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Antigen-specific CD8(+) T cells are required for the clearance of most viral infections and several cancers. However, it is not clear in vivo whether CD8(+) T cells can engage multiple targets simultaneously, engagement results in the formation of an immunologic synapse or molecules involved in CD8 function are redistributed to the synapse. We used here high-resolution microscopy to visualize interactions between virus-specific effectors and target cells in vivo. Using either in situ tetramer staining or green fluorescent protein-labeled virus-specific T cells, we have shown that a single CD8(+) T cell can engage two or three targets, a synapse occurs at the site of engagement and molecules involved in attachment (lymphocyte function-associated antigen 1), signaling (Lck) and lytic activity (perforin) are differentially positioned on the T cell. In addition, we have established an in vivo approach for assessing the intricacies of antigen-specific T cell activation, migration, engagement, memory and other defining elements of adaptive immunity.

Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

Control of primary measles virus (MV) infection in humans and continued maintenance of immune memory that protects against reinfection are mediated primarily through the anti-MV T cell response, as judged by observations of children with defects in antibody formation but competency in making T cells. Further, the failure of T cell responses in those infected with MV most often leads to overwhelming infection. To better define and manipulate the elements involved in human T cell responses to MV, we analyzed the generation of HLA-restricted cytotoxic T lymphocytes (CTL) in a small animal model. Transgenic mice expressing the human class I MHC antigen HLA-B27 in conjunction with human CD8 molecules produced vigorous HLA-restricted CTL responses to MV antigens, paralleling those in MV infection of humans. In addition, such humanized mice generated human CD8 coreceptor-dependent HLA-B27-restricted CTL with the same specificity for recognition of MV fusion (F) peptide RRYPDAVYL as reported for humans during natural MV infection. Neither murine beta(2)-microglobulin nor murine CD8 substituted adequately as coreceptors for the HLA-B27 heavy chain. By contrast, HLA-A2.1-restricted responses to measles could be generated in the absence of expression of human beta(2)-microglobulin or CD8(+) molecules in HLA-A2.1/K(b) transgenic mice. Thus a small animal model is now available for studying strategies for optimizing human CD8(+) T cell responses and for testing vaccines. This model offers the potential, when combined with the newly reported CD46 transgenic mouse model in which MV replicates in cells of the immune system, for uncoding the molecular mechanism of MV-induced immunosuppression., (Copyright 2000 Academic Press.)

The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glycoproteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92-101 (CSANNSHHYI) generated from the lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92-101 bears a glycosylation motif -NXS- that is naturally N-glycosylated in the mature viral glycoprotein, displays high affinity for H-2D(b) molecules, and elicits a CD8(+) cytotoxic T lymphocyte response. By analyzing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demonstrated that multiple forms of LCMV GP92-101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corresponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or alteration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded sequence, thus contributing to increasing the diversity of the T cell repertoire.

Binding of a specific peptide(s) from a viral protein to major histocompatibility complex (MHC) class I molecules is a critical step in the activation of CD8(+) cytotoxic T lymphocytes (CTLs). Once activated, CTLs can cause lethal disease in an infected host, for example, by killing virus-containing ependymal and ventricular cells in the central nervous system or viral protein-expressing beta cells in the pancreatic islets of Langerhans. Here we describe the usage of a designed (not natural) high-affinity peptide to compete with viral peptide(s)-MHC binding. This peptide blocks virus-induced CTL-mediated disease both in the CNS and in the pancreatic islets in vivo. Further, the blocking peptide aborts MHC-restricted killing of target cells by CTLs generated to three separate viruses: lymphocytic choriomeningitis virus, influenza virus, and simian virus 40., (Copyright 1999 Academic Press.)

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-gamma secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-gamma and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

CD8(+) cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8(+) T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8(+) CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8(+) CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8(+) CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8(+) T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8(+) cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4(+) T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8(+) memory CTLp, the probable nature of which is discussed.

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates three well-characterized H-2D(b)-restricted immunodominant epitopes delineated in the NP, GP1, and GP2 proteins. Here we report that the H-2D(b)-restricted GP1 epitope GP33-41/43 (KAVYNFATC/GI) located in the signal sequence of LCMV is also the immunodominant epitope recognized by CTL at the surface of the same infected cells in the context of H-2K(b) restriction. The GP1 epitope bound to H-2D(b) and H-2K(b) molecules with comparable affinities. The respective binding processes involved different sets of peptide anchoring residues and required dramatically different conformations of the peptide backbone as well as rearrangement of residue side chains. The 10-mer peptide GP34-43 (AVYNFATCGI) was the optimal H-2K(b)-binding sequence and the 8-mer peptide GP34-41 (AVYNFATC) the minimal sequence for optimal H-2K(b)-restricted CTL recognition. Comparison of lytic activities of primary splenic anti-LCMV CTL from C57BL/6 (D(b+)/K(b+)), B10A.[5R] (D(b-)/K(b+)), and B10A.[2R] (D(b+)/K(b-)) mice against LCMV-infected or peptide-coated target cells expressing either one or the two MHC alleles revealed that the H-2K(b)-restricted component of the anti-GP1 CTL response was mounted independently of but as efficiently as its H-2D(b) counterpart. Analysis of the immune response against a GP1 variant that escapes CTL recognition showed that the GP1 epitope: (i) was likely the only immunodominant LCMV epitope in the context of H-2K(b), and (ii) could efficiently evade H-2D(b) and H-2K(b)-restricted CTL mediated lysis.

