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. Author manuscript; available in PMC: 2012 Nov 7.
Published in final edited form as: Life Sci. 2011 Sep 10;89(19-20):748–754. doi: 10.1016/j.lfs.2011.08.016

BB rat Gimap gene expression in sorted lymphoid T and B cells

Daniel H Moralejo a,b, Jessica M Fuller c,d, Elizabeth A Rutledge e, Brian Van Yserloo d, Ruth A Ettinger d, Richard Jensen d, William Osborne f, Anne Kwitek g, Åke Lernmark b,c
PMCID: PMC3264544  NIHMSID: NIHMS329237  PMID: 21925515

Abstract

Aims

The Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in Gimap5, one of seven members of the Gimap family, results in lymphopenia and is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the Gimap family members proximal to Gimap5, encompassed within the Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the Gimap family members within the Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D.

Main methods

Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR.

Key findings

Gimap4 expression was reduced in DR.lyp/lyp rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of Gimap8, Gimap6, and Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire Gimap gene family was reduced in DR.lyp/lyp rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.+/+ rats. With the exception of Gimap6, the Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of Gimap9 was only detected in hematopoietic cells of non B cell lineage such as macrophage, dendritic or NK cells.

Significance

These results suggest that lack of the Gimap5 protein in the DR.lyp/lyp congenic rat was associated with impaired expression of the entire family of Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs.

Keywords: Gimap, lymphopenia, autoimmunity, type 1 diabetes

Introduction

The GTPase immunity-associated protein (Gimap) gene family in the rat is comprised of eight genes located within 150 Kb on rat chromosome (RNO) 4 (Krucken, et al. 2004; Rutledge, et al. 2009). The seven functional Gimap genes in rat; Gimap8, Gimap9, Gimap4, Gimap6, Gimap7, Gimap1 and Gimap5 are conserved across multiple vertebrates including human and mouse as well as in Arabidopsis thaliana (MacMurray, et al. 2002; Reuber and Ausubel 1996; Schnell, et al. 2006). These small GTPases regulate pro- and anti- apoptotic T cell pathways (Carter, et al. 2007; Dalberg, et al. 2007; Hernández-Hoyos, et al. 1999; Krücken, et al. 2005; Nitta, et al. 2006; Nitta and Takahama 2007; Pandarpurkar, et al. 2003; Schnell, et al. 2006) as well as thymocyte maturation and differentiation (Filen, et al. 2009; Nitta, et al. 2006) and are characterized by novel guanosine diphosphate/guanosine triphosphate (AIG1) and coiled-coil domains, common binding motifs and variable C termini (Krucken, et al. 2004; MacMurray, et al. 2002).

Peripheral T cell lymphopenia in the BioBreeding (BB) diabetes prone (DP) rat is linked to a single nucleotide deletion in Gimap5 (Iddm2; lyp) resulting in loss of Gimap5 protein expression that is a prerequisite for onset of spontaneous type 1 diabetes (T1D) in the DP rat (Fuller, et al. 2006; Hornum, et al. 2002; MacMurray, et al. 2002; Michalkiewicz, et al. 2004). While the Gimap5 mutation was not associated with T1D in humans (Payne, et al. 2004), the uncommon SNP in the polyadenylation signal of human GIMAP5 was found to be associated with risk for high IA-2 autoantibody levels in T1D patients (Shin, et al. 2007) implicating a possible role in islet autoimmunity. The common SNP at this location was associated with systemic lupus erythematosus underscoring the possible contribution of GIMAP5 to autoimmunity (Hellquist, et al. 2007).

