Search

CD8(+) T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus that occurred in Brazil., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A plethora of work implicates important effects of the vitamin A derivative retinoic acid (RA) in myeloid differentiation, whereas fewer studies explore the role of RA in lymphoid cells. Most work on lymphoid cells has focused on the influence of RA on CD4 T cells. Little information about the role of RA in CD8 T cell differentiation is available, and even less on cell-intrinsic effects in the CD8 T cell. This study explores the role of RA in effector and memory differentiation in a cell-intrinsic manner in the context of vaccinia virus infection. We observed the loss of the short-lived effector cell phenotype (reduced KLRG1(+), T-bet(hi), granzyme B(hi)), accompanied by an enhanced memory precursor phenotype at the effector (increased CD127(hi), IL-2(+)) and contraction phases (increased CD127(hi), IL-2(+), eomesodermin(hi)) of the CD8 response in the absence of RA signaling. The lack of RA also increased the proportion of central memory CD8s. Collectively, these results introduce a new role for RA in CD8 T cell activation and differentiation. This new role may have significant implications for optimal vaccine design in which vitamin A supplementation is used to augment effector responses, but it may be to the detriment of the long-term central memory response.

Patients who survive severe sepsis often display compromised immune function with impairment in innate and adaptive immune responses. These septic patients are highly susceptible to "secondary" infections with intracellular pathogens that are usually controlled by CD8(+) T cells. It is not known when and if this observed immunoparalysis of CD8(+) T cell immunity recovers, and the long-term consequences of sepsis on the ability of naive CD8(+) T cells to respond to subsequent infections are poorly understood. In this study, using the cecal-ligation and puncture mouse model of sepsis, we show that sepsis induces a rapid loss of naive CD8(+) T cells. However, IL-15-dependent numerical recovery is observed a month after initial septic insult. Numerical recovery is accompanied by IL-15-dependent phenotypic changes where a substantial proportion of naive (Ag-inexperienced) CD8(+) T cells display a "memory-like" phenotype (CD44(hi)/CD11a(hi)). Importantly, the impairment of naive CD8(+) T cells to respond to viral and bacterial infection was sustained for month(s) after sepsis induction. Incomplete recovery of naive CD8(+) T cell precursors was observed in septic mice, suggesting that the availability of naive precursors contributes to the sustained impairment in primary CD8(+) T cell responses. Thus, sepsis can result in substantial and long-lasting changes in the available CD8(+) T cell repertoire affecting the capacity of the host to respond to new infections.

Persistent viral infection is often associated with dysfunctional immune responses against unrelated pathogens. Lymphocytic choriomeningitis virus (LCMV) can establish acute or chronic infections in mice and is widely used as a model for persistent virus infections in humans. Mice infected with LCMV develop a transient defect in Ag-specific immunity against heterologous viral infection. Although it has been proposed that LCMV infection induces an immunosuppressed state within the host, our data show that infected mice successfully clear vaccinia virus through a mechanism that involves CD8(+) T cell-derived IFN-γ. This observation demonstrates that chronic LCMV infection does not impair protective immunity against heterologous viral challenge. Rather, a natural sterilizing immunity is induced following a primary infection that prevents a secondary infection. Our findings suggest a need to re-evaluate current thoughts about the immune suppression that might occur during a persistent infection.

Foxp3(+) regulatory T (Treg) cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4(+) T cells overcome the suppressive effects of Treg cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4(+) T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens.

The T cell response possesses a number of inhibitory receptors to regulate the extent of the antiviral response and prevent immune pathology. These receptors are generally transiently upregulated during an effector response and then downregulated during memory. Some inhibitory receptors, such as programmed death 1 (PD-1) and LAG-3, were shown to be aberrantly upregulated during memory to chronic lymphocytic choriomeningitis virus infection, limiting functional capabilities. However, little is known about the impact of inhibitory receptors on memory development during a normal CD8 T cell response to acute virus infection. Our previous data showed that PD-1 is aberrantly upregulated during a secondary response by memory CD8 T cells that were generated without CD4 T cell help. Therefore, we examined the role of PD-1 in memory differentiation during acute vaccinia virus infection in intact mice. In the absence of PD-1, the primary and memory CD8 T cell responses were enhanced. Moreover, there were distinct phenotypic and functional changes in the memory PD-1(-/-) CD8 T cells. Higher levels of CD62L, CD27, and CCR7 were detected; cells produced more IL-2 and made an enhanced secondary response. These changes indicate a skewing of the memory population toward the central memory phenotype in the absence of PD-1 signaling.