The majority of the response of cytotoxic T-lymphocytes (CTL) to lymphocytic choriomeningitis virus (LCMV) in H-2d mice is directed toward one epitope located on the nucleoprotein (NP, aa 118-126), and usually no primary responses to other epitopes are detectable. Previous studies have shown that thymic expression of lymphocytic choriomeningitis virus-nucleoprotein (LCMV-NP) in H-2d transgenic mice (Thy-NP mice) leads to deletion of high-affinity anti-LCMV-NP CTL by negative selection. Selection is incomplete, so that low-affinity NP-specific CTL pass through the thymus and are detectable in the periphery. To analyze the importance of interferon-gamma (IFN-gamma) in the ability of low-affinity antiviral CTL to clear an acute viral infection, double transgenic mice were generated that are IFN-gamma deficient and express the NP of LCMV in the thymus (Thy-NP x IFN-gamma -/- mice). When infected with LCMV, these bigenic mice were unable to clear the infection despite generating low-affinity primary antiviral CTL, and they became persistently infected. In contrast, IFN-gamma competent Thy-NP mice cleared LCMV within 7-8 days and IFN-gamma deficient mice that did not express NP in their thymus generated high-affinity CTL that terminated an acute LCMV infection within 10-12 days post-viral challenge. Persistently infected IFN-gamma deficient mice selectively depleted LCMV-specific CTL and displayed reduced levels of antigen-presenting cells in the spleen, and 60% of these mice died at 2-3 months postinfection. Thus, IFN-gamma is required for clearing an acute viral infection in the absence of a high-affinity CTL response. In the absence of IFN-gamma persistent viral infection results despite the presence of low-affinity CTL.

The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.

CTL play a critical role in immune defense by recognizing and killing virally infected or tumor cells. In this report, the structure of cytoplasm within living CTL was monitored during CTL killing of target cells. Living CTL were simultaneously loaded with fluorescent 70,000- and 10,000-kDa dextran particles. The relative distribution of the large and small dextrans within CTL revealed subcellular heterogeneities in the submicroscopic structure of cytoplasm. Localized alterations in cytoplasmic structure correlated with specific events during CTL killing. Recognition of target cells was accompanied by a transient increase in large dextran accessibility over a broad front near the interface between CTL and target cells. This region narrowed to a smaller area from which pseudopodia were extended toward the target. During extension, there was a large difference between regions of high dextran accessibility within the pseudopod and more structured cytoplasm within the cell body. Areas undergoing structural changes showed localized foci of high dextran accessibility. During retraction, cytoplasmic structure became gradually more uniform throughout the protrusion and cell body. These observations revealed subcellular regions undergoing major changes during early stages of the killing response, and addressed the role of cytoplasmic solation in controlling CTL morphology. They support mechanisms of pseudopod extension driven by hydrostatic pressure and demonstrate a precise regulation of cortical structure to control the direction of pseudopod extension.

Binding of viral antigens to major histocompatibility complex (MHC) class I molecules is a critical step in the activation process of CD8(+) cytotoxic T lymphocytes. In this study, we investigated the impact of structural factors at non-anchor residues in peptide-MHC interaction using the model of lymphocytic choriomeningitis virus (LCMV) infection of its natural host, the mouse. Altering viral genes by making reassortants, recombinants, and using synthetic peptides, CD8(+) cytotoxic T lymphocytes were shown to recognize only three H-2Db-restricted epitopes, GP amino acids 33-41/43, GP 276-286, and NP 396-404. However, LCMV NP and GP proteins contain 31 other peptides bearing the H-2Db motif. These 34 LCMV peptides and 11 other known H2-Db-restricted peptides were synthesized and examined for MHC binding properties. Despite the presence of the H-2Db binding motif, the majority of LCMV peptides showed weak or no affinity for H-2Db. We observed that dominant negative structural elements located at non-anchor positions played a crucial role in peptide-MHC interaction. By comparative sequence analysis of strong versus non-binders and using molecular modeling, we delineated these negative elements and evaluated their impact on peptide-MHC interaction. Our findings were validated by showing that a single mutation of a favorable non-anchor residue in the sequence of known viral epitopes for a negative element resulted in dramatic reduction of antigen presentation properties, while conversely, substitution of one negative for a positive element in the sequence of a non-binder conferred to the peptide an ability to now bind to MHC molecules.

