Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

Dendritic Cells Efficiently Induce Protective Antiviral Immunity

Cytotoxic T lymphocytes (CTL) are essential for effective immunity to various viral infections. Because of the high speed of viral replication, control of viral infections imposes demanding functional and qualitative requirements on protective T-cell responses. Dendritic cells (DC) have been shown to efficiently acquire, transport, and present antigens to naive CTL in vitro and in vivo. In this study, we assessed the potential of DC, either pulsed with the lymphocytic choriomeningitis virus (LCMV)-specific peptide GP33-41 or constitutively expressing the respective epitope, to induce LCMV-specific antiviral immunity in vivo. Comparing different application routes, we found that only 100 to 1,000 DC had to reach the spleen to achieve protective levels of CTL activation. The DC-induced antiviral immune response developed rapidly and was long lasting. Already at day 2 after a single intravenous immunization with high doses of DC (1 × 10⁵ to 5 × 10⁵), mice were fully protected against LCMV challenge infection, and direct ex vivo cytotoxicity was detectable at day 4 after DC immunization. At day 60, mice were still protected against LCMV challenge infection. Importantly, priming with DC also conferred protection against infections in which the homing of CTL into peripheral organs is essential: DC-immunized mice rapidly cleared an infection with recombinant vaccinia virus-LCMV from the ovaries and eliminated LCMV from the brain, thereby avoiding lethal choriomeningitis. A comparison of DC constitutively expressing the GP33-41 epitope with exogenously peptide-pulsed DC showed that in vivo CTL priming with peptide-loaded DC is not limited by turnover of peptide-major histocompatibility complex class I complexes. We conclude that the priming of antiviral CTL responses with DC is highly efficient, rapid, and long lasting. Therefore, the use of DC should be considered as an efficient means of immunization for antiviral vaccination strategies.Dendritic cells (DC) derived from bone marrow (bmDC) are found as antigen-presenting cells (APC) scattered throughout nonlymphoid tissues. After antigen acquisition and processing, DC migrate via lymph vessels or blood to the T-cell areas of regional secondary lymphoid organs, where they present major histocompatibility complex (MHC) class I- and II-restricted peptides to naive T cells (42). The combination of these properties, i.e., antigen uptake, processing, transport, and presentation, makes DC particularly suitable as vehicles for antigens in immunotherapy. DC pulsed with tumor-derived peptides (29, 39, 46), tumor-associated proteins (16), or tumor cell RNA (9) have been shown to activate tumor-specific cytotoxic T lymphocytes (CTL) and to reduce tumor load.The high efficiency of DC in eliciting T-cell responses against infectious agents has been demonstrated with different experimental systems; e.g., DC can prime immune responses against mycobacteria (19), Borrelia burgdorferi (30), and Leishmania major (31). Induction of virus-specific CTL by DC in vitro with virus peptides or virus proteins has been shown for influenza virus (27), Sendai virus (22), and human immunodeficiency virus (26). In vivo, adoptive transfer of human immunodeficiency virus peptide-pulsed DC (43) or hepatitis B virus protein-pulsed DC (10) elicits virus-specific CTL responses. Whether these CTL responses reflect the ability to provide antiviral protection in vivo, however, has not been addressed in these studies. This aspect is particularly important in viral infections, since the high speed of replication and amplification imposes demanding quantitative and functional requirements on protective CTL responses (6, 15, 24).Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a well-characterized model system for studying CTL responses in vivo. Control of an acute virus infection is almost exclusively achieved by CD8⁺ T-cell-mediated, perforin-dependent cytotoxicity (21). Various in vivo and in vitro assays with graded sensitivities allow assessment of the efficiency and biological relevance of an immunization strategy (5, 12). In addition, several CTL immunization strategies, including peptide vaccination (1, 2, 41) and priming with recombinant vaccinia virus (17), have been well characterized for this experimental system. Therefore, the study of DC-induced immunity against LCMV infection allows an interesting qualitative comparison with other vaccination approaches.In the present study, we evaluated the potential of DC to induce CTL-mediated antiviral immunity in vivo using bone marrow-derived DC either pulsed with peptides or obtained from transgenic mice constitutively expressing the immunodominant epitope (GP33-41; hereafter referred to as GP33) of the LCMV (WE strain) glycoprotein (14a). Immunization with these cells allowed us to address the following questions. (i) How many DC are needed to induce protection against a viral infection? (ii) What are the in vivo kinetics of DC-induced CTL activation? (iii) Do DC-induced antiviral CTL emigrate to peripheral tissues to resolve viral infections, and are they protective against virus-induced immunopathology? (iv) Does turnover of peptide-MHC class I complexes influence DC immunogenicity?     

Modelling the dynamics of LCMV infection in mice: II. Compartmental structure and immunopathology

In this study, we develop a mathematical model for analysis of the compartmental aspects and immunopathology of lymphocytic choriomeningitis virus (LCMV) infection in mice. We used sets of original and published data on systemic (extrasplenic) virus distribution to estimate the parameters of virus growth and elimination for spleen and other anatomical compartments, such as the liver, kidney, thymus and lung as well as transfer rates between blood and the above organs. A mathematical model quantitatively integrating the virus distribution kinetics in the host, the specific cytotoxic T lymphocyte (CTL) response in spleen and the re-circulation of effector CTL between spleen, blood and liver is advanced to describe the CTL-mediated immunopathology (hepatitis) in mice infected with LCMV. For intravenous and "peripheral" routes of infection we examine the severity of the liver disease, as a function of the virus dose and the host's immune status characterized by the numbers of precursor and/or cytolytic effector CTL. The model is used to predict the efficacy of protection against virus persistence and disease in a localized viral infection as a function of the composition of CTL population. The modelling analysis suggests quantitative demands to CTL memory for maximal protection against a wide range of doses of infection with a primarily peripheral site of virus replication without the risk of favoring immunopathology. It specifies objectives for CTL vaccination to ensure virus elimination with minimal immunopathology vs. vaccination for disease.     

Absence of CTL responses to early viral antigens facilitates viral persistence

CD8+ T cells are crucial for the control of intracellular pathogens such as viruses and some bacteria. Using lymphocytic choriomeningitis virus (LCMV) infection of mice--the prototypic arenavirus evolutionarily closely related to human Lassa fever and South American hemorrhagic fever viruses, we have shown previously that the kinetics of Ag presentation determine immunodominance of the LCMV-specific CTL response due to progressive exhaustion of LCMV nucleoprotein (NP)-specific CTL upon increasing viral load. In this study, we provide evidence that CTL against early LCMV NP-derived epitopes are more important in virus control than those against late glycoprotein-derived epitopes. We show that mice that are tolerant to all NP-derived T cell epitopes are severely compromised in their ability to control larger inocula of LCMV, supporting our hypothesis that CD8+ T cells specific for early viral Ags play a major role in acute virus control. Thus, the kinetics with which virus-derived T cell epitopes are presented has a strong impact on the efficacy of the antiviral immunity. This aspect should be taken into consideration for the development of vaccines.     

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.     

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.     

DNA vaccination against persistent viral infection.

This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.     

Enhanced establishment of a virus carrier state in adult CD4+ T-cell-deficient mice.

CD4+ T cells play an important role in regulating the immune response; their contribution to virus clearance is variable. Mice that lack CD4+ T cells (CD4-/- mice) and are therefore unable to produce neutralizing antibodies cleared viscero-lymphotropic lymphocytic choriomeningitis virus (LCMV) strain WE when infected intravenously with a low dose (2 x 10(2) PFU) because of an effective CD8+ cytotoxic T-cell (CTL) response. In contrast, infection with a high dose (2 x 10(6) PFU) of LCMV strain WE led to expansion of antiviral CTL, which disappeared in CD4-/- mice; in contrast, CD4+ T-cell-competent mice developed antiviral memory CTL. This exhaustion of specific CTL caused viral persistence in CD4-/- mice, whereas CD4+ T-cell-competent mice eliminated the virus. After infection of CD4-/- mice with the faster-replicating LCMV strain DOCILE, abrogation of CTL response and establishment of viral persistence developed after infection with a low dose (5 x 10(2) PFU), i.e., an about 100-fold lower dose than in CD(4+)-competent control mice. These results show that absence of T help enhances establishment of an LCMV carrier state in selected situations.     

Vaccination against lymphocytic choriomeningitis virus infection in MHC class II-deficient mice

The impact of prophylactic vaccination against acute and chronic infection in a Th-deficient host has not been adequately addressed because of difficulties in generating protective immunity in the absence of CD4(+) T cell help. In this study, we demonstrated that a broad CD8(+) T cell immune response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice, irrespective of the nature of the vaccine. These results indicated that optimized vaccination may lead to efficient protection against acute viral infection, even in Th-deficient individuals, but that the duration of such immunity is limited. Nevertheless, for select immunodeficiencies in which CD4(+) T cell deficiency is incomplete or transient, these results are very encouraging.     

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.     

Immune Senescence: Relative Contributions of Age and Cytomegalovirus Infection

Immune senescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterised by impaired protective immunity and decreased efficacy of vaccines. Recent clinical, epidemiological and immunological studies suggest that Cytomegalovirus (CMV) infection may be associated with accelerated immune senescence, possibly by restricting the naïve T cell repertoire. However, direct evidence whether and how CMV-infection is implicated in immune senescence is still lacking. In this study, we have investigated whether latent mouse CMV (MCMV) infection with or without thymectomy (Tx) alters antiviral immunity of young and aged mice. After infection with lymphocytic choriomeningitis virus (LCMV) or Vaccinia virus, specific antiviral T cell responses were significantly reduced in old, old MCMV-infected and/or Tx mice compared to young mice. Importantly, control of LCMV replication was more profoundly impaired in aged MCMV-infected mice compared to age-matched MCMV-naïve or young mice. In addition, latent MCMV infection was associated with slightly reduced vaccination efficacy in old Tx mice. In contrast to the prevailing hypothesis of a CMV-mediated restriction of the naïve T cell repertoire, we found similar naïve T cell numbers in MCMV-infected and non-infected mice, whereas ageing and Tx clearly reduced the naïve T cell pool. Instead, MCMV-infection expanded the total CD8⁺ T cell pool by a massive accumulation of effector memory T cells. Based on these results, we propose a new model of increased competition between CMV-specific memory T cells and any ‘de novo’ immune response in aged individuals. In summary, our results directly demonstrate in a mouse model that latent CMV-infection impairs immunity in old age and propagates immune senescence.     

Low CD8 T-cell proliferative potential and high viral load limit the effectiveness of therapeutic vaccination

Therapeutic vaccination has the potential to boost immune responses and enhance viral control during chronic infections. However, many therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T-cell responses is not always observed. To examine these issues, we studied the impact of therapeutic vaccination, using a murine model of chronic infection with lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that therapeutic vaccination using a recombinant vaccinia virus expressing the LCMV GP33 CD8 T-cell epitope can be effective at accelerating viral control. However, mice with lower viral loads at the time of vaccination responded better to therapeutic vaccination than did those with high viral loads. Also, the proliferative potential of GP33-specific CD8 T cells from chronically infected mice was substantially lower than that of GP33-specific memory CD8 T cells from mice with immunity to LCMV, suggesting that poor T-cell expansion may be an important reason for suboptimal responses to therapeutic vaccination. Thus, our results highlight the potential positive effects of therapeutic vaccination on viral control during chronic infection but also provide evidence that a high viral load at the time of vaccination and the low proliferative potential of responding T cells are likely to limit the effectiveness of therapeutic vaccination.     

Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation

Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.     

Insufficient APC capacities of dendritic cells in gene gun-mediated DNA vaccination

Gene gun-mediated DNA immunization is a powerful mode of vaccination against infectious diseases and tumors. Many studies have identified dendritic cells (DC) as the central players in inducing immunity upon biolistic DNA vaccination; however, none of these studies directly quantify DC-mediated responses in comparison with immunity triggered by all Ag- and MHC-expressing cells. In this study we use two different approaches to decipher the relative role of DC vs other cell types in gene gun-induced immunity. First, we directly compared the immunization efficacy of different DNA constructs, which allow Ag expression ubiquitously (CMV promoter) or specifically in DC (CD11c promoter) and would encode either for soluble or membrane bound forms of Ag. Second, we immunized transgenic mice in which only DC can present MHC-restricted Ag, and directly compared the magnitudes of CTL activation with those obtained in wild-type mice. Surprisingly, our combined data suggest that, although DC-specific Ag expression is sufficient to induce humoral responses, DC alone cannot trigger optimal CD4 and CD8 T cell responses upon gene gun vaccination. Therefore, we conclude that DC alone are insufficient to mediate optimal induction of T cell immunity upon gene gun DNA vaccination and that broad Ag expression rather than DC-restricted approaches are necessary for induction of complete immune responses.     

Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.     

CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8(+) T cell-independent. In the current study, we sought to determine whether lack of CD8(+) T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8(+) CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8(+) T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8(+) T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8(+) or CD4(+) T cell-mediated tumor clearance.     

Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Absence of vasoactive intestinal peptide expression in hematopoietic cells enhances Th1 polarization and antiviral immunity in mice

Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine CMV (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice had less weight loss and better survival following mCMV infection compared with wild-type (WT) littermates. mCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-γ(+) NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive antiviral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1-polarized T cells, fewer IL-10(+) T cells, and more mCMV-M45 epitope peptide MHC class I tetramer(+) CD8(+) T cells (tetramer(+) CD8 T cells). mCMV-immune VIP-KO mice had enhanced ability to clear mCMV peptide-pulsed target cells in vivo. Enhanced antiviral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked upregulation of MHC-II and CD80 costimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, whereas programmed death-1 and programmed death ligand-1 expression were upregulated in activated CD8(+) T cells and DC, respectively, in WT mice, but not in VIP-KO mice. Because the absence of VIP in immune cells increased innate and adaptive antiviral immunity by altering costimulatory and coinhibitory pathways, selective targeting of VIP signaling represents an attractive therapeutic target to enhance antiviral immunity.     

FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7-21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.