Low-dose remifentanil infusion does not impair natural killer cell function in healthy volunteers.

BACKGROUND: Mu opioid agonists suppress natural killer (NK) cell activity in animal models. Studies in human volunteers, however, have yielded conflicting results, with morphine suppressing and fentanyl increasing NK cell activity. This study evaluated the effect of a constant 8-h infusion of remifentanil on NK cell number and function in human volunteers. METHODS: After IRB approval and informed consent was obtained, 10 healthy volunteers underwent an 11 pm to 7 am infusion of saline, and at least 1 week later an infusion of 0.02-0.04 microg x kg(-1) min(-1) remifentanil. Blood was collected at 7 am for measurement of NK cell cytotoxicity using a (51)Cr release assay and measurement of NK cell number using fluorescent flow cytometry. RESULTS: Median and range of the total NK cell cytotoxicity (KU ml(-1)) was 745.0 (498.3-1483.6) on the control morning and 818.6 (238.5-1454.5) on the morning following the remifentanil infusion. Neither the number of NK cells ml(-1) (2.5 x 10(5) (1.4 x 10(5)-4.2 x 10(5)) vs 2.7 x 10(5) (1.1 x 10(5)-4.4 x 10(5))) nor the cytotoxicity per 1000 NK cells (KU 1000 NK cells(-1)) (3.0 (1.8-5.2) vs 2.9 (0.9-6.7)) changed between the control and remifentanil conditions. CONCLUSIONS: An 8-h infusion of remifentanil did not affect NK cell activity in normal volunteers. This result differs from previous findings of morphine-induced NK cell activity suppression and fentanyl-induced NK cell activity enhancement in normal volunteers.

Mechanisms underlying chemical sympathectomy-induced suppression of herpes simplex virus-specific cytotoxic T lymphocyte activation and function.

Lymphoid tissues are extensively innervated by noradrenergic fibers of the sympathetic nervous system. 6-hydroxydopamine (6-OHDA)-induced chemical sympathectomy is commonly used to assess the impact of this innervation on immune function. Using the glucocorticoid receptor antagonist RU486, the mineralocorticoid receptor antagonist spironolactone, and the beta-adrenergic receptor antagonist nadolol, the roles of corticosterone and norepinephrine in sympathectomy-mediated modulation of both the primary and memory cellular immune responses to herpes simplex virus type 1 (HSV-1) infection was investigated. We demonstrated that both of these immunomodulators play a role in mediating sympathectomy-induced suppression of the generation of HSV-specific primary cytotoxic T lymphocytes (CTL) and the activation of HSV-specific memory CTL (CTLm). Furthermore, we demonstrated a role for both Type I and Type II corticosteroid receptors in the regulation of HSV-specific immunity. Overall, these findings not only further support a role for neuroendocrine-mediated modulation of immune function, but also a need to exercise caution in attributing the effects of chemical sympathectomy to solely the absence of sympathetic innervation of lymphoid tissues.

In vivo effects of chronic treatment with [MET5]-enkephalin on hematological values and natural killer cell activity in athymic mice.

The role of endogenous opioids in immunological mechanisms was examined by subjecting athymic (nu/nu) mice to chronic injections of the opioid agonist [Met5]-enkephalin (MET) or continuous opioid receptor blockade with naltrexone (NTX). After 8 days of treatment, neither excess peptide nor deprivation of opioids from receptors had any effect on body weight, spleen index (spleen to body weight ratio), total and differential white blood cell counts, and natural killer (NK) cell activity in peripheral blood or splenic lymphocytes. At 28 days, chronic treatment with MET or NTX had no effect on any of these parameters with the exception of an elevation from controls in NK cell activity in peripheral blood in mice receiving NTX, and subnormal NK cell activity related to splenic lymphocytes in the MET group. These results suggest that chronic exposure to an opioid agonist, or persistent opioid receptor blockade, have little influence on a variety of immunological properties in athymic mice, suggesting that native opioids such as MET do not play a marked role in defense mechanisms in the athymic mouse.

