Context Severe sepsis is typically characterized by initial cytokine-mediated hyper-inflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting. Objectives To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression. Design, Setting, and Participants Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009–2011). Control spleens (n=29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n=20) were obtained from transplant donors or from lung cancer resections. Main Outcome Measures Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. Results The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1–195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1–40 days). Compared with controls, anti-CD3/anti-CD28–stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327–7485) pg/mL vs 418 (95% CI, 98–738) pg/mL; interferon γ, 1374 (95% CI, 550–2197) pg/mL vs 37.5 (95% CI, −5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313–5070) vs 365 (95% CI, 87–642) pg/mL; and interleukin 10, 633 (95% CI, −269 to 1534) vs 58 (95% CI, −39 to 156) pg/mL; (P<.001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells. Conclusions Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent ...

A current controversy is whether patients with sepsis progress to an immunosuppressed state. We hypothesized that reactivation of latent viruses occurred with prolonged sepsis thereby providing evidence of clinically-relevant immunosuppression and potentially providing a means to serially-monitor patients' immune status. Secondly, if viral loads are markedly elevated, they may contribute to morbidity and mortality. This study determined if reactivation of herpesviruses, polyomaviruses, and the anellovirus TTV occurred in sepsis and correlated with severity. Serial whole blood and plasma samples from 560 critically-ill septic, 161 critically-ill non-septic, and 164 healthy age-matched patients were analyzed by quantitative-polymerase-chain-reaction for cytomegalovirus (CMV), Epstein-Barr (EBV), herpes-simplex (HSV), human herpes virus-6 (HHV-6), and TTV. Polyomaviruses BK and JC were quantitated in urine. Detectable virus was analyzed with respect to secondary fungal and opportunistic bacterial infections, ICU duration, severity of illness, and survival. Patients with protracted sepsis had markedly increased frequency of detectable virus. Cumulative viral DNA detection rates in blood were: CMV (24.2%), EBV (53.2%), HSV (14.1%), HHV-6 (10.4%), and TTV (77.5%). 42.7% of septic patients had presence of two or more viruses. The 50% detection rate for herpesviruses was 5–8 days after sepsis onset. A small subgroup of septic patients had markedly elevated viral loads (>104–106 DNA copies/ml blood) for CMV, EBV, and HSV. Excluding TTV, DNAemia was uncommon in critically-ill non-septic patients and in age-matched healthy controls. Compared to septic patients without DNAemia, septic patients with viremia had increased fungal and opportunistic bacterial infections. Patients with detectable CMV in plasma had higher 90-day mortality compared to CMV-negative patients; p<0.05. Reactivation of latent viruses is common with prolonged sepsis, with frequencies similar to those occurring in transplant patients on immunosuppressive therapy and consistent with development of an immunosuppressive state. Whether reactivated latent viruses contribute to morbidity and mortality in sepsis remains unknown.

Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL-2R/STAT5 responsiveness. GITR-ligand costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire.

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-gamma1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3tau (a phosphorylated serine-binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.

CD28 costimulation regulates a wide range of cellular processes, from proliferation and survival to promoting the differentiation of specialized T-cell subsets. Since first being identified over 20 years ago, CD28 has remained a subject of intense study because of its profound consequences on T cell function and its potential for therapeutic manipulation. In this review we highlight the signaling cascades initiated by the major signaling motifs in CD28, focusing on PI-3 kinase-dependent and -independent pathways and how these are linked to specific cellular outcomes. Recent studies using gene targeted knockin mice have clarified the relative importance of these motifs on in vivo immune responses; however, much remains to be elucidated. Understanding the mechanism behind costimulation holds great potential for development of new clinically relevant reagents, a fact beginning to be realized with the advent of drugs that prevent CD28 ligation and signaling.

IntroductionSevere sepsis is characterized by an initial hyper-inflammatory response that may progress to an immune-suppressed state associated with increased susceptibility to nosocomial infection. Analysis of samples obtained from patients who died of sepsis has identified expression of specific inhibitory receptors expressed on lymphocytes that are associated with cell exhaustion. The objective of this study was to prospectively determine the pattern of expression of these receptors and immune cell function in patients with acute sepsis.MethodsTwenty-four patients with severe sepsis were enrolled within 24 hours of the onset of sepsis, as were 12 age-matched healthy controls. Peripheral blood was obtained at enrollment and again seven days later. Immune cell subsets and receptor expression were extensively characterized by quantitative flow cytometry. Lymphocyte function was assayed by stimulated cytokine secretion and proliferation assays. Results were also correlated to clinical outcome.ResultsAt the onset of severe sepsis, patients had decreased circulating innate and adaptive immune cells and elevated lymphocyte expression of receptors associated with cell activation compared to controls. Samples analyzed seven days later demonstrated increased expression of the inhibitory receptors CTLA4, TIM-3 and LAG-3 on T lymphocytes accompanied by decreased expression of the IL-7 receptor. Functional assays revealed impaired secretion of interferon γ following stimulation in vitro, which was reversible by incubation overnight in fresh media. Impaired secretion of IFNγ correlated with death or development of secondary infection.ConclusionsLymphocytes from patients with acute sepsis upregulate expression of receptors associated with cell exhaustion, which may contribute to the immune suppressed state that occurs in protracted disease. Therapy that reverses T cell exhaustion may restore immune function in immunocompromised patients and improve survival in sepsis.

Sepsis remains the leading cause of death in most intensive care units. Advances in understanding the immune response to sepsis provide the opportunity to develop more effective therapies. The immune response in sepsis can be characterized by a cytokine-mediated hyper-inflammatory phase, which most patients survive, and a subsequent immune-suppressive phase. Patients fail to eradicate invading pathogens and are susceptible to opportunistic organisms in the hypo-inflammatory phase. Many mechanisms are responsible for sepsis-induced immuno-suppression, including apoptotic depletion of immune cells, increased T regulatory and myeloid-derived suppressor cells, and cellular exhaustion. Currently in clinical trial for sepsis are granulocyte macrophage colony stimulating factor and interferon gamma, immune-therapeutic agents that boost patient immunity. Immuno-adjuvants with promise in clinically relevant animal models of sepsis include anti-programmed cell death-1 and interleukin-7. The future of immune therapy in sepsis will necessitate identification of the immunologic phase using clinical and laboratory parameters as well as biomarkers of innate and adaptive immunity.

Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/ Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-X L , or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase C, and glycogen synthase kinase 3␤, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.CD28 and T-cell receptor (TCR)-derived signals act synergistically, leading to optimal T-cell proliferation, cytokine secretion, and cell survival (for a review, see reference 32). The importance of CD28 in vivo is evidenced by impaired responses of CD28-deficient mice in a number of model systems, including allergic airway inflammation and experimental allergic encephalomyelitis (EAE) (13,34). In addition, the recent development of inhibitors of CD28 as effective therapeutics for autoimmune disease and transplant immunosuppression further emphasizes the critical role of this receptor in human disease (21,57).Despite extensive study, the biochemical mechanism(s) that mediates CD28 function remains incompletely understood. Specific motifs within the cytoplasmic tail of CD28 have been identified that trigger distinct signaling pathways. Binding and activation of Src family kinases to the distal proline motif (sequence PYAP) initiates signaling, whereas the proximal tyrosine motif (sequence YMNM) binds and activates the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) as well as other adaptor proteins, including Grb2 and GADS (12,27,28,33,42,48,51). Studies have suggested that both motifs contribute to CD28-dependent interleukin-2 (IL-2) secretion and proliferation but that the upregulation of Bcl-X L is uniquely dependent on PI3-kinase activation by the proximal tyrosine at position 170 (11,25,43). The potential for extensive overlap between pathways initiated by each motif exists, as well as overlap between CD28 and TCR-derived signals, making it unclear as to whether CD28 initiates any critical, nonredundant signaling pathway.We generated gene-targeted knock-in mice expressing either wild-type CD28 or mutations in the proximal tyr...