CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice.

Introduction: A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a αCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results: Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Discussion: Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis.

A Novel Scoring System for Humane Endpoints in Mice with Cecal Ligation and Puncture-Induced Sepsis.

Animal-based research is essential to the study of sepsis pathophysiology, diagnostics, and therapeutics. However, animal models of sepsis are often associated with high mortality because of the difficulty in predicting imminent death based on premortem assessment of the animals. The use of validated visual scoring would allow researchers to systematically identify humane endpoints but visual approaches require high interobserver agreement for accurate results. The objective of this study was to establish a scoring system for mice undergoing cecal ligation and puncture (CLP)-induced sepsis based on 3 visual parameters: respiratory status, activity and response to stimulus (ASR), and eye appearance, with scores ranging from 0 to 3. In the first study, we evaluated interobserver agreement. Veterinary and investigative staff assessed 283 mice with CLP and had substantial to near-perfect agreement for all 3 parameters as evaluated using weighted Cohen κ statistic. The second study assessed the ability of the scoring system and temperature to predict death. The scoring system and subcutaneous transpond- ers were used to monitor C57BL/6J mice (n = 80, male and female) until death or for 7 days after CLP. Results showed that the scoring system discriminates between surviving (n = 26) and nonsurviving (n = 54) septic mice. The scoring system was accurate in predicting death, with an AUC of 0.8997. The sensitivity and specificity of the ASR parameter were 96% and 92%, respectively, and for the eye parameter were 94% and 73%. A sum of the ASR and eye scores that was 5 or more was also predictive of death. Temperature was a quantitative predictor, with sensitivity and specificity of 93% and 92%, respectively. This scoring system refines the CLP model by allowing identification of humane endpoints and avoidance of spontaneous death.

Excellence and duty: Dr. James F. Densler, the first black pediatric surgeon in the United States.

Dr. James F. Densler is a remarkable figure in pediatric surgery for both his pioneering of pediatric operations and, perhaps more importantly, shattering racial barriers. Despite growing up in the segregated South, being barred admission to his initial medical school of choice and experiencing a racially motivated bombing while in medical school, Densler persevered with the desire to fulfill the need he saw in his community, becoming the first practicing Black pediatric surgeon in the United States. His continued devotion to service later led him become a critical figure in the education of students at the newly formed Morehouse School of Medicine in Atlanta, Georgia. Through an extensive literature review and personal interviews with Dr. Densler and his colleagues, this paper outlines Dr. James Densler's life, surgical career, and the indelible mark he leaves behind.

TIGIT modulates sepsis-induced immune dysregulation in mice with preexisting malignancy.

TIGIT is a recently identified coinhibitory receptor that is upregulated in the setting of cancer and functionally contributes to the impairment of antitumor immunity. However, its role during sepsis is unknown. Because patients with cancer are 10 times more likely to die of sepsis than previously healthy (PH) patients with sepsis, we interrogated the role of TIGIT during sepsis in the context of preexistent malignancy. PH mice or cancer (CA) mice inoculated with lung carcinoma cells were made septic by cecal ligation and puncture (CLP). We found that sepsis induced TIGIT upregulation predominantly on Tregs and NK cells in both PH and CA mice. Anti-TIGIT Ab improved the 7-d survival of CA septic mice but not PH mice after CLP. Treatment of CA septic animals but not PH septic animals with anti-TIGIT mAb significantly reversed sepsis-induced loss of CD4+ T cells, CD8+ T cells, Foxp3+ Treg, and CD19+ B cells in the spleen, which was the result of decreased caspase-3+ apoptotic cells. In sum, we found that anti-TIGIT Ab reversed sepsis-induced T cell apoptosis in CA septic mice and led to a significant survival benefit, suggesting its use as a potential immunotherapy to improve outcomes in septic patients with cancer.

Anti-TIGIT differentially affects sepsis survival in immunologically experienced versus previously naive hosts.

Mounting evidence suggests that the balance of T cell costimulatory and coinhibitory signals contributes to mortality during sepsis. Here, we identified a critical role of the coinhibitory molecule T cell Ig and ITIM domain (TIGIT) in regulating sepsis mortality. Because TIGIT is significantly upregulated on memory T cells, we developed a "memory mouse" model to study the role of TIGIT during sepsis in a more physiologically relevant context. Mice received sequential pathogen exposure and developed memory T cell frequencies, similar to those observed in adult humans, and were then subjected to sepsis induction via cecal ligation and puncture. Our results show that targeting the TIGIT pathway during sepsis is fundamentally different in previously naive versus memory mice, in that αTIGIT Ab had no effect on survival in previously naive septic mice but sharply worsened survival in memory septic mice. Mechanistically, αTIGIT increased apoptosis of memory T cells, decreased T cell function, and downregulated the costimulatory receptor DNAM on memory CD8+ T cells in memory septic mice, but not in previously naive septic mice. Additionally, αTIGIT diminished Helios expression in Tregs in memory but not previously naive septic mice. These data highlight fundamental differences in the pathophysiological impact of targeting TIGIT in immunologically experienced versus previously naive hosts during sepsis.

CD28 Agonism Improves Survival in Immunologically Experienced Septic Mice via IL-10 Released by Foxp3+ Regulatory T Cells.

Immune dysregulation during sepsis is mediated by an imbalance of T cell costimulatory and coinhibitory signaling. CD28 is downregulated during sepsis and is significantly altered on memory versus naive T cells. Thus, to study the role of CD28 during sepsis in a more physiologically relevant context, we developed a "memory mouse" model in which animals are subjected to pathogen infections to generate immunologic memory, followed by sepsis induction via cecal ligation and puncture. Using this system, we show that agonistic anti-CD28 treatment resulted in worsened survival in naive septic animals but conferred a significant survival advantage in immunologically experienced septic animals. Mechanistically, this differential response was driven by the ability of CD28 agonism to elicit IL-10 production from regulatory T cells uniquely in memory but not naive mice. Moreover, elevated IL-10 released by activated regulatory T cells in memory mice inhibited sepsis-induced T cell apoptosis via the antiapoptotic protein Bcl-xL. Together, these data demonstrate that immunologic experience is an important parameter that affects sepsis pathophysiology and can fundamentally change the outcome of modulating the CD28 pathway during sepsis. This study suggests that testing therapeutic strategies in immunologically experienced hosts may be one way to increase the physiologic relevance of rodent models in sepsis research.