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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2014 Apr 3;34(6):1290–1297. doi: 10.1161/ATVBAHA.114.303649

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

Anna von Rossum 1, Winnie Enns 1, Yu P Shi 1, Grace E MacEwan 1, Mehrnoush Malekesmaeli 2, Ryan Brinkman 2, Jonathan C Choy 1,*
PMCID: PMC4097606  NIHMSID: NIHMS579913  PMID: 24700126

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.

Keywords: Transplantation, Vascular injury, T cell, Bim

Heart transplantation is a life-saving procedure but its utility is limited by the eventual failure of most grafts due largely to immune-mediated processes (1). As such, understanding the basic mechanisms by which allogeneic immune responses are regulated is essential for the development of strategies that prevent heart transplant failure. Immunological reactions to foreign major histocompatibility complexes (MHC) and their associated peptides on the surface of graft cells drive the activation of effector T cells in lymphoid tissues as well as directly in the graft (2, 3). Effector T cells that accumulate within grafts then cause acute and chronic graft failure by targeting parenchymal and vascular cells. The aggressiveness and persistence of allogeneic effector T cell responses is regulated by a balance between T cell proliferation and death (4). Specifically, defects in T cell proliferation reduce graft-specific immune responses and resultant rejection (5) whereas defects in T cell death augment graft rejection and oppose the development of tolerance (6–10). Bcl-2 proteins are known to control T cell death after transplantation. By using mice in which T cells overexpress the anti-apoptotic Bcl-2 protein Bcl-xL, Wells et al. (6) showed that Bcl-xL increases T cell persistence after heart transplantation, and in this way augments acute and chronic heart transplant rejection.

Bim is a pro-apoptotic BH3-only member of the Bcl-2 family that is a pivotal initiator of T cell death triggered by cytokine deprivation (11). Bim antagonizes the survival actions of Bcl-2 and Bcl-xL, thereby triggering mitochondrial permeabilization and resultant cell death (12). In the mature immune system, Bim is required for the contraction of CD8 T cell responses following both acute and chronic viral infection (12–14). The importance of Bim-mediated T cell death in immune homeostasis is highlighted by the development of certain autoimmune-like manifestations in mice that lack this protein (11). Paradoxically, in addition to regulating T cell death, recent evidence indicates that Bim is also required for optimal T cell activation in autoimmune encephalomyelitis, diabetes, and graft-versus-host disease (15, 16). However, nothing is known about how the opposing effects of Bim on T cell activation and death cooperate to define the outcome of immunopathological conditions, whether Bim differentially affects CD4 and CD8 T cell responses to alloantigens, and how Bim affects immune-mediated vascular injury.

Immune-mediated vascular injury is a main contributor to organ transplant failure through the induction of microvascular damage, which results in thrombosis and hemorrhage, and of macrovascular damage, which disturbs blood flow and contributes to the development of intimal thickening and pathological re-structuring of allograft arteries (17). The latter changes manifest mainly as transplant arteriosclerosis (TA). Although there are distinctions in the acute and chronic immune responses that drive vascular injury, observations that an increased incidence of acute rejection episodes predicts a greater severity of TA suggest an overlapping involvement of both types of immune responses to pathologically important outcomes in the graft vasculature (18) (19, 20) (21, 22). Aspects of immunological vascular injury and rejection that are applicable to organ transplant failure can be modelled using aortic interposition grafting in mice. Using a combination of in vitro experiments and a complete MHC-mismatch model of aortic interposition grafting, we show in the current study that Bim is required for the optimal activation of both CD4 and CD8 T cells in response to allogeneic stimulation. We further identify a quantitative difference in the reliance of T cells on Bim for activation as opposed to cell death, with a partial reduction in Bim expression preventing T cell proliferation but not death, and complete elimination of Bim expression preventing both proliferation and death. Aortic interposition grafting of artery segments into Bim+/+, Bim+/−, and Bim−/− recipients showed that reduction of Bim expression (in Bim+/− mice), but not complete elimination (in Bim−/− mice), reduces immune-mediated vascular injury and rejection. This coincided with attenuated T cell proliferation, but not cell death, in Bim+/− graft recipients. Both T cell proliferation and death were attenuated in Bim−/− graft recipients, likely resulting in offsetting effects on immune activation and inactivation in this setting. Altogether, our findings provide important insight into the control of allogeneic T cell responses, and show that the effect of Bim on alloantigen-induced T cell responses is complexly regulated by its opposing effects on T cell proliferation and death. This has implications for understanding alloimmune-mediated vascular damage that contributes to organ transplant failure.

METHODS AND MATERIALS

Methods and materials are available in the online-only supplement.

