Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity

Abstract

Objectives: We recently reported increased CD4 CD25 T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in TH1-type immunity after serious injury in man and if Treg function is altered by injury.

Methods: Peripheral blood was withdrawn from 19 consenting adult patients (35.1 +/- 16.3 years of age) with Injury Severity Scores (ISS) 36.6 +/- 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4 T cells were purified and sorted into Treg (CD25(high)) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the TH1-type cytokine IFNgamma, TH2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4 T cell proliferation.

Results: All patients survived, 9 (47%) developed infection postinjury. IFNgamma production by patient CD4 T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4 T cells, the IFNgamma production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury.

Conclusions: We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in TH1 cytokine production found after injury.

FIGURE 1. Changes in circulating CD25⁺ CD4⁺ T cells after injury. A, Representative FACS plots showing CD25 staining on circulating human CD4⁺ T cells. CD25⁺ cells were measured by comparison with the isotype Ab (negative control) staining and represent a heterogenous group of cells. CD25^high cells were determined by gating on only the brightly stained cells. B, The percentage of CD25⁺ and CD25^high staining found in CD4⁺ T cells from patients at days 1 and 7 postinjury as well as healthy controls. There were similar percentages of CD25⁺ and CD25^high cells on day 1 after injury compared with controls; however, both populations showed significant increases by day 7 (P < 0.01 and P < 0.05, respectively).

FIGURE 2. T_H1- and T_H2-type cytokine production by CD4⁺ T cell populations. IFNγ production by CD4⁺ T cells after anti-CD3/CD28 Ab stimulation was reduced after injury; however, when Tregs were removed, normal levels of this T_H1-type cytokine were produced by the Treg-depleted CD4⁺ T cells. Tregs produced most IL-4 and IL-5, and the IL-4 production by day 7 Tregs was significantly greater than controls. IL-10 production in response to this stimulus was significantly reduced in patient CD4⁺ cells; however, Tregs were insensitive to this shutdown and showed increased production by day 7. #P < 0.01, control CD4⁺ versus patient CD4⁺. *P < 0.05, patient CD4⁺ day 7 versus patient Treg-depleted cells. **P < 0.01, control Tregs versus patient day 7 Tregs (all ANOVA).

FIGURE 3. In vitro suppression of CD4⁺ T cell proliferation by CD25^high CD4⁺ T cells (Tregs). Representative CFSE FACS plots showing in vitro proliferation. The autologous CD25− CD4⁺ T cells were first labeled with the fluorescent dye, CFSE. The CFSE then distributed evenly among daughter cells, giving peaks on the histogram as the signal decayed with each round of proliferation. Each plot shows events gated on labeled cells only. The control plots indicate proliferation of unstimulated and stimulated naive cells without any additional T cells. Tregs caused a significant suppression of labeled cell proliferation (from 79% to 22%); however, the addition of Treg-depleted cells failed to suppress anti-CD3/CD28-induced cell proliferation (86% proliferating cells).

FIGURE 4. Injury induces increased Treg-mediated suppression of CD4⁺ T cell proliferation. Suppression was defined as the difference between proliferation of naive CFSE-labeled T cells in the presence of CD25⁻ T cells and of Tregs. There was a significant injury-associated increase in the potency of Treg suppression on day 7 after injury (P < 0.05, ANOVA).