David G. Brooks

Interleukin-10 determines viral clearance or persistence in vivo.

Persistent viral infections are a major health concern. One obstacle inhibiting the clearance of persistent infections is functional inactivation of antiviral T cells. Although such immunosuppression occurs rapidly after infection, the mechanisms that induce the loss of T-cell activity and promote viral persistence are unknown. Herein we document that persistent viral infection in mice results in a significant upregulation of interleukin (IL)-10 by antigen-presenting cells, leading to impaired T-cell responses. Genetic removal of Il10 resulted in the maintenance of robust effector T-cell responses, the rapid elimination of virus and the development of antiviral memory T-cell responses. Therapeutic administration of an antibody that blocks the IL-10 receptor restored T-cell function and eliminated viral infection. Thus, we identify a single molecule that directly induces immunosuppression leading to viral persistence and demonstrate that a therapy to neutralize IL-10 results in T-cell recovery and the prevention of viral persistence.

Blockade of chronic type I interferon signaling to control persistent LCMV infection.

INTERFER(ON)ing Persistence During persistent viral infections, a dysregulated immune response fails to control the infection. Wilson et al. (p. 202) and Teijaro et al. (p. 207; see the Perspective by Odorizzi and Wherry) show this occurs because type I interferons (IFN I), critical for early responses to viral infection, contribute to the altered immunity seen during persistent infection. Antibody blockade of IFN I signaling during chronic lymphocytic choriomeningitis virus (LCMV) in mice resulted in reduced viral titers at later stages of infection, reduced expression of inhibitory immune molecules and prevented the disruptions to secondary lymphoid organs typically observed during persistent infection with LCMV. Whether type I IFNs are also detrimental to persistent viral infection humans, such as HIV and hepatitis C virus, remains to be determined. Blockade of type I interferons leads to better control of persistent lymphocytic choriomeningitis virus infection. [Also see Perspective by Odorizzi and Wherry] Type I interferons (IFN-I) are critical for antiviral immunity; however, chronic IFN-I signaling is associated with hyperimmune activation and disease progression in persistent infections. We demonstrated in mice that blockade of IFN-I signaling diminished chronic immune activation and immune suppression, restored lymphoid tissue architecture, and increased immune parameters associated with control of virus replication, ultimately facilitating clearance of the persistent infection. The accelerated control of persistent infection induced by blocking IFN-I signaling required CD4 T cells and was associated with enhanced IFN-γ production. Thus, we demonstrated that interfering with chronic IFN-I signaling during persistent infection redirects the immune environment to enable control of infection.

IL-21 Is Required to Control Chronic Viral Infection

Controlling Chronic Viral Infections Chronic viral infections such as HIV and hepatitis B and C viruses are major public health concerns. T cell—mediated immune responses are critical for controlling viral infections. In contrast to acute infections, chronic viral infections are characterized by “exhausted” cytotoxic CD8+ T cells, cells which exhibit reduced proliferative capacity, cytokine secretion, and cytotoxicity. Treatments that reverse exhaustion result in increased viral control. Despite their exhaustion, these CD8+ T cells eventually help to control chronic infections by killing virally infected cells, and require CD4+ T cell help to do so. How do CD4+ T cells provide help to CD8+ T cells during chronic infection (see the Perspective by Johnson and Jameson)? Elsaesser et al. (p. 1569, published online 7 May), Yi et al. (p. 1572, published online 14 May), and Fröhlich et al. (p. 1576, published online 28 May) now show that the cytokine, interleukin-21 (IL-21), known to be critical for the differentiation of certain CD4+ T cell effector subsets, is an essential factor produced by CD4+ T cells that helps CD8+ T cells to control chronic lymphocytic choriomeningitis virus infection in mice. Acute and chronic infections resulted in differing amounts of IL-21 production by virus-specific CD4+ T cells. CD8+ T cells required IL-21 directly, and when CD8+ T cells were unable to signal through IL-21 or IL-21 was not available, they were reduced in number, exhibited a more exhausted phenotype, and were not able to control the virus. In contrast, the absence of IL-21–dependent signaling did not affect primary CD8+ T cell responses to acute infection or responses to a viral rechallenge, suggesting that differentiation of memory CD8+ T cells is independent of IL-21. Interleukin-21 produced by CD4+ T cells helps CD8+ T cells control viral infection in a mouse model. CD4+ and CD8+ T cell functions are rapidly aborted during chronic infection, preventing viral clearance. CD4+ T cell help is required throughout chronic infection so as to sustain CD8+ T cell responses; however, the necessary factor(s) provided by CD4+ T cells are currently unknown. Using a mouse model of chronic viral infection, we demonstrated that interleukin-21 (IL-21) is an essential component of CD4+ T cell help. In the absence of IL-21 signaling, despite elevated CD4+ T cell responses, CD8+ T cell responses are severely impaired. CD8+ T cells directly require IL-21 to avoid deletion, maintain immunity, and resolve persistent infection. Thus, IL-21 specifically sustains CD8+ T cell effector activity and provides a mechanism of CD4+ T cell help during chronic viral infection.

