T cell exhaustion often occurs during chronic infections and prevents optimal viral control. The molecular pathways involved in T cell exhaustion, however, remain poorly understood. We demonstrate that exhausted CD8+ T cells are subject to complex layers of negative regulation due to co-expression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to 7 inhibitory receptors. Co-expression of multiple distinct inhibitory receptors correlated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by diverse inhibitory pathways was non-redundant since blockade of PD-1 and LAG-3 simultaneously in vivo synergistically improved T cell responses and reduced viral load. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of co-expressed inhibitory receptors.

chronic infection ͉ lymphocytic choriomeningitis virus ͉ T cell exhaustion ͉ PD-1 ͉ T cell memory R ecent studies have revealed an important role for the negative regulatory molecule programmed death-1 (PD-1) in T cell exhaustion during chronic viral infections (1). PD-1, a member of the CD28:CTLA-4 family of costimulatory and coinhibitory receptors, contains both immunotyrosine inhibitory motif (ITIM) and immunotyrosine switch motif (ITSM) signaling motifs, recruits the phosphatase Shp-2, and can deliver inhibitory signals (1). PD-1 interacts with two ligands, PD-ligand 1 (PD-L1), expressed by a wide variety of cells, and PD-L2, expressed mainly by macrophages and DC (1). A role for PD-1 in regulating T cell responses to chronic viral infections was identified by using lymphocytic choriomeningitis virus (LCMV) infection of mice (2). PD-1 was highly overexpressed on exhausted CD8 T cells from chronically infected animals compared with functional memory CD8 T cells from mice that had resolved acute infection. In vivo blockade of the PD-1:PD-L pathway during chronic LCMV infection led to a dramatic increase in the number and functionality of virus-specific CD8 T cells and enhanced control of infection (2). Other animal models of viral infection also supported a major role for the PD-1 pathway in regulating antiviral T cell responses (3, 4). These observations were quickly extended to primates and humans. Simian immunodeficiency virus (SIV)-, HIV-, hepatitis C virus (HCV)-, and hepatitis B virus (HBV)-specific CD8 T cells express elevated levels of PD-1 compared with CD8 T cells specific for nonpersisting pathogens such as influenza virus or vaccinia virus (5-12). In vitro blockade of PD-1:PD-L interactions reverses exhaustion of HIV-, HBV-, and HCV-specific T cells, and the proliferative capacity of these virus-specific T cell populations is dramatically improved. In light of these recent findings, PD-1 has emerged as not only a major regulator of T cell exhaustion during chronic infection, but also as an important potential therapeutic target.At least two models have been proposed for the mechanism of reversal of exhaustion by PD-1:PD-L blockade (13). In one model, PD-1:PD-L blockade reprograms all exhausted T cells, converting them to functional antiviral effector T cells. In a second model, PD-1:PD-L blockade selectively expands and/or enhances function of a subset of exhausted CD8 T cells. In the present work, we have addressed this question directly. We have identified two subsets of exhausted CD8 T cells during chronic LCMV infection in mice that differ in expression of PD-1 and CD44. One subset that expressed intermediate levels of PD-1 and high levels of CD44 (PD-1 Int CD44 Hi ) could be reinvigorated by blocking PD-1:PD-L interactions. In contrast, the second subset was PD-1 Hi but CD44Int and responded poorly to PD-1 pathway blockade. Thus, our data suggest that the cellular mechanism of reinvigorated T cell responses during chronic viral infection is by selective expansion of one subset of exhausted CD8...

Summary T cell exhaustion is common during chronic infections and can prevent optimal immunity. While recent studies have demonstrated the importance of inhibitory receptors and other pathways in T cell exhaustion, the underlying transcriptional mechanisms are unknown. Here, we define a role for the transcription factor Blimp-1 in CD8 T cell exhaustion during chronic viral infection. Blimp-1 repressed key aspects of normal memory CD8 T cell differentiation and promoted high expression of inhibitory receptors during chronic infection. These cardinal features of CD8 T cell exhaustion were corrected by conditionally deleting Blimp-1. While high expression of Blimp-1 fostered aspects of CD8 T cell exhaustion, haploinsufficiency indicated that moderate Blimp-1 expression sustained some effector function during chronic viral infection. Thus, we identify Blimp-1 as a transcriptional regulator of CD8 T cell exhaustion during chronic viral infection and propose that Blimp-1 acts as a transcriptional rheostat balancing effector function and T cell exhaustion.

T cell exhaustion and loss of memory potential occur during many chronic viral infections and cancer. We investigated when during chronic viral infection virus-specific CD8 T cells lose the potential to form memory. Virus-specific CD8 T cells from established chronic infection were unable to become memory CD8 T cells if removed from infection. However, at earlier stages of chronic infection, these virus-specific CD8 T cells retained the potential to partially or fully revert to a memory differentiation program after transfer to infection-free mice. Conversely, effector CD8 T cells primed during acute infection were not protected from exhaustion if transferred to a chronic infection. We also tested whether memory and exhausted CD8 T cells arose from different subpopulations of effector CD8 T cells and found that only the KLRG1 lo memory precursor subset gave rise to exhausted CD8 T cells. Together, these studies demonstrate that CD8 T cell exhaustion is a progressive developmental process. Early during chronic infection, the fate of virus-specific CD8 T cells remains plastic, while later, exhausted CD8 T cells become fixed in their differentiation state. Moreover, exhausted CD8 T cells arise from the memory precursor and not the terminally differentiated subset of effector CD8 T cells. These studies have implications for our understanding of senescence versus exhaustion and for therapeutic interventions during chronic infection.

Efficient maintenance of memory CD8 T cells is central to long-term protective immunity. IL-7– and IL-15–driven homeostatic proliferation is essential for long-term memory CD8 T cell persistence after acute infections. During chronic infections, however, virus-specific CD8 T cells respond poorly to these cytokines. Yet, virus-specific CD8 T cells often persist for long periods of time during chronic infections. We have addressed this apparent paradox by examining the mechanism for maintaining virus-specific CD8 T cells during chronic infection. We find that homeostatic cytokines (e.g., IL-7/15), inflammatory signals, and priming of recent thymic emigrants are not sufficient to maintain virus-specific CD8 T cells over time during chronic infection. Rather, our results demonstrate that viral peptide is required for virus-specific CD8 T cell persistence during chronic infection. Moreover, this viral antigen-dependent maintenance results in a dramatically different type of T cell division than is normally observed during memory T cell homeostasis. Rather than undergoing slow, steady homeostatic turnover during chronic viral infection, CD8 T cells undergo extensive peptide-dependent division, yet cell numbers remain relatively stable. These results indicate that antigen-specific CD8 T cell responses during persisting infection are maintained by a mechanism distinct from that after acute infection.