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Neutrophil responses against pathogens must be balanced between protection and immunopathology. Factors that determine these outcomes are not well-understood. In a mouse model of genital herpes simplex virus-2 (HSV-2) infection, which results in severe genital inflammation, antibody-mediated neutrophil depletion reduced disease. Comparative single-cell RNA-sequencing analysis of vaginal cells against a model of genital HSV-1 infection, which results in mild inflammation, demonstrated sustained expression of interferon-stimulated genes (ISGs) only after HSV-2 infection primarily within the neutrophil population. Both therapeutic blockade of IFNα/β receptor 1 (IFNAR1) and genetic deletion of IFNAR1 in neutrophils concomitantly decreased HSV-2 genital disease severity and vaginal IL-18 levels. Therapeutic neutralization of IL-18 also diminished genital inflammation, indicating an important role for this cytokine in promoting neutrophil-dependent immunopathology. Our study reveals that sustained type I interferon (IFN) signaling is a driver of pathogenic neutrophil responses and identifies IL-18 as a novel component of disease during genital HSV-2 infection., eLife digest Herpes simplex virus (HSV) is a human pathogen that causes genital herpes, an incurable disease that results in recurrent sores and inflammation. Infection with HSV induces a strong antiviral immune response, which results in large numbers of immune cells arriving at these lesions. But while some of these cells help to control viral replication, others might contribute to the inflammation that drives the disease. One of the first immune cells to respond to infection are neutrophils. Although neutrophils are generally protective, especially against bacteria and fungi, they have also been implicated in tissue damage and severe inflammation during viral infections. But what determines whether a neutrophil will help to fight off an infection or increase disease severity is still an open question. To investigate this, Lebratti, Lim et al. studied mice that had been infected with the genital herpes virus HSV-2, which is known to cause significant amounts of inflammation in mice. The experiments revealed that a signaling molecule called type I interferon, which is thought to be antiviral, causes neutrophils at the site of the infection to produce proteins, such as IL-18, which trigger an inflammatory reaction. Lebratti, Lim et al. found that type I interferon and IL-18 had shifting roles during the course of infection. In the early stages, both molecules had a protective effect, confirming results from previous studies. However, as the infection progressed, sustained levels of type I interferon signaling in neutrophils led to excess amounts of IL-18. Lebratti, Lim et al. discovered that blocking interferon signaling or decreasing the levels of IL-18 later during infection unexpectedly reduced the severity of the disease and resulted in less genital tissue damage. Further experiments also showed that mice infected with another genital herpes virus called HSV-1 did not experience sustained levels of type I interferon. This may explain why this virus causes less severe disease in mice. Understanding how the immune system reacts to viruses could reveal new targets for treatments of genital herpes. At the moment, there is little information about IL-18 production during genital herpes in humans. So, the next step is to see whether neutrophils behave in the same way and whether IL-18 can be detected during human disease. It is possible that the same immune components could promote disease in other infections too. If so, this work may help uncover new drug targets for other viral diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2- / - Ifnl3- / - mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1- / - mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1- / - mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens.IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1 Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

Herpes simplex virus-2 (HSV-2) and HSV-1 both can cause genital herpes, a chronic infection that establishes a latent reservoir in the nervous system. Clinically, the recurrence frequency of HSV-1 genital herpes is considerably less than HSV-2 genital herpes, which correlates with reduced neuronal infection. The factors dictating the disparate outcomes of HSV-1 and HSV-2 genital herpes are unclear. In this study, we show that vaginal infection of mice with HSV-1 leads to the rapid appearance of mature DCs in the draining lymph node, which is dependent on an early burst of NK cell-mediated IFN-γ production in the vagina that occurs after HSV-1 infection but not HSV-2 infection. Rapid DC maturation after HSV-1 infection, but not HSV-2 infection, correlates with the accelerated generation of a neuroprotective T cell response and early accumulation of IFN-γ-producing T cells at the site of infection. Depletion of T cells or loss of IFN-γ receptor (IFN-γR) expression in sensory neurons both lead to a marked loss of neuroprotection only during HSV-1, recapitulating a prominent feature of HSV-2 infection. Our experiments reveal key differences in host control of neuronal HSV-1 and HSV-2 infection after genital exposure of mice, and they define parameters of a successful immune response against genital herpes.

Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2(−)(/)(−) Ifnl3(−)(/)(−) mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1(−)(/)(−) mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1(−)(/)(−) mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens. IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1. Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

Zika virus (ZIKV), which can cause devastating disease in fetuses of infected pregnant women, can be transmitted by mosquito inoculation and sexual routes. Little is known about immune protection against sexually transmitted ZIKV. In this study, we show that previous infection through intravaginal or subcutaneous routes with a contemporary Brazilian strain of ZIKV can protect against subsequent intravaginal challenge with a homologous strain. Both routes of inoculation induced high titers of ZIKV-specific and neutralizing antibody in serum and the vaginal lumen. Virus-specific T cells were recruited to and retained in the female reproductive tract after intravaginal and subcutaneous ZIKV infection. Studies in mice with genetic or acquired deficiencies in B and/or T cells demonstrated that both lymphocyte populations redundantly protect against intravaginal challenge in ZIKV-immune animals. Passive transfer of ZIKV-immune IgG or T cells significantly limited intravaginal infection of naive mice, although antibody more effectively prevented dissemination throughout the reproductive tract. Collectively, our experiments begin to establish the immune correlates of protection against intravaginal ZIKV infection, which should inform vaccination strategies in nonpregnant and pregnant women. IMPORTANCE The recent ZIKV epidemic resulted in devastating outcomes in fetuses and may affect reproductive health. Unlike other flaviviruses, ZIKV can be spread by sexual contact as well as a mosquito vector. While previous studies have identified correlates of protection for mosquito-mediated infection, few have focused on immunity against sexual transmission. As exposure to ZIKV via mosquito bite has likely occurred to many living in areas where ZIKV is endemic, our study addresses whether this route of infection can protect against subsequent sexual exposure. We demonstrate that subcutaneous ZIKV infection can protect against subsequent vaginal infection by generating both local antiviral T cell and antibody responses. Our research begins to define the immune correlates of protection for ZIKV infection in the vagina and provides a foundation for testing ZIKV vaccines against sexual transmission.

Memory T cells are an important component of the adaptive immune response. Tissue-resident memory T cells (TRM) are a recently described subset of memory T cells that reside in peripheral tissues and are maintained independently of circulating subsets of memory T cells. Importantly, TRM are frequently found in barrier tissues that commonly serve as entry portals for pathogens such as viruses. Mounting evidence shows that TRM are superior to their circulating counterparts in conferring protective immunity against a wide range of viruses. In this review, we will discuss the role of TRM in controlling viral infection with a focus on CD8+ TRM, the factors that regulate differentiation and a potential role for TRM in future vaccine development.

SummaryEpidemiological evidence suggests that chronic infections impair immune responses to unrelated pathogens and vaccines. The underlying mechanisms, however, are unclear and distinguishing effects on priming versus development of immunological memory has been challenging. We investigated whether bystander chronic infections impact differentiation of memory CD8+ T cells, the hallmark of protective immunity against intracellular pathogens. Chronic bystander infections impaired development of memory CD8+ T cells in several mouse models and humans. These effects were independent of initial priming and were associated with chronic inflammatory signatures. Chronic inflammation negatively impacted the number of bystander CD8+ T cells and their memory development. Distinct underlying mechanisms of altered survival and differentiation were revealed with the latter regulated by the transcription factors T-bet and Blimp-1. Thus, exposure to prolonged bystander inflammation impairs the effector to memory transition. These data have relevance for immunity and vaccination during persisting infections and chronic inflammation.

Tissues such as the genital tract, skin, and lung act as barriers against invading pathogens. To protect the host, incoming microbes must be quickly and efficiently controlled by the immune system at the portal of entry. Memory is a hallmark of the adaptive immune system, which confers long-term protection and is the basis for efficacious vaccines. While the majority of existing vaccines rely on circulating antibody for protection, struggles to develop antibody-based vaccines against infections such as herpes simplex virus (HSV) and human immunodeficiency virus (HIV) have underscored the need to generate memory T cells for robust antiviral control. The circulating memory T-cell population is generally divided into two subsets: effector memory (TEM ) and central memory (TCM ). These two subsets can be distinguished by their localization, as TCM home to secondary lymphoid organs and TEM circulate through non-lymphoid tissues. More recently, studies have identified a third subset, called tissue-resident memory (TRM ) cells, based on its migratory properties. This subset is found in peripheral tissues that require expression of specific chemoattractants and homing receptors for T-cell recruitment and retention, including barrier sites such as the skin and genital tract. In this review, we categorize different tissues in the body based on patterns of memory T-cell migration and tissue residency. This review also describes the rules for TRM generation and the properties that distinguish them from circulating TEM and TCM cells. Finally, based on the failure of recent T-cell-based vaccines to provide optimal protection, we also discuss the potential role of TRM cells in vaccine design against microbes that invade through the peripheral tissues and highlight new vaccination strategies that take advantage of this newly described memory T-cell subset.

