Protective Immunity Does Not Correlate with the Hierarchy of ?Virus-specific Cytotoxic T Cell Responses to Naturally Processed Peptides

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) stimulates major histocompatibility complex class I–restricted cytotoxic T cells (CTLs), which normally resolve the infection. Three peptide epitopes derived from LCMV have been shown to bind the mouse class I molecule H-2 D^b and to stimulate CTL responses in LCMV-infected mice. This report describes the identity and abundance of each CTL epitope after their elution from LCMV-infected cells. Based on this information, peptide abundance was found to correlate with the magnitude of each CTL response generated after infection with LCMV. Subsequent experiments, performed to determine the antiviral capacity of each CTL specificity, indicate that the quantitative hierarchy of CTL activity does not correlate with the ability to protect against LCMV infection. This report, therefore, indicates that immunodominant epitopes should be defined, not only by the strength of the CTL response that they stimulate, but also by the ability of the CTLs to protect against infection.     

T cell memory. Long-term persistence of virus-specific cytotoxic T cells

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.     

Induction and Exhaustion of Lymphocytic Choriomeningitis Virus–specific Cytotoxic T Lymphocytes Visualized Using Soluble Tetrameric Major Histocompatibility Complex Class I–Peptide Complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2D^b, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8⁺ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2D^b in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8⁺ T cells were GP33 tetramer⁺ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8⁺ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.The ability to clear infections with noncytopathic viruses is predominantly attributed to CD8⁺ CTLs. CTLs recognize infected cells via an interaction between TCRs and their corresponding ligands, class I MHC molecules (1). MHC class I molecules are expressed on the cell surface in association with self or pathogen-derived peptides that are generated intracellularly by proteolytic degradation of the parent proteins (2). After recognition of an infected cell, naive CTLs become activated, proliferate, and attain not only the ability to lyse infected cells, but also the ability to produce IFN-γ (3). Although IFN-γ may have a direct antiviral effect (4, 5), it has also been shown to improve the efficiency of antigen presentation by class I molecules (6–9) thereby promoting the induction of a CTL response and improving the efficiency with which CTL can recognize their infected targets.CTLs have been shown to be essential for the recovery of mice from the acute phase of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV; reference 10). So far, it has not been possible to follow the kinetics of appearance and disappearance of antigen-specific effector CTLs during the acute phase of both low- and high-dose LCMV infections in non-TCR-transgenic mice. A recent study by Altman et al. (11) described a method, using tetrameric soluble MHC class I–peptide complexes, for the identification of antigen-specific CD8⁺ cells in the PBMCs of HIV-infected humans. This study describes an adaptation of this method for the identification of antigen-specific CD8⁺ cells in B6 (H-2^b) mice infected with LCMV. In this case, soluble peptide–MHC complexes were generated using the mouse class I heavy chain D^b, chemically biotinylated human β2 microglobulin (β₂M) and the LCMV peptide epitope glycoprotein (GP)33–41 (GP33-KAVYNFATC). Fluorescence-labeled tetrameric complexes were subsequently produced by mixing the biotinylated complexes with phycoerythrin-labeled neutravidin. Peptide GP33–41 (GP33) was used for the purposes of this study since, after LCMV-WE infection of C57BL/6 mice, most CTL activity (∼50–60%) is directed towards this epitope. Two other epitopes, defined by residues 276– 286 of the viral glycoprotein (GP276) and residues 396– 404 of the viral nucleoprotein (NP396), represent 10–20 and 20–30% of the total CTL activity, respectively (12–17).The tetrameric class I–peptide complexes, which stained CTLs specifically, were used to follow the fate of GP33-specific CD8⁺ T cells in mice during the acute phase of LCMV infection. This study demonstrates the accummulation of stained virus-specific CTLs in the CSF and spleens of mice after intracranial infection with a substrain of LCMV-WE called LCMV-DOCILE, and in the spleens of mice infected intravenously with the same virus. In both cases, the accummulation of GP33-specific CTLs, after both low- and high-dose infection with LCMV-DOCILE, was monitored in relation to the capacity of the cells to produce IFN-γ; to mediate cytotoxic activity, and to mediate virus clearance.     

