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.     

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8(+) T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8(+) T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8(+) T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.     

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.     

MyD88 plays a critical T cell-intrinsic role in supporting CD8 T cell expansion during acute lymphocytic choriomeningitis virus infection

During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88(-/-) CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.     

Dynamics of CD8+ T cell responses during acute and chronic lymphocytic choriomeningitis virus infection

Infection of mice with lymphocytic choriomeningitis virus (LCMV) is frequently used to study the underlying principles of viral infections and immune responses. We fit a mathematical model to recently published data characterizing Ag-specific CD8+ T cell responses during acute (Armstrong) and chronic (clone 13) LCMV infection. This allows us to analyze the differences in the dynamics of CD8+ T cell responses against different types of LCMV infections. For the four CD8+ T cell responses studied, we find that, compared with the responses against acute infection, responses against chronic infection are generally characterized by an earlier peak and a faster contraction phase thereafter. Furthermore, the model allows us to give a new interpretation of the effect of thymectomy on the dynamics of CD8+ T cell responses during chronic LCMV infection: a smaller number of naive precursor cells is sufficient to account for the observed differences in the responses in thymectomized mice. Finally, we compare data characterizing LCMV-specific CD8+ T cell responses from different laboratories. Although the data were derived from the same experimental model, we find quantitative differences that can be solved by introducing a scaling factor. Also, we find kinetic differences that are at least partly due to the infrequent measurements of CD8+ T cells in the different laboratories.     

The immune response of the mouse to lymphocytic choriomeningitis virus. III. Differences of numbers of cytotoxic T lymphocytes in spleens of mice of different strains

By use of an accurate procedure for the measurement of relative numbers of murine cytotoxic T lymphocytes (CTL) marked differences of lymphocytic choriomeningitis virus-specific CTL in spleens were found among mouse strains that ranged from low over intermediate and high to very high, suggesting a gradient of responses rather than distinct classes. Differences were also found between mice sharing the major histocompatibility complex.     

The role of proinflammatory cytokines in wasting disease during lymphocytic choriomeningitis virus infection

Infection with pathogens often leads to loss of body weight, but the cause of weight loss during infection is poorly understood. We used the infection of mice with lymphocytic choriomeningitis virus (LCMV) as a model to study how pathogens induce weight loss. If LCMV is introduced into the CNS of CTL-deficient mice, the immune response against the virus leads to a severe weight loss called wasting disease. We planned to determine what components of this antiviral immune response mediate wasting disease. By adoptive transfer, we show that CD4 T cells activated by LCMV infection are sufficient to cause wasting disease. We examined the role of cytokines in LCMV-induced wasting disease using mice lacking specific cytokines or cytokine receptors. Results of adoptive transfer experiments suggest that TNF-alpha is not involved in LCMV-induced wasting disease and show that IFN-gamma contributes to the disease. Consistent with a role for IFN-gamma in wasting, we find that IFN-gamma is necessary for LCMV-specific CD4 T cell responses in the CNS, most likely because it is required to induce MHC class II expression. Our data also indicate that IL-1 is required for LCMV-induced wasting and that IL-6 contributes to the wasting disease. Additionally, our results identify alpha-melanocyte-stimulating hormone as a potential mediator of the disease. Overall, this work defines the critical role of virus-primed CD4 T cells and of proinflammatory cytokines in the pathogenesis of wasting disease induced by LCMV infection.     

Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.     

Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope.

For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CD8+ CTL are essential. In the infected BALB/c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I MHC-encoded molecule L; K- or D-restricted antiviral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant C-H-2dm2 should not be capable of eliminating the virus. The experimental evidence proves the contrary, which is explained by K- and D-restricted CTL that this mouse generates. Why such cells remain undetectable in BALB/c mice is currently unexplained, because there is no lack of precursors and the corresponding virus Ag is presented. Despite the absence of lytic activity in vitro, other than the one associated with L, transfusion of day 8-immune spleen cells from BALB/c into infected C-H-2dm2 (L-deficient) mice results in accelerated virus elimination from the organs of the latter, which was manifest as soon as 8 h after cell transfer. Furthermore, lytic activity did not attain measurable levels in the recipients' spleens. Obviously, this infection can be terminated by CD8+ T lymphocytes even when these cells' lytic activity is below detectability.     

H-2 haplotype specificity of molecular requirements for trinitrophenyl recognition by anti-hapten cytotoxic T lymphocytes

The requirement of direct covalent association of trinitrophenyl (Tnp) groups with cell surface components for functional interactions with anti-Tnp cytotoxic T lymphocytes (CTLs) was analyzed. The question was approached by comparing three different methods of modifying target cells with Tnp groups and analyzing the ability of three anti-Tnp effector populations with different H-2 haplotypes (H-2^k, H-2^d, and H-2^b) to lyse the syngeneic Tnp-modified cells. All effector cell populations were able to lyse in an H-2 restricted manner the appropriate target cell modified with 2,4,6-trinitrobenzenesulfonic acid. As previously shown, H-2^k anti-Tnp CTLs exhibited true H-2 restriction while H-2^d anti-Tnp and H-2^b anti-Tnp CTLs lysed the haptenated syngeneic target cell preferentially but not exclusively. Cells modified by either trintrophenylated bovine serum albumin (Tnp₃₅-BSA) or trinitrophenylated Sendai virus (Tnp-SV) were rendered susceptible to lysis depending upon the H-2 haplotypes of the target cells and the anti-Tnp effector cells. H-2^k anti-Tnp CTLs were able to lyse H-2^k target cells modified with either Tnp₃₅-BSA or Tnp-SV; however, H-2^d anti-Tnp or H-2^b anti-Tnp CTLS did not significantly lyse the H-2^d or H-2^b target cells modified by either Tnp₃₅-BSA or Tnp-SV. The results suggest that Tnp groups not covalently linked with cell surface specific components can be recognized by H-2^k anti-Tnp CTLs, but not by H-2^d or H-2^b anti-Tnp CTLs.     