The ligand for CD40 (CD40L) is expressed on the surface of activated CD4+ T cells and its role in T-B cell collaborations and thymus-dependent humoral immunity is well established. Recently, by generating CD40L-knockout mice, we have confirmed its previously described role in humoral immunity and defined another important function of this molecule in the in vivo clonal expansion of antigen-specific CD4+ T cells. Here, we investigated the potential in vivo role of CD40L in antiviral immunity by examining the immune response mounted by CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV), Pichinde virus, or vesicular stomatitis virus. Humoral immune responses of CD40L-deficient mice to these viruses were severely compromised, although moderate titres of antiviral IgM and some IgG2a were produced by virus-infected CD40L-deficient mice by a CD4+ T cell-independent mechanism. By contrast, CD40L-deficient mice made strong primary CTL responses to all three viruses. Interestingly however, although memory CTL activity was detectable in CD40L-deficient mice two months after infection with LCMV, the memory CTL response was much less efficient than in wild-type mice. Together, the results show that CD40-CD40L interactions are required for strong antiviral humoral immune responses, and reveal a novel role for CD40L in the establishment and/or maintenance of CD8+ CTL memory.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.

We evaluated the role of the costimulatory molecule B7-1 in overcoming peripheral ignorance in transgenic mice, which expressed the glycoprotein (GP) or nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) as the self-antigen in pancreatic beta cells. The viral transgenes or B7-1 alone did not induce autoimmune diabetes (IDDM). However, in bigenic mice expressing B7-1 and LCMV-GP, anti-self (viral) cytotoxic T lymphocytes (CTL) were activated without viral infection and spontaneous IDDM occurred. In contrast, bigenic RIP-B7-1 x RIP-NP mice with thymic expression of the self (viral-NP) antigen deleted the majority of their autoreactive CTL and did not develop spontaneous IDDM. However, these mice developed fast-onset IDDM 14 days after LCMV infection, whereas single-transgenic RIP-NP littermates developed IDDM only within 4-5 months. Rapid IDDM was associated with increased numbers of anti-self CTL and a predominance of IFN gamma produced by islet-infiltrating lymphocytes, whereas single transgenic RIP-NP littermates with slow-onset IDDM displayed less anti-self CTL and more IL-4- and IL-10-producing T lymphocytes in pancreatic infiltrates.

Structural characterization of peptides restricted by major histocompatibility complex (MHC) class I molecules has identified residues critical for MHC class I binding and for T-cell receptor recognition. For example, optimal peptides fitting into the murine MHC class I Db groove are 9 to 11 amino acids long and require as MHC anchor residues an Asn (N) at position 5 and also either a hydrophobic residue, a Met (M) or a Cys (C), at the carboxy terminus. The three known Db-restricted peptides of lymphocytic choriomeningitis virus (LCMV) are glycoproteins GP1 (amino acids [aa] 33 KAVYNFATC), GP2 (aa 276 SGVENPGGYCL), and nucleoprotein NP (aa 396 FQPQNGQFI). In addition to these two GP and one NP peptides, computer search revealed 11 other GP peptide sequences and 20 additional NP sequences that contained the Db binding motif. By Db competitive binding analysis, only two of these 11 GP peptides and 1 of these 20 NP peptides bound to the MHC Db molecule with an affinity equivalent to the measured affinities for the three known GP1, GP2, and NP cytotoxic T-lymphocyte (CTL) epitopes. No CTL specific for these three peptides were generated when H-2b mice were inoculated with viral variants in which either the two known GP epitopes (GP1 and GP2; termed GPV) or the GPV and NP epitopes (termed GPV + NPV) were mutated. However, a novel CD8+ anti-LCMV CTL response ordinarily not seen in H-2b mice inoculated with wild-type virus was noted when such mice were inoculated with the GPV + NPV-mutated variant. This result indicates that (i) despite large numbers of peptides containing the appropriate anchor residues within a viral protein, only a restricted number induce CTL, thereby maintaining a limited CTL repertoire, (ii) despite the limited repertoire, the immune system retains the flexibility to generate an immune response(s) to a previously silent protein(s), suggesting a hierarchial control mechanism, and (iii) identification of a primary amino acid sequence is not sufficient, per se, to predict CTL epitopes, and peptide conformations are likely more complex than indicated by simple linear sequence comparisons.

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.

