Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

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.

Sepsis-Induced T Cell Immunoparalysis: The Ins and Outs of Impaired T Cell Immunity.

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 αß 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 αß 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.

Cutting Edge: Elevated Leptin during Diet-Induced Obesity Reduces the Efficacy of Tumor Immunotherapy.

Various malignancies are reproducibly cured in mouse models, but most cancer immunotherapies show objective responses in a fraction of treated patients. One reason for this disconnect may be the use of young, lean mice lacking immune-altering comorbidities present in cancer patients. Although many cancer patients are overweight or obese, the effect of obesity on antitumor immunity is understudied in preclinical tumor models. We examined the effect of obesity on two immunotherapeutic models: systemic anti-CTLA-4 mAb and intratumoral delivery of a TRAIL-encoding adenovirus plus CpG. Both therapies were effective in lean mice, but neither provided a survival benefit to diet-induced obese BALB/c mice. Interestingly, tumor-bearing leptin-deficient (ob/ob) obese BALB/c mice did respond to treatment. Moreover, reducing systemic leptin with soluble leptin receptor:Fc restored the antitumor response in diet-induced obese mice. These data demonstrate the potential of targeting leptin to improve tumor immunotherapy when immune-modulating comorbidities are present.

Cutting Edge: Evidence for Nonvascular Route of Visceral Organ Immunosurveillance by T Cells.

Lymphocytes enter tissues from blood vessels through a well-characterized three-step process of extravasation. To our knowledge, nonvascular routes of lymphocyte entry have not been described. In this article, we report that Ag-experienced CD8 T cells in mice recirculate from blood through the peritoneal cavity. In the event of infection, Ag-experienced CD8 T cell subsets adhered to visceral organs, indicating potential transcapsular immunosurveillance. Focusing on the male genital tract (MGT), we observed Ag-experienced CD8 T cell migration from the peritoneal cavity directly to the infected MGT across the capsule, which was dependent on the extracellular matrix receptor CD44. We also observed that, following clearance of infection, the MGT retained functional resident memory CD8 T cells. These data suggest that recirculation through body cavities may provide T cells with opportunities for broad immunosurveillance and potential nonvascular mechanisms of entry.

Effects of Modified Parvalbumin EF-Hand Motifs on Cardiac Myocyte Contractile Function.

Cardiac gene delivery of parvalbumin (Parv), an EF-hand Ca(2+) buffer, has been studied as a therapeutic strategy for diastolic heart failure, in which slow Ca(2+) reuptake is an important contributor. A limitation of wild-type (WT) Parv is the significant trade-off between faster relaxation and blunted contraction amplitude, occurring because WT-Parv sequesters Ca(2+) too early in the cardiac cycle and prematurely truncates sarcomere shortening in the facilitation of rapid relaxation. We recently demonstrated that an E → Q substitution (ParvE101Q) at amino acid 12 of the EF-hand Ca(2+)/Mg(2+) binding loop disrupts bidentate Ca(2+) binding, reducing Ca(2+) affinity by 99-fold and increasing Mg(2+) affinity twofold. ParvE101Q caused faster relaxation and not only preserved contractility, but unexpectedly increased it above untreated myocytes. To gain mechanistic insight into the increased contractility, we focused here on amino acid 12 of the EF-hand motif. We introduced an E → D substitution (ParvE101D) at this site, which converts bidentate Ca(2+) coordination to monodentate coordination. ParvE101D decreased Ca(2+) affinity by 114-fold and increased Mg(2+) affinity 28-fold compared to WT-Parv. ParvE101D increased contraction amplitude compared to both untreated myocytes and myocytes with ParvE101Q, with limited improvement in relaxation. Additionally, ParvE101D increased spontaneous contractions after pacing stress. ParvE101D also increased Ca(2+) transient peak height and was diffusely localized around the Z-line of the sarcomere, suggesting a Ca(2+)-dependent mechanism of enhanced contractility. Sarcoplasmic reticulum Ca(2+) load was not changed with ParvE101D, but postpacing Ca(2+) waves were increased. Together, these data show that inverted Ca(2+)/Mg(2+) binding affinities of ParvE101D increase myocyte contractility through a Ca(2+)-dependent mechanism without altering sarcoplasmic reticulum Ca(2+) load and by increasing unstimulated contractions and Ca(2+) waves. ParvE101D provides mechanistic insight into how changes in the Ca(2+)/Mg(2+) binding affinities of parvalbumin's EF-hand motif alter function of cardiac myocytes. These data are informative in developing new Ca(2+) buffering strategies for the failing heart.

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding.

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction in cells, however, decreased the negative-regulatory function of Csk. Csk W47A, a substitution previously reported to block PTPN22 binding, had a secondary effect of impairing homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

Inducing Experimental Polymicrobial Sepsis by Cecal Ligation and Puncture.

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.

Polymicrobial Sepsis Impairs Antigen-Specific Memory CD4 T Cell-Mediated Immunity.