Restricted V-segment usage in T-cell receptors from cytotoxic T lymphocytes specific for a major epitope of lymphocytic choriomeningitis virus.

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.     

Mechanism of recovery from acute virus infection. I. Role of T lymphocytes in the clearance of lymphocytic choriomeningitis virus from spleens of mice

Adult mice were infected by i.v. inoculation with 10(3) mouse infectious doses of lymphocytic choriomeningitis virus (LCM virus). Despite widespread replication of the agent, overt illness did not develop; histopathologic alterations were moderate. High virus concentrations were attained in the spleen, which was chosen for further study. Cytotoxic spleen T cell responses were found to vary among inbred mouse strains, and as a rule, these were correlated with other virus-specific cell-mediated immune phenomena. However, high- and low-responder mice eliminated the virus equally fast and already at times when spleen cytotoxic T lymphocytes were just beginning to appear (and before delayed-type hypersensitivity could be demonstrated). Adoptive transfer experiments showed that very few immune T lymphocytes were capable of reducing virus replication in the recipients' spleens and, furthermore, that protection was rapidly induced; when low numbers of cells were transferred, the effect was apparent 8 hr later, and with higher numbers diminished virus replication was evident after an interval as short as 6 hr. In fact, the data suggest that virus was actually inactivated. In spite of this marked efficiency, morphologic alterations in spleens of adoptively immunized mice were absent. Attempts to reveal expansion of immune cells in the recipients have failed, and the observation that adoptive transfer was as efficient in nude mice as in their furred counterparts makes it unlikely that the recipients' T lymphocytes participated to any extent. The low number of T lymphocytes causing reduction of virus, the short interval after which the effect became measurable, and the lack of histopathologic alterations has led to a working hypothesis in which it is assumed that immunologically activated T lymphocytes secrete lymphokines that directly interfere with virus replication in neighboring cells.     

Cloned lines of human cytotoxic T lymphocytes with specificity for influenza virus

The generation of human cytotoxic T cell clones with specificity for influenza virus and some of their characteristics are described. The clones were generated by limiting dilution of peripheral blood lymphocytes after two in vitro stimulations with autologous influenza A/USSR virus-infected cells and were grown in T cell growth factor. The majority of the virus-specific clones showed cross-reactivity for different influenza A virus subtypes but did not recognize influenza B virus-infected cells. The HLA specificity of two clones was further analyzed. One clone, LL33, was specific for HLA-Bw60, the other, clone WH5, for HLA-A1. Clone WH5 also seemed to recognize the serologically related HLA-A26 as restriction element for the recognition of the viral antigen. Whereas the virus-specific CTL clones had the OKT3+,4-,8+ phenotype, another clone, WH 49, exhibiting natural killer-like activity, was found to have the OKT3+,4+,8- phenotype.     

Opposing effects of CD70 costimulation during acute and chronic lymphocytic choriomeningitis virus infection of mice

T cell costimulation is important for T cell activation. The CD27/CD70 pathway contributes to effector and memory T cell development and is involved in T cell and B cell activation. CD27/CD70 is known for having opposing roles during different models of antigenic challenges. During primary T cell responses to influenza virus infection or during tumor challenges, CD27/CD70 costimulation has a positive role on T cell responses. However, during some chronic infections, constitutive triggering of this signaling pathway has a negative role on T cell responses. It is currently unclear what specific characteristic of an antigen determines the outcome of CD27/CD70 costimulation. We investigated the effect of a transient CD70 blockade during an acute or a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Blockade of this pathway during acute LCMV infection (Armstrong strain) resulted in delayed T cell responses and decreased CD127 (interleukin-7 receptor α [IL-7Rα] chain) conversion. Upregulation of CD127 is an important event in T cell differentiation that heralds the passage of an effector T cell to a long-lived memory T cell. In contrast to the reduced CD8 T cell responses after CD70 blockade during acute infection, CD70 blockade during chronic LCMV infection resulted in increased CD8 T cell responses. Our data show the dual roles of this costimulatory pathway in acute versus persistent antigen challenge. Our findings suggest that antigen persistence may determine the effect of CD27/CD70 signaling on CD8 T cell responses. Tailored triggering or blockade of this costimulatory pathway may be important in vaccination regimens against acute or chronic pathogens.     

Critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells

Under conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8(+) T cells. In this work we studied the down-regulation of the virus-specific CD8(+)-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-alpha/beta]), type II (IFN-gamma), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8(+)-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8(+) T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.