RESULTS

Bim is required for alloantigen-induced activation of T cells

To begin examining the role of Bim in controlling the alloantigen-induced activation of T cells, CD4 and CD8 T cells were isolated from Bim+/+, Bim+/− and Bim−/− mice, labeled with CFSE, and then stimulated with allogeneic macrophages. Proliferation was examined by measuring CFSE dilution after 6 days. There was no proliferation in syngeneic controls but there was robust proliferation of Bim+/+ T cells in response to alloantigen stimulation. Both Bim+/− and Bim−/− CD4 T cells proliferated significantly less than Bim+/+ CD4 T cells, and the defect in proliferation was comparable between Bim+/− and Bim−/− CD4 T cells (Fig. 1A & B). When alloantigen-induced proliferation of CD8 T cells was examined, there was significantly less proliferation of Bim−/− CD8 T cells as compared to Bim+/+ counterparts. There also appeared to be less proliferation of Bim+/− CD8 T cells when compared to Bim+/+ cells, but these results did not reach statistical significance (Fig. 1C & D, p = 0.15).

Figure 1. Bim is required for alloantigen-driven activation of T cells.

Figure 1

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CD4 and CD8 T cells from C57Bl/6 Bim+/+, Bim+/−, and Bim−/− mice were CFSE labelled and cultured with peritoneal macrophages from BALB/c mice. T cells cultured with macrophages from C57Bl/6 Bim+/+ mice served as syngeneic controls. A. Representative dot plots showing proliferation of CD4 T cells in response to allogeneic macrophages. B. Mean ± SE of relative CD4 T cell proliferation over a series of 3 separate experiments. *p<0.05 compared to Bim+/+. C. Representative dot plots showing CD8 T cell proliferation in response to allogeneic macrophages. D. Mean ± SE of relative CD8 T cell proliferation over a series of 3 separate experiments. *p<0.05 compared to Bim+/+. E. Supernatants from allogeneic co-cultures were harvested at 24 hr post-stimulation and IL-2 levels measured by ELISA. * p < 0.01 as compared to Bim+/+ cells. F. Representative histograms of Bim expression in CD4 and CD8 T cells isolated from Bim+/+, Bim+/− and Bim−/− mice. Black line: Bim+/+ cells, Gray line: Bim+/− cells, Shaded area: Bim−/− cells.

IL-2 secretion from CD4 and CD8 T cells was then evaluated. No IL-2 was detected in T cells cultured with syngeneic macrophages and there was substantial IL-2 secretion by Bim+/+ T cells in response to allogeneic stimulation. There was a significant reduction in IL-2 production by both CD4 and CD8 T cells isolated from Bim+/− and Bim−/− mice in response to allogeneic stimulation as compared to cells isolated from Bim+/+ mice (Fig 1E). Thus, Bim is required for the optimal activation of CD4 and CD8 T cells. Moreover, profiling of Bim expression in isolated T cells indicated that cells from Bim+/− mice express ~50% levels of this Bcl-2 protein as compared to Bim+/+ T cells (Fig. 1F), indicating that a partial reduction in Bim expression is sufficient to attenuate alloantigen-induced T cell activation.

Complete elimination of Bim expression prevents cytokine deprivation-induced T cell death

Two modes of T cell death regulate allogeneic responses: activation-induced cell death (AICD) that is caused by continuous exposure of T cells to antigen stimulation and cytokine deprivation-induced cell death (6). T cell viability was therefore analyzed in Bim+/+, Bim+/−, and Bim−/− T cells that were continually stimulated with allogeneic macrophages. There was no difference in the survival of CD4 or CD8 T cells in any of the groups (Fig. 2A&B), indicating that Bim does not affect T cell death in this setting. These findings also support a role for Bim in regulating T cell activation that is independent of its role in controlling cell death.

Figure 2. Bim does not affect cell death of T cells continually exposed to alloantigen but attenuates cytokine deprivation-induced cell death.

Figure 2

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A. CD4 and B. CD8 T cells from Bim+/+, Bim+/− and Bim−/− mice were cultured with peritoneal macrophages from BALB/c mice. After 6 days, cell viability was analyzed by quantifying propidium iodide exclusion by flow cytometry. Data presented is the mean ± SE of cell viability over a series of 3 separate experiments. C. CD4 and D. CD8 T cells from Bim+/+, Bim+/− and Bim−/− mice were cultured in the absence of cytokines or growth factors. Cell viability was analyzed by propidium iodide exclusion over a 72 hr time-course. Data presented is the mean ± SE of cell viability over a series of 4 independent experiments. * p < 0.05 Bim−/− compared to Bim+/+ cells; # p < 0.05 Bim+/− compared to Bim−/− cells; $ p < 0.05 Bim+/− compared to Bim+/+. E. CD4 T cells from Bim+/+, Bim+/− and Bim−/− mice were stimulated with anti-CD3 plus anti-CD28 for 72 h, removed from stimulation and further cultured in the absence of stimulation for 72 h. An IL-2 neutralizing antibody was added to T cells after their removal from stimulation. Cell viability was determined by quantifying propidium iodide exclusion. Data presented is the mean ± SE of cell viability over a series of 3 independent experiments. * p ≤ 0.05 as compared to Bim+/+ cells; # p ≤ 0.05 as compared to Bim +/− cells.

