Frontline Science: OX40 agonistic antibody reverses immune suppression and improves survival in sepsis

Abstract

A defining feature of protracted sepsis is development of immunosuppression that is thought to be a major driving force in the morbidity and mortality associated with the syndrome. The immunosuppression that occurs in sepsis is characterized by profound apoptosis-induced depletion of CD4 and CD8 T cells and severely impaired T cell function. OX40, a member of the TNF receptor superfamily, is a positive co-stimulatory molecule expressed on activated T cells. When engaged by OX40 ligand, OX40 stimulates T cell proliferation and shifts the cellular immune phenotype toward TH1 with increased production of cytokines that are essential for control of invading pathogens. The purpose of the present study was to determine if administration of agonistic Ab to OX40 could reverse sepsis-induced immunosuppression, restore T cell function, and improve survival in a clinically relevant animal model of sepsis. The present study demonstrates that OX40 agonistic Ab reversed sepsis-induced impairment of T cell function, increased T cell IFN-γ production, increased the number of immune effector cells, and improved survival in the mouse cecal ligation and puncture model of sepsis. Importantly, OX40 agonistic Ab was not only effective in murine sepsis but also improved T effector cell function in PBMCs from patients with sepsis. The present results provide support for the use of immune adjuvants that target T cell depletion and T cell dysfunction in the therapy of sepsis-induced immunosuppression. In addition to the checkpoint inhibitors anti-PD-1 and anti-PD-L1, OX40 agonistic Ab may be a new therapeutic approach to the treatment of this highly lethal disorder.

Keywords: immunosuppression; lymphocytes, OX40, programmed cell death, sepsis.

Figure 1:. OX40 expression on CD4 T cells increases during sepsis

A.) Splenocytes of healthy and septic mice (2days and 5 days after the onset of sepsis) were harvested for the determination of OX40 expression on CD4 and CD8 T cells. OX40 expression was significantly increased on septic CD4 T cells compared to naïve healthy control cells but not on CD8 T cells. B.) CD3 and CD28 stimulation overnight induced a general significant increase of OX40 in both healthy and septic T cells versus unstimulated T cells. C.) The gating strategy to measure OX40 on lymphocytes is displayed. A rough lymphocyte gate was drawn on a Forward Scatter (FSC) by Side Scatter (SSC) plot. Cells from within the lymphocyte gate were then examined for CD4 and CD3 double positivity or CD8 and CD3 positivity. CD4+ and CD8+ T cells were then analyzed for OX40 expression. A representative plot from a septic animal is displayed. Data in panel A and B were analyzed by one-way ANOVA with multiple comparison post-tests, CD4 T cell results were significant (overall p<0.0001), Displayed are the p-values correspond to post tests.

Figure 2:. Sepsis induces a marked decrease in the DTH response that is partially restored by OX40 agonistic antibody.

A.) Mice underwent CLP surgery and were divided into 2 treatment groups. One group received 200μg OX40 agonistic antibody 6 hours after CLP and again at 48 hours. The control group received isotype control. 3 days post surgery mice were sensitized with trinitrophenyl (TNP) subcutaneously and challenged into the footpad with TNP four days post sensitization. B.) The degree of footpad swelling was quantified 24 hours post challenge and compared to the swelling of the saline injected control foot (internal control). C.) Example of a recall reaction after sensitization and challenge. A dial gauge in micrometer is used to measure the immune response. Data in Panel B was analyzed by one-way ANOVA (Krusk-Wallace) with multiple comparison post-tests, results were significant (overall p<0.0001), displayed p values correspond to the results of the multiple comparison post-tests.

Figure 3:. OX40 agonist decreases white blood cell (WBC) counts in peripheral blood.

Peripheral blood was harvested 5 days post-surgery by cardiac puncture and the different blood cell subsets were analyzed by Hemavet. White blood cells (WBCs), lymphocytes, and neutrophils were different between groups (p=0.0004, p<0.0001, and p=0.0005 respectively). Total WBC and lymphocytes were significantly decreased in blood following OX40 treatment relative to naïve and control CLP animals. OX40 agonistic antibody treatment had little effect the numbers of neutrophils and monocytes. Results were analyzed by one-way ANOVA, and multiple comparison post-tests were then performed. Displayed p-values correspond to the results of the multiple comparisons.

Figure 4:. OX40 agonist prevents the loss of splenic CD4 T cells and B cellsand increases counts of Granulocytes and Macrophages.

Splenocytes were harvested 5 days post-surgery, counted, and then stained for the diverse cell subsets and analyzed by flow cytometry. With the exception of CD8 T cells, the displayed cell subsets showed a significant increase in cell numbers following OX40 agonistic antibody treatment. Results were analyzed by One-way ANOVA, and multiple comparison post-tests were then performed. Displayed p-values correspond to the results of the multiple comparisons.

Figure 5:. OX40 agonist increases IFN-γ and TNF-α production.

Mice underwent CLP surgery and were either treated with OX40 agonist or isotype control 6 hours post surgery with an additional dose at 48 hours. Splenocytes were harvested at day 5 post surgery. A.) ELISA were performed OX40 agonistic antibody treatment increased the secretion of both IFN-γ and TNF-α relative to either naïve animals or control CLP animals. B.) OX40 agonistic antibody treatment significantly increased the number of IFN-ƴ producing cells compared to isotype control whereas the number of TNF-α expressing cells was unchanged. C.) Representative ELISpot images for the number of IFN-ƴ producing cells for isotype control and OX40 agonistic antibody treatment group are displayed. Data was analyzed by one way ANOVA (p<0.0001 overall for both A and B), displayed p-values correspond to multiple comparison post-tests.

Figure 6:. OX40 agonist improves survival in sepsis.

C57BL/6J mice were injected with 200μg OX40 agonistic antibody 6 hours after CLP and again at 48 hours. Septic control animals received isotype control. Survival was recorded for 7 days. Mice receiving OX40 agonist had a markedly improved survival. Data was analyzed by Gehan-Breslow-Wilcoxon test.

Figure 7:. OX40-ligand increases IFN-γ production in PBMCs from septic and critically ill non-septic patients.

PBMCs isolated from patient blood were plated on 96 well filter plates and stimulated with CD3 and CD28 overnight with and without OX40-ligand. 24 hours later the number of IFN-γ producing cells was determined by ELISpot and expressed as percent increase in IFN-γ production in CD3/CD28 and OX40 agonistic antibody stimulated culture over cell culture stimulated by CD3 and CD28 alone. Comparing samples with and without OX40 treatment showed that OX40 agonistic antibody treatment significantly increased the number of IFN-ƴ producing cells (p<0.0001).