Tcf1 preprograms the mobilization of glycolysis in central memory CD8+ T cells during recall responses.

The mechanisms underlying the heightened protection mediated by central memory CD8+ T (TCM) cells remain unclear. Here we show that the transcription factor Tcf1 was required in resting TCM cells to generate secondary effector CD8+ T cells and to clear pathogens during recall responses. Recall stimulation of CD8+ TCM cells caused extensive reprogramming of the transcriptome and chromatin accessibility, leading to rapid induction of glycolytic enzymes, cell cycle regulators and transcriptional regulators, including Id3. This cluster of genes did not require Tcf1 in resting CD8+ TCM cells, but depended on Tcf1 for optimal induction and chromatin opening in recall-stimulated CD8+ TCM cells. Tcf1 bound extensively to these recall-induced gene loci in resting CD8+ TCM cells and mediated chromatin interactions that positioned these genes in architectural proximity with poised enhancers. Thus, Tcf1 preprogramed a transcriptional program that supported the bioenergetic and proliferative needs of CD8+ TCM cells in case of a secondary challenge.

Peripherally induced brain tissue-resident memory CD8+ T cells mediate protection against CNS infection.

The central nervous system (CNS) is classically viewed as immune-privileged; however, recent advances highlight interactions between the peripheral immune system and CNS in controlling infections and tissue homeostasis. Tissue-resident memory (TRM) CD8+ T cells in the CNS are generated after brain infections, but it is unknown whether CNS infection is required to generate brain TRM cells. We show that peripheral infections generate antigen-specific CD8+ memory T cells in the brain that adopt a unique TRM signature. Upon depletion of circulating and perivascular memory T cells, this brain signature was enriched and the surveilling properties of brain TRM cells was revealed by intravital imaging. Notably, peripherally induced brain TRM cells showed evidence of rapid activation and enhanced cytokine production and mediated protection after brain infections. These data reveal that peripheral immunizations can generate brain TRM cells and will guide potential use of T cells as therapeutic strategies against CNS infections and neurological diseases.

The transcription factor Runx3 guards cytotoxic CD8+ effector T cells against deviation towards follicular helper T cell lineage.

Activated CD8+ T cells differentiate into cytotoxic effector (TEFF) cells that eliminate target cells. How TEFF cell identity is established and maintained is not fully understood. We found that Runx3 deficiency limited clonal expansion and impaired upregulation of cytotoxic molecules in TEFF cells. Runx3-deficient CD8+ TEFF cells aberrantly upregulated genes characteristic of follicular helper T (TFH) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFß transcription factor complex deployed H3K27me3 to Bcl6 and Tcf7 genes to suppress the TFH program. Ablating Tcf7 in Runx3-deficient CD8+ TEFF cells prevented the upregulation of TFH genes and ameliorated their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforced acquisition of cytotoxic functions and protected the cytotoxic lineage integrity by preventing TFH-lineage deviation.

Defining Parameters That Modulate Susceptibility and Protection to Respiratory Murine Coronavirus MHV1 Infection.

Patients infected with SARS-CoV-2 experience variable disease susceptibility, and patients with comorbidities such as sepsis are often hospitalized for COVID-19 complications. However, the extent to which initial infectious inoculum dose determines disease outcomes and whether this can be used for immunological priming in a genetically susceptible host has not been completely defined. We used an established SARS-like murine model in which responses to primary and/or secondary challenges with murine hepatitis virus type 1 (MHV-1) were analyzed. We compared the response to infection in genetically susceptible C3H/HeJ mice, genetically resistant C57BL/6J mice, and genetically diverse, variably susceptible outbred Swiss Webster mice. Although defined as genetically susceptible to MHV-1, C3H/HeJ mice displayed decreasing dose-dependent pathological changes in disease severity and lung infiltrate/edema, as well as lymphopenia. Importantly, an asymptomatic dose (500 PFU) was identified that yielded no measurable morbidity/mortality postinfection in C3H/HeJ mice. Polymicrobial sepsis induced via cecal ligation and puncture converted asymptomatic infections in C3H/HeJ and C57BL/6J mice to more pronounced disease, modeling the impact of sepsis as a comorbidity to ß-coronavirus infection. We then used low-dose infection as an immunological priming event in C3H/HeJ mice, which provided neutralizing Ab-dependent, but not circulating CD4/CD8 T cell-dependent, protection against a high-dose MHV-1 early rechallenge. Together, these data define how infection dose, immunological status, and comorbidities modulate outcomes of primary and secondary ß-coronavirus infections in hosts with variable susceptibility.