In addition to AICD, T cell responses are also regulated by cytokine deprivation-induced cell death in transplantation. This is demonstrated by experiments showing that transplant outcome is affected by the expression of Bcl-xL (which is a key regulator of cell death in response to cytokine deprivation) in T cells (6). This form of cell death is likely to be triggered in the setting of transplantation by local regions of reduced cytokine availability, down-regulation of cytokine receptors from the surface of activated T cells or the elimination of donor-derived antigen presenting cells that directly present alloantigens to T cells in lymphoid tissues and the graft. As such, we characterized the role of Bim in regulating cytokine deprivation-induced cell death of CD4 and CD8 T cells using an established ex vivo protocol which involves culturing freshly isolated T cells in the absence of T cell receptor or cytokine stimulation (11, 23). Bim+/+ CD4 T cells died at a steady rate with less than 10% survival after 72 hrs. As expected, Bim−/−CD4 T cells were significantly protected from cytokine deprivation-induced cell death. Interestingly, Bim+/− CD4 T cells underwent cell death at an identical rate as Bim+/+ CD4 T cells, indicating that a partial reduction in Bim levels is not sufficient to attenuate cytokine deprivation-induced cell death in CD4 T cells (Fig. 2C). Importantly, Bim+/− CD4 T cells were also significantly more susceptible to cytokine deprivation-induced cell death than Bim−/− T cells. With regard to CD8 T cells, Bim+/+ CD8 T cells underwent a steady rate of death in response to cytokine deprivation and, as expected, Bim−/− CD8 T cells were resistant to this form of cell death. However, unlike CD4 T cells, Bim+/− CD8 T cells were partially protected from cell death, undergoing levels of cell death that were intermediate between Bim+/+ and Bim−/− cells (Fig. 2D). These results show that complete elimination of Bim expression protects both CD4 and CD8 T cells from cytokine deprivation-induced cell death, but that a partial reduction in Bim levels does not affect this cell death process in CD4 T cells and partially reduces cell death of CD8 T cells.

We also examined cytokine deprivation-induced cell death of T cell receptor (TCR) activated T cells in vitro. For this, isolated CD4 and CD8 T cells were stimulated with anti-CD3 plus anti-CD28, removed from stimulation and further cultured in the presence of an IL-2 neutralizing antibody for 72 h. IL-2 was neutralized because short-term stimulation of T cells in vitro is known to result in continued proliferation and cytokine secretion for several days after removal from stimulation (24, 25). There was no difference in cell death between any of the groups prior to 72 h after removal from stimulation. At 72 h, activated CD4 T cells from Bim−/− mice were significantly protected from cell death but cells from Bim+/− mice were not. Also, activated CD4 T cells from Bim−/− mice underwent significantly less cell death than those from Bim+/− mice (Fig. 2E). Unexpectedly, Bim did not affect CD8 T cell death in these in vitro experiments (data not shown). This in vitro model may not reflect the in vivo response of CD8 T cells because Bim−/− CD8 T cells are firmly established as being resistant to cell death caused by cytokine deprivation in vivo (13, 26, 27).

Partial reduction, but not complete elimination, of Bim attenuates immune mediated vascular rejection

The role of Bim in regulating T cell-mediated allograft vascular damage and rejection was then examined by interposition grafting of aortic segments from complete MHC-mismatched donors into Bim+/+, Bim+/− and Bim−/− recipients. Immune-mediated vascular injury in this model initiates vascular reparative responses that culminate in rapid intimal thickening (28–30). The grafted arteries were harvested at day 30 post-transplantation for analysis of intimal thickening, which serves as a readout for the severity of vascular injury. Artery segments transplanted into Bim+/− but not Bim−/− mice developed significantly less intimal thickening as compared to Bim+/+ recipients (Fig. 3).

Figure 3. Partial reduction, but not complete elimination, of Bim in graft recipients attenuates intimal thickening of allograft arteries.

Figure 3

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Segments of abdominal aorta from BALB/c mice were interposed into the resected abdominal aortas of C57Bl/6 Bim+/+, Bim+/−, and Bim−/− mice. The grafted arteries were harvested at day 30 post-transplant. A. Representative photomicrographs of elastic van Giesen stained arteries from Bim+/+, Bim+/− and Bim−/− graft recipients. Magnification: 100X. B. Quantification of intimal thickening in allograft arteries from Bim+/+ (n=14), Bim+/− (n=10), and Bim−/− (n=6) mice. * p< 0.05 compared to Bim+/+.

The effect of Bim on the accumulation of leukocytes in allograft arteries was then examined by quantifying the number of CD4 and CD8 T cells, macrophages, and dendritic cells (not shown), within allograft arteries. There was significantly less CD4 T cell accumulation in allograft arteries transplanted into Bim+/− mice as compared to Bim+/+ counterparts, but CD4 T cell accumulation did not differ in arteries harvested from Bim−/− and Bim+/+ mice (Fig. 4A&B). There was no significant difference in the accumulation of CD8 T cells (Fig. 4C&D) or macrophages (Fig. 4E&F) in allograft arteries from Bim+/+, Bim+/− and Bim−/− mice. Staining for dendritic cells revealed too few cells in the intima to conclusively determine differences between the groups (data not shown). The effect of Bim on vascular cells within the grafts was also determined. EC vessel lining and the accumulation of intimal smooth muscle cells was studied by staining sections of the allograft arteries for CD31 and smooth muscle α-actin, respectively. No significant difference was observed in EC lining of the lumen (Fig. 5A&B) or in amount of smooth muscle cells (Fig. 5C&D) in the intima of allograft arteries in any of the groups.

