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.     

Different dynamics of CD4+ and CD8+ T cell responses during and after acute lymphocytic choriomeningitis virus infection

We fit a mathematical model to data characterizing the primary cellular immune response to lymphocytic choriomeningitis virus. The data enumerate the specific CD8(+) T cell response to six MHC class I-restricted epitopes and the specific CD4(+) T cell responses to two MHC class II-restricted epitopes. The peak of the response occurs around day 8 for CD8(+) T cells and around day 9 for CD4(+) T cells. By fitting a model to the data, we characterize the kinetic differences between CD4(+) and CD8(+) T cell responses and among the immunodominant and subdominant responses to the various epitopes. CD8(+) T cell responses have faster kinetics in almost every aspect of the response. For CD8(+) and CD4(+) T cells, the doubling time during the initial expansion phase is 8 and 11 h, respectively. The half-life during the contraction phase following the peak of the response is 41 h and 3 days, respectively. CD4(+) responses are even slower because their contraction phase appears to be biphasic, approaching a 35-day half-life 8 days after the peak of the response. The half-life during the memory phase is 500 days for the CD4(+) T cell responses and appears to be lifelong for the six CD8(+) T cell responses. Comparing the responses between the various epitopes, we find that immunodominant responses have an earlier and/or larger recruitment of precursors cells before the expansion phase and/or have a faster proliferation rate during the expansion phase.     

Complement-dependent enhancement of CD8+ T cell immunity to lymphocytic choriomeningitis virus infection in decay-accelerating factor-deficient mice

Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known. We describe in this study a striking regulatory effect of DAF on the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) infection. Compared with wild-type mice, DAF knockout (Daf-1(-/-)) mice had markedly increased expansion in the spleen of total and viral Ag-specific CD8+ T cells after acute or chronic LCMV infection. Splenocytes from LCMV-infected Daf-1(-/-) mice also displayed significantly higher killing activity than cells from wild-type mice toward viral Ag-loaded target cells, and Daf-1(-/-) mice cleared LCMV more efficiently. Importantly, deletion of the complement protein C3 or the receptor for the anaphylatoxin C5a (C5aR) from Daf-1(-/-) mice reversed the enhanced CD8+ T cell immunity phenotype. These results demonstrate that DAF is an important regulator of CD8+ T cell immunity in viral infection and that it fulfills this role by acting as a complement inhibitor to prevent virus-triggered complement activation and C5aR signaling. This mode of action of DAF contrasts with that of CD59 in viral infection and suggests that GPI-anchored membrane complement inhibitors can regulate T cell immunity to viral infection via either a complement-dependent or -independent mechanism.     

Role of tumor necrosis factor receptors in regulating CD8 T-cell responses during acute lymphocytic choriomeningitis virus infection

The role of tumor necrosis factor (TNF) in regulating various phases of the antiviral T-cell response is incompletely understood. Additionally, despite strong evidence ascribing a role for TNF in protecting against T-cell-dependent autoimmunity, the underlying mechanisms are still obscure. To address these issues, we have investigated the role of tumor necrosis factor receptors (TNFRs) I (p55R) and II (p75R) in regulating CD8 T-cell responses to lymphocytic choriomeningitis virus (LCMV) with wild-type, p55R-deficient (p55(-/-)), p75R-deficient (p75(-/-)), and p55R- and p75R-deficient (DKO) mice. Loss of p55R increased the number of memory CD8 T cells to only one of the two immunodominant epitopes, and p75R deficiency had a minimal impact on the T-cell response to LCMV. Strikingly, deficiency of both p55R and p75R had a more dramatic effect on the LCMV-specific CD8 T-cell response; in the DKO mice, as a sequel to enhanced expansion and a reduction in contraction of CD8 T cells, there was a substantial increase in the number of memory CD8 T cells (specific to the two immunodominant epitopes). While the majority of LCMV-specific memory CD8 T cells in wild-type mice were CD62Lhi CCR7hi (central memory), a major proportion of memory CD8 T cells in DKO mice were CD62Llo CCR7hi. TNFR deficiency did not affect the proliferative renewal of memory CD8 T cells. Taken together, these data suggested that TNFRs p55R and p75R have overlapping roles in downregulating CD8 T-cell responses and establishment of immune homeostasis during an acute viral infection.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Delayed contraction of the CD8+ T cell response toward lymphocytic choriomeningitis virus infection in mice lacking serglycin

We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG(-/-)) mice with lymphocytic choriomeningitis virus (LCMV). Wt and SG(-/-) mice cleared 10(3) PFU of highly invasive LCMV with the same kinetics, and the CD8(+) T lymphocytes from wt and SG(-/-) animals did not differ in GrB, perforin, IFN-gamma, or TNF-alpha content. However, when a less invasive LCMV strain was used, SG(-/-) GrB(+) CD8(+) T cells contained approximately 30% less GrB than wt GrB(+) CD8(+) T cells. Interestingly, the contraction of the antiviral CD8(+) T cell response to highly invasive LCMV was markedly delayed in SG(-/-) mice, and a delayed contraction of the virus-specific CD8(+) T cell response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8(+) T cells in SG(-/-) mice. Moreover, wt LCMV-specific CD8(+) T cells from TCR318 transgenic mice expanded much more extensively in virus-infected SG(-/-) mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics of antiviral CD8(+) T cell responses.     

