Kirsten Richter

Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice

The PD-1–PD-L1 pathway inhibits perforin-mediated killing of PD-L1+ vascular endothelial cells by CD8+ T cells, thereby limiting vascular damage during systemic LCMV infection.

How chronic viral infections impact on antigen-specific T-cell responses

Persistent viral infections are, by definition, associated with ineffective antiviral immunity, in particular those infections caused by viruses that are highly productive and replicative (including HIV, HBV and HCV). The reasons for ineffective antiviral immunity in these types of infections are complex and manifold, and only recently a more comprehensive picture of the parameters responsible for attenuation of immune function is emerging. One reason for poor viral control in these types of infections is the functional deterioration of antiviral T‐cell responses and understanding the underlying mechanisms is of key importance. This review summarizes our current knowledge of cell‐intrinsic and cell‐extrinsic parameters that contribute to T‐cell exhaustion during chronic viral infections and discusses related implications for host survival, immunopathology, and control of infection.

On the role of the inhibitory receptor LAG-3 in acute and chronic LCMV infection

Chronic viral infections are often characterized by CD8 T-cell responses with poor cytokine secretion potential and limited expansion of the CD8 T-cell pool, collectively referred to as CD8 T-cell exhaustion. Exhaustion of lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells was shown to be partially regulated by the inhibitory receptor programmed death 1 (PD-1). Here, we demonstrate that exhausted LCMV-specific CD8 T cells also express the negative regulatory receptor lymphocyte activation gene 3 (LAG-3) which is mainly expressed on cells co-expressing the negative regulatory receptors PD-1 and Tim-3. Expression levels of LAG-3 on anti-viral CD8 T cells remain stable over short-term in vitro stimulations in presence of antigenic peptide. Nevertheless, in vitro and in vivo blockade of LAG-3 did not rescue cytokine production by virus-specific CD8 T cells and did not alter the virus titer in various organs. Likewise, chronic LCMV infection of LAG-3-/- mice led to a comparable degree of T-cell exhaustion as observed in C57BL/6 controls and to similar virus titers. Further, LAG-3 did not influence T-cell activation or cell division during chronic LCMV infection. These data suggest that even though LAG-3 is continuously up-regulated on LCMV-specific exhausted CD8 T cells, it alone does not significantly contribute to T-cell exhaustion.

Virus‐specific CD8 T cells: activation, differentiation and memory formation

CD8 T cells are pivotal for the control of many intracellular pathogens, and besides their role in immediate control of infections, CD8 T cells have the capacity to differentiate into long‐lived antigen‐independent memory CD8 T cells, at least in situations of acute and resolved infections. The population of memory cells is heterogeneous with respect to their phenotype, their anatomical localization and their functional capacities in order to afford optimal protection against secondary infections. In the past years, it has become clear that multiple in vivo parameters are involved in shaping the composition of the memory CD8 T cell population, including antigen load, duration and strength of CD8 T cell stimulation, the level of inflammation, availability of CD4 T cell help and CD8 T cell precursor frequencies. With respect to the timing when CD8 T cells are committed to become memory cells, several models have been proposed. In contrast to acute, resolved infection, the continued in vivo exposure to high levels of antigen during persistent chronic viral infection precludes the development of long‐lived antigen‐independent memory CD8 T cells and might even result in severe dysfunction of virus‐specific CD8 T cells.

IL-10 suppression of NK/DC crosstalk leads to poor priming of MCMV-specific CD4 T cells and prolonged MCMV persistence.

IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10 −/− mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.

Antigen amount dictates CD8+ T-cell exhaustion during chronic viral infection irrespective of the type of antigen presenting cell

Chronic viral infections lead to CD8+ T‐cell exhaustion, characterized by impaired cytokine secretion and loss of proliferative capacity. While viral load and T‐cell dysfunction correlate, it is currently unclear whether the quality of a cell type presenting antigen determines the degree of T‐cell exhaustion or if the overall amount of antigen recognized by T cells promotes exhaustion. We found that chronic lymphocytic chorio‐meningitis virus infection led to decreased CD8+ T‐cell exhaustion in DC‐MHC class I (MHCI) mice, in which CD8+ T cells can only recognize antigen on DCs. However, this increase in CD8+ T‐cell function came at the expense of fatal immunopathology. Additional antigen recognition on nonhematopoietic cells in DC‐MHCI mice promoted T‐cell exhaustion and avoidance of immunopathology. Likewise, increased numbers of antigen‐expressing hematopoietic cells, as well as a selective elevation of the number of DCs as the only cell type presenting antigen in DC‐MHCI mice, resulted in compromised T‐cell function. These results favor a scenario in which the overall amount of antigen exposure, rather than the type of cell engaging with virus‐specific CD8+ T cells, is responsible for their functional exhaustion. Furthermore, exhaustion of virus‐specific CD8+ T cells leads to avoidance of life‐threatening immunopathology.

Macrophage and T Cell Produced IL-10 Promotes Viral Chronicity

Chronic viral infections lead to CD8+ T cell exhaustion, characterized by impaired cytokine secretion. Presence of the immune-regulatory cytokine IL-10 promotes chronicity of Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 infection, while absence of IL-10/IL-10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine producing T cells. IL-10 is produced by several cell types during LCMV infection but it is currently unclear which cellular sources are responsible for induction of viral chronicity. Here, we demonstrate that although dendritic cells produce IL-10 and overall IL-10 mRNA levels decrease significantly in absence of CD11c+ cells, absence of IL-10 produced by CD11c+ cells failed to improve the LCMV-specific T cell response and control of LCMV infection. Similarly, NK cell specific IL-10 deficiency had no positive impact on the LCMV-specific T cell response or viral control, even though high percentages of NK cells produced IL-10 at early time points after infection. Interestingly, we found markedly improved T cell responses and clearance of normally chronic LCMV Clone 13 infection when either myeloid cells or T cells lacked IL-10 production and mice depleted of monocytes/macrophages or CD4+ T cells exhibited reduced overall levels of IL-10 mRNA. These data suggest that the decision whether LCMV infection becomes chronic or can be cleared critically depends on early CD4+ T cell and monocyte/macrophage produced IL-10.

Non-neutralizing antibodies protect from chronic LCMV infection independently of activating FcγR or complement

Chronic viral infections lead to CD8+ T cell exhaustion, characterized by impaired cytokine secretion. The presence of the immune‐regulatory cytokine IL‐10 promotes chronic lymphocytic choriomeningitis virus (LCMV) Clone 13 infection in mice, whereas the absence of IL‐10/IL‐10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine‐producing T cells. However, it is currently unclear which cell populations and effector molecules are crucial to protect against chronic infection. In this study, we demonstrate that antiviral, LCMV‐binding, non‐neutralizing antibodies are needed, in addition to CD4+ and CD8+ T cells, to clear a high‐dose LCMV infection in mice, in the absence of IL‐10. The interaction between CD4+ T cells and B cells in B‐cell follicles via CD40/CD40L, in addition to class switch and/or somatic hypermutation, is crucial for viral control in the absence of IL‐10. Interestingly, transfer of LCMV‐binding non‐neutralizing antibodies protected recipients from chronic infection. In addition, viral clearance in the absence of IL‐10R signaling was independent of activating Fcγ receptors and complement. These data highlight that non‐neutralizing antibodies effectively contribute to the control of LCMV infection when present prior to infection, suggesting that the induction of neutralizing antibodies is not implicitly necessary for the generation of successful vaccines.

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8+ T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8+ T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8+ T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.

CD4+ T-cell help is required for effective CD8+ T cell-mediated resolution of acute viral hepatitis in mice.

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.