Figure 4. Partial reduction, but not complete elimination, of Bim reduces CD4 T cell accumulation in allograft arteries but does not affect CD8 T cell or macrophage accumulation.

Figure 4

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Representative photomicrographs of allograft arteries from Bim+/+, Bim+/−, and Bim−/− graft recipients immunohistochemically stained for A. CD4, C. CD8. and E. Mac-3. The percentage of B. CD4-positive, D. CD8-positive, and F. Mac-3-positive cells within the intima of allograft arteries was quantified by manual counting. Data presented in B, D, and E is the mean ± SE of the percentage of all intimal cells that are CD4-, CD8- and Mac-3-positive in arteries from Bim+/+ (n=14), Bim+/− (n=10) and Bim−/− (n=6) mice. * p < 0.05 compared to Bim+/+. The inset photomicrographs depict isotype staining controls.

Figure 5. Bim does not affect endothelial lining or smooth muscle cell accumulation in the intima of allograft arteries.

Figure 5

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Representative photomicrographs of allograft arteries from Bim+/+, Bim+/−, and Bim−/− grafts recipients immunohistochemically stained for A. CD31 and C. smooth muscle α-actin. Quantification of B. CD31 positivity and D. smooth muscle a-actin positivity was performed. The data presented in B. and D. is the mean ± SE of the percent luminal lining that is CD31 positive or the percent smooth muscle a-actin positive area in the intima.

Partial reduction of Bim levels attenuates T cell proliferation, but not death, in response to allograft arteries

Because a decrease in Bim expression levels reduced intimal thickening and attenuated the accumulation of CD4 effector T cells in allograft arteries, T cell proliferation and death in response to allograft arteries was examined. Segments of abdominal aorta were transplanted into Bim+/+, Bim+/−, or Bim−/− mice, and splenocytes isolated at day 7 post-transplant. Flow cytometric analysis was performed for CD4, CD8, CD44, Ki67 and active caspase-3 to quantify CD4 and CD8 T cell proliferation and death, respectively. Significantly fewer proliferating CD4 T cells were observed in both Bim+/− and Bim−/− mice in response to allograft arteries as compared to Bim+/+ counterparts (Fig 6A&B). Also, there was no difference in cell death of CD4 T cells in Bim+/− as compared to Bim+/+ artery graft recipients, but T cell death was significantly reduced in Bim−/− mice as compared to either Bim+/+ or Bim+/− counterparts (Fig 6C&D). Proliferation of CD8 T cells was also reduced in both Bim+/− and Bim−/− mice in response to allograft arteries (Fig. 6E&F). Cell death of CD8 T cells was significantly reduced only in Bim−/− as compared to Bim+/+ graft recipients (Fig. 6G&H).

Figure 6. Partial reduction in Bim levels is sufficient to attenuate T cell proliferation in response to allograft arteries but complete elimination of Bim is needed to prevent T cell death.

Figure 6

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Segments of abdominal aorta from BALB/c mice were interposed into the resected abdominal aortas of C57Bl/6 Bim+/+, Bim+/−, or Bim−/− mice. Syngrafts (syn) were performed as controls. Splenocytes were isolated at day 7 post-transplant and examined by flow cytometry. A. Representative dot plots showing the frequency of CD4 T cells that are Ki67-positive in Bim+/+, Bim+/− and Bim−/− graft recipients. B. Mean ± SE of the percentage of CD4 T cells that are Ki67-positive in Bim+/+ (n=9), Bim+/− (n=8) and Bim−/− (n=8) graft recipients. * p < 0.05 compared to Bim+/+ mice. C. Representative dot plots showing the frequency of CD4+CD44+ effector T cells that are active caspase-3-positive in Bim+/+, Bim+/− and Bim−/− graft recipients. D. Mean ± SE of the percentage of CD4+CD44+ effector T cells that are active caspase-3-positive in Bim+/+ (n=9), Bim+/− (n=9) and Bim−/− (n=9) graft recipients. * p < 0.05 compared to Bim+/+ mice, # p < 0.05 compared to Bim+/− mice. E. Representative dot plots showing the frequency of CD8 T cells that are Ki67-positive in Bim+/+, Bim+/− and Bim−/− graft recipients. F. Mean ± SE of the percentage of CD8 T cells that are Ki67-positive in Bim+/+ (n=9), Bim+/− (n=7) and Bim−/− (n=8) graft recipients. * p ≤ 0.05 compared to Bim+/+ mice. G. Representative dot plots showing the frequency of CD8+CD44+ effector T cells that are active caspase-3-positive in Bim+/+, Bim+/− and Bim−/− graft recipients. H. Mean ± SE of the percentage of CD8+CD44+ effector T cells that are active caspase-3-positive in Bim+/+ (n=9), Bim+/− (n=7) and Bim−/− (n=9) graft recipients. *p<0.05 as compared to Bim+/+.

