Bim Regulates Alloimmune-Mediated Vascular Injury Through Effects on T-Cell Activation and Death

Abstract

Objective Bim is a pro-apoptotic Bcl-2 protein known to down-regulate immune responses and to also be required for antigen-induced T cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T cell responses in a model of vascular rejection. Approach and Results Bim was required for proliferation of CD4 and CD8 T cells, and for IL-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T cell activation whereas a complete elimination of Bim was required to prevent CD4 T cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim+/−, but not Bim−/−, graft recipients. T cell proliferation in response to allograft arteries was significantly reduced in both Bim+/− and Bim−/− mice, but cell death was attenuated only in Bim−/− animals. Conclusions Bim controls both T cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries.

Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells That Drive Alloimmune-Mediated Vascular Rejection

“…21,22 Briefly, segments of abdominal aorta from Balb/c (H2 d ) IL-6 +/+ or IL-6 −/− donor mice were interposed into the resected infra-renal aorta of C57Bl/6 (H2 b ) recipient mice. Arteries from C57Bl/6 donors served as syngraft controls.…”

“…Luminal narrowing and intimal area was quantified using Image J software (NIH) as described. [21][22][23] Immunohistochemistry Cross-sections of arteries were stained by immunohistochemistry as described previously using rabbit polyclonal antibodies to CD4 (Abnova, Taipei City, Taiwan), CD8 (Abnova), smooth muscle cell (SMC) α-actin (Abnova), CD31 (Abnova), Foxp3 (Abcam, Cambridge, MA), and a rat monoclonal antibody to Mac-3 (BD Biosciences, Franklin Lakes, NJ). 21 Staining for antidonor antibodies was performed using an antibody to mouse IgG (Jackson ImmunoResearch, West Grove, PA).…”

“…[21][22][23] Immunohistochemistry Cross-sections of arteries were stained by immunohistochemistry as described previously using rabbit polyclonal antibodies to CD4 (Abnova, Taipei City, Taiwan), CD8 (Abnova), smooth muscle cell (SMC) α-actin (Abnova), CD31 (Abnova), Foxp3 (Abcam, Cambridge, MA), and a rat monoclonal antibody to Mac-3 (BD Biosciences, Franklin Lakes, NJ). 21 Staining for antidonor antibodies was performed using an antibody to mouse IgG (Jackson ImmunoResearch, West Grove, PA). The accumulation of T cells and macrophages in the intima was quantified by manual counting of positive cells, then dividing the respective values by the total number of intimal cells (which was determined by counting the number of hematoxylin-positive nuclei).…”

“…21,24 CD4 and CD8 T cells were purified from splenocytes by negative selection using CD4 and CD8 EasySep T Cell Enrichment kits (StemCell Technologies, Vancouver, BC, Canada). The isolated T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), resuspended in Roswell Park Memorial Institute medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), and cultured with macrophages.…”

“…21,22 We focused on the role of IL-6 from graft cells because it has been shown that graft-derived, but not recipient-derived, IL-6 contributes to the activation of allogeneic responses that causes acute heart transplant rejection. 18 Also, the role of graft-derived IL-6 in the development of vascular rejection and TA has not been examined.…”