance their lytic activity.

Generation of IL-2 Dependent Bovine Cytotoxic T Lymphocyte Clones Reactive Against BHV-1 Infected Target Cells: Loss of Genetic Restriction and Virus Specificity

To gain insight into the mechanisms of immunity to bovine herpesvirus type-1 (BHV-1) in particular the importance of the T-cell response, we attempted to clone bovine cytotoxic T lymphocytes that were specific for BHV-1 infected autologous target cells. A number of bovine T cell clones were generated by limiting dilution in the presence of bovine recombinant IL-2 and BHV-1 infected target cells as feeder layers. These clones were maintained in culture on crude IL-2 containing supernatants. In functional studies, 4 of the 16 T cell clones were shown to have high levels of cytotoxic activity specific for autolgous BHV-1 infected target cells with significantly lower cytotoxic activity against uninfected target cells and heterologous BHV-1 infected target cells. Continuous culturing of these 4 T cell clones, using either the crude IL-2 or high concentrations of recombinant bovine IL-2, resulted in the loss of both MHC restricted and BHV-1 specific cytotoxic activity. These clones now exhibit promiscuous type cytotoxic activity with the ability to lyse a variety of target cells. Using flow cytometric analysis, the phenotype of the T cell clones were shown to have bovine T lymphocyte characteristics including expression of the BoT8 marker. This is the first report of cloned bovine cytotoxic T lymphocytes reactive against BHV-1 and the generation from these clones of promiscuous cytotoxic activity against both virus-infected and non-infected bovine target cells.

The immune response of the mouse to lymphocytic choriomeningitis virus. V. High numbers of cytolytic T lymphocytes are generated in the spleen during acute infection.

In the spleens of C57BL/6J (B6) and CBA/J (CBA) mice undergoing acute infection with lymphocytic choriomeningitis (LCM) virus, lymphocytes with the ability to develop in vitro into LCM virus-specific cytolytic clones were enumerated by use of the limiting dilution method. At intervals after virus inoculation, defined numbers of cells were cultivated with virus-infected syngeneic stimulator cells and T cell growth factor in multiple wells of microculture plates. After 7 days, individual cell cultures were tested for their ability to cause release of 51Cr from infected and uninfected syngeneic target cells. In cultures seeded with spleen cells from uninfected mice or from mice infected 3 days previously, no cytolytic activity was observed. On day 5, cells developing into LCM virus-specific cytolytic effector cells were detected. They rose in numbers, and on days 8 to 9 after infection, values of approximately 1/10 and 1/200 in B6 and CBA mice, respectively, were calculated. A low proportion of microcultures proved cytolytic also for noninfected syngeneic target cells, but the counts thus released were consistently much lower than the counts set free from infected targets, and no regular dose-response relationships existed between seeded cells and positive cultures. Determination of cell surface antigens of responder cells by negative and positive selection procedures disclosed that they were predominantly T lymphocytes and expressed Lyt-2 but not L3T4 surface markers. Lysis by the great majority of LCM virus-specific clones was restricted by products of the major histocompatibility gene complex (MHC), but a few lysed, in addition, allogeneic infected or uninfected targets; however, a consistent pattern of alloreactivity was not observed. Furthermore, cells of a proportion of the cultures also lysed uninfected YAC cells. Probably this natural killer-like activity was acquired by T lymphocytes during prolonged cultivation. We conclude that most spleen cells that during acute infection with LCM virus attained the ability to develop in vitro into LCM virus-specific cytolytic clones were derived from MHC-restricted Lyt-2+, L3T4- antigen-specific cytolytic T lymphocytes and their activated precursors.

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide.

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.

Cell-mediated cytotoxicity in hepatitis A virus infection.

We studied cell-mediated cytotoxicity to hepatitis A virus-infected cells in seven patients with acute type A hepatitis and two controls. Skin fibroblast cultures obtained from the skin biopsies of seven patients after acute hepatitis A virus infection and from two persons without history of current or past hepatitis A virus infection were inoculated with hepatitis A virus. Infection of fibroblast cultures always resulted in an inapparent, persistent infection with production and release of infectious hepatitis A virus. Peripheral blood lymphocytes were collected from the same patients at different times after onset of icterus and were stored in liquid nitrogen. Cytolytic activity of peripheral blood lymphocytes was determined by a microcytotoxicity assay using autologous 51Cr-labeled hepatitis A virus-infected and uninfected target cells. Cytotoxic peripheral blood lymphocytes capable of lysing autologous hepatitis A virus-infected skin fibroblasts were detected in all patients with hepatitis A but were not demonstrable in the controls without antibodies against hepatitis A virus. The clinical course of the hepatitis A virus infection was normal in five patients; and in these patients, cytolytic activity of peripheral blood lymphocytes against hepatitis A virus-infected autologous targets peaked 2 to 3 weeks after onset of icterus. A clinically protracted form of the disease with persistent elevation of aminotransferases for at least 5 months after onset was present in two patients. In these cases, the highest cytolytic activity was demonstrated in peripheral blood lymphocytes collected 8 to 12 weeks after onset of icterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine thymocytes mediate a natural killer-like activity against herpes virus-infected target cells but not YAC-1 target cells.

In this report, we demonstrated a natural killer (NK)-like activity against HSV-1 infected cells mediated by CD-1 mouse thymocytes. This cytolytic activity is specific for HSV-1-infected MCN cells, since both uninfected MCN and YAC-1 target cells are not susceptible to thymocyte lysis. Antibody plus complement depletion experiments indicate that a portion of the activity is associated with the Lyt 2-/L3T4- thymocyte subpopulation. This NK-like activity cannot be enhanced by addition of interleukin 2 in vitro.

Cross-reactive recognition of mouse cells expressing the bm3 and bm11 mutations within H-2Kb by H-2Kb-restricted herpes simplex virus-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes, generated in C57BL/6 mice in response to herpes simplex virus type 1 (HSV) and known to be restricted in their recognition of HSV-encoded antigen(s) in association with the class I H-2Kb gene product, were consistently found to contain a subpopulation that recognized and lysed uninfected, SV40-transformed cells that expressed the H-2Kbm3 and H-2Kbm11 mutant class I gene products on their cell surface. The mutant cell lines, designated Lgbm3SV and Kbm11SV, share a common amino acid substitution at position 77, with the bm3 mutation having an additional amino acid substitution at position 89. Cross-reactive lysis was observed only after in vivo priming with HSV, suggesting an important role for an antigen-dependent driving step in the expansion of these cross-reactive CTL. The phenotype of the cross-reactive effector population was further confirmed as a T lymphocyte by negative-selection techniques. Limiting dilution analysis of the frequency of cross-reactive CTL precursors suggested that cross-reactivity was mediated by a subpopulation of HSV-specific CTL, and this was confirmed by clonal analysis of the reactivity patterns of short-term, HSV-specific CTL clones. However, analysis of the specificity of the cross-reactive CTL population by cold-target inhibition of bulk culture-derived CTL, or by Spearman ranking analysis of limiting dilution-derived CTL, indicated that the specificity of the cross-reactive population for HSV-infected H-2b target cells and for uninfected bm3 or bm11 target cells was quite distinct. These findings suggested that the cross-reactive CTL population played little, if any, role in the HSV-specific CTL response as measured in vitro. The findings also suggested that the HSV-specific CTL clones able to mediate cross-reactive recognition of the bm3 and bm11 targets had a higher intrinsic avidity for the foreign target than for the inducing antigen.