In addition to the manual analysis of flow cytometric data using isotype staining controls to identify positive gates, positive events in all the data were also identified using an automated, unbiased approach in order to eliminate potential human error and to standardize the analysis. This analysis confirmed that CD4 and CD8 T cell proliferation was reduced in Bim+/− and Bim−/− graft recipients, respectively, and that CD4 T cell death was reduced only in Bim−/− graft recipients (Supplemental Figure I & II). There was a strong trend towards a reduction in CD8 T cell death in Bim−/− graft recipients compared to Bim+/+ counterparts (p = 0.09). All together, our data show that a partial reduction in Bim expression is sufficient to attenuate T cell proliferation but not death in response to allograft arteries, and that complete elimination of Bim attenuates both processes. Therefore, allogeneic immune responses are diminished in Bim+/−, but not Bim−/−, mice because defects in T cell activation in the latter condition are likely offset by increased T cell survival.

DISCUSSION

In the current study, we have shown that Bim is required for the activation of T cells in response to stimulation by alloantigen and that this process is involved in immune-mediated vascular damage and rejection. We further report that the effect of Bim on T cell activation and death can be separated by differential reductions in Bim levels. Specifically, a partial reduction in Bim expression, as seen in Bim+/−T cells, is sufficient to attenuate T cell activation but complete elimination of Bim expression is required to prevent CD4 T cell death. To our knowledge, our data are the first to demonstrate that the regulation of allogeneic immune responses by Bim is regulated by its opposing effects on T cell activation and death as well as to establish a role for this Bcl-2 family member in the regulation of allograft vascular injury.

Our in vitro data shows that, in response to allogeneic stimulation, both Bim+/− and Bim−/−T cells display reduced proliferation as compared to Bim+/+ cells. Interestingly, a half reduction in Bim levels, as occurs in Bim+/− T cells, is sufficient to produce this effect. Cell death was measured in these cultures and no difference was observed between any of the groups, indicating that the observed effect on the accumulation of proliferated cells was not due to altered cell survival. Since T cells in this setting are exposed to continuous antigen and there is persistent production of IL-2, cell death in these experiments was likely a result of activation-induced cell death. Bim has been reported to be involved in the induction of activation-induced cell death (31), although its role in driving T cell death in response to cytokine deprivation is more extensively understood (11, 13). We have also characterized the role of Bim in cytokine deprivation-induced T cell death and confirmed that both CD4 and CD8 T cells from Bim−/− mice are protected from this form of cell death. Interestingly, a partial reduction in Bim expression, as occurs in Bim+/− mice, did not inhibit CD4 T cell death indicative of Bim haplosufficiency. CD8 T cells from Bim+/− mice underwent cell death at an intermediate level between the Bim−/− and the Bim+/+ CD8 T cells. This cell death pathway may regulate T cell death in organ transplantation because graft-derived professional antigen presenting cells that directly present alloantigens to T cells are eliminated as allogeneic immune responses progress over time and this may result in localized regions in which graft-reactive T cells experience a paucity of survival signals (32, 33). Also, down-regulation of certain cytokine receptors, such as CD25 and CD127, by effector T cells may render them unable to respond to survival signals even in settings of persistent antigen (34).

An unexpected role for Bim in regulating T cell activation has recently been reported in animal models of autoimmune encephalomyelitis, diabetes, and graft-versus-host disease (15, 16). However, the interplay between the regulation of T cell activation and death by Bim was not examined in these studies. Our findings extend existing knowledge by establishing 1) a role for Bim in controlling both the activation and death of T cells in response to alloantigen stimulation, 2) that a partial reduction of Bim expression inhibits T cell proliferation without affecting CD4 T cell death, for which complete elimination of Bim is necessary, and 3) that both processes control immune responses that lead to allograft vascular injury and rejection. It is important to note that, although Bim is required for certain types of clonal deletion in the thymus (35), we did not observe splenomegaly in Bim+/− mice in our studies (data not shown). Also, systemic elimination of Bim has been reported to result in an increase in dysfunctional Foxp3-expressing T regs (36), but we did not observe an increase in Foxp3-expressing T cells in Bim+/− or Bim−/− graft recipients (data not shown).

In our experiments we observe a very early defect in T cell activation in the absence or reduced levels of Bim. Although we examined IL-2 secretion as a surrogate indicator of effector cytokine production in vitro, other cytokines are clearly involved in supporting the expansion of effector T cells in the setting of transplantation in vivo (37). Bim is likely to affect the acquisition of a large number of T cell effector functions because it has been suggested to act very proximally in T cell receptor signalling pathways (15). Ludwuinski et al. (15) reported that Bim−/− T cells activated with agonistic CD3 antibody had significantly reduced levels of calcium influx as compared to Bim+/+ T cells, and that this resulted in reduced activation of nuclear factor of activated T cells. Bcl-2 and Bcl-xL are known to localize to the endoplasmic reticulum and interact with the inositol 1,4,5 triphosphate receptor to either inhibit or potentiate calcium release (38, 39). Given this, antagonism of Bcl-2 effects by Bim may serve to augment T cell receptor-mediated calcium signaling in T cells. Further studies are needed to define these processes in T cells.