Stability and Diversity of ?T Cell Receptor Repertoire Usage during Lymphocytic Choriomeningitis Virus Infection of Mice

Numerous studies have examined T cell receptor (TCR) usage of selected virus-specific T cell clones, yet little information is available regarding the stability and diversity of TCR repertoire usage during viral infections. Here, we analyzed the Vβ8.1 TCR repertoire directly ex vivo by complementarity-determining region 3 (CDR3) length spectratyping throughout the acute lymphocytic choriomeningitis virus (LCMV) infection, into memory, and under conditions of T cell clonal exhaustion. The Vβ8 population represented 30–35% of the LCMV-induced CD8⁺ T cells and included T cells recognizing several LCMV-encoded peptides, allowing for a comprehensive study of a multiclonal T cell response against a complex antigen. Genetically identical mice generated remarkably different T cell responses, as reflected by different spectratypes and different TCR sequences in same sized spectratype bands; however, a conserved CDR3 motif was found within some same sized bands. This indicated that meaningful studies on the evolution of the T cell repertoire required longitudinal studies within individual mice. Such longitudinal studies with peripheral blood lymphocyte samples showed that (a) the virus-induced T cell repertoire changes little during the apoptosis period after clearance of the viral antigens; (b) the LCMV infection dramatically skews the host T cell repertoire in the memory state; and (c) continuous selection of the T cell repertoire occurs under conditions of persistent infections.     

Virus-triggered immune suppression in mice caused by virus-specific cytotoxic T cells

Normal mice infected with 10(5) infectious doses of lymphocytic choriomeningitis virus (LCMV, WE isolate) generated a reduced or no T cell-independent IgM and/or T cell-dependent IgG response to a subsequent vesicular stomatitis virus Indiana (VSV-IND) injection; this transient immune suppression lasted for weeks to months. Connatally infected LCMV-carrier mice or acutely infected T cell-deficient nude mice had normal anti-VSV IgM and IgG or IgM responses respectively. LCMV-infected nude mice transfused with helper cell-depleted LCMV-specific immune spleen cells were immunosuppressed. Normal mice infected with LCMV but treated with a rat anti-CD8 mAb (that had been shown previously to eliminate cytotoxic T cells in vivo) and then infected with VSV exhibited a normal anti-VSV IgM and IgG response. Since no IFN-alpha or -beta was detected on, or after, day 6 of LCMV infection, neither LCMV alone, nor IFN induced by it caused the observed immune suppression; the presented evidence suggests that LCMV-immune CD8+ T cells were responsible for it. It is conceivable that a similar pathogenesis where virus-specific cytotoxic T cells may destroy virus-infected cells essentially involved in an immune response (APC, T helper cells, etc.) may be involved in other virally triggered immune suppression or in AIDS.     

Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence

We studied the mechanism of lymphocytic choriomeningitis virus (LCMV) persistence and the suppression of cytotoxic T lymphocyte (CTL) responses in BALB/c WEHI mice infected at birth with LCMV Armstrong strain. Using adoptive transfer experiments we found that spleen cells from persistently infected (carrier) mice actively suppressed the expected LCMV-specific CTL response of spleen cells from normal adult mice. The suppression was specific for the CTL response and LCMV - specific antibody responses were not affected. Associated with the specific CTL suppression was the establishment of persistent LCMV infection. The transfer of spleen or lymph node cells containing LCMV - specific CTL resulted in virus clearance and prevented establishment of the carrier state. The suppression of LCMV -specific CTL responses by carrier spleen cells is not mediated by a suppressor cell, but is due to the presence of genetic variants of LCMV in spleens of carrier mice. Such virus variants selectively suppress LCMV-specific CTL responses and cause persistent infections in immunocompetent mice. In striking contrast, wild-type LCMV Armstrong, from which these variants were generated, induces a potent CTL response in immunocompetent mice and the LCMV infection is rapidly cleared. Our results show that LCMV variants that emerge during infection in vivo play a crucial role in the suppression of virus-specific CTL responses and in the maintenance of virus persistence.     

CD8 T cell memory in B cell-deficient mice

Antigen presentation by B cells and persistence of antigen-antibody complexes on follicular dendritic cells (FDC) have been implicated in sustaining T cell memory. In this study we have examined the role of B cells and antibody in the generation and maintenance of CD8+ cytotoxic T lymphocyte (CTL) memory. To address this issue we compared CTL responses to lymphocytic choriomeningitis virus (LCMV) in normal (+/+) versus B cell-deficient mice. The CTL response to acute LCMV infection can be broken down into three distinct phases: (a) the initial phase (days 3-8 after infection) of antigen-driven expansion of virus- specific CD8+ T cells and the development of effector CTL (i.e., direct ex vivo killers); (b) a phase of death (between days 10 and 30 after infection) during which >95% of the virus-specific CTL die and the direct effector activity subsides; and (c) the phase of long-term memory (after day 30) that is characterized by a stable pool of memory CTL that persist for the life span of the animal. The role of B cells in each of these three phases of the CTL response was analyzed. We found that B cells were not required for the expansion and activation of virus-specific CTL. The kinetics and magnitude of the effector CTL response, as measured by direct killing of infected targets by ex vivo isolated splenocytes, was identical in B cell-deficient and +/+ mice. Also, the expansion of CD8+ T cells was not affected by the absence of B cells and/or antibody; in both groups of mice there was an approximately 10,000-fold increase in the number of LCMV-specific CTL and a greater than 10-fold increase in the total number of activated (CD44hi) CD8+ T cells during the first week after virus infection. Although no differences were seen during the "expansion" phase, we found that the "death" phase was more pronounced in B cell-deficient mice. However, this increased cell death was not selective for LCMV- specific CTL, and during this period the total number of CD8+ T cells also dropped substantially more in B cell-deficient mice. As a result of this, the absolute numbers of LCMV-specific CTL were lower in B cell- deficient mice but the frequencies were comparable in both groups of mice. More significantly, the memory phase of the CTL response was not affected by the absence of B cells and a stable number of LCMV-specific CTL persisted in B cell-deficient mice for up to 6 mo. Upon reinfection, B cell-deficient mice that had resolved an acute LCMV infection were able to make accelerated CTL responses in vivo and eliminated virus more efficiently than naive B cell-deficient mice. Thus, CTL memory, as assessed by frequency of virus-specific CTL or protective immunity, does not decline in the absence of B cells. Taken together, these results show that neither B cells nor antigen-antibody complexes are essential for the maintenance of CD8+ CTL memory.     

Induction of Protective Cytotoxic T Cell Responses in the Presence of High Titers of Virus-neutralizing Antibodies: Implications for Passive and Active Immunization

The effect of preexistent virus-neutralizing antibodies on the active induction of antiviral T cell responses was studied in two model infections in mice. Against the noncytopathic lymphocytic choriomeningitis virus (LCMV), pretreatment with neutralizing antibodies conferred immediate protection against systemic virus spread and controlled the virus below detectable levels. However, presence of protective antibody serum titers did not impair induction of antiviral cytotoxic T lymphocyte (CTL) responses after infection with 10² PFU of LCMV. These CTLs efficiently protected mice independent of antibodies against challenge with LCMV–glycoprotein recombinant vaccinia virus; they also protected against otherwise lethal lymphocytic choriomeningitis caused by intracerebral challenge with LCMV-WE, whereas transfused antibodies alone did not protect, and in some cases even enhanced, lethal lymphocytic choriomeningitis. Against the cytopathic vesicular stomatitis virus (VSV), specific CTLs and Th cells were induced in the presence of high titers of VSV-neutralizing antibodies after infection with 10⁶ PFU of VSV, but not at lower virus doses. Taken together, preexistent protective antibody titers controlled infection but did not impair induction of protective T cell immunity. This is particularly relevant for noncytopathic virus infections since both virus-neutralizing antibodies and CTLs are essential for continuous virus control. Therefore, to vaccinate against such viruses parallel or sequential passive and active immunization may be a suitable vaccination strategy to combine advantages of both virus-neutralizing antibodies and CTLs.     