Chemical sympathectomy alters cytotoxic T lymphocyte responses to herpes simplex virus infection.

Numerous studies have sought to delineate the impact of neuroendocrine function on overall immune responsiveness. Using various murine models, we and others have previously shown that both adrenal-dependent and adrenal-independent mechanisms regulate components of the primary and memory cellular immune responses to herpes simplex virus type 1 (HSV-1) infection. We have extended these studies by determining the impact of 6-hydroxydopamine (6-OHDA)-induced peripheral sympathetic denervation on these responses. C57BL/6 mice treated with 6-OHDA (200 mg/kg) were inhibited in their ability to generate primary, HSV-specific cytotoxic T lymphocytes (CTL) in response to HSV infection. Sympathectomy also suppressed the activation and function of HSV-specific memory CTL (CTLm). In addition, administration of 6-OHDA resulted in a transient but substantial increase in levels of circulating corticosterone and hypothalamic Fos expression. Together, these findings suggest that peripheral sympathetic denervation may modulate immune function via activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Immunogenicity of herpes simplex virus type 1 mutants containing deletions in one or more alpha-genes: ICP4, ICP27, ICP22, and ICP0.

Replication defective mutants of HSV have been proposed both as vaccine candidates and as vehicles for gene therapy because of their inability to produce infectious progeny. The immunogenicity of these HSV replication mutants, at both qualitative and quantitative levels, will directly determine their effectiveness for either of these applications. We have previously reported (Brehm et al., J. Virol., 71, 3534, 1997) that a replication defective mutant of HSV-1, which expresses a substantial level of viral genes without producing virus particles, is as efficient as wild-type HSV-1 in eliciting an HSV-specific cytotoxic T-lymphocyte (CTL) response. In this report, we have further evaluated the immunogenic potential of HSV-1-derived replication defective mutants by examining the generation of HSV-specific CTL following immunization with viruses that are severely restricted in viral gene expression due to mutations in one or more HSV alpha genes (ICP4, ICP27, ICP22, and ICP0). To measure the CTL responses induced by the HSV alpha-mutants, we have targeted two H-2Kb-restricted CTL epitopes: an epitope in a virion protein, gB (498-505), and an epitope in a nonvirion protein, ribonucleotide reductase (RR1 822-829). The HSV mutants used in this study are impaired in their ability to express gB while a majority of them still express RR1. Our findings demonstrate that a single immunization with these mutants is able to generate a strong CTL response not only to RR1 822-829, but also to gB498-505 despite their inability to express wild-type levels of gB. Furthermore, a single immunization with any individual mutant can also provide immune protection against HSV challenge. These results suggest that mutants which are restricted in gene expression may be used as effective immunogens in vivo.

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity.

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

Peripheral sympathetic denervation alters both the primary and memory cellular immune responses to herpes simplex virus infection.

Numerous studies have sought to delineate the impact of neuroendocrine function on overall immune responsiveness. Using various murine models, we and others have previously shown that both adrenal-dependent and adrenal-independent mechanisms associated with psychological stress modulate components of both the primary and memory cellular immune responses to herpes simplex virus type 1 (HSV-1) infection. We have extended these studies by determining the impact of 6-hydroxydopamine (6-OHDA)-mediated peripheral sympathetic denervation on both responses. C57BL/6 mice treated with 6-OHDA (200 mg/kg) exhibited reduced generation of both primary lymph node- and splenic-derived cytotoxic T lymphocytes (CTL) following a local (footpad) and systemic HSV infection, respectively. 6-OHDA also suppressed activation of HSV-specific memory CTL (CTLm). In both models, alterations in cytokine production and lymphocyte subset distribution were also observed. Administration of 6-OHDA also resulted in substantial but transient activation of the hypothalamic-pituitary-adrenal (HPA) axis as was indicated by a dramatic elevation of serum corticosterone and hypothalamic Fos expression. Moreover, the corticosterone levels were directly correlated with the extent of CTLm activation. Together, these findings suggest that peripheral sympathetic denervation alters immune function through activation of the HPA axis.

