Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo.

Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent 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.     

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

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

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

T-cell receptors from virus-specific cytotoxic T lymphocytes recognizing a single immunodominant nine-amino-acid viral epitope show marked diversity.

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.     

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

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

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

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.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Molecular analyses of a five-amino-acid cytotoxic T-lymphocyte (CTL) epitope: an immunodominant region which induces nonreciprocal CTL cross-reactivity

The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.     

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

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

Efficiency and effectiveness of cloned virus-specific cytotoxic T lymphocytes in vivo

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.     

The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes.

Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.     

The virus-specific and allospecific cytotoxic T-lymphocyte response to lymphocytic choriomeningitis virus is modified in a subpopulation of CD8(+) T cells coexpressing the inhibitory major histocompatibility complex class I receptor Ly49G2

The role of negatively signaling NK cell receptors of the Ly49 family on the specificity of the acute CD8(+) cytotoxic T-lymphocyte (CTL) response was investigated in lymphocytic choriomeningitis virus (LCMV)-infected C57BL/6 mice. Activated CD8(+) T cells coexpressing Ly49G2 expanded during LCMV infection, and T-cell receptor analyses by flow cytometry and CDR3 spectratyping revealed a unique polyclonal T-cell population in the Ly49G2(+) fraction. These cells lysed syngeneic targets infected with LCMV or coated with two of three LCMV immunodominant peptides examined. Transfection of these sensitive targets with H2D(d), a ligand for Ly49G2, inhibited lysis. This was reversed by antibody to Ly49G2, indicating effective negative signaling. LCMV characteristically induces an anti-H2(d) allospecific T-cell response that includes T-cell clones cross-reactive between allogeneic and LCMV-infected syngeneic targets. The CD8(+) Ly49G2(+) population mediated no allospecific killing, nor was any NK-like killing observed against YAC-1 cells. This study shows that CD8(+) Ly49G2(+) cells participate in the virus-induced CTL response but lyse a more restricted range of targets than the rest of the virus-induced CTL population.     

Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses

Intestinal mucosa is a portal for many infectious pathogens. Systemic immunization, in general, does not induce a cytotoxic T-lymphocyte (CTL) response at the mucosal surface. Because papillomavirus (PV) naturally infects mucosa and skin, we determined whether PV pseudovirus, i.e., PV-like particles in which unrelated DNA plasmids are packaged, could generate specific mucosal immunity. We found that the pseudovirus that encoded the lymphocytic choriomeningitis virus gp33 epitope induced a stronger CTL response than a DNA vaccine (plasmid) encoding the same epitope given systemically. The virus-like particles that were used to make the pseudoviruses provided an adjuvant effect for induction of CTLs by the DNA vaccine. The PV pseudovirus pseudoinfected mucosal and systemic lymphoid tissues when administered orally. Oral immunization with the pseudovirus encoding human PV type 16 mutant E7 induced mucosal and systemic CTL responses. In comparison, a DNA vaccine encoding E7, when given orally, did not induce a CTL response in intestinal mucosal lymphoid tissue. Further, oral immunization with the human PV pseudovirus encoding E7 protected mice against mucosal challenge with an E7-expressing bovine PV pseudovirus. Thus, PV pseudovirus can be used as a novel vaccine to induce mucosal and systemic CTL responses.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

Abrogation of anti-Pichinde virus cytotoxic T cell memory by cyclophosphamide and restoration by coinfection or interleukin 2

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomeningitis virus

The role of gamma interferon (IFN-gamma) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-gamma-deficient (IFN-gamma -/-) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-gamma -/- mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-gamma -/- mice. The prolonged effector phase in infected IFN-gamma -/- mice was associated with elevated numbers of CD8(+) T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8(+) T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-gamma -/- mice than in wild-type mice. These findings indicate that in the absence of IFN-gamma, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-gamma-mediated antiviral activity on the dynamics between virus replication and CTL activity.