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.     

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.     

CD8+ T cell immunodominance in lymphocytic choriomeningitis virus infection is modified in the presence of toll-like receptor agonists

Currently, we have limited understanding of how Toll-like receptor (TLR) engagement by microbial products influences the immune response during a concurrent virus infection. In this study, we established that dual TLR2 plus TLR3 (designated TLR2+3) stimulation alters the immunodominance hierarchies of lymphocytic choriomeningitis virus (LCMV) epitopes by reducing NP396-specific CD8+ T cell responses and shifting it to a subdominant position. The shift in immunodominance occurred due to a reduction in antigen uptake and the reduced cross-presentation of NP396, a major LCMV immunodominant epitope that is efficiently cross-presented. Moreover, the altered immunodominance was dependent on TLR stimulation occurring at the site of infection. Finally, as lipopolysaccharide failed to induce the same phenomenon, the data suggest that these findings are dependent not only on the dual engagement of the TRIF/MyD88 pathways but also on how TLR agonists activate antigen-presenting cells. Taken together, our data demonstrate a novel role for TLR ligands in regulating antiviral CD8+ T cell responses due to the regulation of the cross-presentation of cell-associated antigens.     

Virus specificity of cytotoxic T lymphocytes generated during acute lymphocytic choriomeningitis virus infection: role of the H-2 region in determining cross-reactivity for different lymphocytic choriomeningitis virus strains

We have compared the relatedness of five different strains of lymphocytic choriomeningitis virus (LCMV) as assessed by LCMV-specific cytotoxic T lymphocytes (CTL). Several different mouse strains were injected with each of the five LCMV strains, and the cross-reactivity of virus-specific CTL generated during the acute infection was tested by killing on a panel of target cells infected with the various LCMV strains. We found that the cross-reactivity pattern of LCMV-specific CTL generated in mice of H-2d haplotype (BALB/c WEHI and DBA/2) was strikingly different from that in mice of H-2b haplotype (C57BL/6 and C3H.Sw/Sn), suggesting that the fine specificity of LCMV-specific CTL is a function of the H-2 region. The characteristic cross-reactivity patterns were also observed in (C57BL/6 X DBA/2)F1 mice, demonstrating that the repertoire of the H-2b- and H-2d-restricted LCMV-specific CTL is not changed as a result of complementation by gene products of the other major histocompatibility haplotype. Studies with congenic BALB.B10 and (BALB.B10 X BALB/c)F1 mice firmly established that the characteristic cross-reactivity patterns of LCMV-specific CTL map to the H-2 region and are not influenced by background genes outside the major histocompatibility locus. These results suggest that LCMV determinants seen in the context of H-2d-restricting elements are different from those seen in the context of H-2b-restricting elements. Moreover, our studies show that CTL can be used as probes for dissecting differences among various LCMV strains, but the degree of relatedness between the different LCMV strains is not absolute when measured by CTL recognition. Since the H-2 region regulates the fine specificity of CTL generated during LCMV infection in its natural host, the degree of cross-protective immunity developed during a viral infection apparently depends on the major histocompatibility haplotype. The importance of these findings lies in understanding susceptibility or resistance of various host populations to viral infections and in designing vaccination programs to provide immunity.     

CD8 T cell-intrinsic GITR is required for T cell clonal expansion and mouse survival following severe influenza infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-x(L). In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphocyte survival, they clearly have nonredundant functions in protection from severe infection.     

Conditional CD8+ T cell escape during acute simian immunodeficiency virus infection

CD8⁺ T cell responses rapidly select viral variants during acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. We used pyrosequencing to examine variation within three SIV-derived epitopes (Gag_386-394GW9, Nef_103-111RM9, and Rev_59-68SP10) targeted by immunodominant CD8⁺ T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev_59-68SP10 was associated with delayed accumulation of variants in Gag_386-394GW9 but had no effect on variation within Nef_103-111RM9. This demonstrates that the entire T cell repertoire, rather than a single T cell population, influences the timing of immune escape, thereby providing the first example of conditional CD8⁺ T cell escape in HIV/SIV infection.     

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.     

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.     

Protracted course of lymphocytic choriomeningitis virus WE infection in early life: induction but limited expansion of CD8+ effector T cells and absence of memory CD8+ T cells

Viral infections in human infants frequently follow a protracted course, with higher viral loads and delayed viral clearance compared to viral infections in older children. To identify the mechanisms responsible for this protracted pattern of infection, we developed an infant infection murine model using the well-characterized lymphocytic choriomeningitis virus (LCMV) WE strain in 2-week-old BALB/c mice. In contrast to adult mice, in which viral clearance occurred as expected 8 days after infection, LCMV titers persisted for several weeks after infection of infant mice. LCMV-specific effector CD8(+) T cells were elicited in infant mice and fully functional on day 7 but rapidly waned and could not be recovered from day 12 onwards. We show here that this results from the failure of LCMV-specific CD8(+) T cells to expand and the absence of protective LCMV-specific memory CD8(+) T cells. Under these early life conditions, viral control and clearance are eventually achieved only through LCMV-specific B cells that contribute to protect infant mice from early death or chronic infection.     

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.     

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.