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. 2008 Dec 15;112(13):4905-14.
doi: 10.1182/blood-2008-03-146555. Epub 2008 Sep 16.

Impaired survival of peripheral T cells, disrupted NK/NKT cell development, and liver failure in mice lacking Gimap5

Ryan D Schulteis  1 , Haiyan Chu, Xuezhi Dai, Yuhong Chen, Brandon Edwards, Dipica Haribhai, Calvin B Williams, Subramaniam Malarkannan, Martin J Hessner, Sanja Glisic-Milosavljevic, Srikanta Jana, Edward J Kerschen, Soumitra Ghosh, Demin Wang, Anne E Kwitek, Ake Lernmark, Jack Gorski, Hartmut Weiler
Affiliations

Impaired survival of peripheral T cells, disrupted NK/NKT cell development, and liver failure in mice lacking Gimap5

Ryan D Schulteis et al. Blood. .

Abstract

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5(-/-) bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5(-/-) bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.

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Figures

Figure 1
Figure 1
Generation of gimap5 knockout mice. (A) A replacement-type gene-targeting vector was constructed from a thymidine kinase expression cassette (TK); a 5.4-kb EcoR1-BamH1 fragment (5′-homology) of gimap5 intron 1; a pkg-promoter-neomycin resistance gene cassette (NEOR, orientation opposite to gimap5) and a 1.3-kb fragment comprising the last 713 bp of exon 3 plus 597 bp of 3′-flanking region. Homologous recombination replaces a 2466-bp fragment of the Gimap5 gene containing exon 2 and a fragment of exon 3, thereby deleting the translation start signal within exon 2 and the first 130 amino acids of gimap5 protein. The figure is not drawn to scale. (B) Recombination in the 5′ and 3′ region of the Gimap5 gene was confirmed by amplification of a 4-kb genomic fragment with primers A and B (ii), and a 2.2-kb fragment with primers C and D (iii), respectively. * indicates ES clones having undergone the predicted recombination events in both the 5′ and 3′ homology. (iv) Southern blot hybridization of Xba1-digested genomic DNA from Gimap+/− ES clones (lanes 1,2) or wild-type ES cells (lane 3) with a 32P-labeled neomycin probe detects the predicted 3.1-kb fragment, consistent with a single locus integration of the NEO cassette. (v) Southern blot hybridization analysis of Bgl2-digested genomic DNA from wild-type (lane 1) and Gimap5−/− mice (lane 2) with probe E. The probe detects in wild-type mice a 2.6-kb fragment comprising exon 3 of gimap5 and a 1.8-kb fragment containing a homologous region in exon 5 of Gimap3; in Gimap5−/− DNA, the Gimap3 fragment detected is identical to wild-type, whereas the Gimap5 fragment shifts to 4.1 kb resulting from insertion of the NEO gene. (C) Measurements of gimap gene family mRNA abundance by real-time PCR. The structure of the gimap gene cluster on chromosome 6 is shown on top. Bars indicate the average plus or minus SEM from triplicate determinations from 3 mice each. Data are normalized for gimap abundance in wild-type C57BL/6J mice. Only gimap5 mRNA is significantly reduced in Gimap5+/− mice and is undetectable in Gimap5−/− mice. (D) Survival of Gimap5−/− mice (n = 35) over 42 weeks. The median age of death lies between 14 and 15 weeks. By 42 weeks, no surviving Gimap5−/− mice remained.
Figure 2
Figure 2
Erythrocyte abnormalities and peripheral lymphopenia in Gimap5−/− mice. (A) Peripheral blood smear (Wright-Giemsa stain, original magnification ×400). Bars represent 10 μm. wt indicates wild-type; −/−, Gimap5-knockout. Images were acquired on a Nikon Eclipse E600 microscope with 10×, 20×, and 40× PlanFluor objectives and a SPOT Insight 11.2 color mosaic digital camera with Spot software version 4.1 (Diagnostic Instruments, Sterling Heights, MI). (B) Representative FACS analysis of wild-type (wt) and Gimap5-deficient (−/−) splenocytes and inguinal lymph node cells (INL) for expression of CD8 and CD4. Boxes indicate gates used to calculate frequencies presented in Table 1. Gimap5−/− mice exhibit peripheral lymphopenia, with reduced abundance of CD4 T cells and almost complete absence of CD8 T cells from the spleen and inguinal lymph nodes (INL).
Figure 3
Figure 3
Characterization of peripheral T cells in Gimap5−/− mice. (A) Representative histograms of CD69 and CD62L expression in CD4+ (top) and CD8+ splenocytes (bottom). Increased expression of CD69 and shedding of CD62L indicate a state of increased activation, compared with wild-type mice. (B) The frequency of CD4+CD25+FoxP3+ regulatory T cells in the spleen or the inguinal lymph node (ILN) is unaltered in Gimap5−/− mice. (C) Apoptosis is increased among peripheral T cells from Gimap5−/− mice. CD4+ (left) and CD8+ (right) splenocytes were stained with the dyes YO-PRO-1 and 7-AAD to identify early apoptotic (YO-PRO-1+7-AAD−) and dead (YO-PRO-1+7-AAD+) T cells. (D) Abundance of CD45.2+ gimap5−/− CD4 and CD8 T-cell populations in lethally irradiated CD45.1+ wild-type recipients 8 weeks after transplantation with unfractionated BM from CD45.2 Gimap5−/− mice. Overall representation of splenic CD45.2 T cells in the example shown was approximately 60%. The relative abundance of donor-derived CD8 and CD4 cells captured by the indicated gates is given as average percentage plus or minus SD (n = 3). The ratio between CD4 and CD8 cells is significantly different between Gimap5−/− donor cells and residual wt recipient cells (P < .05). (E) Jak3−/− mice were sublethally irradiated and transplanted with unfractionated BM from wild-type (wt) or Gimap5−/− mice (−/−). Relative abundance of CD4 and CD8 cells represents average plus or minus SD (n = 3). The ratio between CD4 and CD8 cells is significantly different between Gimap5−/− and wild-type donor cells (P < .05).
Figure 4
Figure 4
Thymic development in Gimap5−/− mice. Representative FACS plots of wild-type (wt) or Gimap5-deficient (−/−) thymocytes. (A) Expression of CD4 and CD8 in unfractionated thymocytes. Boxes indicate gates used to calculate the average frequencies included in the text. (B) Expression of CD44 and CD25 in DN thymocytes contained in the DN gate of panel A. (C) Expression of CD69 and H57/TCR-β (TCR) in DP thymocytes contained in the DP gate of panel A. (D) Expression of CD69 and Qa2 in CD4SP and CD8SP thymocytes. The relative accumulation of CD69LOWQa2− versus CD69LOWQa2+ cells indicates a defect in the final stages of intrathymic T-cell maturation in Gimap5−/− mice.
Figure 5
Figure 5
Gimap5−/− mice lack NK and NKT cells. Development of NK and NKT cells is severely impaired in Gimap5−/− mice. (A) Representative analysis of BM from Gimap5−/− and WT mice stained with anti–CD3-ϵ and anti-CD122; numbers indicate percentage of cells (average ± SD) in gate. CD3-ϵ−CD122+ NK and CD3-ϵ+CD122+ NKT are significantly reduced in Gimap5−/− mice (NK: P = .004; NK-T: P = .002). (B) Representative analysis of BM from Gimap5−/− and WT mice stained with anti–CD3-ϵ and CD49b. (C) The absolute numbers (average ± SD) of CD3-ϵ−CD49b+ NK or CD3-ϵ+CD49b+ NKT cells were significantly reduced in the Gimap5−/− mice. Absolute cell numbers were calculated as percentage cells times percentage lymphocyte times cellularity of spleen (Spl) or BM (n = 6/group). P values calculated by the Student t test. (D) Expression of developmental markers in BM-derived fresh Gimap5−/− CD3-ϵ−CD49b+ NK cells is severely reduced. The frequencies of cells positive for each marker among CD3-ϵ−CD49b+ NK cells are shown along with representative histograms. Gates were set using unstained or nonspecific isotype antibody controls (not shown). Data are representative of 4 mice/genotype. (E) Gimap5−/− mice lack CD1-αGalCer+ NKT cells. Single-cell suspensions prepared from the indicated tissues of wild-type mice (WT) and Gimap5−/− mice (−/−) were stained with anti–CD3-ϵ mAb and α-GalCer–loaded CD1d dimers. Numbers represent percentage Vα14+ CD3-ϵ+ iNKT cells in the gates shown. Anti–CD3-ϵ mAb and unloaded CD1d dimers were used as background controls. Data presented are representative of at least 3 independent analyses.
Figure 6
Figure 6
Liver pathology of Gimap5−/− mice. (A) In situ appearance of the liver in a 7-week-old Gimap5−/− mouse. The gallbladder (brown, lower left) appears normal. Image was acquired with a Nikon Coolpix digital camera. (B,C) Histologic sections (periodic acid Schiff [PAS] stain). Large areas appear necrotic (pale PAS stain, swollen hepatocytes with finely granular cytoplasm, sparse nuclear staining) but are largely devoid of mononuclear cells. (C) Other areas of the liver are characterized by massive, centrilobular, and periportal accumulation of mononuclear cells (blue nuclear stain, original magnification ×100). Bars represent 100 μm. (D) Liver histology of a 3-week-old gimap5−/− mouse (Trichome stain, original magnification ×200). Bar represents 100 μm. External appearance and histology of the liver parenchyma appear normal; the Gimap5−/− liver contains a large number of hematopoietic foci (→; inset shows high power view; original magnification ×400). Bar represents 10 μm. (B-D) Images were acquired on a Nikon Eclipse E600 microscope with 10×, 20×, and 40× PlanFluor objectives and a SPOT Insight 11.2 color mosaic digital camera with Spot software version 4.1 (Diagnostic Instruments, Sterling Heights, MI). (E) TUNEL stain of liver shown in panel B; areas of TUNEL positivity correlate with the pale-staining areas in PAS-stained sections. (F) DNA fragmentation is not detected in young Gimap5−/− livers shown in panel D. (E,F) TUNEL staining with fluorescein-labeled deoxyuridine triphosphate (dUTP) was recorded in the FITC channel.
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