The proteins of lymphocytic choriomeningitis virus (LCMV) contain only three known peptide regions that are processed and then held in place by the MHC class I H-2b (Db) glycoprotein on the cell's surface for recognition by LCMV-specific Db-restricted cytotoxic T lymphocytes (CTL). These peptides are from the glycoprotein (GP), amino acids 33-41 KAVYNFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleoprotein (NP), 396-404. We have used CTL clones that recognized only GP1, GP2, and NP to select viral variants that upon infecting cells bearing H-2b molecules escaped recognition by virus-specific CTL directed against the viral GP (GP1 + GP2) mutant, termed GPV, or the viral GP and NP (GP1 + GP2 + NP) mutant, termed GPV+NPV. These CTL "escape" variants nevertheless elicited sufficient host-protective activity in vivo to abort acute infection and prevent the occurrence of persistent infection. This protection was CD8+ lymphocyte mediated and associated with the generation of a novel (for H-2b mice) CTL response to the viral L protein. Hence CTL epitopes form a hierarchy, in which responses to "weak" epitopes are suppressed in the presence of "stronger" epitopes. Mutation in the strong epitopes may be of limited biological significance since the host can mount a protective response directed against the second level (weak) epitopes.

Cytotoxic T lymphocytes (CTL) play a pivotal role in preventing persistent viral infections and aborting acute infections. H-2Db-restricted CTL optimally recognize a specific peptide of 9 to 11 amino acids (aa) derived from a viral protein and held in place (restricted) by a MHC class I glycoprotein on the surfaces of infected cells. Only three peptide sequences with the appropriate Db motif from lymphocytic choriomeningitis virus Armstrong strain (LCMV) are known to be presented to CTL by H-2Db molecules; they are from the glycoproteins (GP), residues 33-41 KAVYNFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleoprotein (NP), 396-404 FQPQNGQFI. Incubation of virally infected H-2b cells with CTL clones that recognize only GP1, GP2, or NP leads to the selection of viral variants which upon infecting cells bearing H-2b molecules, escape recognition by CTL of the appropriate specificity. Nucleic acid sequencing showed a single mutation in GP1 (aa 38 F-->L), GP2 (aa 282 G-->D), or NP (aa 403 F-->L) in the variant viruses. When wild-type (wt) LCMV peptides and the three variant peptides (GP1, GP2, NP) were synthesized and subjected to a competitive inhibition binding assay, no differences in binding affinity for H-2Db were found between the wt and variant peptides. Uninfected cells coated with the wt peptide were recognized and lysed by the appropriate CTL clone or by in vivo-primed bulk CTL, but similar targets coated with the GP1, GP2, or NP variant peptides were not. This result, coupled with computer graphic analysis of these variant peptides with the recently solved three-dimensional structure for the Db MHC class I molecule, placed the side chain of the mutated residues on the outer surface of the MHC-peptide complex and accessible to the T cell receptor. Ala substitution at GP residue 38 or 282 or at NP 403 also abrogated CTL recognition and lysis. Inoculation of any one of the mutated viral variants into mice produced an effective CTL response to the other two nonmutated GP or NP peptides, suggesting that production of biologically relevant CTL escape virus variants in vivo requires selection of mutations in more than one and likely all the CTL epitopes, a low probability event.

Simian immunodeficiency virus (SIV)-specific CD8+ CTL were isolated from blood, cerebrospinal fluid, and brains of rhesus macaques infected i.v. with SIV. CTL were found as early as 1 wk postinfection and their appearance correlated with a decrease of viral Ag (p27) found in the blood. CTL isolated from cerebrospinal fluid and/or brain often recognized different viral proteins than CTL isolated from blood, suggesting either a unique migratory pattern to the central nervous system or a difference in activation/retention of lymphocytes in these compartments.

Infection with lymphocytic choriomeningitis virus induces the generation of CD8+ cytotoxic T lymphocytes (CTL). In the H-2b mouse, this cellular immune response is directed against three viral structural epitopes (GP1, GP2, and NP) presented by the major histocompatibility complex (MHC) class I H-2Db molecules. This study was undertaken to delineate which sequence of each of these three epitopes is optimal for MHC binding and CTL recognition. The first step was to synthesize the relevant peptides truncated at the N or C terminus and flanking the crucial H-2Db-anchoring Asn residue in position 5. These peptides were then tested (i) for their binding properties in two H-2Db-specific assays with viable cells (upregulation of H-2Db expression on the surface of RMA-S cells and competition against the Db-restricted peptide 125I-gp276-286 on T2-Db cells) and (ii) for their abilities to sensitize H-2b target cells for CTL lysis in vitro. For optimal antigenic presentation, all three epitopes required the MHC-anchoring Asn residue at position 5 of their sequences. The results clearly and unambiguously delineated optimal lengths for two of the epitopes and two options for the third. NP appeared as a conventional 9-amino-acid (aa)-long peptide, np396-404 (FQPQNGQFI). GP2 was defined as a longer peptide (11 aa), gp276-286 (SGVENPGGYCL). Characterization of the GP1 epitope was more complex: the 9-aa-long peptide gp33-41 (KAVYNFATC) and the carboxyl-extended 11-aa-long peptide gp33-43 (KAVYN FATCGI) were both established as possible optimal sequences depending on the cell line used to test binding and lysis.