Sublethal whole-body irradiation induces permanent loss and dysfunction in pathogen-specific circulating memory CD8 T cell populations.

The increasing use of nuclear energy sources inevitably raises the risk of accidental or deliberate radiation exposure and associated immune dysfunction. However, the extent to which radiation exposure impacts memory CD8 T cells, potent mediators of immunity to recurring intracellular infections and malignancies, remains understudied. Using P14 CD8 T cell chimeric mice (P14 chimeras) with an lymphocytic choriomeningitis virus (LCMV) infection model, we observed that sublethal (5Gy) whole-body irradiation (WBI) induced a rapid decline in the number of naive (TN) and P14 circulating memory CD8 T cells (TCIRCM), with the former being more susceptible to radiation-induced numeric loss. While TN cell numbers rapidly recovered, as previously described, the number of P14 TCIRCM cells remained low at least 9 mo after radiation exposure. Additionally, the remaining P14 TCIRCM in irradiated hosts exhibited an inefficient transition to a central memory (CD62L+) phenotype compared to nonirradiated P14 chimeras. WBI also resulted in long-lasting T cell intrinsic deficits in memory CD8 T cells, including diminished cytokine and chemokine production along with impaired secondary expansion upon cognate Ag reencounter. Irradiated P14 chimeras displayed significantly higher bacterial burden after challenge with Listeria monocytogenes expressing the LCMV GP33-41 epitope relative to nonirradiated controls, likely due to radiation-induced numerical and functional impairments. Taken together, our findings suggest that sublethal radiation exposure caused a long-term numerical, impaired differentiation, and functional dysregulation in preexisting TCIRCM, rendering previously protected hosts susceptible to reinfection.

Sepsis leads to lasting changes in phenotype and function of naïve CD8 T cells.

Sepsis, an amplified immune response to systemic infection, is characterized by a transient cytokine storm followed by chronic immune dysfunction. Consequently, sepsis survivors are highly susceptible to newly introduced infections, suggesting sepsis can influence the function and composition of the naïve CD8 T cell pool and resulting pathogen-induced primary CD8 T cell responses. Here, we explored the extent to which sepsis induces phenotypic and functional changes within the naïve CD8 T cell pool. To interrogate this, the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis was used. In normal, non-septic mice, we show type-I interferon (IFN I)-mediated signaling plays an important role in driving the phenotypic and functional heterogeneity in the naïve CD8 T cell compartment leading to increased representation of Ly6C+ naïve CD8 T cells. In response to viral infection after sepsis resolution, naïve Ly6C+ CD8 T cells generated more primary effector and memory CD8 T cells with slower conversion to a central memory CD8 T cell phenotype (Tcm) than Ly6C- naïve CD8 T cells. Importantly, as a potent inducer of cytokine storm and IFN I production, sepsis leads to increased representation of Ly6C+ naïve CD8 T cells that maintained their heightened ability to respond (i.e., effector and memory CD8 T cell accumulation and cytokine production) to primary LCMV infection. Lastly, longitudinal analyses of peripheral blood samples obtained from septic patients revealed profound changes in CD8 T cell subset composition and frequency compared to healthy controls. Thus, sepsis has the capacity to alter the composition of naïve CD8 T cells, directly influencing primary CD8 T cell responses to newly introduced infections.

Sifting through CD8+ T Cell Memory.

In this issue of Immunity, Gerlach et al. (2016) describe three distinct memory CD8+ T cell subsets based upon expression of the fractalkine receptor CX3CR1. Their findings revise the paradigm of effector and central memory T cells by revealing a subset of CD8+ memory T cells defined by intermediate levels of expression of CX3CR1 that conducts tissue surveillance.

CD8+ T Cells Utilize Highly Dynamic Enhancer Repertoires and Regulatory Circuitry in Response to Infections.