Certain virus infections depend on the presence of T cell help for the generation of primary CD8(+) T cell responses. However, the mechanisms that render these particular viral infections T cell help dependent is largely unknown. In this study, we compared CD8(+) T cell responses elicited by lymphocytic choriomeningitis virus infection, as prototype of a T cell help independent infection, with T cell help dependent CD8(+) T cell responses induced by vaccinia virus infection. In this paper, we show that a key parameter decisive for T cell help independence is the ability of an infectious agent to stimulate early and robust production of type I IFN. Experimental provision of type I IFN during VV infection rendered the ensuing CD8(+) T cell response completely T cell help independent. Our results support a model in which type I IFN has to be present during the first 3 d of Ag encounter and has to act directly on the responding CD8(+) T cells to promote their survival and effector differentiation. We show that type I IFN signaling on responding CD8(+) T cells induces profound upregulation of CD25 and increased IL-2 expression; however, neither this nor IL-15 accounts for the type I IFN effects on responding CD8(+) T cells. Thus, type I IFN can effectively replace the requirement of T cell help by directly promoting CD8(+) T cell survival and differentiation independent of the type I IFN-induced cytokines IL-2 and IL-15.

CD8 T cells can acquire cytokine-secreting phenotypes paralleling cytokine production from Th cells. IL-17-secreting CD8 T cells, termed Tc17 cells, were shown to promote inflammation and mediate immunity to influenza. However, most reports observed a lack of cytotoxic activity by Tc17 cells. In this study, we explored the anti-viral activity of Tc17 cells using a vaccinia virus (VV) infection model. Tc17 cells expanded during VV infection, and TCR transgenic Tc17 cells were capable of clearing recombinant VV infection. In vivo, adoptively transferred Tc17 cells lost the IL-17-secreting phenotype, even in the absence of stimulation, but they did not acquire IFN-gamma-secreting potential unless stimulated with a virus-encoded Ag. However, examination of cells following infection demonstrated that these cells acquired cytotoxic potential in vivo, even in the absence of IFN-gamma. Cytotoxic potential correlated with Fasl expression, and the cytotoxic activity of postinfection Tc17 cells was partially blocked by the addition of anti-FasL. Thus, Tc17 cells mediate VV clearance through expression of specific molecules associated with cytotoxicity but independent of an acquired Tc1 phenotype.

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

The diversity of the pathogen-specific T cell repertoire is believed to be important in allowing recognition of different pathogen epitopes and their variants and thereby reducing the opportunities for mutation-driven pathogen escape. However, the extent to which the TCR repertoire can be manipulated by different vaccine strategies so as to obtain broad diversity and optimal protection is incompletely understood. We have investigated the influence of the infectious/inflammatory context on the TCR diversity of the CD8(+) T cell response specific for the immunodominant epitope in C57BL/6 mice, derived from glycoprotein B of HSV-1. To that effect, we compared TCR V segment utilization, CDR3 length, and sequence diversity of the response to natural HSV-1 infection with those elicited by either Listeria monocytogenes or vaccinia virus expressing the immunodominant epitope in C57BL/6 mice. We demonstrate that although the type of infection in which the epitope was encountered can influence the magnitude of the CD8(+) T cell responses, TCR beta-chain repertoires did not significantly differ among the three infections. These results suggest that widely different live vaccine vectors may have little impact upon the diversity of the induced CTL response, which has important implications for the design of live CTL vaccine strategies against acute and chronic infections.

TNF has been shown to be important for controlling many pathogens. Here, we directly demonstrate using wild-type TNF(-/-) and TNFR1(-/-) mice that TNF does play a role in protection against vaccinia virus (VV) infection in naive mice. Since VV replication is also partially controlled in lymphocytic choriomeningitis virus (LCMV)-immune C57BL/6J mice through the process of heterologous immunity, we questioned whether TNF was required in mediating this protection. VV-infected LCMV-immune mice that were TNF-deficient as a consequence of genetic deletion or receptor blockade demonstrated normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells and controlled VV infection similar to LCMV-immune mice having TNF function. This indicates that neither TNF nor lymphotoxin, which uses the same receptor, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV made the role of TNF in protection against VV infection much less important, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of patients with anti-TNF compounds is reasonably well tolerated with relatively few infectious complications.