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2-positive LCMV-NP-expressing T lymphocytes in the periphery.

Human immunodeficiency virus type 1 (HIV-1) Env-, Gag-, Pol-, Nef-, and Tat-specific cytotoxic T-lymphocyte (CTL) activities were quantitated temporally in five patients with symptomatic primary HIV-1 infection. A dominant CD8(+)-mediated, major histocompatibility complex class I-restricted CTL response to the HIV-1 envelope glycoprotein, gp160, was noted in four of the five patients studied. The level of HIV-1-specific CTL activity in the five patients paralleled the efficiency of control of primary viremia. Patients who mounted strong gp160-specific CTL responses showed rapid reduction of acute plasma viremia and antigenemia, while in contrast, primary viremia and antigenemia were poorly controlled in patients in whom virus-specific CTL activity was low or undetectable. These results suggest that HIV-1-specific CTL activity is a major component of the host immune response associated with the control of virus replication following primary HIV-1 infection and have important implications for the design of antiviral vaccines.

Astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. We have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. The major histocompatibility complex (MHC) class I molecule, Db, was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (GFAP) gene. Baseline cerebral MHC class I mRNA levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral MHC class I expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. FACS analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the GFAP-Db fusion gene at the protein level. Functional antigen-presenting capacity was conferred by the Db transgene, as virus-infected primary astrocytes obtained from transgenic BALB/c mice (KdIdDdLd) expressing the Db molecule were lysed by Db-restricted anti-viral CTL.

Following infection of the H-2d mouse by lymphocytic choriomeningitis virus, the newly generated cytotoxic T lymphocyte (CTL) response is focused to a single 9-amino-acid peptide sequence (epitope) of the virus. More than 96% of the primary, secondary, and clonal CTL respond to this lymphocytic choriomeningitis virus nucleoprotein epitope. This unique system affords the opportunity to evaluate the T-cell response to a single viral CTL epitope in a case in which the outcome of infection, either viral clearance or host death, is mediated by the CTLs. Specifically, the molecular structure of the T-cell receptors (TCRs) of CTLs responding to this epitope was analyzed. By using an anchored polymerase chain reaction, the TCR chains of three CTL clones cDNAs were amplified, sequenced, and found to have unique V alpha of V beta chains relative to each other as well as to lack restriction to any particular variable chain. These data indicate that the highly diverse antiviral CTL response is pleomorphic and probably provides an advantage to the host as it limits the emergence of viral variants that could more easily arise if the TCR response were homogeneous.

The major host response to many viral infections is the generation of virus-specific CTL. Many protein molecules on the surfaces of both CTL and target cells interact to mediate adhesion of the cells and generate signals that lead to T cell activation and proliferation of virus-specific CTL that then mediate lysis of infected cells. One such protein, CD2, has been shown to increase the binding affinity of CTL to infected cells, and, in addition, enhance CTL activation signals. To determine whether virus-specific CTL could be generated in the absence of CD2, mice lacking a functional CD2 gene were infected with lymphocytic choriomeningitis virus (LCMV), and the responses to the virus were monitored. CD2-deficient mice infected intracerebrally with LCMV died as a consequence of CTL-mediated choriomeningitis, similar to control littermates. Additionally, CD2-deficient mice inoculated i.p. with LCMV cleared the infection by 2 wk postinfection, as did control mice. Viral clearance in these mice was shown to be due to the generation of a vigorous virus-specific MHC-restricted CTL response. Finally, to determine whether CD2 is essential for the generation of memory CTL, we examined the ability of CD2-deficient mice to generate memory CTL to LCMV and found normal memory CTL responses. Our results indicate that CD2 is not required for the generation of an LCMV-specific CTL response in vivo, nor is CD2 required for the maintenance or activation of memory CTL.