Most successful existing vaccines rely on neutralizing antibodies, which may not require specific anatomical localization of B cells. However, efficacious vaccines that rely on T cells for protection have been difficult to develop, as robust systemic memory T-cell responses do not necessarily correlate with host protection. In peripheral sites, tissue-resident memory T cells provide superior protection compared to circulating memory T cells. Here we describe a simple and non-inflammatory vaccine strategy that enables the establishment of a protective memory T-cell pool within peripheral tissue. The female genital tract, which is a portal of entry for sexually transmitted infections, is an immunologically restrictive tissue that prevents entry of activated T cells in the absence of inflammation or infection. To overcome this obstacle, we developed a vaccine strategy that we term 'prime and pull' to establish local tissue-resident memory T cells at a site of potential viral exposure. This approach relies on two steps: conventional parenteral vaccination to elicit systemic T-cell responses (prime), followed by recruitment of activated T cells by means of topical chemokine application to the restrictive genital tract (pull), where such T cells establish a long-term niche and mediate protective immunity. In mice, prime and pull protocol reduces the spread of infectious herpes simplex virus 2 into the sensory neurons and prevents development of clinical disease. These results reveal a promising vaccination strategy against herpes simplex virus 2, and potentially against other sexually transmitted infections such as human immunodeficiency virus.

The requirements for tonic T-cell receptor (TCR) signaling in CD8+ memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain–containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76–dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.

CD8(+) T cells in chronic viral infections such as HIV develop functional defects including loss of interleukin-2 (IL-2) secretion and decreased proliferative potential that are collectively termed 'exhaustion'. Exhausted T cells express increased amounts of multiple inhibitory receptors, such as programmed death-1 (PD-1), that contribute to impaired virus-specific T cell function. Although reversing PD-1 inhibition is therefore an attractive therapeutic strategy, the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 also acts by upregulating genes in exhausted T cells that impair their function. Here we analyzed gene expression profiles from HIV-specific CD8(+) T cells in individuals with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8(+) T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, whereas BATF knockdown reduced PD-1 inhibition. Silencing BATF in T cells from individuals with chronic viremia rescued HIV-specific T cell function. Thus, inhibitory receptors can cause T cell exhaustion by upregulating genes--such as BATF--that inhibit T cell function. Such genes may provide new therapeutic opportunities to improve T cell immunity to HIV.

T cell exhaustion often occurs during chronic infection and prevents optimal viral control. The molecular pathways involved in T cell exhaustion remain poorly understood. Here we show that exhausted CD8+ T cells are subject to complex layers of negative regulation resulting from the coexpression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to seven inhibitory receptors. Coexpression of multiple distinct inhibitory receptors was associated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by various inhibitory pathways was nonredundant, as blockade of the T cell inhibitory receptors PD-1 and LAG-3 simultaneously and synergistically improved T cell responses and diminished viral load in vivo. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of coexpressed inhibitory receptors.

Programmed death-1 (PD-1) regulates T cell exhaustion during chronic infections. Blocking the PD-1:PD-ligand (PD-L) pathway reinvigorates exhausted CD8 T cells. Exactly how blocking PD-1:PD-L interactions improves T cell immunity, however, remains unclear. PD-1:PD-L blockade could reprogram all exhausted T cells to become antiviral effectors. Alternatively, this blockade might selectively expand a subset of exhausted T cells. We have identified two subpopulations of exhausted CD8 T cells during chronic viral infection in mice. One subset of exhausted CD8 T cells is rescued by αPD-L1 blockade, whereas the other subset appears more terminally differentiated and responds poorly to PD-1:PD-L blockade. Blocking PD-1:PD-L interactions reduces spontaneous apoptosis and enhances expansion and protective immunity of the rescuable subset, but not the more terminally differentiated subset of exhausted CD8 T cells. These results have implications for predicting clinical responses to PD-1-based therapeutic interventions and for understanding T cell dynamics during persisting infections.

