Gennadiy Zelinskyy

The regulatory T-cell response during acute retroviral infection is locally defined and controls the magnitude and duration of the virus-specific cytotoxic T-cell response

Cytotoxic CD8(+) T cells control acute viremia in many viral infections. However, most viruses that establish chronic infections evade destruction by CD8(+) T cells, and regulatory T cells (Treg) are thought to be involved in this immune evasion. We have infected transgenic mice, in which Treg can be selectively depleted, with Friend retrovirus (FV) to investigate the influence of Treg on pathogen-specific CD8(+) T-cell responses in vivo. We observed that Treg expansion during acute infection was locally defined to organs with high viral loads and massive activation of virus-specific effector CD8(+) T cells. Experimental ablation of Treg resulted in a significant increase of peak cytotoxic CD8(+) T-cell responses against FV. In addition, it prevented the development of functional exhaustion of CD8(+) T cells and significantly reduced FV loads in lymphatic organs. Surprisingly, despite the massive virus-specific CD8(+) T-cell response after temporary Treg depletion, no evidence of immunopathology was found. These results demonstrate the important role of Treg in controlling acute retrovirus-specific CD8(+) T-cell responses, and suggest that temporary manipulation of Treg might be a possible therapeutic approach in chronic infectious diseases.

Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection.

Cytolytic CD8+ T cells are critical for the control of acute Friend virus (FV) infection yet they fail to completely eliminate the virus during chronic infection because they are functionally impaired by regulatory T cells (Treg). We performed a kinetic analysis of T cell responses during FV infection to determine when dysfunction of CD8+ T cells and suppressive activity of CD4+ regulatory T cells develops. At 1 week post infection, virus‐specific CD8+ T cells with effector phenotype and cytolytic potential expanded. Peak expansion was found at 12 days post infection, correlating with peak viral loads. After 2 weeks when viral loads dropped, numbers of activated CD8+ T cells started to decline. However, a population of virus‐specific CD8+ T cells with effector phenotype was still detectable subsequently, but these cells had lost their ability to produce granzymes and to degranulate cytotoxic molecules. Contemporaneous with the development of CD8+ T cell dysfunction, different CD4+ T cell populations expressing cell surface markers for Treg and the Treg‐associated transcription factor Foxp3 expanded. Transfer as well as depletion experiments indicated that regulatory CD4+ cells developed during the second week of FV infection and subsequently suppressed CD8+ T cell functions, which was associated with impaired virus clearance.

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.

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.

Regulatory T Cells Suppress Antiviral Immune Responses and Increase Viral Loads during Acute Infection with a Lymphotropic Retrovirus