Aplastic Anemia Rescued by Exhaustion of Cytokine-secreting CD8+ T Cells in Persistent Infection with Lymphocytic Choriomeningitis Virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P^0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8⁺, but not of CD4⁺, T cells and accelerated by increasing the frequency of LCMV-specific CD8⁺ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8⁺ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8⁺ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P^0/0 and P^0/0 × TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-α and interferon (IFN)-γ produced by CD8⁺ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P^0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P^0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-γ–producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-γ–producing CD8⁺ T cells. Thus, in the absence of IFN-γ and/or TNF/LT-α, exhaustion of virus-specific T cells was not hampered.     

Bystander Activation of Cytotoxic T Cells: Studies on the Mechanism and Evaluation of In Vivo Significance in a Transgenic Mouse Model

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I–restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8⁺ T cells from specific pathogen-free maintained, unimmunized “naive” TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not α/β-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.Specificity is one of the hallmarks of the adaptive immune system. For CTL, specific activation requires the interaction of the TCR with its nominal peptide bound to MHC class I molecules. The questions of whether, how, and to what extent CTL can also be nonspecifically activated in the absence of this cognate interaction are of obvious importance; nonspecific activation of potentially self-reactive T cells during immune responses to foreign antigens may trigger autoimmune diseases. In addition, reactivation of primed T cells by heterologous viruses or cytokines has been postulated to contribute to maintenance of immunological memory (1–5).Unspecific polyclonal stimulation of alloreactive CTL has been described to occur during viral infections (6–8) in mice and in humans (9–11). The high precursor frequency of alloreactive CTL clones allows detection of this nonspecific stimulation in a cytotoxicity assay. Also, nonspecific activation of antiviral memory CTL, displaying an intermediate precursor frequency, has been demonstrated, mainly using limiting dilution assays (2, 3). In contrast, the frequency of naive precursor CTL against viral or self antigens is comparatively low. Whether these cells may be nonspecifically activated has, therefore, been difficult to study in functional assays.The mechanisms postulated to be involved in nonspecific T cell activation are poorly characterized; they include the following. (a) Cross-reactivity at the level of the TCR, which recognizes MHC molecules presenting the nominal peptide, but could also bind to a nonnominal peptide with sufficient avidity for the T cell to be activated. Several examples for this “molecular mimicry” have been postulated and presented (12). (b) Activation of a given T cell by a virus-specific TCR, which could lead to effector function of this T cell via a second TCR of different (e.g., self) specificity (13). It has been shown that allelic exclusion of the α chain of the T cell receptor is incomplete (14, 15), and T cells carrying two different TCRs have been demonstrated in mice (16, 17) as well as in humans (18). (c) “Bystander activation” via cytokines secreted by antigen-responsive cells such as type I IFN (5) or combinations of other cytokines (4) which could act independently of the TCR.When discussing the biological effects of this nonspecific T cell activation, a strict definition of “activation” is indispensable. Whereas proliferation or upregulation of certain surface markers are measurable signs of activation, the extent these phenomena reflect in vivo effector function varies between experimental systems. In the lymphocytic choriomeningitis virus (LCMV)¹ model used in this study, CTLmediated antiviral effector function is almost exclusively mediated by contact-dependent perforin-mediated cytotoxicity. Within this system, the question of whether bystander activation is of biological significance in vivo, can therefore be studied by assessing whether nonspecific activation can induce cytolytically active effector CTL, which can mediate antiviral protection, immunopathology, or autoimmunity.In this study, we analyzed the mechanisms of how TCR transgenic CD8⁺ cytotoxic T cells specific for an LCMVderived peptide presented by H-2D^b may differentiate into LCMV-specific cytolytic effector cells during infections with unrelated pathogens in vivo or mixed lymphocyte cultures in vitro. The high precursor frequency of CTL of a defined specificity in these transgenic mice allowed us to address the following questions: (a) Are these “bystander” CTL protective against LCMV infection in vivo? (b) Which mechanisms are responsible for the nonspecific activation?, (c) How many CTL of a defined specificity are activated?, (d) Does bystander activation have a functional impact on CTL memory in nontransgenic mice?, and (e) Can bystander CTL cause autoimmune disease in a corresponding transgenic mouse model of diabetes mellitus?     

Viral Immune Evasion Due to Persistence of Activated T Cells Without Effector Function

We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69^hi, CD44^hi, CD62L^lo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8– CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.     

Cross-reactivities in memory cytotoxic T lymphocyte recognition of heterologous viruses

Analyses of the relationships between different viruses and viral proteins have focused on homologies between linear amino acid sequences, but cross-reactivities at the level of T cell recognition may not be dependent on a conserved linear sequence of several amino acids. The CTL response to Pichinde virus (PV) and vaccinia virus (VV) in C57BL/6 mice previously immunized with lymphocytic choriomeningitis virus (LCMV) included the reactivation of memory cytotoxic T lymphocyte (CTL) specific to LCMV. Limiting dilution assays (LDA) demonstrated that at least part of this reactivation of memory cells in LCMV-immune mice related to cross-reactivity at the clonal level, even though acute infections with these viruses in nonimmune mice elicited CTL responses that did not cross-react in conventional bulk CTL assays. Precursor CTL (pCTL) to LCMV were generated in splenic leukocytes from LCMV-immune mice acutely infected with PV or VV when stimulated in vitro with only the second virus but not with uninfected peritoneal exudate cells (PECs). Cytotoxicity mediated by LCMV-specific CTL clones activated by PV infection was greatly inhibited by anti-CD8 antibody, suggesting that these memory CTL clones recognizing LCMV-infected targets were of low affinity. LCMV-immune splenocytes stimulated in vitro with PV or VV demonstrated a low but significant precursor frequency (p/f) to the heterologous viruses, and splenocytes from PV- or VV-immune mice when stimulated in vitro against LCMV generated a low but significant p/f to LCMV. Short-term CTL clones cross-reactive between LCMV and PV were derived from splenic leukocytes from LCMV-immune mice acutely infected with PV. To distinguish whether the cross-reactivity was directed against a viral peptide or a virus-induced endogenous cellular neoantigen, we demonstrated that a pCTL frequency to PV about 1/4-1/7 that of the frequency to LCMV could be generated from LCMV-immune splenic leukocytes stimulated with the immunodominant LCMV NP peptide. A partially homologous PV peptide generated from the equivalent site to the LCMV NP peptide did not sensitize targets to lysis by either LCMV- or PV-specific CTLs, suggesting that the cross-reactivity in killing was not due to evolutionarily conserved equivalent sequences. Experiments also indicated that prior immunity to one virus could modulate future primary immune responses to a second virus. Elevated pCTL frequencies to PV were seen after acute PV infection of LCMV- immune mice, and elevated pCTL frequencies to LCMV were seen after acute LCMV infection of PV- and VV-immune mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Infection of lymphocytes by a virus that aborts cytotoxic T lymphocyte activity and establishes persistent infection

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.     

Self Antigens Expressed by Solid Tumors Do Not Efficiently Stimulate Naive or Activated T Cells: Implications for Immunotherapy

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic β-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP × Tag2) mice (137 ± 18 d) as opposed to control RIP-Tag2 mice (88 ± 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP–specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP × Tag2) mice further prolonged survival (168 ± 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.Tumor-specific cytotoxic T lymphocytes (CTLs) can prevent or eradicate tumors in a number of experimental systems (1–5). However, in other models CTLs failed to control tumor growth, which resembles the situation for most cancer patients. Possible reasons for the lack of antitumor CTL activity are selection of tumor antigen or MHC-negative tumor cells (6–9), defective lymphocyte homing to the tumor (10), lack of immunogenicity and costimulation for CTL activation (11–15), or functional or physical absence of tumor antigen–specific CTLs (16). Alternatively, efficient antitumor CTL activity may initially be induced but subsequently declines. It is of interest to characterize why tumor immunosurveillance fails so that antitumor immunotherapy can be improved.Tumor antigens recognized by CTLs may be encoded by viral genes or by mutated cellular genes (1–3). These antigens are foreign to the tumor-bearing host and are thus tumor specific. However, detailed analyses have shown that most defined human tumor antigens are tumor selective but not entirely tumor specific, since physiological expression is also detected in testis, placenta, or melanocytes (17– 19). Since such antigens are expressed only in selected tissues and/or at low levels they may be less accessible for lymphocytes, but it is still possible that autoimmune disease may occur if CTLs are activated for therapeutic purposes. Nonetheless, if tissue-specific self antigens were used as targets for immunotherapy, the development and application of tumor vaccines might be simplified by targeting the same protein or major epitopes in different patients. Therefore, it is important to evaluate whether CTL activation against such antigens results in damage of healthy tissues and whether this would trigger chronic autoimmune disease.To assess these questions in an in vivo model, we took advantage of the simian virus 40 tumor T antigen (Tag)¹ which is a potent oncogene, largely due to its ability to inactivate two tumor suppressors, Rb and p53 (20, 21). We used transgenic mice expressing Tag under the control of the rat insulin promoter (RIP). The RIP-Tag2 line of transgenic mice develops pancreatic β-cell tumors leading to progressive autonomous insulin secretion and hypoglycemia, which is lethal when the mice reach the age of 3–4 mo (22). Histological sections show both hyperplasia and tumor formation by proliferating β cells. The transgenic mouse line RIP-Tag2 used in this study expresses Tag early during embryogenesis leading to immune tolerance such that Tag-specific lymphocytes are not detectable in the T cell repertoire (16).As a tumor-associated antigen, we introduced the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), also expressed under the control of the RIP (24). Like the RIP-Tag2 mice (23), these RIP-GP mice do not mount a spontaneous immune response against the transgenic product. However, in contrast to the RIP-Tag2 mice, there is no specific T cell tolerance to the transgenic neoantigen. As a consequence, infection with LCMV activates GP-specific CTLs which destroy the pancreatic β cells and induce type I autoimmune diabetes (24). LCMV is a natural mouse pathogen which replicates rapidly in vivo. It induces a strong immune response dominated by LCMV-specific cytotoxic T cells which proliferate to high numbers (∼25% of splenic mononuclear cells). These CTLs are responsible for rapid virus elimination occurring within ∼10 d and give rise to a long-term protective CTL memory (25–27). This strong CTL response was well suited for an in vivo model of tumor immunotherapy.The two transgenic lines were crossed to obtain mice with β-islet cell tumors expressing LCMV-GP. The effectiveness of antitumor responses was evaluated by addressing the following questions in vivo: (a) Are LCMV-GP specific T cells spontaneously activated in tumor-bearing mice? (b) Does immunological unresponsiveness to LCMV-GP develop? (c) Do these mice mount a cytotoxic T cell response after immunization? (d) Will chronic autoimmune disease develop after induction of the tumor-specific LCMV-GP response? Our data show that the tumor antigen–specific CTLs were not spontaneously activated but efficiently reduced the tumor mass upon activation by virus infection. The tumor cells were profoundly incapable of stimulating an LCMV-GP–specific response in vivo since the antitumor CTL activity was not maintained but declined in parallel with the virus specific response.     

