Sepsis-induced apoptosis leads to active suppression of delayed-type hypersensitivity by CD8+ regulatory T cells through a TRAIL-dependent mechanism

Abstract

Patients who survive severe sepsis often display severely compromised immune function. One hallmark of such immune suppression in septic patients is an impaired delayed-type hypersensitivity (DTH) response, manifested by a loss of skin testing to recall Ags. Because sepsis induces significant apoptosis in lymphoid and myeloid cells, and apoptotic cells are themselves tolerogenic, we tested the hypothesis that suppression of DTH is mediated by tolerogenic properties of the apoptotic cells generated during sepsis. Mice subjected to cecal ligation and puncture demonstrated a loss of DTH for the 7 d following cecal ligation and puncture; however, the immune response returned to normal by day 10. Blocking sepsis-induced apoptosis via Bcl-2 overexpression or Bim deficiency prevented the loss of DTH. Importantly, injection of apoptotic cells into Bim-/- mice prevented an effective DTH response, thereby suggesting a causal link between apoptotic cells and immune suppression. Surprisingly, when TRAIL null mice were examined, we found that these animals had significant apoptosis but retained their DTH responses. Further studies revealed that apoptotic cells generated during sepsis induced a CD8+ regulatory T cell that suppressed DTH by TRAIL production. These results establish a link between apoptotic cells and immune suppression during sepsis and suggest TRAIL may be a viable therapeutic target for boosting the adaptive immune response following sepsis.

FIGURE 1

DTH response during sepsis. C57BL/6 mice were subjected to CLP or sham surgery. On various days postsurgery, the mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right (Ag challenge) and left footpad (PBS challenge). The data are reported as immune response to TNP. *Significant difference from sham-treated mice measured on the same day (p < 0.01).

FIGURE 2

Role of cellular apoptosis in the DTH response following sepsis. Bcl2-Tg or littermate C57BL/6 mice (A) or Bim^−/− and Bim^+/+ (C57BL/6 littermates) mice (B) were subjected to CLP or sham surgery. Four days postsurgery, mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right (Ag challenge) and left footpad (PBS challenge). The data are reported as immune response to TNP. C, Bim^−/− mice and Bim^+/+ (C57BL/6 littermates) were subjected to CLP or sham surgery. Two days later, mice were given 10⁷ γ-irradiated apoptotic spleen cells i.v. After an additional 2 d (4 d postsurgery), mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. *Significant difference from C57BL/6 or Bim^+/+ control mice (p < 0.01).

FIGURE 3

Apoptosis and cell loss in Trail^−/− mice. A, Trail^+/− or Trail^+/+ (C57BL/6 littermates) were subjected to CLP or sham surgery. Twenty-four hours later, spleens were harvested, and TUNEL stains were performed and analyzed by flow cytometry on gated CD3⁺ cells. Results are reported as % TUNEL⁺ CD3⁺ splenocytes. B–D, Trail^−/− or Trail^+/− littermates were subjected to CLP or sham surgery. The number of CD4⁺ and CD8⁺ CD3⁺ T cells (B), splenic macrophages (CD11b⁺, F4/80⁺) (C), and DCs (CD11c⁺, MHC II⁺) (D) were quantified by flow cytometry. *Significant reduction versus Trail^+/− mice.

FIGURE 4

TRAIL regulates DTH in naive and sensitized CLP mice. A, Trail^−/− or Trail^+/+ (C57BL/6 littermates) were subjected to CLP or sham surgery. Four days postsurgery, mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. B, Trail^−/− or Trail^+/+ (C57BL/6 littermates) were immunized s.c. with TNBS. Four days later, they were subjected to CLP. After an additional 3 d, they were challenged with TNBS in the right and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right (Ag challenge) and left footpad (PBS challenge). The data are reported as immune response to TNP and are compared with an immune control that received only s.c. immunization. *Significant difference from immune control mice measured on the same day (p < 0.01).

FIGURE 5

CD8⁺ T cells suppress DTH. A, C57BL/6 mice were depleted of CD8⁺ T cells by i.v. injection of 100 μg anti-CD8 Ab beginning 3 d prior to CLP or sham surgery. Four days postsurgery, mice were immunized to the hapten TNP by s.c. injection TNBS. After 4 additional days, mice were challenged with TNBS in the right footpad and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right footpad (Ag challenge) and left footpad (PBS challenge). The data are reported as immune response to TNP. *Significant difference from sham nondepleted mice measured on the same day (p < 0.01). B, C57BL/6 mice were subjected to CLP or sham surgery. Four days following surgery, spleens were isolated, and CD4⁺ and CD8⁺ T cells were purified by negative selection. Mice received an equivalent number (one donor into one recipient) of cells i.v. On the day of cell transfer, mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right (Ag challenge) and left footpad (PBS challenge). *Significant difference from immune control mice measured on the same day (p < 0.05). C, Trail^−/− or Trail^+/+ (C57BL/6 littermates) were subjected to CLP or sham surgery. Four days following surgery, spleens were isolated, and CD4⁺ and CD8⁺ T cells were purified by negative selection. Mice (Trail^+/+) received an equivalent number (one donor into one recipient) of cells i.v. On the day of cell transfer, mice were immunized s.c. with TNBS. After an additional 4 d, mice were challenged with TNBS in the right and PBS in the left footpad. Measurements (μm ± SE) were taken 24 h later and represent the difference between right (Ag challenge) and left footpad (PBS challenge). The data are reported as immune response to TNP. *Significant difference from immune control mice measured on the same day (p < 0.05).