In a recent paper in Science, Lund et al. [1] investigated the effect of regulatory T cells (Tregs) on the antiviral immune response to an acute herpes virus infection. Previous studies on the role of Tregs in different viral infections suggested that this T cell population suppresses antiviral effector T cell responses or local immune activation at the sites of viral replication [2],[3], which might subsequently facilitate viral immune evasion and the establishment of chronic infections [4],[5],[6]. Thus, it was quite surprising that ablation of Tregs in HSV-2-infected mice resulted in an accelerated fatal infection with increased viral loads instead of enhanced immunity to the virus [1]. In their fascinating paper, the authors showed that depletion of Tregs amplified the immune responses in draining lymph nodes at the site of infection, but at the same time delayed the entry of the immune cells into the HSV-2-infected tissue. As a general concept, Lund et al. postulated that Tregs promote immune responses in acute infections in which the pathogen replicates in non-lymphoid tissues. However, a number of viruses that induce severe human diseases, like HIV or the measles virus, replicate in primary or secondary lymphoid organs. Therefore, the question arises whether Tregs have a suppressive or promoting effect on antiviral immune responses in infections with viruses that are lymphotropic. We have used the Friend retrovirus (FV) mouse model to address this question. FV is a lymphotropic retroviral complex that replicates efficiently in the spleen of infected mice and can induce a lethal erythroleukemia [7]. It was demonstrated in previous studies that the virus induces a significant expansion of Tregs during acute infection [8]. Similar to Lund et al. [1], we used a transgenic mouse expressing the diphtheria toxin (DT) receptor and GFP under the control of the Foxp3 promoter [9] to selectively deplete Tregs by DT injection during acute FV infection. DT injection into FV-infected mice at days 0, 2, 4, 6, and 8 postinfection depleted all GFP-expressing CD4+ Foxp3+ T cells in the spleen (Figure S1A), which resulted in a slight reduction of the overall CD4+ T cell counts (Figure S1B). However, a small population of cells positive for CD4 and Foxp3 but negative for GFP remained detectable. The DT injection did not significantly influence the overall numbers of CD8+ T cells and CD19+ B cells in the spleen of infected mice, indicating that the general lymphocyte population was not affected. In addition, a recent study clearly indicates that a DT injection into mice expressing a DT receptor/GFP cassette under the control of the Foxp3 promoter leads to a specific ablation of Tregs [10], but not to the depletion of epithelial cells as previously suggested by Liu et al. [11]. Since virus-specific CD8+ T cells are the most important immune cells that control acute FV replication [12], we determined the quantity and quality of these cells in the spleen of mice experimentally ablated of Tregs. At 10 days postinfection, significantly more CD8+ T cells expressing markers of effector T cells (CD43+) were found in the spleen of Treg-depleted mice compared to non-depleted controls (Figure 1A). Treg ablation also enhanced the number of FV-specific CD8+ T cells in the spleen of infected mice, which were stained with a FVgag MHC class I tetramer [4] (Figure 1B). In addition to the magnitude of the CD8+ T cell response, we analyzed the functional properties of these T cells. After Treg ablation, the expression of the cytotoxic molecules granzyme A and B in splenic CD8+ T cells was significantly enhanced during acute FV infection (Figure 1C and 1D), suggesting an improved cytotoxic potential of these cells. To verify that the enhanced granzyme production was associated with an improved degranulation of cytotoxic molecules by CD8+ T cells, expression of the degranulation marker CD107a [13],[14] was also determined. Figure 1E shows that Treg cell depletion significantly increased the number of effector CD8+ T cells expressing CD107a. In addition, Treg ablation not only influenced the production of cytotoxic molecules by antiviral CD8+ T cells, but also influenced their cytokine response. After FV infection, subpopulations of the CD8+ T cells produced IFNγ, TNFα (Figure S2 and [15]), or low amounts of IL-2. Treg depletion increased the percentage of CD8+ T cells producing these cytokines after FV infection (Figure S2). In depleted mice most of the responding CD8+ T cells were multifunctional. Multifunctional T cells simultaneously produce two or more cytokines and have enhanced antiviral effector functions [16]. Most cytokine-producing CD8+ T cells from DT-treated DEREG mice expressed two or three different cytokines, whereas in non-depleted mice the majority of the effector CD8+ T cells expressed only one of the three cytokines measured (unpublished data). In contrast to the results from Lund et al. [1], the augmented virus-specific CD8+ T cell response decreased viral loads in the spleen more than ten times (Figure 1F). Thus, targeted depletion of Treg during acute FV infection resulted in superior control of viral replication rather than an accelerated infection as reported for HSV-2 [1]. The results clearly show that the suppressive activity of Tregs on antiviral immunity is the predominant effect in viral infections in which the primary targets of the virus are cells of the lymphoid organs. This suggests that the concept derived from the study of Lund et al. [1] might mainly apply to pathogens replicating in non-lymphoid tissue during acute infection. Therefore, inhibiting Treg responses therapeutically in infections with lymphotropic viruses might still be an interesting approach for antiviral treatment. Figure 1 Cytotoxic CD8+ T cell responses and spleen viral loads in mice infected with FV and experimentally depleted of Tregs.

