Kim J. Hasenkrug

Functional Impairment of CD8+ T Cells by Regulatory T Cells during Persistent Retroviral Infection

The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.

HIV vaccine design: insights from live attenuated SIV vaccines

The International AIDS Vaccine Initiative has established a consortium to elucidate mechanisms of protection conferred by live attenuated simian immunodeficiency virus vaccines in monkeys. Here, the strategies defining key components of the protective immune response elicited by these vaccines are discussed.

Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection.

Normal levels of CD4+ regulatory T cells are critical for the maintenance of immunological homeostasis and the prevention of autoimmune diseases. However, we now show that the expansion of CD4+ regulatory T cells in response to a chronic viral infection can lead to immunosuppression. Mice persistently infected with Friend retrovirus develop approximately twice the normal percentage of splenic CD4+ regulatory T cells and lose their ability to reject certain tumor transplants. The role of CD4+ regulatory T cells was demonstrated by the transmission of immunosuppression to uninfected mice by adoptive transfers of CD4+ T cells. CD4+ T cells from chronically infected mice were also immunosuppressive in vitro, inhibiting the generation of cytolytic T lymphocytes in mixed lymphocyte cultures. Inhibition occurred at the level of blast-cell formation through a mechanism or mechanisms involving transforming growth factor-β and the cell surface molecule CTLA-4 (CD152). These results suggest a possible explanation for HIV- and human T cell leukemia virus-I-induced immunosuppression in the absence of T cell depletion.

Apobec3 Encodes Rfv3, a Gene Influencing Neutralizing Antibody Control of Retrovirus Infection

Recovery from Friend virus 3 (Rfv3) is a single autosomal gene encoding a resistance trait that influences retroviral neutralizing antibody responses and viremia. Despite extensive research for 30 years, the molecular identity of Rfv3 has remained elusive. Here, we demonstrate that Rfv3 is encoded by Apobec3. Apobec3 maps to the same chromosome region as Rfv3 and has broad inhibitory activity against retroviruses, including HIV. Not only did genetic inactivation of Apobec3 convert Rfv3-resistant mice to a susceptible phenotype, but Apobec3 was also found to be naturally disabled by aberrant messenger RNA splicing in Rfv3-susceptible strains. The link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV and other human retroviral infections.

Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection.

Infection by live attenuated retroviruses provides excellent protection from challenge with pathogenic viruses in several animal models, but little is known about which immune effectors are necessary for protection. We examined this using adoptive transfer experiments in the Friend virus mouse model. Transfers of immune spleen cells into naive mice conferred complete protection, and transfers of purified lymphocyte subsets demonstrated that this effect required complex immune responses involving CD4+ and CD8+ T cells and also B cells. In addition, passive immunization experiments demonstrated that antibodies alone reduced virus loads but did not prevent infection. These findings may have implications for retroviral vaccine design in general.

Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points

Although chronic infections with viruses such as HIV and hepatitis C virus have been associated with regulatory T cell (Treg)-mediated suppression of virus-specific CD8+ T-cell activity, no causal relationship between Tregs and chronic viral set points has been established. Using transgenic mice in which Tregs can be selectively ablated, we now show that transient depletion of Tregs during a chronic retroviral infection allows exhausted CD8+ T cells to regain antiviral functions, including secretion of cytokines, production of cytotoxic molecules, and virus-specific cytolytic activity. Furthermore, short-term Treg ablation resulted in long-term reductions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases.

In Vitro Suppression of CD8 + T Cell Function by Friend Virus-Induced Regulatory T Cells

Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.

Suppression of Acute Anti-Friend Virus CD8+ T-Cell Responses by Coinfection with Lactate Dehydrogenase-Elevating Virus

ABSTRACT Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8+ T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8+ T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8+ T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4+ T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

CD8+ T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection

ABSTRACT Virus-specific CD8+ T cells are critical for the control of acute Friend virus (FV) infections, but are rendered impotent by CD4+ regulatory T cells during the chronic phase of infection. The current study examines this CD8+ T-cell dysfunction by analyzing the production and release of cytolytic molecules by CD8+ T cells. CD8+ T cells with an activated phenotype (CD43+) from acutely infected mice produced all three key components of lytic granules: perforin, granzyme A, and granzyme B. Furthermore, they displayed evidence of recent degranulation and in vivo cytotoxicity. In contrast, activated CD8+ T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation and poor in vivo cytotoxicity. Evidence from tetramer-positive CD8+ T cells with known virus specificity confirmed the findings from the activated subset of CD8+ T cells. Interestingly, perforin and granzyme A mRNA levels were not significantly reduced during chronic infection, indicating control at a posttranscriptional level. Granzyme B deficiency was associated with a significant decrease in mRNA levels, but posttranscriptional control also appeared to contribute to deficiency. These results demonstrate a broad impairment of cytotoxic CD8+ T-cell effector function during chronic retroviral infection and explain the inability of virus-specific CD8+ T cells to eliminate persistent virus.

Reduction of Retrovirus-Induced Immunosuppression by In Vivo Modulation of T Cells during Acute Infection

ABSTRACT Chronic infection with Friend retrovirus is associated with suppressed antitumor immune responses. In the present study we investigated whether modulation of T-cell responses during acute infection would restore antitumor immunity in persistently infected mice. T-cell modulation was done by treatments with DTA-1 anti- glucocorticoid-induced tumor necrosis factor receptor monoclonal antibodies. The DTA-1 monoclonal antibody is nondepleting and delivers costimulatory signals that both enhance the activation of effector T cells and inhibit suppression by regulatory T cells. DTA-1 therapy produced faster Th1 immune responses, significant reductions in both acute virus loads and pathology and, most importantly, long-term improvement of CD8+ T-cell-mediated antitumor responses.