Our data establishes that the reduction, but not the elimination, of Bim expression in graft recipients attenuates vascular injury and rejection. Partial reduction in Bim levels also resulted in reduced accumulation of effector CD4 T cells in the intima of allograft arteries and in reduced proliferation of alloreactive T cells but did not affect CD4 T cell death. On the other hand, both T cell activation and death were attenuated when Bim expression was completely absent in graft recipients. When assessed in combination with our in vitro findings which show that Bim expression in T cells regulates T cell activation and death in a similar manner, our data indicate that in the setting of Bim+/− graft recipients, CD4 T cell activation is compromised but death is not and this results in reduced allograft damage. However, when Bim expression is completely absent in Bim−/− recipients, attenuated T cell activation is offset by increased survival of activated T cells and this results in no net effect on rejection. Interestingly, partial reduction of Bim levels in Bim+/− mice did not appear to affect CD8 T cell accumulation in allograft arteries in our studies. In our studies Bim is likely to affect vascular rejection through its actions in CD4 T cells because intimal thickening in complete MHC-mismatched aortic interposition grafts depends mainly on this T cell subset (40).

We have evaluated the effect of Bim on alloantigen-driven immune responses and associated allograft vascular injury using aortic interposition grafting across a complete MHC barrier. This is an antigen mismatch situation that is reflective of that which occurs in almost all heart transplants and permits a comprehensive evaluation of T cell responses that are known to be involved in organ transplant rejection. This includes direct T cell recognition and targeting of foreign peptide-MHC presented by graft-derived cells, indirect T cell recognition and targeting of graft-derived alloantigens presented by recipient antigen presenting cells, and CD4 T cell-dependent antibody-mediated targeting of foreign MHC molecules on vascular cell surfaces (17). However, immune-mediated arterial damage is severe in this model. This results in the rapid population of the intima by smooth muscle cells that are derived from circulating stem cells from the recipient (28), a feature different from that which occurs clinically where almost all of the intimal smooth muscle cells are of donor origin (41). As such, our analysis is applicable to understanding the basic immune mechanisms that result in allograft vascular injury and rejection but the specific vascular cell responses that contribute to intimal thickening in our model are partially distinct from those that drive similar changes in a clinical setting. (17, 42)

In summary, we have shown that reduction, but not elimination, of Bim in T cells inhibits T cell activation in response to alloantigen stimulation and attenuates allograft vascular injury. The regulation of allogeneic immune responses by Bim is complex and involves the control of both T cell activation and death, and these processes are differentially susceptible to reductions in Bim expression. Our findings have implications for understanding the regulation of immune responses in transplantation, and may have clinical implications given the investigation of BH3 mimetics for therapeutic applications (43).

Supplementary Material

Methods and Materials section
Supplemental Figures

SIGNIFICANCE.

We have identified a new role for the cell death regulatory protein, Bim, in controlling alloantigen-induced T cell activation and vascular injury in transplantation. We have also determined that Bim controls both T cell activation and death in response to alloantigens and that the balance of the effect of Bim on these opposing cellular processes determines the outcome of immunopathological responses.

Acknowledgments

We are grateful to Ms. Mary Dearden and Kim Buettner for expert assistance with animal care as well as Andy Johnson for assistance with flow cytometry.

Sources of Funding: This work was supported by an Operating Grant from the Canadian Institutes of Health Research (CIHR; J.C.C), a Grant-in-Aid from the Heart & Stroke Foundation of BC & Yukon (J.C.C), a Venture Grant from the Transplant Research Foundation of British Columbia (J.C.C), the Natural Sciences and Engineering Research Council (R.B.), NIH/NIBIB grant R01 EB008400 (R.B.), and a CIHR Training Grant in Transplantation (A.V.). J.C.C. is a recipient of CIHR New Investigator and Michael Smith Foundation for Health Research Scholar awards.

Nonstandard abbreviations and acronyms

AICD

Activation-induced cell death

CFSE

5-(and-6)-carboxyfluorecein diacetate succinimidyl ester

MHC

Major histocompatibility complex

TA

Transplant arteriosclerosis

TCR

T cell receptor

Footnotes

Disclosure: None

References

  • 1.Taylor DO, Edwards LB, Boucek MM, Trulock EP, Waltz DA, Keck BM, Hertz MI. Registry of the International Society for Heart and Lung Transplantation: twenty-third official adult heart transplantation report--2006. J Heart Lung Transplant. 2006;25:869–879. doi: 10.1016/j.healun.2006.05.002. [DOI] [PubMed] [Google Scholar]
  • 2.Chalasani G, Dai Z, Konieczny BT, Baddoura FK, Lakkis FG. Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs. Proc Natl Acad Sci U S A. 2002;99:6175–6180. doi: 10.1073/pnas.092596999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Pober JS, Tellides G. Participation of blood vessel cells in human adaptive immune responses. Trends Immunol. 2012;33:49–57. doi: 10.1016/j.it.2011.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jameson SC. Maintaining the norm: T-cell homeostasis. Nat Rev Immunol. 2002;2:547–556. doi: 10.1038/nri853. [DOI] [PubMed] [Google Scholar]
  • 5.Sevigny CP, Li L, Awad AS, Huang L, McDuffie M, Linden J, Lobo PI, Okusa MD. Activation of adenosine 2A receptors attenuates allograft rejection and alloantigen recognition. J Immunol. 2007;178:4240–4249. doi: 10.4049/jimmunol.178.7.4240. [DOI] [PubMed] [Google Scholar]
  • 6.Wells AD, Li XC, Li Y, Walsh MC, Zheng XX, Wu Z, Nunez G, Tang A, Sayegh M, Hancock WW, Strom TB, Turka LA. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med. 1999;5:1303–1307. doi: 10.1038/15260. [DOI] [PubMed] [Google Scholar]
  • 7.Wekerle T, Kurtz J, Sayegh M, Ito H, Wells A, Bensinger S, Shaffer J, Turka L, Sykes M. Peripheral deletion after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death. J Immunol. 2001;166:2311–2316. doi: 10.4049/jimmunol.166.4.2311. [DOI] [PubMed] [Google Scholar]
  • 8.Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med. 1999;5:1298–1302. doi: 10.1038/15256. [DOI] [PubMed] [Google Scholar]
  • 9.Choy JC, Wang Y, Tellides G, Pober JS. Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature. Proc Natl Acad Sci U S A. 2007;104:1313–1318. doi: 10.1073/pnas.0607731104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Choy JC, Pober JS. Generation of NO by bystander human CD8 T cells augments allogeneic responses by inhibiting cytokine deprivation-induced cell death. Am J Transplant. 2009;9:2281–2291. doi: 10.1111/j.1600-6143.2009.02771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM, Strasser A. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science. 1999;286:1735–1738. doi: 10.1126/science.286.5445.1735. [DOI] [PubMed] [Google Scholar]
  • 12.Hildeman DA, Zhu Y, Mitchell TC, Kappler J, Marrack P. Molecular mechanisms of activated T cell death in vivo. Curr Opin Immunol. 2002;14:354–359. doi: 10.1016/s0952-7915(02)00335-7. [DOI] [PubMed] [Google Scholar]
  • 13.Hughes PD, Belz GT, Fortner KA, Budd RC, Strasser A, Bouillet P. Apoptosis regulators Fas and Bim cooperate in shutdown of chronic immune responses and prevention of autoimmunity. Immunity. 2008;28:197–205. doi: 10.1016/j.immuni.2007.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Weant AE, Michalek RD, Khan IU, Holbrook BC, Willingham MC, Grayson JM. Apoptosis regulators Bim and Fas function concurrently to control autoimmunity and CD8+ T cell contraction. Immunity. 2008;28:218–230. doi: 10.1016/j.immuni.2007.12.014. [DOI] [PubMed] [Google Scholar]
  • 15.Ludwinski MW, Sun J, Hilliard B, Gong S, Xue F, Carmody RJ, DeVirgiliis J, Chen YH. Critical roles of Bim in T cell activation and T cell-mediated autoimmune inflammation in mice. J Clin Invest. 2009;119:1706–1713. doi: 10.1172/JCI37619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yu Y, Yu J, Iclozan C, Kaosaard K, Anasetti C, Yu XZ. Bim is required for T-cell allogeneic responses and graft-versus-host disease in vivo. Am J Blood Res. 2012;2:77–85. [PMC free article] [PubMed] [Google Scholar]
  • 17.Choy JC. Granzymes and perforin in solid organ transplant rejection. Cell Death Differ. 2010;17:567–576. doi: 10.1038/cdd.2009.161. [DOI] [PubMed] [Google Scholar]
  • 18.Nakagawa T, Sukhova GK, Rabkin E, Winters GL, Schoen FJ, Libby P. Acute rejection accelerates graft coronary disease in transplanted rabbit hearts. Circulation. 1995;92:987–993. doi: 10.1161/01.cir.92.4.987. [DOI] [PubMed] [Google Scholar]
  • 19.Matas AJ, Gillingham KJ, Payne WD, Najarian JS. The impact of an acute rejection episode on long-term renal allograft survival (t1/2) Transplantation. 1994;57:857–859. doi: 10.1097/00007890-199403270-00015. [DOI] [PubMed] [Google Scholar]
  • 20.Almond PS, Matas A, Gillingham K, Dunn DL, Payne WD, Gores P, Gruessner R, Najarian JS. Risk factors for chronic rejection in renal allograft recipients. Transplantation. 1993;55:752–756. doi: 10.1097/00007890-199304000-00013. [DOI] [PubMed] [Google Scholar]
  • 21.Taylor DO, Edwards LB, Boucek MM, Trulock EP, Deng MC, Keck BM, Hertz MI. Registry of the International Society for Heart and Lung Transplantation: twenty-second official adult heart transplant report--2005. J Heart Lung Transplant. 2005;24:945–955. doi: 10.1016/j.healun.2005.05.018. [DOI] [PubMed] [Google Scholar]