Virus-Specific CD8+ T Cells Upregulate Programmed Death-1 Expression during Acute Friend Retrovirus Infection but Are Highly Cytotoxic and Control Virus Replication

It was recently reported that inhibitory molecules such as programmed death-1 (PD-1) were upregulated on CD8+ T cells during acute Friend retrovirus infection and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8+ T cells upregulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1hi and PD-1lo subsets. More PD-1lo cells produced antiviral cytokines such as IFN-γ and TNF-α, whereas more PD-1hi cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8+ T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus, direct ex vivo analyses and in vivo experiments revealed high CD8+ T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.

Combining regulatory T cell depletion and inhibitory receptor blockade improves reactivation of exhausted virus-specific CD8+ T cells and efficiently reduces chronic retroviral loads.

Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8+ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8+ T cells and expansion of regulatory T cells (Tregs) that suppress CD8+ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8+ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8+ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8+ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.

Anti-retroviral effects of type I IFN subtypes in vivo

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti‐viral efficacy in vivo. Treatment of FV‐infected mice with the IFN‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8+ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.

The Level of Friend Retrovirus Replication Determines the Cytolytic Pathway of CD8+ T-Cell-Mediated Pathogen Control

ABSTRACT Cytotoxic T cells (CTL) play a central role in the control of viral infections. Their antiviral activity can be mediated by at least two cytotoxic pathways, namely, the granule exocytosis pathway, involving perforin and granzymes, and the Fas-FasL pathway. However, the viral factor(s) that influences the selection of one or the other pathway for pathogen control is elusive. Here we investigate the role of viral replication levels in the induction and activation of CTL, including their effector potential, during acute Friend murine leukemia virus (F-MuLV) infection. F-MuLV inoculation results in a low-level infection of adult C57BL/6 mice that is enhanced about 500-fold upon coinfection with the spleen focus-forming virus (SFFV). Both the low- and high-level F-MuLV infections generated CD8+ effector T cells that were essential for the control of viral replication. However, the low-level infection induced CD8+ T cells expressing solely FasL but not the cytotoxic molecules granzymes A and B, whereas the high-level infection resulted in induction of CD8+ effector T cells secreting molecules of the granule exocytosis pathway. By using knockout mouse strains deficient in one or the other cytotoxic pathway, we found that low-level viral replication was controlled by CTL that expressed FasL but control of high-level viral replication required perforin and granzymes. Additional studies, in which F-MuLV replication was enhanced experimentally in the absence of SFFV coinfection, supported the notion that only the replication level of F-MuLV was the critical factor that determined the differential expression of cytotoxic molecules by CD8+ T cells and the pathway of CTL cytotoxicity.

TLR1/2 Ligand–Stimulated Mouse Liver Endothelial Cells Secrete IL-12 and Trigger CD8+ T Cell Immunity In Vitro

Liver sinusoidal endothelial cells (LSECs) are unique organ-resident APCs capable of Ag cross-presentation and subsequent tolerization of naive CD8+ T cells. Under certain conditions, LSECs can switch from a tolerogenic to an immunogenic state and promote the development of T cell immunity. However, little is known about the mechanisms of LSECs to induce T cell immunity. In this study, we investigated whether functional maturation of LSECs can be achieved by TLR ligand stimulation and elucidated the mechanisms involved in LSEC-induced T cell immunity. We demonstrate that pretreatment of LSECs with palmitoyl-3-cysteine-serine-lysine-4 (P3C; TLR1/2 ligand) but not poly(I:C) (TLR3 ligand) or LPS (TLR4 ligand) reverted their suppressive properties to induce T cell immunity. Importantly, P3C stimulation caused functional maturation of Ag-presenting LSECs and enabled them to activate virus-specific CD8+ T cells. The LSEC-mediated CD8+ T cell immunity was initiated by soluble mediators, one of which was IL-12 secreted at a low but sustained level after P3C stimulation. P3C stimulation did not induce programmed death ligand 1 expression on LSECs, thereby favoring T cell proliferation and activation instead of suppression. Our data suggest that LSECs undergo maturation exclusively in response to TLR1/2 ligand stimulation and that the immunological status of LSECs was dependent upon the balance between programmed death ligand 1 and IL-12 expression. These results have implications for our understanding of liver-specific tolerance and autoimmunity and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections or liver cancer.