Viral persistence redirects CD4 T cell differentiation toward T follicular helper cells.

Persistent virus infection drives follicular T helper cell differentiation.

Effects of Prostratin on T-Cell Activation and Human Immunodeficiency Virus Latency

ABSTRACT Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.

Molecular Characterization, Reactivation, and Depletion of Latent HIV

Antiretroviral therapy is unable to eliminate HIV infection in a small, long-lived population of latently infected T cells, providing a source for renewed viral replication following cessation of therapy. Analysis of individual latently infected cells generated in the SCID-hu (Thy/Liv) mouse demonstrated no functional viral RNA produced in the latent state. Following reactivation viral expression was dramatically increased, rendering the infected cells susceptible to an anti-HIV immunotoxin. Treatment with the immunotoxin in conjunction with agents that activate virus expression without inducing cell division (IL-7 or the non-tumor-promoting phorbol ester prostratin) depleted the bulk of the latent reservoir and left uninfected cells able to respond to subsequent costimulation. We demonstrate that activation of latent virus is required for targeting by antiviral agents and provide the basis for future therapeutic strategies to eradicate the latent reservoir.

Generation of HIV latency during thymopoiesis.

The use of combination antiretroviral therapy results in a substantial reduction in viremia, a rebound of CD4+ T cells and increased survival for HIV-infected individuals. However, this treatment does not result in the total eradication of HIV. Rather, the virus is thought to remain latent in a subset of cells, where it avoids elimination by the immune system. In this state the virus is capable of reactivation of productive infection following cessation of therapy. These latently infected cells are very few in number and it has thus been difficult to determine their origin and to study the molecular nature of the latent viral genome. HIV replication is linked to cellular gene transcription and requires target cell activation. Therefore, should an activated, infected cell become transcriptionally inactive prior to cytopathic effects, the viral genome might be maintained in a latent state. We used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis, a process where previously activated cells mature towards quiescence. Moreover, we showed that these cells can be exported into the periphery where the virus remains latent until T-cell receptor stimulation, indicating that the thymus might be a source of latent HIV in humans.

Interleukin-7 Induces Expression of Latent Human Immunodeficiency Virus Type 1 with Minimal Effects on T-Cell Phenotype

ABSTRACT Latent human immunodeficiency virus type 1 (HIV-1) persists even in patients treated with antiretroviral therapy. New treatment strategies are therefore needed to eradicate this latent viral reservoir without reducing immune cell function. We characterize the interleukin-7 (IL-7)-induced stimulation of primary human T cells and thymocytes and demonstrate, using the SCID-hu model, that IL-7 induces substantial expression of latent HIV while having minimal effects on the cell phenotype. Thus, IL-7 is a viable candidate to activate expression of latent HIV and may facilitate immune clearance of latently infected cells.

Intrinsic Functional Dysregulation of CD4 T Cells Occurs Rapidly following Persistent Viral Infection

ABSTRACT Effective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.

Sterol regulatory element–binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals that mediate metabolic reprogramming remain poorly defined. Here we demonstrate an essential role for sterol regulatory element–binding proteins (SREBPs) in the acquisition of effector-cell metabolism. Without SREBP signaling, CD8+ T cells were unable to blast, which resulted in attenuated clonal expansion during viral infection. Mechanistic studies indicated that SREBPs were essential for meeting the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs were dispensable for homeostatic proliferation, which indicated a context-specific requirement for SREBPs in effector responses. Our studies provide insights into the molecular signals that underlie the metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation.