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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.

Sepsis, an amplified immune response to systemic infection that leads to life-threatening organ dysfunction, affects >125,000 people/day worldwide with 20% mortality. Modest therapeutic progress for sepsis has been made, in part because of the lack of therapeutic translatability between mouse-based experimental models and humans. One potential reason for this difference stems from the extensive use of immunologically naive specific pathogen-free mice in preclinical research. To address this issue, we used sequential infections with well-defined BSL-2 pathogens to establish a novel immune-experienced mouse model (specific pathogen experienced [SPexp]) to determine the extent to which immunological experience and/or inflammation influences the host capacity to respond to subsequent infections, including sepsis. Consistent with their immunological experience, SPexp inbred or outbred mice had significant changes in the composition and activation status of multiple leukocyte populations known to influence the severity of cecal ligation and puncture–induced sepsis. Importantly, by varying the timing of sepsis induction, we found the level of basal inflammation controls sepsis-induced morbidity and mortality in SPexp mice. In addition, although a beneficial role of NK cells in sepsis was recently demonstrated in specific pathogen-free mice, NK cell depletion before cecal ligation and puncture induction in SPexp mice lead to diminished mortality, suggesting NK cells may have beneficial or detrimental roles in the response to septic insult dependent on host immune status. Thus, data highlight the importance of utilizing immune-experienced models for preclinical studies to interrogate the cellular/molecular mechanism(s) that could be therapeutically exploited during severe and dysregulated infection-induced inflammatory responses, such as sepsis.

The dysregulated host response and organ damage following systemic infection that characterizes a septic event predisposes individuals to a chronic immunoparalysis state associated with severe transient lymphopenia and diminished lymphocyte function, thereby reducing long-term patient survival and quality of life. Recently, we observed lasting production of reactive oxygen species (ROS) in mice that survive sepsis. ROS production is a potent mechanism for targeting infection, but excessive ROS production can prove maladaptive by causing organ damage, impairing lymphocyte function, and promoting inflammaging, concepts paralleling sepsis-induced immunoparalysis. Notably, we observed an increased frequency of ROS-producing immature monocytes in septic hosts that was sustained for greater than 100 days postsurgery. Recent clinical trials have explored the use of vitamin C, a potent antioxidant, for treating septic patients. We observed that therapeutic vitamin C administration for sepsis limited ROS production by monocytes and reduced disease severity. Importantly, we also observed increased ROS production by immature monocytes in septic patients both at admission and ∼28 days later, suggesting a durable and conserved feature that may influence the host immune response. Thus, lasting ROS production by immature monocytes is present in septic patients, and early intervention strategies to reduce it may improve host outcomes, potentially reducing sepsis-induced immunoparalysis.

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.

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.

NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of “Ag-specific” NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains that exhibit broad genetic diversity across strains. Our assessment identified and validated a putative “ideal” CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57BL/6 mice, are capable of developing enhanced NK cell–mediated protective responses to murine CMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models.

The global health burden due to sepsis and the associated cytokine storm is substantial. While early intervention has improved survival during the cytokine storm, those that survive can enter a state of chronic immunoparalysis defined by transient lymphopenia and functional deficits of surviving cells. Memory CD8 T cells provide rapid cytolysis and cytokine production following re-encounter with their cognate antigen to promote long-term immunity, and CD8 T cell impairment due to sepsis can pre-dispose individuals to re-infection. While the acute influence of sepsis on memory CD8 T cells has been characterized, if and to what extent pre-existing memory CD8 T cells recover remains unknown. Here, we observed that central memory CD8 T cells (TCM) from septic patients proliferate more than those from healthy individuals. Utilizing LCMV immune mice and a CLP model to induce sepsis, we demonstrated that TCM proliferation is associated with numerical recovery of pathogen-specific memory CD8 T cells following sepsis-induced lymphopenia. This increased proliferation leads to changes in composition of memory CD8 T cell compartment and altered tissue localization. Further, memory CD8 T cells from sepsis survivors have an altered transcriptional profile and chromatin accessibility indicating long-lasting T cell intrinsic changes. The sepsis-induced changes in the composition of the memory CD8 T cell pool and transcriptional landscape culminated in altered T cell function and reduced capacity to control L. monocytogenes infection. Thus, sepsis leads to long-term alterations in memory CD8 T cell phenotype, protective function and localization potentially changing host capacity to respond to re-infection., eLife digest A dirty cut, a nasty burn, a severe COVID infection; there are many ways for someone to develop sepsis. This life-threatening condition emerges when the immune system overreacts to a threat and ends up damaging the body. Even when patients survive, they are often left with a partially impaired immune system that cannot adequately protect against microbes and cancer; this is known as immunoparalysis. Memory CD8 T cells, a type of immune cell that is compromised by sepsis, are a long-lived population of cells that ‘remember’ previous infection or vaccination, and then react faster to prevent the same illness if the person ever encounters the same threat again. Yet it is unclear how exactly sepsis harms the function and representation of memory CD8 T cells, and the immune system in general. Jensen et al. investigated this question, first by showing that sepsis leads to a profound loss of memory CD8 T cells, but that surviving memory CD8 T cells multiply quickly – especially a subpopulation known as central memory CD8 T cells – to re-establish the memory CD8 T cell population. Since the central memory CD8 T cells proliferate better than the other memory T cells this alters the overall composition of the pool of memory CD8 T cells, with central memory cells becoming overrepresented. Further experiments revealed that this biasing toward central memory T cells, due to sepsis, created long-term changes in the distribution of memory CD8 T cells throughout the body. The way the genetic information of these cells was packaged had also been altered, as well as which genes were switched on or off. Overall, these changes reduced the ability of memory CD8 T cells to control infections. Together, these findings help to understand how immunoparalysis can emerge after sepsis, and what could be done to correct it. These findings could also be applied to other conditions – such as COVID-19 – which may cause similar long-term changes to the immune system.

We recently demonstrated how sepsis influences the subsequent development of experimental autoimmune encephalomyelitis (EAE) presented a conceptual advance in understanding the postsepsis chronic immunoparalysis state. However, the reverse scenario (autoimmunity prior to sepsis) defines a high-risk patient population whose susceptibility to sepsis remains poorly defined. In this study, we present a retrospective analysis of University of Iowa Hospital and Clinics patients demonstrating increased sepsis prevalence among multiple sclerosis (MS), relative to non-MS, patients. To interrogate how autoimmune disease influences host susceptibility to sepsis, well-established murine models of MS and sepsis and EAE and cecal ligation and puncture, respectively, were used. EAE, relative to non-EAE, mice were highly susceptible to sepsis-induced mortality with elevated cytokine storms. These results were further recapitulated in LPS and Streptococcus pneumoniae sepsis models. This work highlights both the relevance of identifying highly susceptible patient populations and expands the growing body of literature that host immune status at the time of septic insult is a potent mortality determinant.

Background The epidermal growth factor receptor (EGFR) monoclonal IgG1 antibody cetuximab is approved for first-line treatment of recurrent and metastatic (R/M) HNSCC as a part of the standard of care EXTREME regimen (platinum/5-fluorouracil/cetuximab). This regimen has relatively high response and disease control rates but is generally not curative and many patients will experience recurrent disease and/or metastasis. Therefore, there is a great need to identify predictive biomarkers for recurrence and disease progression in cetuximab-treated HNSCC patients to facilitate patient management and allow for treatment modification. The goal of this work is to assess the potential of activating interleukin-1 (IL-1) ligands (IL-1 alpha [IL-1α], IL-1 beta [IL-1β]) as predictive biomarkers of survival outcomes in HNSCC patients treated with cetuximab-based chemotherapy. Methods Baseline gene, serum and tumor expression of interleukin-1 (IL-1) ligands were analyzed from The Cancer Genome Atlas (TCGA) database or clinical trials of cetuximab-based therapies and interrogated for associations with clinical outcome data. Results High tumor gene expression of IL-1β was associated with a more favorable overall survival in cetuximab-treated HNSCC patients but not in non-cetuximab-treated patients. In HNSCC patients treated with cetuximab-based chemotherapy, higher gene and circulating levels of IL-1α and IL-1β were correlated with a more favorable progression free survival compared to patients with low or undetectable levels of IL-1 ligands. Conclusions These findings suggest that IL-1 ligands may function as predictive biomarkers for tumor response to cetuximab-based chemotherapy in HNSCC patients and warrants further investigation and validation in larger clinical studies.

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.

Summary Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory “sensing-and-alarm” function and ∼42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections.

Protective lung tissue-resident memory CD8+T cells (Trm) form after influenza A virus (IAV) infection. We show that IAV infection of mice generates CD69+CD103+and other memory CD8+T cell populations in lung-draining mediastinal lymph nodes (mLNs) from circulating naive or memory CD8+T cells. Repeated antigen exposure, mimicking seasonal IAV infections, generates quaternary memory (4M) CD8+T cells that protect mLN from viral infection better than 1M CD8+T cells. Better protection by 4M CD8+T cells associates with enhanced granzyme A/B expression and stable maintenance of mLN CD69+CD103+4M CD8+T cells, vs the steady decline of CD69+CD103+1M CD8+T cells, paralleling the durability of protective CD69+CD103+4M vs 1M in the lung after IAV infection. Coordinated upregulation in canonical Trm-associated genes occurs in circulating 4M vs 1M populations without the enrichment of canonical downregulated Trm genes. Thus, repeated antigen exposure arms circulating memory CD8+T cells with enhanced capacity to form long-lived populations of Trm that enhance control of viral infections of the mLN.

Sepsis is a life-threatening condition characterized by an acute cytokine storm followed by prolonged dysfunction of the immune system in the survivors. Post-septic lymphopenia and functional deficits of the remaining immune cells lead to increased susceptibility to secondary infections and other morbid conditions causing late death in the patients. This state of post-septic immunoparalysis may also influence disorders stemming from inappropriate or overactive immune responses, such as autoimmune and immunoinflammatory diseases, including multiple sclerosis. In addition, ongoing autoimmunity likely influences the susceptibility to and outcome of sepsis. This review article addresses the bidirectional relationship between sepsis and multiple sclerosis, with a focus on the immunologic mechanisms of the interaction and potential directions for future studies.

The persistence and maintenance of immunological memory is critical for long-term immunity generated in response to infection and vaccination. Aging of memory T cells in humans has predominantly been studied in the peripheral blood, and considerably less is known about the aging of memory T cells within tissues. Using tissues obtained from human organ donors, we investigated how tissue localization, expression of the CD103 integrin, and age influence the phenotype of tissue resident memory T cells (TRM). Using multi-parameter flow cytometry, we characterized diverse tissue sites from donors ages 9 to 93 years old and demonstrate that age-associated changes in the T cell compartment exhibit tissue- and subset- specific dynamics. CD8 T cells in blood-rich tissues spleen and lung have elevated expression of replicative senescence markers CD57 and KLRG1 relative to lung-associated lymph nodes (LLN), jejunum, and mesenteric lymph nodes (MLN). CD103+ TRM have the lowest expression of CD57 and KLRG1 across all tissue sites relative to both CD103− TRM and circulating TEM. CD8 T cells in lymphoid tissues spleen, LLN, and MLN exhibit the largest age-associated increases in expression of CD57, KLRG1, CD244, and PD-1. Together, these results demonstrate that age-associated phenotypic changes are most prominent in secondary lymphoid organs, while TRM and mucosal sites exhibit few senescent changes. Supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) and grants from NIH (P01 AI106697, U19 AI057266)

NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of ‘antigen-specific’ NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains which exhibit broad genetic diversity across strains. Our assessment identified and validated a putative ‘ideal’ CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57Bl/6 mice, are capable of developing enhanced NK cell-mediated protective responses to MCMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models. Supported by T32AI007511 (IJJ), T32AI007485 (IJJ), 5R01AI114543 (VPB), 1R35GM134880 (VPB), U01-AI150680 (DLF)

Immunization with novel mRNA vaccines against SARS-CoV-2 administered worldwide protects against COVID-19 through the generation of neutralizing antibodies directed against the viral spike protein. While immune responses generated from vaccines have been evaluated in blood, the cellular stores of memory T and B cells are maintained in tissues. Using tissues from vaccinated, but previously uninfected organ donors, we surveyed lymphoid and barrier sites for the presence of SARS-CoV-2 vaccine specific memory B and T cells. Fluorescently labeled, full length Spike and RBD probes were used to identify vaccine specific memory B cells, while vaccine specific T cells were identified by expression of activation induced markers following stimulation with peptide pools spanning the entire spike protein. All donors were confirmed negative for anti-nucleocapsid antibodies and positive for anti-spike and anti-RBD antibodies via ELISA. Spike specific T cells were identified in the blood, spleen, lung, and lung associated lymph nodes (LLN), but not bone marrow (BM). In all tissues, these vaccine specific T cells were primarily CD4 and exhibited a mixed central- and effector-memory phenotype; in the LLN the majority (up to 55%) were T-follicular helper cells (CXCR5+/PD-1+). Spike and RBD specific memory B cells were detected in blood, lung, spleen, BM, and LLN. These vaccine specific B cells in the LLN specifically expressed the tissue residency marker CD69, but only a minority had features of germinal center B cells. These results indicate that memory B and T cells generated from mRNA vaccination localize primarily to lymphoid organs, including those draining the lung, which is important for understanding their longevity and protective capacity. Supported by grants from NIH (U19 AI128949, PO1 AI106697, T32 GM007367)

Evaluation of sepsis-induced immunoparalysis has highlighted how decreased lymphocyte number/function contribute to worsened infection/cancer. Yet, an interesting contrast exists with autoimmune disease development, wherein diminishing pathogenic effectors may benefit the post-septic host. Within this framework, the impact of cecal ligation and puncture (CLP)-induced sepsis on the development of experimental autoimmune encephalomyelitis (EAE) was explored. Notably, CLP mice have delayed onset and reduced disease severity, relative to sham mice. Reduction in disease severity was associated with reduced number, but not function, of autoantigen (MOG)-specific pathogenic CD4 T cells in the CNS during disease and draining lymph node during priming. Numerical deficits of CD4 T cell effectors are associated with the loss of MOG-specific naive precursors. Critically, transfer of MOG-TCR transgenic (2D2) CD4 T cells after, but not before, CLP led to EAE disease equivalent to sham mice. Thus, broad impairment of antigenic responses, including autoantigens, is a hallmark of sepsis-induced immunoparalysis., eLife digest Sepsis is a life-threatening condition that can happen when the immune system overreacts to an infection and begins to damage tissues and organs in the body. It causes an extreme immune reaction called a cytokine storm, where the body releases uncontrolled levels of cytokines, proteins that are involved in coordinating the body’s response to infections. This in turn activates more immune cells, resulting in hyperinflammation. People who survive sepsis may have long-lasing impairments in their immune system that may leave them more vulnerable to infections or cancer. But scientists do not know exactly what causes these lasting immune problems or how to treat them. The fact that people are susceptible to cancer and infection after sepsis may offer a clue. It may suggest that the immune system is not able to attack bacteria or cancer cells. One way to explore this clue would be to test the effects of sepsis on autoimmune diseases, which cause the immune system to attack the body’s own cells. For example, in the autoimmune disease multiple sclerosis, the immune system attacks and destroys cells in the nervous system. If autoimmune disease is reduced after sepsis, it would suggest the cell-destroying abilities of the immune system are lessened. Using this approach, Jensen, Jensen et al. show that sepsis reduces the number of certain immune cells, called CD4 T cells, which are are responsible for an autoimmune attack of the central nervous system. In the experiments, mice that survived sepsis were evaluated for their ability to develop a multiple sclerosis-like disease. Mice that survived sepsis developed less severe or no autoimmune disease. After sepsis, these animals also had fewer CD4 T cells. However, when these immune cells were reinstated, the autoimmune disease emerged. The experiments help explain some of the immune system changes that occur after sepsis. Jensen, Jensen et al. suggest that rather than being completely detrimental, these changes may help to block harmful autoimmune responses. The experiments may also hint at new ways to combat autoimmune diseases by trying to replicate some of the immune-suppressing effects of sepsis. Studying the effect of sepsis on other autoimmune diseases in mice might provide more clues.

Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (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 disease course (RR-EAE). 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 RR-EAE, PLP(178–191) CD8 T cells suppress disease, while PLP(139–151) CD8 T cells lack this function. In this study, we utilized this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP(178–191) CD8 T cells vs. non-regulatory PLP(139–151) or OVA(323–339) CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation amongst activated regulatory CD8 T cells, relative to non-regulatory counterparts. This subset exhibited increased degranulation and IFNγ and IL-10 co-production. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP(178–191) CD8 T cells, but not within non-regulatory OVA(323–339) 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.

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.

Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC. Basic Protocol: Cecal ligation and puncture in the mouse.

Sepsis results in a deluge of pro- and anti-inflammatory cytokines, leading to lymphopenia and chronic immunoparalysis. Sepsis-induced long-lasting immunoparalysis is defined, in part, by impaired CD4 and CD8 αβ T cell responses in the postseptic environment. The dysfunction in T cell immunity affects naive, effector, and memory T cells and is not restricted to classical αβ T cells. Although sepsis-induced severe and transient lymphopenia is a contributory factor to diminished T cell immunity, T cell–intrinsic and -extrinsic factors/mechanisms also contribute to impaired T cell function. In this review, we summarize the current knowledge of how sepsis quantitatively and qualitatively impairs CD4 and CD8 T cell immunity of classical and nonclassical T cell subsets and discuss current therapeutic approaches being developed to boost the recovery of T cell immunity postsepsis induction.

Summary: Microbial exposures can define an individual’s basal immune state. Cohousing specific pathogen-free (SPF) mice with pet store mice, which harbor numerous infectious microbes, results in global changes to the immune system, including increased circulating phagocytes and elevated inflammatory cytokines. How these differences in the basal immune state influence the acute response to systemic infection is unclear. Cohoused mice exhibit enhanced protection from virulent Listeria monocytogenes (LM) infection, but increased morbidity and mortality to polymicrobial sepsis. Cohoused mice have more TLR2+ and TLR4+ phagocytes, enhancing recognition of microbes through pattern-recognition receptors. However, the response to a TLR2 ligand is muted in cohoused mice, whereas the response to a TLR4 ligand is greatly amplified, suggesting a basis for the distinct response to Listeria monocytogenes and sepsis. Our data illustrate how microbial exposure can enhance the immune response to unrelated challenges but also increase the risk of immunopathology from a severe cytokine storm. : Cohousing of laboratory mice with pet store animals changes the immune system and alters responsiveness to future challenges. Huggins et al. demonstrate that microbial exposure results in alterations to immune cells, serum cytokines, and microbiome composition. This study shows that cohousing alters the ability to detect pathogens through pattern-recognition receptors. Keywords: infection, Listeria monocytogenes, polymicrobial sepsis, Toll-like receptors, physiological microbial exposure, microbiome

Induction of memory CD8(+) T cells is important for controlling infections such as malaria and HIV/AIDS and for cancer immunotherapy. Accurate assessment of antigen-specific (Ag-specific) CD8(+) T cells is critical for vaccine optimization and for defining correlates of protection. However, conditions for determining Ag-specific CD8(+) T cell responses ex vivo using intracellular cytokine staining (ICS) may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influence the magnitude of Ag-specific and bystander T cell responses. Indeed, after immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ–producing memory CD8(+) T cells comprising Ag-specific and bystander responses were detected when the duration of Ag stimulation prior to addition of BFA was increased. Mechanistic analyses of virus-specific CD8(+) T cells in mice revealed that the increase in IFN-γ–producing CD8(+) T cells was due to bystander activation of Ag-experienced memory CD8(+) T cells, and correlated with the proportion of Ag-experienced CD8(+) T cells in the stimulated populations. Incubation with anti-cytokine antibodies (e.g., IL-12) improved accuracy in detecting bona fide memory CD8(+) T cell responses, suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8(+) T cells.

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.

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 (T

Background Despite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab. Methods Parental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS). Results Cetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels. Conclusions Altogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs. Electronic supplementary material The online version of this article (10.1186/s40425-019-0550-z) contains supplementary material, which is available to authorized users.

The central nervous system (CNS) is classically viewed as an immune privileged site; however, recent advances in the field highlight the importance between the interaction of the peripheral immune system and the CNS in maintaining tissue homeostasis. Tissue resident memory (Trm) CD8 T cells have been described in almost every organ and are shown to provide a first line of defense against secondary invading pathogens. Trm CD8 T cells in the CNS have been described using multiple models of brain infections, but it is unknown whether CNS infection is required for development of brain Trm populations. Here, we show that peripheral infections and immunizations generate robust antigen (Ag)-specific CD8 memory T cells in the brain that adopt a Trm phenotype with a unique brain resident signature not prevalent in other organs. Upon peripheral antibody mediated depletion of circulating memory T cells, this brain signature was enriched without reducing proportion or number of CNS residing memory CD8 T cells. 2-photon microscopy revealed peripherally-induced Trm are highly dynamic in the CNS. CD8 T cells surveyed the tissue with multiple mechanisms including moving along the blood vessels, traveling long distances in apparently random fashion or remaining stationary. Most importantly, peripherally-induced Trm cells in the CNS showed enhanced cytokine production and provided protection against brain infections by reducing both bacterial and viral loads and morbidity and mortality associated with infections. These data increase our understanding of how peripheral immunization strategies can generate brain Trm populations and will guide potential use of T cells as therapeutic strategies against CNS infections and neurological diseases.

The sepsis-induced cytokine storm leads to severe lymphopenia and reduced effector capacity of remaining/surviving cells. This results in a prolonged state of immunoparalysis, that contributes to enhanced morbidity/mortality of sepsis survivors upon secondary infection. The impact of sepsis on several lymphoid subsets has been characterized, yet its impact on NK-cells remains underappreciated–despite their critical role in controlling infection(s). Here, we observed numerical loss of NK-cells in multiple tissues after cecal-ligation-and-puncture (CLP)-induced sepsis. To elucidate the sepsis-induced lesions in surviving NK-cells, transcriptional profiles were evaluated and indicated changes consistent with impaired effector functionality. A corresponding deficit in NK-cell capacity to produce effector molecules following secondary infection and/or cytokine stimulation (IL-12,IL-18) further suggested a sepsis-induced NK-cell intrinsic impairment. To specifically probe NK-cell receptor-mediated function, the activating Ly49H receptor, that recognizes the murine cytomegalovirus (MCMV) m157 protein, served as a model receptor. Although relative expression of Ly49H receptor did not change, the number of Ly49H+ NK-cells in CLP hosts was reduced leading to impaired in vivo cytotoxicity and the capacity of NK-cells (on per-cell basis) to perform Ly49H-mediated degranulation, killing, and effector molecule production in vitro was also severely reduced. Mechanistically, Ly49H adaptor protein (DAP12) activation and clustering, assessed by TIRF microscopy, was compromised. This was further associated with diminished AKT phosphorylation and capacity to flux calcium following receptor stimulation. Importantly, DAP12 overexpression in NK-cells restored Ly49H/D receptors-mediated effector functions in CLP hosts. Finally, as a consequence of sepsis-dependent numerical and functional lesions in Ly49H+ NK-cells, host capacity to control MCMV infection was significantly impaired. Importantly, IL-2 complex (IL-2c) therapy after CLP improved numbers but not a function of NK-cells leading to enhanced immunity to MCMV challenge. Thus, the sepsis-induced immunoparalysis state includes numerical and NK-cell-intrinsic functional impairments, an instructive notion for future studies aimed in restoring NK-cell immunity in sepsis survivors., Author summary Sepsis is an exaggerated host response to infection that can initially lead to significant morbidity/mortality and a long-lasting state of immunoparalysis in sepsis survivors. Sepsis-induced immunoparalysis functionally impairs numerous lymphocyte populations, including NK-cells. However, the scope and underlying mechanisms of NK-cell impairment and the consequences for NK-cell-mediated pathogen control remain underappreciated. NK-cells contribute to early host control of pathogens through a balance of activating and inhibitory receptors, and alterations in the number and capacity of NK-cells to exert receptor-mediated immunity can lead to dramatic impairment in host control of infection. The present study defines sepsis-induced numerical and cell-intrinsic functional impairments in NK-cell response to cytokine stimulation and receptor signaling that contribute to impaired host capacity to mount NK-cell-mediated effector responses and provide protection to bacterial and/or viral pathogens. Impairments in receptor signaling were due to reduced expression of adaptor protein DAP12. Importantly, the diminished ability of NK-cells from CLP hosts to provide anti-viral (MCMV) immunity is partially restored by IL-2 complex (IL-2c) therapy, which increased the number, but not function, of protective Ly49H+ NK-cells. Thus, these findings define sepsis-induced changes of the NK-cell compartment and provide insight into potential therapeutic interventions aimed at resolving sepsis-induced immunoparalysis in sepsis survivors.

The central nervous system (CNS) is classically viewed as an immune privileged site; however recent advances in the field highlight the importance between the interaction of the peripheral immune system and the CNS in maintaining tissue homeostasis. Tissue resident memory (Trm) CD8 T cells have been described in almost every organ and are shown to provide a first line of defense against secondary invading pathogens. Trm cell generation generally involves active infection at the site of tissue residence. Using multicolor flow cytometry, we describe an increased representation of antigen-specific (Ag) CD8 T cells in the CNS after peripheral systemic immunizations or infections. Utilizing unbiased SPADE analysis, the intravascular (IV) stain negative Ag-specific tissue residing cells isolated from the CNS were phenotypically distinct from the IV stain positive cells in the vasculature. Further characterization indicated peripherally-induced CD8 T cells residing in the brain adopted a Trm phenotype and were resistant to systemic antibody-mediated depletion. The increased representation and phenotype of CD8 T cells in the brain after systemic immunization was similarly found in the meninges but not in peripheral organs including liver or lungs. 2-photon microscopy revealed peripherally-induced Trm are highly dynamic in the brain. CD8 T cells surveyed the tissue with multiple mechanisms including moving along the blood vessels, traveling long distances in apparently random fashion or remaining stationary. Most importantly, systemically-induced Trm cells in the CNS provided protection against neurotropic challenges by reducing bacterial and viral loads and limiting morbidity and mortality associated with the infections.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of head and neck squamous cell carcinoma (HNSCC), limited to no long-term changes in overall survival are observed in HNSCC patients even though EGFR is expressed at high levels in these tumors. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity specifically via natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which is also an important mechanism of action of cetuximab. The goal of our work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab. We found that blockade of IL-1 signaling using an IL-1-receptor antagonist (IL-1RA, anakinra), neutralizing IL-1α/IL-1β antibodies, and genetic knockdown of the IL-1R all suppressed the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α enhanced HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to modulation of ADCC, as we found that IL-1 blockade significantly reduced cetuximab-mediated ADCC in vitro. Additionally, we found that HNSCC patients with high baseline circulating levels of IL-1 ligands (IL-1α, IL-1β) were significantly more likely to respond favorably to cetuximab monotherapy compared to patients with low or no baseline circulating IL-1 ligands. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Therefore, IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as an immunologic biomarker for cetuximab response. Citation Format: Madelyn M. Espinosa-Cotton, Rachel A. Dahl, Elana Fertig, Isaac Jensen, Ayana J. McLaren, Kenley Miller, Samuel N. Rodman, Sandra Schmitz, Andrean L. Simons. Interleukin-1 signaling is required for HNSCC tumor response to cetuximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5734.

Sepsis is a systemic infection that enhances host vulnerability to secondary infections normally controlled by T cells. Using CLP sepsis model, we observed that sepsis induces apoptosis of circulating memory CD8 T-cells (TCIRCM) and diminishes their effector functions, leading to impaired CD8 T-cell mediated protection to systemic pathogen re-infection. In the context of localized re-infections, tissue resident memory CD8 T-cells (TRM) provide robust protection in a variety of infectious models. TRM rapidly ‘sense’ infection in non-lymphoid tissues and ‘alarm’ the host by enhancing immune cell recruitment to the site of the infection to accelerate pathogen clearance. Here, we show that compared to pathogen-specific TCIRCM, sepsis does not invoke significant numerical decline of Vaccinia virus induced skin-TRM keeping their effector functions (e.g., Ag-dependent IFN-γ production) intact. IFN-γ-mediated recruitment of immune cells to the site of localized infection was, however, reduced in CLP hosts despite TRM maintaining their ‘sensing and alarming’ functions. The capacity of memory CD8 T-cells in the septic environment to respond to inflammatory cues and arrive to the site of secondary infection/antigen exposure remained normal suggesting T-cell-extrinsic factors contributed to the observed lesion. Mechanistically, we showed that IFN-γ produced rapidly during sepsis-induced cytokine storm leads to reduced IFN-γR1 expression on vascular endothelium. As a consequence, decreased expression of adhesion molecules and/or chemokines (VCAM1 and CXCL9) on skin endothelial cells in response to TRM-derived IFN-γ was observed, leading to sub-optimal bystander-recruitment of effector cells and increased susceptibility to pathogen re-encounter. Importantly, as visualized by intravital 2-photon microscopy, exogenous administration of CXCL9/10 was sufficient to correct sepsis-induced impairments in recruitment of effector cells at the localized site of TRM antigen recognition. Thus, sepsis has the capacity to alter skin TRM anamnestic responses without directly impacting TRM number and/or function, an observation that helps to further define the immunoparalysis phase in sepsis survivors., Author summary Infectious pathogens are generally relegated within barrier tissues; however, when infections enter the bloodstream the host enters a septic state that (when severe enough) can lead to widespread tissue damage and death. After resolution of acute stage of sepsis, patients often display enhanced susceptibility to secondary infections resulting from quantitative and qualitative alterations in the immune response. Previously, we and others have shown that sepsis dramatically reduces the number and function of memory CD8 T cells within the host, contributing to the state of immunoparalysis early after sepsis induction. The present study directly examines the impact of sepsis on tissue resident memory CD8 T cells (TRM) that are restricted to barrier tissues and provide protection to localized infections. In contrast to circulating memory T cells found within lymphoid tissues, the number and function of TRM within non-lymphoid peripheral tissue (such as the skin) was maintained during sepsis suggesting a protective niche for memory CD8 T cells within barrier tissues of septic hosts. However, the capacity of TRM to provide protection upon re-infection was severely diminished in septic hosts, which was attributed to a sepsis-induced lesion in TRM-extrinsic factors. Thus, this report supports the notion that sepsis has the capacity to influence host response to pathogen re-infection either by directly influencing memory CD8 T cell populations or by preventing other cell types to properly recognize localized pathogen-induced alarming signals delivered by resident memory CD8 T cells.