Lymphocytes and/or monocytes/macrophages carry viral genetic information in most, if not all, persistent and latent viral infections, and serve as potential reservoirs for maintaining or reintroducing the infection. Similarly, neurons can be persistently infected by several DNA and RNA viruses whose continued presence can alter the physiologic function of these cells, leading to disorders in neurotransmitters and disease. Here, we document that adoptive transfer of virus-specific cytotoxic T lymphocytes clears virus and viral nucleic acid sequences, in vivo, from individually infected lymphocytes, macrophages, and neurons. By plaquing, infectious center, Northern blot, and in situ hybridization at the single cell level, virus was efficiently removed from these cells.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease marked by hyperglycemia and mononuclear cell infiltration of insulin-producing beta islet cells. Predisposition to IDDM in humans has been linked to the class II major histocompatibility complex (MHC), and islet cells often become aberrantly class II positive during the course of the disease. We have used two recently described transgenic lines to investigate the role of class II molecules and CD4+ T cells in the onset of autoimmune insulitis. Mice that are class II deficient secondary to a targeted disruption of the A beta b gene were bred to mice carrying a transgene for the lymphocytic choriomenigitis virus (LCMV) glycoprotein (GP) targeted to the endocrine pancreas. Our results indicate that class II-deficient animals with and without the GP transgene produce a normal cytotoxic T lymphocyte response to whole LCMV. After infection with LCMV, GP-transgenic class II-deficient animals develop hyperglycemia as rapidly as their class II-positive littermates. Histologic examination of tissue sections from GP-transgenic class II-deficient animals reveals lymphocytic infiltrates of the pancreatic islets that are distinguishable from those of their class II-positive littermates only by the absence of infiltrating CD4+ T cells. These results suggest that in this model of autoimmune diabetes, CD4+ T cells and MHC class II molecules are not required for the development of disease.

We have used cell or tissue-specific promoters to express lymphocytic choriomeningitis virus (LCMV) proteins in selected cells in independent lines of transgenic mice. Upon adoptive transfers into these mice, MHC-restricted LCMV-specific cytotoxic T lymphocytes homed specifically to either the choroid plexus (SV40 promoter) or beta cells of the islets of Langerhans (rat insulin promoter). The availability of promoters specific for neurons, oligodendrocytes and astrocytes makes this approach compelling for evaluating T cell trafficking into the CNS and for analyzing antigen presentation in vivo in the CNS.

Viral antigens are presented to cytotoxic T lymphocytes (CTLs) by H-2-restricted major histocompatibility complex (MHC) glycoproteins. Binding of the endogenously processed viral peptides (epitopes) to their specific MHC molecules is an early intracellular event in the recognition process and is necessary for subsequent killing of virus-infected cells by virus-specific CTLs. It is now well established that interaction between a viral antigenic peptide and MHC is dependent on the primary structure (length and amino acid sequence) of that antigen. Here we show, using the H-2Db-restricted epitope GP277-286 of lymphocytic choriomeningitis virus as a model, that the secondary structure (conformation) of the viral sequence also plays a crucial role in the binding of a viral antigen to MHC glycoprotein and in its subsequent presentation to virus-specific CTLs.

Cytotoxic T lymphocytes (CTLs) recognize viral antigens presented by infected cells in the context of their major histocompatibility complex glycoproteins. The irreversible killing of virus-infected cells by virus-specific CTLs can be the cause of serious disease, particularly in the central nervous, hepatic, and cardiovascular systems. Design of molecules controlling (blocking) interaction between CTLs and infected cells, and their further use to inhibit (or antagonize) T-lymphocyte activity, is an important pharmacologic goal. In this report, we describe the design of a new family of peptides which selectively inhibit activity of lymphocytic choriomeningitis virus-specific CD8+ T lymphocytes, which recognize endogenously processed viral epitopes presented by major histocompatibility complex class I molecules.

H-2k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93-100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.

Of nine established murine haplotypes, mice of three types (H-2d, H-2u, and H-2q) possess major histocompatibility complex class I glycoproteins able to present an identical viral peptide for recognition and lysis by virus-specific cytotoxic T lymphocytes. Incorporation of this viral epitope into a recombinant vaccinia vaccine and administration of a single dose protects mice with these three haplotypes from an ordinarily lethal challenge of virus. Hence, a common epitope can exist. The sharing of the ability to bind such epitopes among different MHC haplotypes underscores the feasibility of developing an effective cytotoxic T-lymphocyte vaccine for outbred populations like humans.