Differentiation of effector and memory CD8+ T cells is accompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naive, differentiated effector, and central memory CD8+ T cells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ T cell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitry at each CD8+ T cell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ T cells.

The Calm after the Storm: Implications of Sepsis Immunoparalysis on Host Immunity.

The immunological hallmarks of sepsis include the inflammation-mediated cytokine storm, apoptosis-driven lymphopenia, and prolonged immunoparalysis. Although early clinical efforts were focused on increasing the survival of patients through the first phase, studies are now shifting attention to the long-term effects of sepsis on immune fitness in survivors. In particular, the most pertinent task is deciphering how the immune system becomes suppressed, leading to increased incidence of secondary infections. In this review, we introduce the contribution of numerical changes and functional reprogramming within innate (NK cells, dendritic cells) and adaptive (T cells, B cells) immune cells on the chronic immune dysregulation in the septic murine and human host. We briefly discuss how prior immunological experience in murine models impacts sepsis severity, immune dysfunction, and clinical relevance. Finally, we dive into how comorbidities, specifically autoimmunity and cancer, can influence host susceptibility to sepsis and the associated immune dysfunction.

Inefficient Recovery of Repeatedly Stimulated Memory CD8 T Cells after Polymicrobial Sepsis Induction Leads to Changes in Memory CD8 T Cell Pool Composition.

Long-lasting sepsis-induced immunoparalysis has been principally studied in primary (1°) memory CD8 T cells; however, the impact of sepsis on memory CD8 T cells with a history of repeated cognate Ag encounters is largely unknown but important in understanding the role of sepsis in shaping the pre-existing memory CD8 T cell compartment. Higher-order memory CD8 T cells are crucial in providing immunity against common pathogens that reinfect the host or are generated by repeated vaccination. In this study, we analyzed peripheral blood from septic patients and show that memory CD8 T cells with defined Ag specificity for recurring CMV infection proliferate less than bulk populations of central memory CD8 T cells. Using TCR-transgenic T cells to generate 1° and higher-order (quaternary [4°]) memory T cells within the same host, we demonstrate that the susceptibility and loss of both memory subsets are similar after sepsis induction, and sepsis diminished Ag-dependent and -independent (bystander) functions of these memory subsets equally. Both the 1° and 4° memory T cell populations proliferated in a sepsis-induced lymphopenic environment; however, due to the intrinsic differences in baseline proliferative capacity, expression of receptors (e.g., CD127/CD122), and responsiveness to homeostatic cytokines, 1° memory T cells become overrepresented over time in sepsis survivors. Finally, IL-7/anti-IL-7 mAb complex treatment early after sepsis induction preferentially rescued the proliferation and accumulation of 1° memory T cells, whereas recovery of 4° memory T cells was less pronounced. Thus, inefficient recovery of repeatedly stimulated memory cells after polymicrobial sepsis induction leads to changes in memory T cell pool composition, a notion with important implications in devising strategies to recover the number and function of pre-existing memory CD8 T cells in sepsis survivors.

Cutting Edge: Influenza-Induced CD11alo Airway CD103+ Tissue Resident Memory T Cells Exhibit Compromised IFN-γ Production after In Vivo TCR Stimulation.

Although tissue resident memory T cells (TRM) in the lung confer robust protection against secondary influenza infection, their in vivo production of IFN-γ is unknown. In this study, using a mouse model, we evaluated production of IFN-γ by influenza-induced TRM (defined as CD103+) that localize to the airways or lung parenchyma. Airway TRM consist of both CD11ahi and CD11alo populations, with low CD11a expression signifying prolonged airway residence. In vitro, high-dose peptide stimulation evoked IFN-γ from most CD11ahi airway and parenchymal TRM, whereas most CD11alo airway TRM did not produce IFN-γ. In vivo production of IFN-γ was clearly detectable in CD11ahi airway and parenchymal TRM but essentially absent in CD11alo airway TRM, irrespective of airway-instilled peptide concentration or influenza reinfection. The majority of IFN-γ-producing airway TRM in vivo were CD11ahi, suggesting recent airway entry. These results question the contribution of long-term CD11alo airway TRM to influenza immunity and reinforce the importance of defining TRM tissue compartment-specific contributions to protective immunity.

A Functionally Distinct CXCR3+/IFN-γ+/IL-10+ Subset Defines Disease-Suppressive Myelin-Specific CD8 T Cells.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP178-191 CD8 T cells suppress disease, whereas PLP139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP178-191 CD8 T cells versus nonregulatory PLP139-151 or OVA323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP178-191 CD8 T cells but not within nonregulatory OVA323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.

NK Cell-Derived IL-10 Supports Host Survival during Sepsis.

The dysregulated sepsis-induced cytokine storm evoked during systemic infection consists of biphasic and interconnected pro- and anti-inflammatory responses. The contrasting inflammatory cytokine responses determine the severity of the septic event, lymphopenia, host survival, and the ensuing long-lasting immunoparalysis state. NK cells, because of their capacity to elaborate pro- (i.e., IFN-γ) and anti-inflammatory (i.e., IL-10) responses, exist at the inflection of sepsis-induced inflammatory responses. Thus, NK cell activity could be beneficial or detrimental during sepsis. In this study, we demonstrate that murine NK cells promote host survival during sepsis by limiting the scope and duration of the cytokine storm. Specifically, NK cell-derived IL-10, produced in response to IL-15, is relevant to clinical manifestations in septic patients and critical for survival during sepsis. This role of NK cells demonstrates that regulatory mechanisms of classical inflammatory cells are beneficial and critical for controlling systemic inflammation, a notion relevant for therapeutic interventions during dysregulated infection-induced inflammatory responses.

Severity of Sepsis Determines the Degree of Impairment Observed in Circulatory and Tissue-Resident Memory CD8 T Cell Populations.

Sepsis reduces the number and function of memory CD8 T cells within the host, contributing to the long-lasting state of immunoparalysis. Interestingly, the relative susceptibility of memory CD8 T cell subsets to quantitative/qualitative changes differ after cecal ligation and puncture (CLP)-induced sepsis. Compared with circulatory memory CD8 T cells (TCIRCM), moderate sepsis (0-10% mortality) does not result in numerical decline of CD8 tissue-resident memory T cells (TRM), which retain their "sensing and alarm" IFN-γ-mediated effector function. To interrogate this biologically important dichotomy, vaccinia virus-immune C57BL/6 (B6) mice containing CD8 TCIRCM and skin TRM underwent moderate or severe (∼50% mortality) sepsis. Severe sepsis led to increased morbidity and mortality characterized by increased inflammation compared with moderate CLP or sham controls. Severe CLP mice also displayed increased vascular permeability in the ears. Interestingly, skin CD103+ CD8 TRM, detected by i.v. exclusion or two-photon microscopy, underwent apoptosis and subsequent numerical loss following severe sepsis, which was not observed in mice that experienced moderate CLP or sham surgeries. Consequently, severe septic mice showed diminished CD8 T cell-mediated protection to localized skin reinfection. Finally, the relationship between severity of sepsis and demise in circulatory versus tissue-embedded memory CD8 T cell populations was confirmed by examining tumor-infiltrating and nonspecific CD8 T cells in B16 melanoma tumors. Thus, sepsis can differentially affect the presence and function of Ag-specific CD8 T cells that reside inside tissues/tumors depending on the severity of the insult, a notion with direct relevance to sepsis survivors and their ability to mount protective memory CD8 T cell-dependent responses to localized Ag re-encounter.

Cutting Edge: Antitumor Immunity by Pathogen-Specific CD8 T Cells in the Absence of Cognate Antigen Recognition.

Cancer prognosis often correlates with the number of tumor-infiltrating CD8 T cells, but many of these cells recognize pathogens that commonly infect humans. The contribution of pathogen-specific "bystander" CD8 T cells to antitumor immunity remains largely unknown. Inflammatory cytokines are sufficient for memory CD8 T cell activation and gain of effector functions, indicating tumor-derived inflammation could facilitate pathogen-specific CD8 T cells to participate in tumor control. In this study, we show in contrast to tumor-specific CD8 T cells that pathogen-specific primary memory CD8 T cells inside tumor were not able to exert their effector functions and influence tumor progression. However, infection-induced memory CD8 T cells with defined history of repeated Ag encounters (i.e., quaternary memory) showed increased sensitivity to tumor-derived inflammation that resulted in activation, gain of effector functions, and better control of tumor growth. Thus, memory CD8 T cells with heightened ability to recognize environmental inflammatory stimuli can contribute to antitumor immunity in the absence of cognate Ag recognition.