  • 22.Roussel JC, Baron O, Perigaud C, et al. Outcome of heart transplants 15 to 20 years ago: graft survival, post-transplant morbidity, and risk factors for mortality. J Heart Lung Transplant. 2008;27:486–493. doi: 10.1016/j.healun.2008.01.019. [DOI] [PubMed] [Google Scholar]
  • 23.Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM, Strasser A. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 2003;302:1036–1038. doi: 10.1126/science.1090072. [DOI] [PubMed] [Google Scholar]
  • 24.Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol. 2001;2:415–422. doi: 10.1038/87720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Raue HP, Beadling C, Haun J, Slifka MK. Cytokine-mediated programmed proliferation of virus-specific CD8(+) memory T cells. Immunity. 2013;38:131–139. doi: 10.1016/j.immuni.2012.09.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hildeman DA, Zhu Y, Mitchell TC, Bouillet P, Strasser A, Kappler J, Marrack P. Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim. Immunity. 2002;16:759–767. doi: 10.1016/s1074-7613(02)00322-9. [DOI] [PubMed] [Google Scholar]
  • 27.Strasser A, Pellegrini M. T-lymphocyte death during shutdown of an immune response. Trends Immunol. 2004;25:610–615. doi: 10.1016/j.it.2004.08.012. [DOI] [PubMed] [Google Scholar]
  • 28.Johnson P, Carpenter M, Hirsch G, Lee T. Recipient cells form the intimal proliferative lesion in the rat aortic model of allograft arteriosclerosis. Am J Transplant. 2002;2:207–214. doi: 10.1034/j.1600-6143.2002.20304.x. [DOI] [PubMed] [Google Scholar]
  • 29.Mennander A, Tiisala S, Halttunen J, Yilmaz S, Paavonen T, Hayry P. Chronic rejection in rat aortic allografts. An experimental model for transplant arteriosclerosis. Arterioscler Thromb. 1991;11:671–680. doi: 10.1161/01.atv.11.3.671. [DOI] [PubMed] [Google Scholar]
  • 30.Shi C, Lee WS, He Q, Zhang D, Fletcher DL, Jr, Newell JB, Haber E. Immunologic basis of transplant-associated arteriosclerosis. Proc Natl Acad Sci U S A. 1996;93:4051–4056. doi: 10.1073/pnas.93.9.4051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Snow AL, Oliveira JB, Zheng L, Dale JK, Fleisher TA, Lenardo MJ. Critical role for BIM in T cell receptor restimulation-induced death. Biol Direct. 2008;3:34. doi: 10.1186/1745-6150-3-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Pietra BA, Wiseman A, Bolwerk A, Rizeq M, Gill RG. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J Clin Invest. 2000;106:1003–1010. doi: 10.1172/JCI10467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Game DS, Lechler RI. Pathways of allorecognition: implications for transplantation tolerance. Transpl Immunol. 2002;10:101–108. doi: 10.1016/s0966-3274(02)00055-2. [DOI] [PubMed] [Google Scholar]
  • 34.Kalia V, Sarkar S, Subramaniam S, Haining WN, Smith KA, Ahmed R. Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity. 2010;32:91–103. doi: 10.1016/j.immuni.2009.11.010. [DOI] [PubMed] [Google Scholar]
  • 35.Suen AY, Baldwin TA. Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens. Proc Natl Acad Sci U S A. 2012;109:893–898. doi: 10.1073/pnas.1114834109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhan Y, Zhang Y, Gray D, Carrington EM, Bouillet P, Ko HJ, O’Reilly L, Wicks IP, Strasser A, Lew AM. Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells. J Immunol. 2011;187:1566–1577. doi: 10.4049/jimmunol.1100027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Gaston RS. Cytokines and transplantation: a clinical perspective. Transplant Sci. 1994;4 (Suppl 1):S9–19. [PubMed] [Google Scholar]
  • 38.Oakes SA, Scorrano L, Opferman JT, Bassik MC, Nishino M, Pozzan T, Korsmeyer SJ. Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2005;102:105–110. doi: 10.1073/pnas.0408352102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chen R, Valencia I, Zhong F, McColl KS, Roderick HL, Bootman MD, Berridge MJ, Conway SJ, Holmes AB, Mignery GA, Velez P, Distelhorst CW. Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate. J Cell Biol. 2004;166:193–203. doi: 10.1083/jcb.200309146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Shi C, Lee WS, He Q, Zhang D, Fletcher DL, Jr, Newell JB, Haber E. Immunologic basis of transplant-associated arteriosclerosis. Proc Natl Acad Sci U S A. 1996;93:4051–4056. doi: 10.1073/pnas.93.9.4051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Minami E, Laflamme MA, Saffitz JE, Murry CE. Extracardiac progenitor cells repopulate most major cell types in the transplanted human heart. Circulation. 2005;112:2951–2958. doi: 10.1161/CIRCULATIONAHA.105.576017. [DOI] [PubMed] [Google Scholar]
  • 42.Stevenson S, Shaffer JW, Goldberg VM. The humoral response to vascular and nonvascular allografts of bone. Clin Orthop Relat Res. 1996:86–95. doi: 10.1097/00003086-199605000-00011. [DOI] [PubMed] [Google Scholar]
  • 43.Labi V, Grespi F, Baumgartner F, Villunger A. Targeting the Bcl-2-regulated apoptosis pathway by BH3 mimetics: a breakthrough in anticancer therapy? Cell Death Differ. 2008;15:977–987. doi: 10.1038/cdd.2008.37. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Methods and Materials section
NIHMS579913-supplement-Methods_and_Materials_section.pdf (117.1KB, pdf)
Supplemental Figures
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