Viruses frequently persist in neurons, suggesting that these cells can evade immune surveillance. In a mouse model, 5 x 10(6) cytotoxic T lymphocytes (CTLs), specific for lymphocytic choriomeningitis virus (LCMV), did not lyse infected neurons or cause immunopathologic injury. In contrast, intracerebral injection of less than 10(3) CTL caused disease and death when viral antigens were expressed on leptomeningeal and choroid plexus cells of the nervous system. The neuronal cell line OBL21 expresses little or no major histocompatibility (MHC) class I surface glycoproteins and when infected with LCMV, resisted lysis by virus-specific CTLs. Expression of MHC heavy chain messenger RNA was limited, but beta 2-microglobulin messenger RNA and protein was made normally. OBL21 cells were made sensitive to CTL lysis by transfection with a fusion gene encoding another MHC class I molecule. Hence, neuronal cells probably evade immune surveillance by failing to express MHC class I molecules.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

For viruses to establish persistent infections in their hosts, they must possess some mechanism for evading clearance by the immune system. When inoculated into adult immunocompetent mice, wild-type lymphocytic choriomeningitis virus (LCMV ARM) induces a CD8(+)-mediated cytotoxic T lymphocyte (CTL) response that clears the infection within 7-14 d (CTL+ [P-]). By contrast, variant viruses isolated from lymphoid tissues of persistently infected mice fail to induce a CTL response and are thus able to establish a persistent infection in adult mice (CTL- [P+]). This report compares the interaction of CTL+ (P-) and CTL- (P+) viruses with cells of the immune system. Both types of virus initially bind to 2-4% of CD4+ and CD8+ T lymphocytes and replicate within cells of both subsets. The replication of CTL- (P+) and CTL+ (P-) viruses in lymphocytes in vivo is similar for the first 5 d after initiating infection. Thereafter, in mice infected with CTL- (P+) variants, lymphocytes retain viral genetic information, and infectious virus can be recovered throughout the animals' lives. In contrast, when adult mice are infected with wild-type CTL+ (P-) LCMV ARM, virus is not recovered from lymphocytes for greater than 7 d after infection. A CD8(+)-mediated anti-LCMV CTL response is induced in such mice. Clearance of infected lymphocytes is produced by these LCMV-specific CTLs, as shown by their ability to lyse lymphocytes expressing LCMV determinants in vitro and the fact that depletion of CD8+ lymphocytes before infection with CTL+ (P-) viruses results in levels of infected lymphocytes similar to those found in undepleted CTL- (P+)-infected mice. Hence, CTL-mediated lysis of T lymphocytes carrying infectious virus is a critical factor determining whether virus persists or the infection is terminated.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.

Cytotoxic T lymphocytes (CTL) play an important role in recovery from a number of viral infections. They are also implicated in virus-induced immunopathology as best demonstrated in lymphocytic choriomeningitis virus (LCMV) infection of adult immunocompetent mice. In the present study, the structure of the T-cell receptor (TCR) in LCMV-specific CTL in C57BL/6 (B6) mice was investigated. Spleen T cells obtained from LCMV-infected mice were cultured in vitro with virus-infected stimulator cells and then stained with anti-TCR V beta antibodies. A skewing of V beta usage was noticeable in T cells enriched for their reactivity to LCMV, suggesting that particular V segments are important for the recognition of LCMV T-cell epitopes in B6 mice. To gain more detailed information on the structure of the TCR specific for LCMV epitopes, we studied CTL clones. It has been shown that approximately 90% of LCMV-reactive CTL clones generated in H-2b mice are specific for a short peptide fragment of the LCMV glycoprotein, residues 278 to 286, recognized in the context of the class I major histocompatibility complex molecule, Db. Four CTL clones possessing the specificity were randomly selected from a collection of clones, and their TCR genes were isolated by cDNA cloning or by the anchored polymerase chain reaction. All four clones were found to use V alpha gene segments belonging to the V alpha 4 subfamily. By RNA blot analysis, two more clones with the same specificity were also shown to express the V alpha 4 mRNA. In contrast, three different V beta gene segments were used among the four clones examined. J beta 2.1 was used by three of the clones. Although amino acid sequences in the V(D)J junctional regions were dissimilar, aspartic acid was found in the V alpha J alpha and/or V beta D beta J beta junctions of all four of these clones, suggesting that this residue is involved in binding the LCMV fragment. Restricted usage of V alpha and possibly J beta segments in the CTL response to a major T-cell epitope of LCMV raises the possibility that immunopathology in LCMV infection can be treated with antibodies directed against such TCR segments. Thus, similar analysis of the TCR in other virus infections is warranted and may lead to therapeutic strategies for immunopathology due to virus infections.

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) is determined largely by the cytotoxic T lymphocyte (CTL) response of the host. In H-2b mice, the anti-glycoprotein (GP) response is directed to at least two epitopes, one located at GP aa 272-286 and a second in GP-1. Here we show that the second epitope can be minimally identified by amino acid residues GP 34-40 (AVYNFAT). The epitope is restricted by the Db class I glycoprotein. Characterization of these CTL epitopes allowed us to address the role(s) played by each epitope when expressed singly in the control of a lethal challenge with LCMV. Here we show that a single immunization with a recombinant vaccinia virus (VV) vaccine expressing LCMV GP aa 1-59 confers protection to H-2b mice from lethal LCMV infection. In contrast, a VV expressing LCMV GP aa 272-293, although recognized by CTL, does not protect. We show that the success or failure of protective immunization is determined by the ability of the immunizing sequences to prime for CTL in vivo. Although the GP 278-286 epitope when contained as a "minigene" fails to induce CTL, when incorporated in the normal GP "backbone" it successfully elicits CTL. These observations suggest that the "minimal" recognition sequence alone may not be sufficient to induce a protective CTL response in vivo. Thus a single CTL epitope can protect against a lethal virus infection, but to achieve an effective vaccine, the immunizing sequences must be carefully selected.

