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

Abstract

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.

Keywords: T lymhpocyte; costimulation; memory T cell; regulatory T Cell; sepsis.

Figure 1

Depletion of CD8⁺, but not CD4⁺ or CD25⁺, T cells worsens the survival of CD28-agonist treated immunologically-experienced septic mice. (A) Experimental design. Immunologically experienced mice underwent CLP and received anti-CD28 Ab on days 0, 2, 4, and 6 (white syringes) and either anti-CD25, andti-CD4, or anti-CD8 on days -1 and 1 (blue syringes). (B) Immunologically experienced mice underwent CLP and received anti-CD28 Ab and either anti-CD25 (n=10) or PBS (n=10). (C) Immunologically experienced mice underwent CLP and received anti-CD28 Ab and either anti-CD4 (n=19) or PBS (n=20). (D) Immunologically experienced mice underwent CLP and received anti-CD28 Ab and either anti-CD8 (n=20) or PBS (n= 20). Mice were monitored for 7-day survival. Data are pooled from 3 independent experiments with a total of 10-20 mice/group as indicated. Mantel-Cox log-rank test was used to determine significance. A p value of less than or equal to 0.05 was considered significant.

Figure 2

αCD28-agonist treatment accrues CD44^hi memory T cells following CLP in immunologically-experienced mice. B6 Immunologically experienced mice were rendered septic by CLP. αCD28 (n=9) or PBS (n=10) was administered immediately following surgery. Mice were sacrificed 24hr after surgery, spleens were harvested, and CD8 and CD44 expression were determined by flow cytometry. (A) Representative flow plots and (B) summary data depicting frequencies and absolute numbers of Increased expression of bulk and CD44^hi CD4⁺ and CD8⁺ T cells in vehicle vs αCD28-treated animals. Data are pooled from 2 independent experiments with a total of 9-10 mice/group as indicated. Significance was determined by Mann Whitney U test. A p value of less than or equal to 0.05 was considered significant. *p<0.05.

Figure 3

αCD28-agonist treatment does not affect markers of exhaustion, activation, or proliferation of CD8⁺ T cells, in immunologically-experienced septic mice. B6 Immunologically experienced mice were rendered septic by CLP. αCD28 (n=9) or PBS (n=10) was administered immediately following surgery. Mice were sacrificed 24hr after surgery, spleens were harvested, and expression of activation, exhaustion, and proliferation markers were determined by flow cytometry. (A) CD28 agonism during CLP did not change the expression of the indicated markers on bulk CD8⁺ T cells (B) CD28 agonism during CLP did not change the expression of the indicated markers on CD44^hi CD8⁺ T cells. Data are pooled from 3 independent experiments with a total of 9-10 mice/group as indicated. Significance was determined by Mann Whitney U test. A p value of less than or equal to 0.05 was considered significant.

Figure 4

αCD28-agonist treatment significantly enhances IL-2 responses in CD8⁺ T cells, in immunologically-experienced septic mice. B6 Immunologically experienced mice were rendered septic by CLP. αCD28 (n=9) or PBS (n=10) was administered immediately following surgery. Mice were sacrificed 24hr after surgery and spleens were harvested. Intracellular IL-1β (A), TNF (B), IFN-γ (C), and IL-2 (D) expression was determined on bulk CD8⁺, CD44^lo CD8⁺, and CD44^hi CD8⁺ by flow cytometry following ex vivo restimulation with PMA/ionomycin. Data are pooled from 3 independent experiments with a total of 9 mice/group as indicated. Significance was determined by Mann Whitney U test. A p value of less than or equal to 0.05 was considered significant. *p<0.05. ****p<0.0001.

Figure 5

αCD28-administration promotes Foxp3⁺ CD8⁺ T cell accumulation in immunologically-experienced septic mice. B6 Immunologically experienced mice were rendered septic by CLP. αCD28 (n=9) or PBS (n=10) was administered immediately following surgery. Mice were sacrificed 24hr after surgery and spleens were harvested and stained for Foxp3. Intracellular cytokine expression was determined by flow cytometry following ex vivo restimulation with PMA/iono. (A, B) Summary data showing that CD28 agonism during sepsis significantly increased the frequency of Foxp3⁺ CD8⁺ T cells, and Foxp3⁺ CD8⁺ exhibited increased IL-10 expression with CD28 agonism. Data are pooled from 3 independent experiments with a total of 9-10 mice/group as indicated. (C-E) Phenotypic analysis of Foxp3⁺ CD8⁺ T cells isolated from vehicle vs CD28 agonist-treated mice. Data are pooled from 2 independent experiments with a total of 5 mice/group. Significance was determined by Mann Whitney U test. A p value of less than or equal to 0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

Significant increase of serum IL-1β, TNF, and IFN-γ after αCD28-agonist treatment in immunologically-experienced septic mice depleted of CD8⁺ T cells. αCD8 (n=10) or PBS (n=10) was administered to B6 Immunologically experienced mice. They all were then rendered septic by CLP and given αCD28 immediately following surgery. Mice were sacrificed 24hr after surgery and whole blood was collected. (A) Serum cytokine concentrations. (B) Blood and peritoneal fluid bacterial load. Data are pooled from 2 independent experiments with a total of 10 mice/group. Significance was determined by Mann Whitney U test. A p value of less than or equal to 0.05 was considered significant. *p<0.05, **p<0.01.