Innate immunity is required for effective control of poxvirus infections, but cellular receptors that initiate the host response to these DNA viruses remain poorly defined. Given this information and the fact that functions of TLRs in immunity to DNA viruses remain controversial, we investigated effects of TLR3 on pathogenesis of vaccinia virus, a prototype poxvirus. We used a recombinant strain Western Reserve vaccinia virus that expresses firefly luciferase to infect wild-type C57BL/6 and TLR3-/- mice through intranasal inoculation. Bioluminescence imaging showed that TLR3-/- mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. Mice lacking TLR3 had reduced disease morbidity, as measured by decreased weight loss and hypothermia after infection. Importantly, TLR3-/- mice also had improved survival relative to wild-type mice. Infected TLR3-/- mice had significantly reduced lung inflammation and recruitment of leukocytes to the lung. Mice lacking TLR3 also had lower levels of inflammatory cytokines, including IL-6, MCP-1, and TNF-alpha in serum and/or bronchoalveolar lavage fluid, but levels of IFN-beta did not differ between genotypes of mice. To our knowledge, our findings show for the first time that interactions between TLR3 and vaccinia increase viral replication and contribute to detrimental effects of the host immune response to poxviruses.

Recent studies have defined vaccinia virus (VACV)-specific CD8(+) T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4(+) T cell responses. In this study, we identified 14 I-A(b)-restricted VACV-specific CD4(+) T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for approximately 20% of the total anti-VACV CD4(+) T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4(+) T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4(+) T cell targets. These results are in contrast to what was previously found in CD8(+) T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4(+) and CD8(+) T cell responses to a complex pathogen.

Type-I IFNs (IFN-I) provide direct survival signals to T cells during Ag-driven proliferation. Because IFN-I production differs depending on the pathogen, we assessed CD8 T cell requirement for direct IFN-I signals during responses to vaccinia virus (VV), vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), and Listeria monocytogenes (LM) immunizations in vivo. IFN-I-receptor-deficient (IFN-IR(o)) CD8 T cells expanded 3- to 5-fold less and formed a diminished memory pool compared with wild-type (WT) CD8 T cells in response to VV, VSV, or LM. WT CD8 T cells expanded more robustly in response to LCMV-encoded Ags than to Ags encoded by the other three pathogens, and under these conditions the lack of direct IFN-I signals inhibited their expansion by approximately 100-fold. To test whether the high antigenic-load provided by LCMV caused greater expansion and greater IFN-I dependency, we primed WT and IFN-IR(o) OVA-specific OT-1 CD8 T cells with a fixed-number of OVA-peptide-pulsed dendritic cells along with adjuvant effect provided by LCMV, VV, VSV, or LM. Both WT and IFN-IR(o) OT-1 cells were recruited, proliferated, and differentiated into effectors in all the four cases. However, WT OT-1 cells expanded similarly in all four cases. IFN-IR(o) OT-1 cells expanded approximately 20-fold less than the WT OT-1 CD8 T cells when LCMV was used as adjuvant, whereas their expansion was affected only marginally when VV, VSV, or LM were used as adjuvants. Thus, innate/inflammatory signals induced by different pathogens contribute to CD8 T cell expansion and memory formation via distinct levels of IFN-I dependence.

Virus-specific CD8(+) T cells produce IFN-gamma after Ag contact and, in the absence of this cytokine, the host often cannot eradicate infection. However, our ability to identify cells that are actively expressing this critical effector function in vivo is limited, because the protein is rapidly secreted. In this study, we describe a simple approach that circumvents the need for ex vivo Ag stimulation and allows the enumeration of CD8(+) T cells that are actively synthesizing IFN-gamma in vivo during primary and secondary virus infections. The proportion of Ag-specific primary CD8(+) T cells producing IFN-gamma peaks at 5 days postinfection, when the T cell population is still expanding exponentially. In vivo IFN-gamma synthesis by memory cells is explosive, peaking at approximately 12 h after secondary infection and terminating hours thereafter. This technique will be useful when evaluating in vivo immune cell activity in many situations, including a variety of noninfectious (e.g., autoimmune) diseases.

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8(+) T cells vs that of CD4(+) T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-gamma responses in CD4(+) and CD8(+) T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4(+) T cells or B cells in IgH(-/-) mice, but was not sensitive to CD8(+) T cell depletion alone. However, a role for CD8(+) T cells in primary protection was demonstrated in MHC class II(-/-) mice, where depleting CD8(+) T cells lead to increase severity of disease. Unlike control MHC class II(-/-) mice, the group depleted of CD8(+) T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8(+) T cells can mediate protective memory. These results collectively show that both CD4(+) and CD8(+) T cell-mediated immunity can contribute to protection against VV infection. However, CD4(+) T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8(+) T cells can contribute to protection against disease.

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.

CD8+ T lymphocytes play an important role in the control of intracellular pathogens during both acute and persistent infections. This is particularly true in the case of persistent herpesviruses such as human CMV, which are typified by large virus-specific CD8+ T cell populations during viral latency. To understand the origin of these populations and the factors shaping them over time, we investigated the CD8+ T cell response after murine CMV (MCMV) infection. The kinetics of the acute response were characterized by rapid expansion of activated T cells, followed by a contraction phase. Thereafter, we observed a striking pattern, where MCMV-specific memory CD8+ T cells steadily accumulated over time, with 20% of all CD8+ T cells at 1 year specific for one MCMV epitope. Accumulation of MCMV-specific CD8+ T lymphocytes was seen in all organs tested and was associated with continuous activation of specific CD8+ T lymphocytes, primarily within lymph nodes. The pattern of accumulation was observed in only two of five epitopes tested, and was accompanied by a gradual restriction in usage of the variable region of the TCR beta-chain over time. This novel pattern of a virus-specific CD8+ T cell response suggests that continuous or repetitive exposure to Ag can slowly mold memory T cell populations over time. This may be relevant for understanding the evolution of the large human CMV-specific CD8+ T cell populations seen in humans.

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.

Previous studies have shown that heterologous viral infections have a significant impact on pre-existing memory T cell populations in secondary lymphoid organs through a combination of cross-reactive and bystander effects. However, the impact of heterologous viral infections on effector/memory T cells in peripheral sites is not well understood. In this study, we have analyzed the impact of a heterologous influenza virus infection on Sendai virus-specific CD8(+) effector/memory cells present in the lung airways. The data show a transient increase in the numbers of Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells in the airways of the influenza-infected mice peaking around day 4 postinfection. Intratracheal transfer studies and 5-bromo-2'-deoxyuridine incorporation demonstrate that this increase is due to the recruitment of resting memory cells into the airways. In addition, the data show that these immigrating memory cells are phenotypically distinct from the resident memory T cells of the lung airways. A similar influx of nonproliferating Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells is also induced by a secondary (homologous) infection with Sendai virus. Together, these data suggest that inflammation can accelerate memory T cell migration to nonlymphoid tissues and is a part of the normal recall response during respiratory infections.

Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were approximately 50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.

The existence of gammadelta T cells has been known for over 15 years, but their significance in innate immunity to virus infections has not been determined. We show here that gammadelta T cells are well suited to provide a rapid response to virus infection and demonstrate their role in innate resistance to vaccinia virus (VV) infection in both normal C57BL/6 and beta TCR knockout (KO) mice. VV-infected mice deficient in gammadelta T cells had significantly higher VV titers early postinfection (PI) and increased mortality when compared with control mice. There was a rapid and profound VV-induced increase in IFN-gamma-producing gammadelta T cells in the peritoneal cavity and spleen of VV-infected mice beginning as early as day 2 PI. This rapid response occurred in the absence of priming, as there was constitutively a significant frequency of VV-specific gammadelta T cells in the spleen in uninfected beta TCR KO mice, as demonstrated by limiting dilution assay. Also, like NK cells, another mediator of innate immunity to viruses, gammadelta T cells in uninfected beta TCR KO mice expressed constitutive cytolytic activity. This cytotoxicity was enhanced and included a broader range of targets after VV infection. VV-infected beta TCR KO mice cleared most of the virus by day 8 PI, the peak of the gammadelta T cell response, but thereafter the gammadelta T cell number declined and the virus recrudesced. Thus, gammadelta T cells can be mediators of innate immunity to viruses, having a significant impact on virus replication early in infection in the presence or absence of the adaptive immune response.

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.

Vaccinia virus employs multiple mechanisms to evade the immune system, yet is highly immunogenic. We studied the interaction between vaccinia and human dendritic cells (DCs), potent APCs. DCs develop from precursor cells in two stages: an immature stage in which Ag uptake and processing occur, and a mature stage in which there is up-regulation of costimulatory and HLA molecules and efficient T cell activation. Vaccinia virus undergoes an abortive replication in both stages of DCs and induces apoptotic cell death. Furthermore, maturation of immature DCs and consequently T cell activation are inhibited. Obstruction of DC maturation may constitute a novel mechanism by which vaccinia attempts to evade the immune response.

Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 x 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60-80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells.