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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 Tregmediated 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.cytotoxic T cells | retrovirus | friend virus

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, virusspecific 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8+ T cell subsets. PD-1-expressing CD8+ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. IMPORTANCE Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients.

It was recently reported that inhibitory molecules such as PD-1 were up-regulated 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 up-regulated 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 anti-viral cytokines such as IFNγ and TNFα, while 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.

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BackgroundMyeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells.MethodsWe developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders.ResultsWe observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC.ConclusionsThis study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.

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.