Thymic selection of H-2-incompatible bone marrow cells in SCID mice. Differences in T help for induction of B cell IgG responses versus cytotoxic T cells

Mice with congenital severe combined immunodeficiency disease (SCID) failed to mount either a T cell-independent IgM or T cell-dependent IgG anti-vesicular stomatitis virus (VSV) Indiana (IND) response. They did not generate cytotoxic T cells against lymphocytic choriomeningitis virus (LCMV) or vaccinia virus, but exhibited NK cell-like activities. When SCID mice were given bone marrow from syngeneic BALB/c (H-2d) nu/nu mice, all immune responses were expressed at control levels. If SCID mice were reconstituted with allogeneic H-2b C57BL/6 nu/nu bone marrow, the following primary anti-viral immune responses were measured. T-independent IgM anti-VSV-IND were normal, but T-dependent IgG anti-VSV-IND responses were absent. Cytotoxic T cell responses to LCMV and vaccinia virus were within normal ranges, were donor cell mediated, and were specific exclusively for the recipient SCID H-2d type. Since antigen presentation by spleen cells was functional in these chimaeras, the presented results indicate that (a) thymic selection of T cell restriction is strict; and (b) the type of T help necessary for B cells depends upon H-2-restricted contact between T and B cells, whereas, such contact-dependent help is not mandatory for the induction of virus-specific cytotoxic T cells.     

Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:D^b complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.     

Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans

Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.     

Cytotoxic T-cell response to ectromelia virus-infected cells. Different H-2 requirements for triggering precursor T-cell induction or lysis by effector T cells defined by the BALB/c-H-2(db) mutation

The T(c)-cell response to ectromelia virus infection was studied in BALB/c-H-2(db) mice which carry a loss mutation in the H-2D region that results in the absence from cell surfaces of a molecule (D’) bearing certain public H-2 specificities. When infected, these mice showed a poor response of T(c) cells that recognize H-2D(d) plus virus-specific determinants on infected macrophage targets, but gave a normal response to H-2K d plus virus-specific antigens. However, their own infected macrophages do display wild-type antigenic patterns involving virus and H-2D(d) since they were killed as efficiently as wild-type (BALB/c,H- 2(d))-infected cells by T(c) cells specific only for H-2D(d) plus viral antigens. When tested in vitro, infected BALB/c-H-2(db) cells stimulated a poor T(c)-cell response to H-2D plus virus-specific antigens, but stimulated a normal response (in comparison with infected BALB/c macrophages) to H-2K(d) plus viral antigens. Uninfected BALB/c-H-2(db) cells stimulated a normal T(c)-cell response to minor H antigens or trinitrophenyl in association with H-2D(d), thus suggesting that the defective response to infection may reside in a failure of the relevant H-2D(d) antigens of mutant cells to physically associate with viral antigens. Close association of viral and H-2D-coded molecules was also suggested by ability of specific anti-H-2K or -H-2D to partially block T(c)-cell-mediated lysis of infected targets. These results were interpreted to mean that H-2Dd-dependent, virus- immune T(c) cells recognized an antigenic pattern consisting of virus- specific and H-2D(d) determinants with the latter borne on an H-2D molecule carrying serologically-defined H-2D(d) private specificities. A second H-2D(d)-coded molecule (D’) was not required for recognition and lysis by activated T(c) cells, but was apparently necessary for efficient stimulation of precursor T(c) cells, perhaps by promoting appropriate physical association of viral and H-2D(d) molecules.     

c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 signaling pathways have divergent roles in CD8(+) T cell-mediated antiviral immunity

