In this study we analyzed the ER stress response in T cells from Gimap5−/− BBDP rats. We demonstrate that absence of functional full length Gimap5 protein in T cells leads to ER stress response signaling as evidenced by increased GRP78 protein expression. In addition, our data show that BBDP T cells initiate ER stress-induced apoptotic signaling through upregulation of CHOP protein. Finally, decreasing the expression of this ER stress apoptotic factor, protected BBDP T cells from ER stress-induced death. These observations suggest that Gimap5 protein plays an essential role in the maintenance of ER homeostasis and is indispensable for T cell survival.
Lymphopenia in the BBDP rat was originally linked to a recessive mutation in a diabetes susceptibility locus designated lyp/Iddm1
, which encodes for Gimap5 protein 
. Although spontaneous diabetes development in the BBDP rat depends on the presence of lyp/Iddm1
, diabetes susceptibility and lymphopenia are traits that can be inherited independently 
. To ensure that ER stress response signaling in T cells from the BBDP rat is due to absence of functional Gimap5 protein and not a byproduct of diabetes development, we only used nondiabetic BBDP rats for studies. Furthermore, flow cytometry analyses on Gimap5−/−
T cells from congenic WF.ART2a.Gimap5−/−
rats that do not develop spontaneous diabetes but carry the Gimap5
null mutation have increased expression of ER stress response proteins, including GRP78 and CHOP (Figure S1E,F
). Overall, this indicates that ER stress response signaling in T cells from the BBDP rat is a result of the absence of functional Gimap5 protein product and not a secondary consequence of spontaneous diabetes development.
Within the immune system, the establishment of immunological tolerance involves mechanisms to delete self-reactive T cells in order to avoid autoimmune diseases such as type 1 diabetes 
. The interaction of TCRs on thymocytes with thymic stromal cells are important for proper T cell development and eventually allow for CD4+
double positive thymocytes to differentiate into mature CD4+
single positive thymocytes 
. Our data reveal that the percentage of double positive and single positive thymocytes does not differ between BBDR and BBDP rats, despite the absence of Gimap5 in the BBDP rat. Furthermore, ER stress response signaling is similar in the thymocyte populations from both BBDR and BBDP rats indicating the main ER regulatory functions for Gimap5 protein may occur during T cell post-thymic development. In general, our data indicate that ER stress response and ER stress-mediated signaling may only occur minimally during thymocyte development as levels of ER chaperones remained constant as double positive thymocytes matured to the single positive stage.
Following thymic development, T cells still expressing CD90, known as RTEs, enter the circulation. In the BBDP rat, a limited number of RTEs survive, down-regulate CD90 expression and become mature T cells, as others have reported and we have confirmed () 
. Because RTEs undergo apoptosis rapidly in the Gimap5−/−
BBDP rat, we initially hypothesized that ER stress protein levels would be higher in RTEs than in CD90−
mature T cells from the same animal. Our data suggest that ER chaperone expression is similar in all CD90+
and CD90− Gimap5−/−
T cell subpopulations in the BBDP rat. Our data further demonstrate that the percentage of the peripheral CD90+
T cells that express the activation marker CD25 is higher in the BBDP rat than in the BBDR rat (). Although activation of T cells is known to enhance ER stress 
T cell populations from the Gimap5−/−
BBDP rat expressed ER stress levels significantly higher than their counterparts from the Gimap5+/+
BBDR rat. These data indicate a global absence of the protein is sufficient to initiate ER stress response signaling throughout peripheral T cell development and that activation signals are not required for ER stress induction.
ER stress response signaling has been shown to be required for numerous physiological functions in several cell types 
. In addition, pathological signals from the ER have been attributed to cell death and apoptosis and linked to many diseases, including diabetes 
. In this report, we show that absence of functional Gimap5 protein leads to these pathological signals from the ER and subsequent ER stress-induced apoptosis in T cells from the BBDP rat. We suggest that triggering of the ER stress-induced apoptotic pathway in the T cells of BBDP rats is the underlying mechanism for their lymphopenia. This inference is supported by our data demonstrating that decreasing CHOP protein expression reduces the number of apoptotic T cells. Interestingly, although B cells normally express Gimap5, we found that ER stress response signaling in B cells is similar between Gimap5+/+
BBDR and Gimap5−/−
BBDP rats (). These data suggest that modulation of ER stress response signaling by Gimap5 is required for the maintenance of T cell survival.
As documented above, this report demonstrates that ER stress-induced apoptosis occurs in Gimap5−/−
T cells. However, we and others have previously shown a role for Gimap5 protein in the maintenance of mitochondrial integrity for T cell survival 
. These two Gimap5-dependent processes are both linked through CHOP, a protein that is both highly expressed in cells undergoing ER stress-induced cell death and also capable of sensitizing mitochondria to numerous apoptotic factors 
. This sensitization occurs via downregulation of antiapoptotic Bcl-2 protein, activation of JNK and its associated proapoptotic downstream kinases 
, and activation of caspase-12 
. We hypothesize that CHOP protein through its role in ER stress-induced apoptotic signaling may lead to the disruption of mitochondrial integrity that is observed in Gimap5−/−
T cells 
. We propose that loss of mitochondrial integrity in Gimap5−/−
T cells may be secondary to ER dysfunction and subsequent ER stress-induced apoptotic signaling.
The intracellular distribution of Gimap5 protein remains controversial, but recent research indicates localization to the ER 
. Our data support an intimate link between wild type Gimap5 and the ER due to ER stress response signaling in Gimap5−/−
T cells. Accumulation of intracellular proteins is known to cause ER stress and can lead to cell death through CHOP signaling 
. This association has been demonstrated in pancreatic beta cells of the Akita mouse in which accumulation of mutant insulin causes ER stress and eventual apoptosis through CHOP protein induction 
. We observed an accumulation of αβTCR protein within Gimap5−/−
BBDP rat lymphocytes that correlates with increased GRP78 levels (unpublished observations).
In the BBDP rat there is the frameshift mutation in Gimap5
which results in a truncated protein product 
. We can not rule out the possibility that the presence of this truncated protein product or increased intracellular αβTCR protein may accumulate in the ER and lead to the pathological ER stress that is observed in Gimap5−/−
T cells. In either case, disruption of CHOP was effective in delaying apoptosis and preventing cell death, thus indicating the importance of CHOP in ER stress-associated T cell death signaling. Interestingly, overexpression of either wild type rat Gimap5 or the truncated BBDP rat Gimap5 in a rat T cell line is associated with enhanced apoptosis 
. In this report, the apoptosis-inducing effect of mutant Gimap5 was much greater than that of the overexpressed wild type protein. In the context of our data, this may indicate that the expression of Gimap5 in T cells is both required for survival and tightly regulated.
In conclusion, our findings suggest a role for ER stress in mediating the spontaneous apoptosis found in T cells from Gimap5−/−
rats. It will be of interest to determine if the same mechanisms are responsible for the T cell lymphopenia and hepatic dysfunction observed in gimap5−/−
. We provide the first evidence of a link between Gimap5 protein and the maintenance of ER homeostasis and cellular integrity. Our results indicate that proper functioning of the ER in T cells is critical for their survival. Investigations into the transient lymphopenia which occurs following virus infections in humans 
and in the blood of patients with type 1 diabetes 
may reveal a role for ER regulatory pathways in mediating human immunity. Further studies of wild type and mutant Gimap5 protein are needed to precisely define the mechanisms by which they affect T cell survival and function. Such studies may uncover novel pathways capable of modulating the survival and function of T cells and other tissues.