A genetic basis for neuroendocrine-immune interactions.

Psychoneuroimmunology is an exciting, complex field that elucidates interactions among the nervous, endocrine, and immune systems. The contribution of psychosocial factors and behavioral processes to these interactions has been the focus of numerous studies designed to investigate the intricate pathways that are involved in the "mind-body connection." In addition, the effects of this connection on the development and progression of various disease conditions are of considerable interest. Although efforts have been made to identify the cellular and molecular mechanisms underlying these relationships, the impact of genetic makeup on the communication among these systems has yet to be fully realized. The development of sophisticated genetic analytical methods and gene mapping techniques now provide the "tools" to determine the influence of genetics on behavior-neuroendocrine-immune interactions--an area of study that may represent the next frontier in psychoneuroimmunology.

Differential effects of stress-induced adrenal function on components of the herpes simplex virus-specific memory cytotoxic T-lymphocyte response.

We have previously demonstrated in a murine model system that psychological stress, applied in the form of physical restraint, suppresses both the activation of splenic-derived, herpes simplex virus (HSV)-specific memory cytotoxic T-lymphocytes (CTLm) to the lytic phenotype and the production of cytokines associated with CTL activation and function. In the studies described herein, we investigated the hypothesis that an adrenal-dependent event is responsible, either in whole or in part, for these observations. While adrenalectomy was shown to abrogate stress-induced suppression of both HSV-specific CTLm activation and the production of IL-6 and IFN-gamma, the reduction in splenic cellularity associated with restraint stress remained, In addition, a role for adrenal function in the regulation of splenic cellularity and IFN-gamma production in non-stressed mice was observed. Together, these results indicate that both adrenal-dependent and adrenal-independent events, operative under both baseline and stress conditions, mediate control of the memory component of the cellular immune response to HSV infection. Overall, these studies provide insight into the mechanisms by which psychological stress modulates immune responsiveness to viral pathogens.

A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.

The impact of psychological stress on the efficacy of anti-viral adoptive immunotherapy in an immunocompromised host.

Adoptive immunotherapy represents a potentially effective approach by which to control the extent of viral infections in an immunocompromised host. However, the impact of psychological stress and its associated neuroendocrine components on the efficacy of such a treatment strategy has yet to be determined. In the studies described herein, we have developed and utilized a model of primary, local herpes simplex virus (HSV) infection in radiation-induced, immunosuppressed C57BL/6 mice to investigate the role of stress in altering the protective capacity of adoptively transferred lymphocytes that contribute to the resolution of primary HSV infection. The sublethal dose of irradiation chosen for this model was shown to abrogate the local, adaptive immune response to HSV infection as measured by the degree of in vivo lymphoproliferation, development of HSV-specific cytotoxic T lymphocytes (CTL), and production of gamma interferon (IFN-gamma). Both short- and long-term acute stress, applied in the form of physical restraint, diminished the effectiveness of adoptively transferred lymphocytes as was indicated by an enhancement of viral replication in the footpad tissue and an increased rate of mortality. A reduction in the levels of IFN-gamma at the site of primary HSV infection represented at least one mechanism underlying this suppression of anti-viral immunity. Furthermore, the time-dependent restoration of immune function following irradiation was shown to be compromised in mice subjected to the restraint stress procedure. Together, these findings emphasize the potential role of psychological stress in suppressing both the capability of adoptive immunotherapeutic procedures to combat viral infection and the reestablishment of immune function in individuals who have undergone immunosuppressive therapy.

Immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1) induces a CD8+ cytotoxic T-lymphocyte response and confers a level of protection comparable to that of wild-type HSV-1.

Replication-deficient viruses provide an attractive alternative to conventional approaches used in the induction of antiviral immunity. We have quantitatively evaluated both the primary and memory cytotoxic T-lymphocyte (CTL) responses elicited by immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1). In addition, we have examined the potential role of these CTL in protection against HSV infection. Using bulk culture analysis and limiting-dilution analysis, we have shown that a replication-deficient virus, d301, generates a strong primary CTL response that is comparable to the response induced by the wild type-strain, KOS1.1. Furthermore, the CTL induced by d301 immunization recognized the immunodominant, H-2Kb-restricted, CTL recognition epitope gB498-505 to a level similar to that for CTL from KOS1.1-immunized mice. The memory CTL response evoked by d301 was strong and persistent, even though the frequencies of CTL were slightly lower than the frequencies of CTL induced by KOS1.1. Adoptive transfer studies indicated that both the CD8+ and the CD4+ T-cell responses generated by immunization with d301 and KOS1.1 were able to limit the extent of a cutaneous HSV infection to comparable levels. Overall, these results indicate that viral replication is not necessary to elicit a potent and durable HSV-specific immune response and suggest that replication-deficient viruses may be effective in eliciting protection against viral pathogens.

Transgenic mice expressing the thymus leukemia antigen fail to control cutaneous herpes simplex virus infection.

TL-transgenic mice expressing the thymus leukemia antigen demonstrate a lack of viral clearance following cutaneous HSV infection of the footpad. In this study, both uninfected and HSV-infected TL-transgenic mice demonstrate increased concentrations of IL-4 as well as decreased concentrations of IFN-gamma which may possibly underlie the impairment of viral clearance. Furthermore, lymphocytes from HSV-infected nontransgenic mice, adoptively transferred into HSV-infected TL-transgenic mice, promoted viral clearance and led to an increase in IFN-gamma production. Transgenic mice which were subcutaneously injected with IFN-gamma in the right footpad were also capable of clearing the viral challenge; however, clearance was restricted solely to the right footpad. These studies support the possibility of perturbations in the immune system of TL-transgenic mice and effectively demonstrate the utility of this model system in the study of HSV clearance, persistence, and potential spontaneous reactivation. Moreover, the TL-transgenic animals may provide a useful model system for additional studies requiring a host system skewed toward a Th2 phenotype.

Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a herpes simplex virus-specific CTL epitope expressed in a cellular protein.

CD8+ cytotoxic T-lymphocytes recognize small epitope peptides in association with MHC class I molecules expressed on the cell surface. In this study, we have determined whether an 8 amino acid viral CTL epitope, when expressed in a cellular protein, can be appropriately processed, presented, and recognized by the corresponding epitope-specific CTL and whether it is capable of inducing a CTL response in vivo. An H-2Kb-restricted CTL epitope from herpes simplex virus type 1 (HSV-1) glycoprotein B (gB epitope, residues 498-505) was cloned into the mouse dihydrofolate reductase protein (DHFR) at amino acid position 87. The recombinant DHFRs were expressed in vaccinia virus recombinants. To distinguish the recombinant DHFR proteins from the endogenous DHFR, an antibody epitope, recognized by monoclonal antibody PAb 901 and derived from simian virus 40 (SV40) T antigen was tagged to the C-termini of recombinant DHFR proteins. In vivo expression of recombinant DHFR was demonstrated by immunoprecipitation with the monoclonal antibody PAb 901. The H-2b cells infected with recombinant vaccinia virus expressing the recombinant DHFR were specifically lysed by gB epitope-specific CTL. Furthermore, the recombinant DHFR was functional in inducing a long lasting HSV gB epitope-specific CTL response upon immunization of C57BL/6 (B6) mice. These results indicate that a viral epitope expressed in a cellular protein can be efficiently processed, presented, and recognized by epitope-specific CTL and show that cellular proteins expressing CTL epitopes can be used for induction of CD8+ T lymphocyte responses.

Stress-induced effects on integral immune components involved in herpes simplex virus (HSV)-specific memory cytotoxic T lymphocyte activation.

We have previously shown that restraint stress suppresses the activation of a polyclonal population of herpes simplex virus (HSV)-specific memory cytotoxic T lymphocytes (CTLm) to the lytic phenotype. We have extended these findings by demonstrating that this suppression occurs in two distinct HSV-specific CTLm populations that are generated in C57BL/6 mice in response to HSV infection and that recognize distinct epitopes expressed early in the HSV infection cycle. Moreover, these CTLm exhibited different levels of susceptibility to stress-induced suppression of activation. To elucidate the mechanisms responsible for this suppression, we have examined the effect of restraint on immunological components that are necessary for CTLm activation. We demonstrated that the expression of the T cell receptor (TCR), IL-2 receptor (IL-2R), and other accessory molecules involved in T cell activation were similar on CD8(+) T cell populations from both control and restrained groups of mice. However, splenic lymphoid cells from restrained mice generated significantly lower levels of IL-2, IL-4, IL-6, and gamma interferon (IFN-gamma) than did those cells from control, nonrestrained mice. The reduced ability to activate HSV-specific CTLm from mice subjected to restraint could be overcome by increasing the cell density and, thus, the lymphokine concentrations in these cultures. Overall, these findings suggest that restraint stress does not affect the inherent ability of an HSV-specific CTLm to be activated to the lytic phenotype; rather, the availability of lymphokines necessary to drive the activation process may be the limiting factor as to whether or not CTLm activation occurs. This stress-induced suppression of lymphokine production may not only play a role in inhibiting HSV-specific CTL activation but may also contribute to a diminution in the responsiveness and function of other components of immunological memory that are dependent on the presence of lymphokines.

Thymus-leukemia antigen interacts with T cells and self-peptides.

The functional role of the class Ib thymus-leukemia (TL) Ag expressed within the thymic cortex and intestinal mucosa of the mouse remains unknown. In an approach to elucidate the potential functionality of TL, we developed transgenic mice that ectopically express the H-2T18d gene product on essentially all nucleated cells through the control of a heterologous H-2Kb gene promoter. Transgenic mice demonstrated an increase in the number of CD4+ lymphocytes within the thymus and lymph nodes; these cells displayed an altered T cell receptor repertoire possibly suggesting a role for the ectopically expressed TL protein. The TL protein additionally displayed the characteristics of a bona fide transplantation Ag, because skin grafts from transgenic animals onto MHC- and minor histocompatibility Ag-matched nontransgenic recipient mice resulted in a rapid and vigorous immunologic rejection of the allograft. In MLR studies, transgenic stimulator cells induced the proliferation of responders to a level intermediate between genetically identical and H-2-disparate responder-stimulator combinations. The TL protein was also capable of stimulating cytotoxic T lymphocytes, thereby resulting in specific lysis of TL+ target cells. Further data demonstrated that the TL protein assembles with peptides that are modified at the amino terminus, and that TL retains these molecules at the cell surface. Together, these data suggest that H-2T18d is capable of interacting with T cells via a bound peptide. These data further support the possibility that TL may subserve a specialized function within the immunologic system.

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.

Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2Kb-restricted determinant in simian virus 40 tumor antigen.