c-Jun NH(2)-terminal kinases (JNK) play important roles in T helper cell (Th) proliferation, differentiation, and maintenance of Th1/Th2 polarization. To determine whether JNKs are involved in antiviral T cell immunity, and whether JNK1 and JNK2 bear biological differences, we investigated the immune responses of JNK1-deficient and JNK2-deficient mice to lymphocytic choriomeningitis virus (LCMV). After LCMV infection, wild-type (JNK(+/+)) mice had a 5- to 10-fold increase in splenic CD8(+) T cells. In contrast, infected JNK1(-/-) mice showed a significantly lower virus-specific CD8(+) T cell expansion. However, JNK1(-/-) mice cleared LCMV infection with similar kinetics as JNK(+/+) mice. Splenic T cells from LCMV-infected JNK1(-/-) animals produced interferon gamma after stimulation with viral peptides. However, fewer JNK1(-/-) T cells acquired an activated phenotype (CD44(hi)) and more JNK1(-/-)CD8(+)CD44(hi) cells underwent apoptosis than JNK(+/+) cells at the peak of the primary response. In contrast, LCMV-infected JNK2(-/-) mice generated more virus-specific CD8(+) T cells than JNK(+/+) mice. These results indicate that JNK1 and JNK2 signal pathways have distinct roles in T cell responses during a viral infection. JNK1 is involved in survival of activated T cells during immune responses, and JNK2 plays a role in control of CD8(+) T cell expansion in vivo.     

Phase-variable Expression of Lipopolysaccharide Contributes to the Virulence of Legionella pneumophila

The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8⁺ T cells using a cytolytic T cell line specific for ovalbumin (OVA)_257-264 presented by H-2K^b. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon γ and tumor necrosis factor α required stimulation of the CTL with E.G7-OVA. The kinetics of lymphokine secretion after stimulation by E.G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and β₂-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8⁺ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.Memory is a hallmark of the immune response to T cell–dependent antigens. Memory, in both B and T cells, is manifest by stronger and quicker responses upon secondary exposure to antigen. The mechanisms that maintain memory are not completely understood. In part, T cell memory reflects an increase in the frequency of precursor cells. Memory T cells are also qualitatively distinct from naive T cells in that they have less stringent requirements for activation and respond more rapidly than naive T cells (for review see reference 1). Naive and memory T cells are distinct from effector T cells in that the latter are actively engaged in lymphokine secretion and lytic function in the case of CD8⁺ T cells. By contrast, both naive and memory T cells must be stimulated with antigen to express these effector functions. Naive T cells can be distinguished from effector T cells by differences in the expression of several cell-surface antigens. However, effector T cells cannot be easily distinguished from memory T cells because they both express activation antigens and increased density of adhesion molecules.Several important questions remain concerning the maintenance of memory T cells. The question of whether antigen is required for memory T cell persistence has been the subject of considerable debate. The majority of studies on the requirement for antigen in maintaining CD8⁺ memory T cells have been performed using polyclonal T cells activated by viruses such as lymphocytic choriomeningitis virus (LCMV), Sendai virus, or influenza. One of the major obstacles in studying memory in virus-specific, CD8⁺ T cells is ruling out restimulation by persisting Ags. However, transfer of CD8⁺ T cells from infected mice into irradiated, syngeneic recipients showed that memory persisted in the absence of virus, as measured by PCR (2–4). Another important issue is the relationship between effector and memory T cells. Whether memory T cells are derived from effector T cells in a linear fashion or whether they differentiate along separate pathways is not known. Signals that regulate development of memory T cells have not been identified.We have recently demonstrated that soluble OVA emulsified in CFA (5) or nonionic block copolymer adjuvants primed CD8⁺ CTL precursors in mice (6). These OVA-specific CTLs (OVA-CTLs) produced IFN-γ and TNF-α upon stimulation with E.G7-OVA or with OVA_257-264 in association with H-2K^b (6). However, these OVA-CTL cells do not produce IL-2 or -4. We reasoned that such CD8⁺ T cells would provide a good model for studying the memory function of CTLs because persistence of Ags would not be a problem. The results presented below compare the requirements for activation of CTLs versus maintenance of memory CTLs in vitro and in vivo. Our results show that CD8⁺ CTLs specific for the exogenous Ag, OVA, have the same potential to persist as memory T cells specific for viruses and tumors. Moreover, persistence of memory T cells was more dependent upon IL-2 than cross-reactive complexes of peptides and MHC class I antigens.