Patients who survive sepsis display prolonged immune dysfunction and heightened risk of secondary infection. CD4 T cells support a variety of cells required for protective immunity, and perturbations to the CD4 T cell compartment can decrease overall immune system fitness. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we investigated the impact of sepsis on endogenous Ag-specific memory CD4 T cells generated in C57BL/6 (B6) mice infected with attenuated Listeria monocytogenes (Lm) expressing the I-Ab-restricted 2W1S epitope (Lm-2W). The number of 2W1S-specific memory CD4 T cells was significantly reduced on day 2 after sepsis induction, but recovered by day 14. In contrast to the transient numerical change, the 2W1S-specific memory CD4 T cells displayed prolonged functional impairment after sepsis, evidenced by a reduced recall response (proliferation and effector cytokine production) after restimulation with cognate Ag. To define the extent to which the observed functional impairments in the memory CD4 T cells impacts protection to secondary infection, B6 mice were infected with attenuated Salmonella enterica-2W (Se-2W) 30 days before sham or CLP surgery, and then challenged with virulent Se-2W after surgery. Pathogen burden was significantly higher in the CLP-treated mice compared to shams. Similar reductions in functional capacity and protection were noted for the endogenous OVA323-specific memory CD4 T cell population in sepsis survivors upon Lm-OVA challenge. Our data collectively show CLP-induced sepsis alters the number and function of Ag-specific memory CD4 T cells, which contributes (in part) to the characteristic long-lasting immunoparalysis seen after sepsis.

Sepsis impedes EAE disease development and diminishes autoantigen-specific naive CD4 T cells.

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.

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.

Microbial Exposure Enhances Immunity to Pathogens Recognized by TLR2 but Increases Susceptibility to Cytokine Storm through TLR4 Sensitization.

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.

Polymicrobial Sepsis Chronic Immunoparalysis Is Defined by Diminished Ag-Specific T Cell-Dependent B Cell Responses.

Immunosuppression is one hallmark of sepsis, decreasing the host response to the primary septic pathogens and/or secondary nosocomial infections. CD4 T cells and B cells are among the array of immune cells that experience reductions in number and function during sepsis. "Help" from follicular helper (Tfh) CD4 T cells to B cells is needed for productive and protective humoral immunity, but there is a paucity of data defining the effect of sepsis on a primary CD4 T cell-dependent B cell response. Using the cecal ligation and puncture (CLP) mouse model of sepsis induction, we observed reduced antibody production in mice challenged with influenza A virus or TNP-KLH in alum early (2 days) and late (30 days) after CLP surgery compared to mice subjected to sham surgery. To better understand how these CD4 T cell-dependent B cell responses were altered by a septic event, we immunized mice with a Complete Freund's Adjuvant emulsion containing the MHC II-restricted peptide 2W1S56-68 coupled to the fluorochrome phycoerythrin (PE). Immunization with 2W1S-PE/CFA results in T cell-dependent B cell activation, giving us the ability to track defined populations of antigen-specific CD4 T cells and B cells responding to the same immunogen in the same mouse. Compared to sham mice, differentiation and class switching in PE-specific B cells were blunted in mice subjected to CLP surgery. Similarly, mice subjected to CLP had reduced expansion of 2W1S-specific T cells and Tfh differentiation after immunization. Our data suggest CLP-induced sepsis impacts humoral immunity by affecting the number and function of both antigen-specific B cells and CD4 Tfh cells, further defining the period of chronic immunoparalysis after sepsis induction.

Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A-mediated C-terminal dystrophin cleavage fragment.

Enterovirus infection can cause severe cardiomyopathy in humans. The virus-encoded 2A protease is known to cleave the cytoskeletal protein dystrophin. It is unclear, however, whether cardiomyopathy results from the loss of dystrophin or is due to the emergence of a dominant-negative dystrophin cleavage product. We show for the first time that the 2A protease-mediated carboxyl-terminal dystrophin cleavage fragment (CtermDys) is sufficient to cause marked dystrophic cardiomyopathy. The sarcolemma-localized CtermDys fragment caused myocardial fibrosis, heightened susceptibility to myocardial ischemic injury, and increased mortality during cardiac stress testing in vivo. CtermDys cardiomyopathy was more severe than in hearts completely lacking dystrophin. In vivo titration of CtermDys peptide content revealed an inverse relationship between the decay of membrane-bound CtermDys and the restoration of full-length dystrophin at the sarcolemma, in support of a physiologically relevant loss of dystrophin function in this model. CtermDys gene titration and dystrophin replacement studies further established a target threshold of 50% membrane-bound intact dystrophin necessary to prevent mice from CtermDys cardiomyopathy. Conversely, the NtermDys fragment did not compete with dystrophin and had no pathological effect. Thus, CtermDys must be localized to the sarcolemma, with intact dystrophin <50% of normal levels, to exert dominant-negative peptide-dependent cardiomyopathy. These data support a two-hit dominant-negative disease mechanism where membrane-associated CtermDys severs the link to cortical actin and inhibits both full-length dystrophin and compensatory utrophin from binding at the membrane. Therefore, membrane-bound CtermDys is a new potential translational target for virus-mediated cardiomyopathy.

LynA regulates an inflammation-sensitive signaling checkpoint in macrophages.

Clustering of receptors associated with immunoreceptor tyrosine-based activation motifs (ITAMs) initiates the macrophage antimicrobial response. ITAM receptors engage Src-family tyrosine kinases (SFKs) to initiate phagocytosis and macrophage activation. Macrophages also encounter nonpathogenic molecules that cluster receptors weakly and must tune their sensitivity to avoid inappropriate responses. To investigate this response threshold, we compared signaling in the presence and absence of receptor clustering using a small-molecule inhibitor of Csk, which increased SFK activation and produced robust membrane-proximal signaling. Surprisingly, receptor-independent SFK activation led to a downstream signaling blockade associated with rapid degradation of the SFK LynA. Inflammatory priming of macrophages upregulated LynA and promoted receptor-independent signaling. In contrast, clustering the hemi-ITAM receptor Dectin-1 induced signaling that did not require LynA or inflammatory priming. Together, the basal-state signaling checkpoint regulated by LynA expression and degradation and the signaling reorganization initiated by receptor clustering allow cells to discriminate optimally between pathogens and nonpathogens.