Lymphocytic choriomeningitis virus (LCMV) infection prevents the usual insulin-dependent diabetes mellitus of aged BB rats (Dyrberg, Schwimmbeck & Oldstone, 1988; Schwimmbeck, Dyrberg & Oldstone, 1988). In this study earlier observations are extended by noting that LCMV infection substantially alters the immune responsesof BB diabetic-prone (dp) rats. The control, uninfected rats make vigorous primary and secondary antibody responses when challenged with keyhole limpet haemocyanin (KLH), human immunoglobulin (HuIg) or sheep red blood cells (SRBC). Such rats fail to mount a primary response to bovine serum albumin (BSA) but do produce a moderate secondary response. They mount good antibody responses to LCMV but fail to generate either primary or secondary LCMV-specific cytotoxic T-lymphocyte (CTL) responses or CTL responses to Pichinde virus. In contrast, BB dp rats acutely infected with LCMV generate no primary immune responses to SRBC, KLH or BSA and only meager responses when challenged with HuIg. They mount secondary responses to KLH, HuIg and BSA that approximate those of their uninfected litter mates, but have a comparatively lower response to SRBC. LCMV binds to and infects lymphocytes of the BB dp rat. Binding is enhanced over that observed with lymphocytes from BB diabetic-resistant (dr) rats, which are able to generate CTL immune responses to LCMV and Pichinde viruses. Hence, lymphocytes from BB dp rats are uniquely susceptible to binding and replication of LCMV. During the acute stage of LCMV infection, a general primary T-cell immunosuppression occurs with respect to a variety of viral and non-viral antigens. Over time, responsiveness to T-cell dependent antigens returns except for the ability to generate CTL responses to LCMV or the autoimmune response(s) required to cause insulin-dependent diabetes mellitus.

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 x 10(7) p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies. In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) in the natural murine host is determined in large part by the cytotoxic T lymphocyte response (CTL) mounted by the host. In order to define the specificities of CTL induced by LCMV infection, we have cloned and expressed the full-length nucleoprotein (NP) gene and 75% of the glycoprotein (GP) gene of LCMV in vaccinia virus vectors and have used these recombinant viruses to sensitize syngeneic target cells to lysis by anti-LCMV CTL. We have studied the anti-LCMV CTL responses induced on three different mouse H2 (major histocompatibility complex) backgrounds. First, we find that the relative recognition of the two LCMV proteins differs markedly on different H2 haplotypes; both proteins are seen on the H2bb background, while only NP is recognized on two other haplotypes (H2dd and H2qq). Second, we show that on the H2bb background the anti-GP CTL response comprises a major component of the overall CTL response, in marked contrast to several other viruses, e.g., influenza virus, vesicular stomatitis virus, and respiratory syncytial virus where anti-GP responses, if present, comprise only a minor portion of the whole. Third, LCMV GP can be a major target antigen for CTL induced by a serotypically distinct strain of LCMV, again in contrast to the above virus systems in which CTL cross-reactivity among different serotypes is dependent largely on the recognition of "internal" proteins.

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.

Cytotoxic T lymphocytes (CTL) generated in mice against a particular serotype of vesicular stomatitis virus (VSV) were previously shown to cross-reactively lyse syngeneic target cells infected with serologically distinct types of VSV. To analyze the antigenic basis of this T cell cross-reactivity, we generated CTL against VSV-Indiana (VSV-Ind) and established them by limiting dilution as cloned in vitro cell lines. The cells continuously proliferate in medium containing concanavalin A-induced T cell growth factors. All of the cells are Thy-1.2+ and Lyt-2.2+. Lysis by these cells is H-2Dd-restricted, no natural killer cell activity is detectable, and all the clones cross-reactively lyse target cells infected with either VSV-Ind or VSV-New Jersey (VSV-NJ). In addition, no specific blocking of primary, secondary, or cloned anti-VSV CTL was achieved with the use of several monoclonal antibodies specific for the glycoprotein of VSV and capable of neutralizing either VSV-Ind or VSV-NJ. These results suggest that VSV serotype-specific neutralizing antibodies may recognize immunodominant determinants of VSV glycoprotein that are distinct from those recognized by the majority of VSV-specific CTL.