When intracellular pathogens invade mammalian hosts, naïve CD8 + T cells differentiate into cytotoxic killers, which lyse infected target cells and secrete cytokines that activate intracellular microbicides. We show that CD8 + T cells deficient in the transcription factors T-bet and eomesodermin (Eomes) fail to differentiate into functional killers required for defense against lymphocytic choriomeningitis virus. Instead, virus-specific CD8 + T cells lacking both T-bet and Eomes differentiate into an interleukin-17–secreting lineage, reminiscent of the helper T cell fate that has been implicated in autoimmunity and extracellular microbial defense. Upon viral infection, mice with T cells lacking both T-bet and Eomes develop a CD8 + T cell–dependent, progressive inflammatory and wasting syndrome characterized by multi-organ infiltration of neutrophils. T-bet and Eomes, thus, ensure that CD8 + T cells adopt an appropriate course of intracellular rather than extracellular destruction.

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.

Genital herpes is an incurable, chronic disease that affects millions of people worldwide. Not only does genital herpes cause painful, recurrent symptoms, it is also a significant risk factor for the acquisition of other sexually transmitted infections such as HIV-1. Antiviral drugs are used to treat herpes simplex virus (HSV) infection, but they cannot stop viral shedding and transmission. Thus, developing a vaccine that can prevent or clear infection will be critical in limiting the spread of disease. In this review, we outline recent studies that improve our understanding of host responses against HSV infection, discuss past clinical vaccine trials and highlight new strategies for vaccine design against genital herpes.

Clearance of primary infection often leads to the development of highly functional memory T cells capable of rapid and long-lasting protective immunity. By contrast, chronic infections can result in T cell dysfunction and poor pathogen control. In this review, we will discuss recent work that highlights two main types of T cell dysfunction during chronic infection: exhaustion of effector functions and altered memory T cell development.

Chronic infections impair immune responses to unrelated pathogens and vaccines but the underlying mechanisms are not clear. We investigated whether bystander chronic infections impact the differentiation of memory CD8 T cells. We demonstrate that diverse chronic viral and parasitic infections substantially impair both the quantity and quality of developing memory CD8 T cells, independently of initial priming. Fully-formed memory CD8 T cells were less susceptible, though not impervious, to the effects of bystander chronic infection, suggesting that the impact of inflammation on memory development is more prominent during the critical period of transitioning from effector to memory. Prolonged induction of type I IFN recapitulated the effects of chronic bystander infection on memory CD8 T cell development, though these effects were independent of IFNAR signaling on the CD8 T cells during chronic infection. However, systemic blockade of IFNAR signaling after the priming phase enhanced the survival and the numerical seeding of the developing memory CD8 T cell pool. In contrast to survival, altered CD8 T cell memory differentiation was regulated by the transcription factors Tbx21 (T-bet) and Prdm1 (Blimp-1), specifically in the post-effector phase. Thus, exposure to chronic bystander infections and inflammation impairs the transition from effector to memory. These results have relevance for immunity and vaccination during persisting infections and chronic inflammation.

Accumulating epidemiological evidence suggests that chronic infections compromise immunity against antigenically unrelated infections, but the underlying mechanisms are unclear. We investigated whether antigenically unrelated “bystander” chronic infections impacted the development of memory CD8 T cells, the hallmarks of protective immunity against intracellular pathogens. We demonstrate that chronic bystander infection and inflammation substantially impair memory CD8 T cell responses in several mouse models with similar changes in non-human primates and in chronically HCV -infected humans. Type I IFN -induced molecular pathways were specifically dysregulated in memory CD8 T cells in inflammatory environments and induction of type I IFN by chronic poly(I:C) administration similarly impaired memory CD8 T cell development and function. Chronic inflammation increased the expression of the proapoptotic protein Bim in bystander CD8 T cells, while inhibiting their IL15-induced rescue from apoptosis ex vivo. Two transcriptional regulators of T cell development, Blimp-1 and T-bet, integrated the effects of inflammatory mediators in impairing optimal memory CD8 T cell development. Thus, exposure to chronic bystander infections deregulates the normal effector to memory CD8 T cell transition. These results have direct relevance concerning current therapeutic approaches and vaccination strategies for patients with persisting infections or chronic inflammatory conditions.

Some of the world’s most pressing health concerns include sexually transmitted infections such as HIV and HSV. Thus far, vaccines against these pathogens, even those that elicit robust systemic cytotoxic responses, have been unsuccessful. The genital tract is a highly restrictive tissue that does not allow entry of activated CD8 T cells in the absence of infection or inflammation. Thus, it is possible that the failure of previous vaccination attempts reflects an inability to establish a tissue-resident population of cytotoxic T cells at the front line of infection within the genital tract. Thus, to address this issue, we have designed a novel vaccination method called ‘prime and pull’ to recruit antigen-specific effector CD8 T cells into the vaginal mucosa. After subcutaneous immunization (prime), very few effector CD8 T cells are observed in the genital tissue. However, a single topical application of the chemokines CXCL9 and CXCL10 in the vagina (pull) leads to robust recruitment of effector CD8 T cells, which is independent of CD4 T cell help. Furthermore, our ‘prime and pull’ method may generate a long-term population of memory CD8 T cells within the tissue that appears to enhance viral control after a lethal challenge. Thus, our studies suggest that ‘prime and pull’ is a promising method not only for providing protection against sexually transmitted infections, but also for directing immune responses to other restrictive microenvironments such as tumors.

Abstract 916 T cells responding to chronic infections such as HIV lose the ability to secrete cytokines or to proliferate, functions critical for control of viral replication, in a process termed exhaustion. However the molecular mechanisms of T cell exhaustion are not understood, and few therapeutic targets to reinvigorate exhausted T cells have been identified. We therefore conducted a comprehensive genomic analysis of HIV-specific CD8+ T cells to identify the mechanisms underlying defective function in T cell exhaustion. We used Affymetrix microarrays to study gene expression profiles from sorted Gag-specific tetramer+ CD8+ T cells in two cohorts of HIV-infected individuals that differed in their extent of T cell exhaustion: a) Progressors (n=24), who show chronic elevation of viral load and have defects in HIV-specific T cell cytokine secretion, proliferation and survival; and b) Controllers (n=18), who show spontaneous control of viral replication and have relatively good HIV-specific T cell function. Profiles of Gag-specific CD8+ T cells (median 21,500 cells/sample) from progressors showed marked alterations in gene expression compared with those from controllers (n=518 genes upregulated in progressors, moderated t-statistic >2.0). There was highly significant similarity at the whole-genome level between dysfunctional Gag-specific CD8+ T cells from progressors and exhausted CD8+ T cells in the mouse model of chronic LCMV infection (gene set enrichment analysis, P=4.8e−005), suggesting that T cell exhaustion is associated with an evolutionarily conserved program of gene expression. Next, we determined whether this exhausted signature was influenced by inhibitory signaling via the receptor PD-1, an inhibitory receptor known to be upregulated in expression on exhausted T cells. We developed an in vitro model of PD-1 signaling and identified a unique signature of genes upregulated by PD-1 ligation. The signature of PD-1 induced genes was highly significantly upregulated in profiles from Gag-specific CD8+ T cells in HIV progressors compared to controllers (P=5e−006), and in exhausted CD8+ T cells from the LCMV mouse model (P=2e−004). Thus the signature of T cell exhaustion in humans and mice is driven in part by the consequences of PD-1 signaling. Finally, we asked whether the genes upregulated by PD-1 in exhausted T cells directly inhibit T cell function. PD-1 ligation upregulated the transcription factor BATF in HIV-specific CD8+ T cells and in exhausted CD8+ T cells from the mouse model of LCMV infection. Enforced expression of BATF, an inhibitory member of the AP-1 family, in normal human T cells inhibited proliferation (P=0.02) and IL2 secretion (P=4.5e-05). Infection with LCMV in BATF transgenic mice resulted in marked acceleration of T cell exhaustion compared to wild-type animals, indicating that BATF represses T cell effector functions. Silencing of BATF using shRNA in primary human T cells showed that it was required for PD-1 mediated inhibition of T cell function. In summary, our results demonstrate that 1) PD-1 ligation induces a conserved transcriptional program in exhausted HIV-specific CD8+ T cells and in exhausted LCMV-specific CD8+ T cells in the mouse; 2) this transcriptional program includes the upregulation of genes such as BATF that directly inhibit T cell function. Our data suggest that BATF causes the functional defects seen in T cell exhaustion, and represents a new therapeutic target to rescue T cell function in HIV infection. Disclosures: No relevant conflicts of interest to declare.

T cell exhaustion is common during chronic infections and can prevent optimal immunity. Although 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. Although 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.