New Insights into the Immune System Using Dirty Mice.

The mouse (Mus musculus) is the dominant organism used to investigate the mechanisms behind complex immunological responses because of their genetic similarity to humans and our ability to manipulate those genetics to understand downstream function. Indeed, our knowledge of immune system development, response to infection, and ways to therapeutically manipulate the immune response to combat disease were, in large part, delineated in the mouse. Despite the power of mouse-based immunology research, the translational efficacy of many new therapies from mouse to human is far from ideal. Recent data have highlighted how the naive, neonate-like immune system of specific pathogen-free mice differs dramatically in composition and function to mice living under barrier-free conditions (i.e., "dirty" mice). In this review, we discuss major findings to date and challenges faced when using dirty mice and specific areas of immunology research that may benefit from using animals with robust and varied microbial exposure.

Cutting Edge: Polymicrobial Sepsis Has the Capacity to Reinvigorate Tumor-Infiltrating CD8 T Cells and Prolong Host Survival.

Malignancy increases sepsis incidence 10-fold and elevates sepsis-associated mortality. Advances in treatment have improved survival of cancer patients shortly after sepsis, but there is a paucity of information on how sepsis impacts cancer growth, development, and prognosis. To test this, cecal ligation and puncture surgery was performed on B16 melanoma-bearing mice to show that sepsis has detrimental effects in hosts with advanced tumors, leading to increased mortality. Surprisingly, mice experiencing cecal ligation and puncture-induced sepsis earlier during tumor development exhibited CD8 T cell-dependent attenuation of tumor growth. Sepsis-resistant CD8 tumor-infiltrating T cells showed increased in vivo activation, effector IFN-γ cytokine production, proliferation, and expression of activation/inhibitory PD-1/LAG-3 receptors because of a sepsis-induced liberation of tumor Ags. Sepsis-reinvigorated CD8 tumor-infiltrating T cells were also amenable to (anti-PD-L1/LAG-3) checkpoint blockade therapy, further prolonging cancer-associated survival in sepsis survivors. Thus, sepsis has the capacity to improve tumor-specific CD8 T cell responses, leading to better cancer prognosis and increased survival.

Sepsis-Induced State of Immunoparalysis Is Defined by Diminished CD8 T Cell-Mediated Antitumor Immunity.

Patients who survive sepsis experience long-term immunoparalysis characterized by numerical and/or functional lesions in innate and adaptive immunity that increase the host's susceptibility to secondary complications. The extent to which tumor development/growth is affected in sepsis survivors remains unknown. In this study, we show cecal ligation and puncture (CLP) surgery renders mice permissive to increased B16 melanoma growth weeks/months after sepsis induction. CD8 T cells provide partial protection in this model, and tumors from sepsis survivors had a reduced frequency of CD8 tumor-infiltrating lymphocytes (TILs) concomitant with an increased tumor burden. Interestingly, the postseptic environment reduced the number of CD8 TILs with high expression of activating/inhibitory receptors PD-1 and LAG-3 (denoted PD-1hi) that define a tumor-specific CD8 T cell subset that retain some functional capacity. Direct ex vivo analysis of CD8 TILs from CLP hosts showed decreased proliferation, IFN-γ production, and survival compared with sham counterparts. To increase the frequency and/or functional capacity of PD-1hi CD8 TILs in tumor-bearing sepsis survivors, checkpoint blockade therapy using anti-PD-L1/anti-LAG-3 mAb was administered before or after the development of sepsis-induced lesions in CD8 TILs. Checkpoint blockade did not reduce tumor growth in CLP hosts when therapy was administered after PD-1hi CD8 TILs had become reduced in frequency and/or function. However, early therapeutic intervention before lesions were observed significantly reduced tumor growth to levels seen in nonseptic hosts receiving therapy. Thus, sepsis-induced immunoparalysis is defined by diminished CD8 T cell-mediated antitumor immunity that can respond to timely checkpoint blockade, further emphasizing the importance of early cancer detection in hosts that survive sepsis.