Gimap8, Gimap9, Gimap4, and Gimap7 are transcribed in the same direction while Gimap6 runs opposite to the rest of the family. Each gene is separated by approximately 12 Kb of non-coding sequence. Gimap8, unique with three AIG1 binding domains (Dion, et al. 2005; Krucken, et al. 2004; Nitta, et al. 2006; Rutledge, et al. 2009), localizes to the endoplasmic reticulum, Golgi and mitochondria and may play an anti-apoptotic role in immune cells (Krücken, et al. 2005). Gimap6 and Gimap7 are transmembrane proteins containing weak endoplasmic reticulum (ER) localization signals (Krucken, et al. 2004). Gimap4, a cytosolic lymphoid specific signaling molecule, is associated with β-selection during maturation of CD4−CD8− double negative (DN) thymocytes into CD4+CD8+ double positive (DP) thymocytes (Fehlingvon Boehmer 1997; Schnell, et al. 2006) positive selection of T cells and accelerated T cell apoptosis (Nitta, et al. 2006; Poirier, et al. 1999; Schnell, et al. 2006).

Our previous study of the Gimap family showed that all seven members are differentially expressed in DR.+/+ and DR.lyp/lyp spleen, thymus and mesenteric lymph node (MLN) (Rutledge, et al. 2009). However, as whole organs of mixed cell populations were studied, it remains uncertain to what extent the observed differences were due to the cellular composition of these organs which are markedly affected by the reduction of T cells and a concordant increase in B cells and monocytes. To investigate these Gimap gene expression variations in sorted subpopulations of cells, we performed quantitative real time (qRT) PCR analysis on purified T and B cells from DR.+/+ and DR.lyp/lyp rat thymus, spleen and MLN. We chose to study lymphocytes from MLN in DR.lyp/lyp rats because their defective intestinal permeability may permit passage of environmental antigens initiating an autoimmune response, triggering T1D and changes in expression of Gimap genes (Graham, et al. 2004; Malaisse, et al. 2004; Vaarala 2008). Clarifying Gimap gene expression in specific populations of cells will aid in our understanding of the role of the Gimap family not only in thymocyte development, T cell survival in the periphery, or both, but also in T1D.

Materials and Methods

DR.lyp Rats

The DR.lyp (BBDR.BBDP-(D4Rhw17- SS99306861)(D4Rhw11-D4Rhw10)/Rhw) congenic rat line was derived from animals with two independent recombination events developed from our previously described introgression of the lymphopenia locus by cyclic cross-intercross breeding of BBDP with BBDR rats (Bieg, et al. 1998). The first recombination event was flanked by simple sequence length polymorphism (SSLP) marker D4Rhw11 (75.81 Mb) and the second flanked by SSLP marker D4Rhw10 (77.81 Mb) (Fuller, et al. 2006). Thus, the DP DNA in the DR.lyp rat line encompasses the lyp critical interval from D4Rhw6 (76.83 Mb) to IIsnp3 (77.16 Mb) (MacMurray, et al. 2002). In addition, the DR.lyp congenic rat line used in the present study also contains BBDP DNA at D4Rat102 (66.22 Mb) and D4Rat26 (69.18 Mb). The DR.lyp congenic rat line is kept in heterozygous sister-brother breeding and produces Mendelian proportions of the DR.lyp/lyp (25%), DR.lyp/+ (50%) and DR.+/+ (25%) genotypes. One hundred percent of DR.lyp/lyp rats are lymphopenic and diabetic.

Housing

Rats were housed in a specific pathogen–free facility at the University of Washington, Seattle, Washington, on a 12-h light/dark cycle with 24-h access to food (Harlan Teklad, Madison, WI) and water. The University of Washington Rodent Health Monitoring Program was used to track infectious agents via a quarterly sentinel monitoring system. The University of Washington institutional animal use and care committee approved all of the protocols used in the present study.

Genotyping Chromosome 4

Between 25 and 30 days of age, 2 mm tail snips were obtained and DNA isolated as previously described (Fuller, et al. 2009). The isolated DNA samples were diluted to 25 ng/μL in ddH2O and 2μL of this genomic DNA used in the following 10 μL reaction: 2 μL 5X reaction buffer (Promega, Madison, WI), 0.2 μL 10 mmol/L dNTP (Promega), 1.0 μL 1 μmol/L IRDye 700 labeled primer (LiCor Biosciences, Lincoln, NE), 0.5 μL 20 μmol/L unlabeled reverse primer (Invitrogen, Carlsbad, CA), 0.20 μL GoTaq DNA Polymerase (Promega) and 4.1 μL ddH2O. The PCR protocol was 95 °C for 5 min, 95 °C for 20 sec, 62 °C for 20 sec, 72 °C for 30 sec, steps 2–4 repeated 30 times, then 72 °C for 3 min. PCR products were diluted to 25% with STOP solution (LiCor Biosciences) and analyzed using a NEN Global IR2 DNA Analyzer System (Model 4200S-2) with a 6.5% gel matrix (LiCor Biosciences).

Cell Preparation and Purification

Rat thymocytes, spleen, and MLN were minced in PBS containing 10% FCS. The single cell suspensions from spleen were washed in lysing buffer (BD Pharm Lyse™ ) (BD Biosciences, San Diego, CA) to eliminate red blood cells. B cell suspensions were purified by positive selection of CD45+ and CD45RA+ cells with the respective biotinylated monoclonal antibody (mAb) from BD Bioscience Pharmingen (San Diego, CA) using streptavidin-conjugated MACS microbeads according to the manufacturer’s instructions Miltenyi MicroBead (Invitrogen, Carlsbad, CA).

FACS Sorting Of Lymphocyte Subsets

Single cell suspensions of rat thymocytes, spleen and MLN cells were washed three times in PBS containing 10% FCS and then incubated for 30 min at 4°C with primary mAbs in the dark. Cells were washed twice and after the final wash filtered through a 40-μm cell-strainer immediately before cell sorting. The following mouse anti rat mAbs obtained from BD Bioscience Pharmingen, were used R-phycoerythrin (R-PE)-labeled anti-CD3 (G4.18), Cy-chrome labeled anti-CD4 (OX35), FITC labeled anti-CD8 (OX8), and FITC labeled anti-alpha-beta T cell receptor (R73). Cell populations were sorted on a FACSAria cell sorter (BD Biosciences, Mountain View, CA). Purities were checked by flow cytometry to be between 90 and 99.9%.

RNA Isolation

FACS sorted thymus, spleen and MLN cells from 45–50 day old rats were homogenized in RNA lysis solution (Stratagene, La Jolla, CA or Qiagen, Valencia, CA) using a Kinematica Polytron PT 10/35 (Brinkmann, Westbury, NY) then aliquoted to minimize degradation from freezing and thawing. Total RNA was isolated using either RNeasy (Qiagen) or Absolutely RNA Miniprep Kit (Stratagene) followed by treatment with DNase according to the manufacturer’s instructions.

Quantitative RT-PCR

Quantitative RT-PCR was performed on an Mx4000® Multiplex QPCR System (Stratagene) with samples run in duplicate using approximately 10 ng of total RNA. Twenty-five μL reactions were run using a Brilliant® Single-Step qRT-PCR Kit (2.5 μL 10x core RT-PCR buffer, 5.0 mmol/L MgCl2, 400 nmol/L each primer, 200 nmol/L each probe, 0.8 mmol/L dNTP mix, 75 nmol/L passive reference dye, 2.0 units Stratascript RT, 1.25 units SureStart Taq DNA-polymerase). The PCR cycling conditions were as follows; 45°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 30 sec, 60°C for 1 min. Resulting Ct values were converted to copies, normalized to GAPDH and expressed as the mean ± standard error of the mean (SEM). Probes were positioned in the 3′ regions of the transcripts where there is more variation between the different Gimap genes and subjected to BLAST alignment to ensure specificity. The primers and probes used for each gene are detailed elsewhere (Rutledge, et al. 2009). Primers were obtained from Integrated DNA Technologies (Coralville, IA) or Qiagen Operon (Valencia, CA). Fluorescently labeled probes were obtained from Integrated DNA Technologies. Representative qRT-PCR products for each gene were run on an agarose gel to check for primer pair binding specificity. These PCR amplicons (used to convert to copy number) were quantitated in triplicate wells with the PicoGreen® RNA Quantitation Kit (Molecular Probes, Eugene, OR) using standards supplied by the manufacturer. Results from each assay were validated and normalized against GAPDH. The standard curves showed the following reaction efficiencies; Gimap8: 96% ± 3 SD, Gimap9: 94% ± 1 SD, Gimap4: 92% ± 2 SD, Gimap6: 90% ± 3 SD, Gimap7: 91% ± 4 SD, Gimap1: 94% ± 2 SD, Gimap5: 92% ± 2 SD, Lr8: 94% ± 3 SD, and GAPDH: 92% ± 1 SD.

Data analysis

Analyses of Gimap gene expression were carried out using linear mixed effect models (Pinheiro and Bates 2000) with S-PLUS (Insightful Corp., Seattle, WA). Comparisons of expression between cell types consisted of twelve rats with two repeated measures for each rat. Comparison between DR.lyp/lyp and DR.+/+ genotypes consisted of six rats from three litters consisting of one DR.lyp/lyp and one DR.+/+ rat in each litter and two repeated measures for each rat. All of these analyses contained random effects for each of the three litters and two measures for each rat nested within those litters. Likelihood ratio tests were used to test the need for random intercepts. The significance for the fixed effects were assessed by F test for the coefficient or the group of coefficients (Lehmann 1986). A Bonferroni correction was applied to p-values to adjust for multiple comparisons and adjusted values were considered significant at p < 0.05.

Results

Thymic T cells

Expression of the five Gimap genes within the Iddm39 QTL; Gimap8, Gimap9, Gimap4, Gimap6 and Gimap7, was compared to Gimap1, Gimap5 and Lr8, a gene unassociated with the Gimap family but located within the 33 Kb lymphopenia critical interval (Fig. 1). The number of thymocytes, sorted based on double staining with CD4 (T helper) and CD8 (T cytotoxic) monoclonal antibodies, did not differ between DR.+/+ and DR.lyp/lyp rats (Fig. 2). The sorted cells showed a high degree (>90%) of purity (Fig. 2, Panels A&B). Expression of Gimap4 in CD4−CD8− DN, CD4 single positive (SP), and CD8 SP thymocytes differed between DR.+/+ and DR.lyp/lyp rats (p<0.05 for all) while expression of Gimap8 (p<0.05), Gimap6 (p<0.05) and Gimap7(p<0.001) only differed in CD8 SP cells (Fig. 3). It should be noted that all statistical tests have been corrected for both litter and multiple comparisons. Within the lyp critical interval, Gimap5 expression was reduced in DR.lyp/lyp rat CD4 and CD8 SP thymocytes when compared to DR.+/+ (p<0.05 for both). Gimap9, Gimap1 and Lr8 were not appreciably expressed at any stage of T cell development in the thymus.

Fig. 1. The Gimap Gene Family on RNO4.

Fig. 1

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An expanded map of the 2 Mb of DP DNA in the congenic DR.lyp rat line is shown along with the Gimap family chromosomal locations on RNO4. The 33 Kb lyp critical interval is indicated between the SSLP markers D4Rhw6 and IIsnp3. Arrows indicate the orientation of the full length mRNA with exons shown in black and introns in white. Ian aliases are in parentheses underneath the corresponding Gimap name.

Fig. 2. T cell Profile and Purity.

Fig. 2

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Scatter plots are shown from one DR.+/+ and one DR.lyp/lyp rat FACS sorted T cells from thymus, spleen and MLN following staining with CD4 and CD8 monoclonal antibodies. Cells from spleen and MLN were first gated based on staining with a CD3 monoclonal antibody. The percent gated cells are shown within each quadrant. Panels A–D show the purity of sorted cells from one DR.lyp/lyp rat.

Fig. 3. Gimap Gene Expression in DR.+/+ and DR.lyp/lyp Sorted Thymic Cells.

Fig. 3

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The mean ± standard error of the mean (SEM) is shown for Gimap family expression in CD4 and CD8 FACS sorted thymocytes following normalization to GAPDH. DN is double negative (CD4−CD8−) and DP is double positive (CD4+CD8+). Black columns represent DR.+/+ rats (+, n=3) and hatched columns represent DR.lyp/lyp (l, n=3). Significant differences are * for p<0.05 and ** for p<0.001. Genes appear in the order at which they appear on RNO4.

Spleen and Mesenteric Lymph Node Peripheral T cells

Peripheral T cells from spleen and MLN were triple stained with CD3 (T cell receptor), CD4 and CD8 monoclonal antibodies. The number of sorted cells gated for CD3 and then sorted based on staining with CD4 and CD8 did not differ between DR.+/+ and DR.lyp/lyp rats and showed a high degree of purity (Fig. 2, panel C and D). Correcting for litter and multiple testing, we found that the expression of all Gimap genes in CD4+ splenic T cells did not differ between DR.+/+ and DR.lyp/lyp rats (Fig. 4). In contrast, the expression of all of the Gimap genes within the Iddm39 QTL was reduced in DR.lyp/lyp CD8+ splenic T cells.

Fig. 4. Gimap Gene Expression in Peripheral T cells.

Fig. 4

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The mean ± standard error of the mean (SEM) is shown for Gimap family expression in CD4+ and CD8+ FACS sorted T cells from spleen (Spl) and MLN following normalization to GAPDH. Cells from spleen and MLN were first gated based on staining with a CD3 monoclonal antibody. Black columns represent DR.+/+ rats (+, n=3) and hatched columns represent DR.lyp/lyp (l, n=3). Significant differences are * for p<0.05 and ** for p<0.001. Genes appear in the order at which they appear on RNO4.

The expression of Gimap4 and Gimap9 genes in the MLN CD4+ T cells showed no difference between DR.lyp/lyp and DR.+/+ rats, however, there was a reduction in Gimap8, Gimap6 and Gimap7. The MLN CD8+ T cells showed a reduction in expression of Gimap8, Gimap4 and Gimap7 while there was no difference in Gimap9 and Gimap6 (Fig. 4). As in the thymus, Gimap1 and Lr8 in the lyp critical interval did not express appreciably in either DR.+/+ or DR.lyp/lyp rat peripheral T cells (Fig. 4).

Spleen and MLN derived B cells

Hematopoietic B and non B cells (including T cells, macrophages, dendritic or NK cells) from spleen and MLN were double stained with CD45 (hematopoietic) and CD45RA (B cell) monoclonal antibodies and sorted based on staining with CD45RA (+/−). With the exception of Gimap6, the Gimap family in did not express appreciably in DR.+/+ or DR.lyp/lyp rat B cells (CD45+CD45RA+) from spleen or MLN (Fig. 5). Expression of the Gimap genes within the Iddm39 QTL; Gimap8, Gimap9, Gimap4, Gimap6 and Gimap7 was reduced in non B cells (CD45+CD45RA−) from DR.lyp/lyp rat spleen and MLN. The comparisons which remained statistically significant after correction for litter and multiple testing are indicated (Fig. 5). Interestingly, Gimap9 was expressed in both DR.+/+ and DR.lyp/lyp rat non B cells suggesting that Gimap9 is expressed in hematopoietic cells of non T or B cell lineage such as NK, macrophage or dendritic cells. Within the lyp critical interval (Fig. 1), Gimap5 showed a similar pattern with little or no expression in B cells and a reduced expression in DR.lyp/lyp rat non B cells (p<0.05). As in the thymus, spleen and MLN, Gimap1 did not express appreciably in either DR.+/+ or DR.lyp/lyp rat B or non B cells (Fig. 5).

Fig. 5. Gimap Gene Expression in Peripheral B cells.

Fig. 5

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The mean ± standard error of the mean (SEM) is shown for Gimap family expression in FACS sorted hematopoietic B cells (CD45+CD45RA+) and non B cells (CD45+CD45RA−) from spleen and MLN after normalization to GAPDH. Black columns represent DR.+/+ rats (+, n=3) and hatched columns represent DR.lyp/lyp (l, n=3). Significant differences are * for p<0.05. Genes appear in the order at which they appear on RNO4.

Discussion

The Gimap5 mutation within the lyp critical interval has been documented to result in peripheral T cell lymphopenia in the DR.lyp/lyp rat (Dion, et al. 2005; Hornum, et al. 2002; MacMurray, et al. 2002). In our previous qRT-PCR analysis we showed reduced expression of all seven Gimap genes; Gimap8, Gimap9, Gimap4, Gimap6, Gimap7, Gimap1 and Gimap5 in DR.lyp/lyp rat spleen and MLN when compared to DR.+/+ while only four; Gimap9, Gimap4, Gimap1, and Gimap5 were reduced in thymus (Rutledge, et al. 2009). The present study resolves these previously observed expression differences to specific cell types both during thymocyte development and to mature T cells in the periphery. As lymphopenia impacts the cellular composition of the spleen and MLN, but not necessarily the thymus, the present qRT-PCR analysis of sorted cells further substantiates the Gimap5 frameshift mutation as the primary defect in the periphery. Three of the Gimap genes within the Iddm39 QTL; Gimap8, Gimap6 and Gimap7 as well as the Gimap5 gene itself were expressed in DN and DP thymocytes with no significant difference in expression between DR.+/+ and DR.lyp/lyp rats. Interestingly, while these genes were turned on during maturation of DN to DP thymocytes, no impact of the Gimap5 mutation on gene expression was observed, as was seen in the periphery. Upon transition from DP to SP thymocytes, expression of four of the Iddm39 genes; Gimap8, Gimap4, Gimap6 and Gimap7 as well as Gimap5 increased in CD4 SP thymocytes and showed the highest overall expression as compared to the other three stages of thymocyte development however only Gimap4 and Gimap5 showed expression variation between DR.+/+ and DR.lyp/lyp rats. These same five Gimap genes were poorly expressed in CD8 SP thymocytes and all showed a significant difference in expression between DR.+/+ and DR.lyp/lyp rats. We believe this apparent reduction in expression of Gimap8, Gimap4, Gimap6, Gimap7and Gimap5 in DR.lyp/lyp CD8+ cells before leaving the thymus is a significant observation that may aid in understanding the possible role of the Gimap genes in T cells as well as their possible role in the pathogenesis of T1D.

The possible role of the Gimap genes in either protection from T cell apoptosis, such as Gimap5 (Nitta, et al. 2006; NittaTakahama 2007; Pandarpurkar, et al. 2003) and Gimap8 (Krücken, et al. 2005), or to induce apoptosis such as Gimap4 (Nitta, et al. 2006) would perhaps explain the severe lack of CD8+ T cells in peripheral blood and tissues. Gimap4 and Gimap5 interact with the Bcl-2 family of signal transduction molecules to regulate the mitochondrial mediated T cell apoptosis pathway (Nitta, et al. 2006). The lower observed expression of Gimap4 in the DR.lyp/lyp CD4+ SP thymocytes would potentially explain that more CD4+ and less CD8+ T cells are detected in peripheral blood and tissues. It is however not understood why CD8+ cells are expressing less of all the Gimap genes, whether from DR.+/+ or DR.lyp/lyp rats, or how this low expression impacts the ability of CD8+ T cells to survive in the periphery.

The major finding from the analysis of CD3+ spleen and MLN T cells sorted for either CD4 or CD8 positivity was that all the Gimap genes, except Gimap9 and Gimap1, whether in the Iddm39 QTL or the lyp critical interval showed reduced expression in the cells from the DR.lyp/lyp rats. While the Gimap genes showed the highest expression levels overall in cells sorted from the spleen and MLN, the difference in Gimap gene expression between DR.+/+ and DR.lyp/lyp appeared more pronounced than in CD4 or CD8 SP thymocytes. The study of lymphocytes from MLN is justified because it is well known that the defective intestinal permeability associated with DR.lyp/lyp rats may permit passage of environmental antigens initiating an autoimmune response and triggering T1D (Graham, et al. 2004; Malaisse, et al. 2004; Vaarala 2008). It should be noted that the same number of cells were sorted from both the spleen and the MLN whether the cells were from a DR.+/+ (typical sorting time for CD8+ T cells was 90 minutes) or DR.lyp/lyp (typical sorting time for CD8+ T cells was 4 hours) rat. The higher level of expression of all the Gimap genes, except Gimap9 and Gimap1, in peripheral compared to thymic CD8+ cells suggests that only the highest Gimap expressing cells survive the transition from the thymus to the periphery.

Our observation in sorted peripheral CD4+ and CD8+T cells was that the loss of the Gimap5 protein is associated with a reduction in expression of all the Gimap family members (except Gimap1 and Gimap9) suggesting that despite an increase in expression from the thymus to the periphery, CD8+ T cells from the DR.lyp/lyp rat are prone to die. It is tempting to speculate that the coordinate expression of Gimap8, Gimap4, Gimap6, Gimap7 and Gimap5 may be due to the absence of the Gimap5 protein. However, analyses of the Gimap5 amino acid sequence show no indication that this protein would represent a transcription factor. The alternative explanation would be that Gimap8, Gimap4, Gimap6, Gimap7 and Gimap5 share a common transcriptional element. Bioinformatic analyses of our own and available database sequences have failed to detect a common promoter region or other sequences that would indicate the presence of a common regulator. Further studies are therefore required to uncover the mechanisms by which Gimap8, Gimap4, Gimap6, Gimap7 and Gimap5 are regulated in a coordinate fashion in CD4+ and CD8+ cells from DR.lyp/lyp rats.

The nature of the Gimap5 frame shift mutation leading to the absence of the Gimap5 protein (MacMurray, et al. 2002; Michalkiewicz, et al. 2004) suggests that the loss of this anti-apoptotic protein contributes to lymphopenia. The importance of the Gimap5 mutation for the DR.lyp/lyp rat lymphopenia was amply illustrated by the marked reduction in expression of the Gimap5 in the non B cells sorted from the spleen or the MLN. As the Gimap5 mutation is comparable to a null mutation, i.e. no Gimap5 protein is made (MacMurray, et al. 2002; Michalkiewicz, et al. 2004) it is of interest to note that the Gimap5 knock out mouse that we recently reported (Schulteis, et al. 2008) resulted in impaired thymocyte maturation as well as survival of peripheral CD4 and CD8 positive T cells. Gimap5 deficiency also blocked natural killer and NKT cell differentiation which could be restored on transfer of Gimap5 deficient bone marrow into a wild-type environment. Although the phenotype of the Gimap5 knockout mouse is comparable to that of the DR.lyp/lyp rat, it remains to be clarified if the other Gimap genes are affected as demonstrated in the present study. As many of these proteins have been implicated in cell survival (Nitta, et al. 2006; Pandarpurkar, et al. 2003; Sandal, et al. 2003), it cannot be excluded that the concomitant reduction in several of the Gimap genes outside the lyp critical interval may contribute to the severe lymphopenia observed in the periphery (Dion, et al. 2005; Hornum, et al. 2002; MacMurray, et al. 2002).

In conclusion, a frameshift mutation in Gimap5 results in lymphopenia and precedes onset of spontaneous T1D in the diabetes prone BB rat. Reduced expression of all seven Gimap genes in peripheral lymphoid organs suggests that the lack of Gimap5 protein in the DR.lyp/lyp congenic rat line impairs expression of the entire Gimap gene family and regulates T cell homeostasis in the peripheral lymphoid organs. The major findings in the present study both confirm our initial observations as well as resolve Gimap gene expression in specific thymic lineages as well as peripheral T cell and B cell subsets. Further elucidating the role of the Gimap family, specifically Gimap5, in T cell survival, development and/or activation will aid in our understanding of the pathways in onset of spontaneous T1D in the BB rat and ultimately the pathways leading to development of human disease.

Acknowledgments

This work was supported by the National Institutes of Health (AI42380- D.H.M., J.M.F., R.A.E., A.K., Å.L.) and the Molecular and Genetics Core of the University of Washington Diabetes and Endocrinology Research Center (DK17047- E.A.R., B.V., W.R.O.), a Junior Faculty Award (D.H.M), a Mentor Based Postdoctoral Fellowship Award (Å.L.) from the American Diabetes Association, the Robert H Williams Endowment at the University of Washington and the National Heart, Lung, and Blood Institute Training Grant T32HL007902 (R.A.J.).

Footnotes

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