Immunization of C57BL/6 mice with syngeneic cells transformed by simian virus 40 large T antigen (SV40 T ag) induces the generation of T antigen-specific cytotoxic T lymphocytes (CTL) which are restricted by the major histocompatibility class I antigens H-2Db and H-2Kb. Previous studies have shown that the H-2Db-restricted CTL response is directed to at least three distinct epitopes (I, II/III, and V) in the SV40 T antigen which have been precisely mapped using deletion mutagenesis and overlapping synthetic peptides. Although in vivo the CTL response to SV40 T antigen is dominated by the H-2Kb class I antigen, the precise location of the H-2Kb-restricted epitope(s) was not known, and whether there was multiplicity of H-2Kb-restricted epitopes remained unclear. In this study, we have defined the minimal recognition epitope for the SV40-specific H-2Kb-restricted CTL clone Y-4 as T antigen residues 404-411 by using T antigen deletion and point mutants and synthetic peptides. DNA sequence analysis of the region encoding residues 404-411 from the T antigens expressed in three independently isolated CTL clone Y-4 escape variants identified inactivating mutations capable of abrogating CTL recognition. Estimation of CTL precursor (CTLp) frequencies by limiting dilution analysis revealed that CTLp specific for epitope IV represent a large percentage of the total CTL response elicited by the intact T antigen in H-2b mice. Immunization of B6 mice with cells expressing a T antigen derivative deleted of residues 404-411 revealed that site IV represents the only immunodominant H-2Kb-restricted epitope within T antigen.

Polymorphism within the herpes simplex virus (HSV) ribonucleotide reductase large subunit (ICP6) confers type specificity for recognition by HSV type 1-specific cytotoxic T lymphocytes.

A panel of herpes simplex virus type 1 (HSV-1)-specific, CD8+, major histocompatibility complex class I (H-2Kb)-restricted cytotoxic T-lymphocyte (CTL) clones was derived from HSV-1-immunized C57BL/6 (H-2b) mice in order to identify the HSV-1 CTL recognition epitope(s) which confers type specificity. HSV-1 x HSV-2 intertypic recombinants were used to narrow the region encoding potential CTL recognition epitopes to within 0.51 to 0.58 map units of the HSV-1 genome. Using an inhibitor of viral DNA synthesis and an ICP6 deletion mutant, the large subunit of ribonucleotide reductase (ICP6, RR1) was identified as a target protein for these type-specific CTL. Potential CTL recognition epitopes within RR1 were located on the basis of the peptide motif predicted to bind to the MHC class I H-2Kb molecule. A peptide corresponding to residues 822 to 829 of RR1 was shown to confer susceptibility on H-2Kb-expressing target cells to lysis by the type 1-specific CTL. On the basis of a comparison of the HSV-1 RR1 epitope (residues 822 to 829) with the homologous sequence of HSV-2 RR1 (residues 828 to 836) and by the use of amino acid substitutions within synthetic peptides, we identified HSV-1 residue 828 as being largely responsible for the type specificity exhibited by HSV-1-specific CTL. This HSV-1 RR1 epitope, when expressed in recombinant simian virus 40 large T antigen in primary C57BL/6 cells, was recognized by the HSV-1 RR1-specific CTL clones. These results indicate that an early HSV protein with enzymatic activity provides a target for HSV-specific CTL and that type specificity is dictated largely by a single amino acid.

In vivo priming and activation of memory cytotoxic T-lymphocytes (CTL) by a chimeric simian virus 40 T antigen expressing an eight amino acid residue herpes simplex virus gB CTL epitope.

The simian virus 40 (SV40) large T antigen was used as an immunogenic vector to express a herpes simplex virus type 1 (HSV-1) glycoprotein B (gB), H-2Kb-restricted cytotoxic T lymphocyte (CTL) recognition epitope corresponding to amino acid residues 498-505. Immunization of naive, C57BL/6 mice with a cell line, B6/350gB, expressing the chimeric T antigen was able to induce the generation of gB498-505-specific CTL in both the lymph nodes and the spleen. Splenic-derived, gB498-505-specific memory CTL (CTLm) were detected in these mice for at least 6 months following immunization at a slightly lower frequency than in those mice immunized with infectious HSV-1. B6/350gB was also able to activate in vitro gB498-505-specific memory CTL obtained from mice previously challenged with HSV. Overall, these findings support the use of a chimeric T antigen as a vector in determining the immunogenic potential of individual CTL epitopes and to assess their potential contribution in inducing a protective immune response in vivo.