Generation and Characterisation of Mice Deficient in the Multi-GTPase Domain Containing Protein, GIMAP8

<div><p>Background</p><p>GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function.</p><p>Principal Findings</p><p>We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell <i>in vitro</i> in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen.</p><p>Conclusions</p><p>Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.</p></div

GIMAP8 protein expression during T and B lymphocyte development.

<p>Thymocyte, bone marrow, and splenic subpopulations of developing lymphocytes were sorted and subjected to Western blotting to determine GIMAP8 expression during T cell development (A), B cell subsets in the bone marrow (B), and the spleen (C). Actin was used as a loading control. Intracytoplasmic flow cytometry was performed on developing T (D) and B (E) lymphocytes. All cells were stained with extracellular markers to define distinct subpopulations prior to intracytoplasmic staining for GIMAP8 with mAb MAC 418. Results are representative of two independent experiments. Median levels of fluorescence were determined by dividing levels of fluorescence in wild type cells by the average median fluorecence for the same cell type from GIMAP8-deficient animals. Results show mean levels of fluorescence for 3 individual animals ± S.D.</p

Bone marrow cell analysis.

<p>Bone marrow cells from <i>GIMAP8^−/−</i> mice and littermate controls were stained for flow cytometric analysis to identify B-lineage subsets. (A) Representative flow cytometry plots for each genotype. The numbers given inside the component panels in (A) are the percentages of different bone marrow B cell populations. (B) Numbers of B-lineage subsets in BM are shown where each dot represents a single mouse.*P <0.05 (unpaired 2-tailed Student's t test).</p

T cell-dependent immune responses in <i>GIMAP8^−/−</i> mice.

<p>Day 14 titers of anti-NP₂₃ IgM (A) and anti-NP₂₃ IgG₁ (B). Five weeks after primary immunization, mice were immunized with NP-KLH to examine secondary responses. Titers of anti-NP₂₃ IgM (C), anti-NP₂₃ IgG₁ (D), and high affinity anti-NP₂ IgG1 7 days after secondary immunization. Each dot represents a single mouse.</p

Targeted deletion of exons three and four of the GIMAP8 gene disrupts production of GIMAP8 protein.

<p>(A) Summary of the structure of the mouse GIMAP8 gene, the derived targeting vector and the recombined GIMAP8 gene after homologous recombination. Note that for both exons 1 and 2 and exons 3 and 4, the gene structure is represented by single solid blocks as the intervening introns are too small to show at the same scale. Numbered arrows indicate the approximate location of oligonucleotide primers used to identify wild-type and recombined alleles. (B) Agarose gel analysis of PCR products derived using the indicated primer pairs from wild type, heterozygous and homozygous GIMAP8-targeted mice. (C) Upper panel – Western blot of GIMAP8 expression in splenocytes from wild type, heterozygous and homozygous GIMAP8-targeted mice using rat anti-mouse monoclonal antibody MAC 443; Lower panel – the GIMAP8 blot re-probed with an anti β-actin antibody. In both panels, the mobilities of molecular weight standards resolved on the same gel are indicated.</p

Thymocyte development in GIMAP8-deficient mice.

<p>(A) Representative flow cytometry plots of thymocytes from <i>GIMAP8^−/−</i> mice and littermate controls stained for CD4 and CD8. (B) Numbers of thymocyte subsets are shown where each dot is representing a single mouse. (C) Numbers of CD4 and CD8 splenocytes are shown where each dot is representing a single mouse.</p

Splenic and peritoneal B cell analysis.

<p>(A) Representative flow cytometry plots of splenic cells for each genotype. The numbers given inside the component panels in A and D are the percentages of lymphocyte events contained within the lymphocyte gate. Numbers of B-lineage subsets in spleen and peritoneum are shown where each dot represents a single mouse. Numbers of mature B cell subsets in spleen (Figures B & C). Representative flow cytometry plots of peritoneal cells for each genotype (D). Numbers of B-lineage subsets in spleen and peritoneum are shown where each dot represents a single mouse (Figures E & F).</p

B cell survival and respiration <i>ex vivo</i>.

<p>(A) the mean number (± SD) of live cells as a percentage of the original number of cells plated for three individual mice. (B) Mean (± SD) OCR for triplicate wells containing purified splenic B cells from WT WT (▪) and GIMAP8-and deficient mice (□) mice following injection of oligomycin, CCCP, and antimycin A plus rotenone and is representative of two independent experiments. (C) T cells from either WT (•) and GIMAP8-and deficient mice (□) were incubated in complete medium (control), dexamethasone, or following gamma irradiation for 8 hr. The number of live (Annexin V- and DAPI-), apoptotic (Annexin V+ and DAPI-), and dead (AnnexinV+ or - and DAPI+) cells were enumerated by flow cytometry. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110294#pone-0110294-g006" target="_blank">Figure 6C</a> shows the average count for duplicate samples from three mice and is representative of two independent experiments. *p<0.05, ** P<0.005 (unpaired 2-tailed Student's t test).</p

Effect on the expression of selected proteins of Gimap6 ablation in vitro from GIMAP6^fl/flERT2Cre CD4⁺ T cells.

<p>Panel A) Enriched CD4⁺ cell fractions were prepared from spleens of ERT2Cre or GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro for five days in the presence or absence of 200 nM 4HT. Cell lysates were prepared and analysed by SDS polyacrylamide gel electrophoresis with the indicated antibodies. Panel B) Enriched CD4⁺ cell fractions were prepared from spleens of GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro in the presence or absence of 4HT for the indicated times before preparation of cell lysates and western analysis with the indicated antibodies. The electrophoretic mobilities of marker proteins are indicated. Panel C) Enriched CD4⁺ cell fractions were prepared and incubated with or without 200 nM 4HT for 4 days. TBK1 inhibitor BX795 (1 μM) was then added to the cells as indicated and incubation continued for 6h. Cell lysates were prepared and proteins analysed by western blotting. In all panels, the electrophoretic mobility of marker proteins is indicated. All results were confirmed in at least two experiments.</p

The reduced number of peripheral CD4⁺ T cells in GIMAP6^fl/flCD2Cre mice reflects an increase in apoptosis.

<p>A) Splenocytes isolated from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre animals were stained with fluorescent antibodies to CD4 and CD24. The left-hand panel shows the distribution of CD24 staining on CD4⁺ cells from single mice of the two genotypes (GIMAP6^fl/fl–dotted line; GIMAP6^fl/flCD2Cre–solid line). The right-hand panel shows the median fluorescence intensity of CD24 staining of CD4⁺ cells. n = 4; **P<0.01. B) Freshly isolated splenocytes were stained with an APC-conjugated antibody to CD4 and PE- conjugated annexin V. The cells were then washed and stained with DAPI before flow analysis. The two dot-plots show typical staining patterns for a control (C) and knockout (KO) animal. The quadrants are as follows: Q1 –annexin V^- DAPI⁺; Q2 –annexin V⁺ DAPI⁺; Q3 –annexin V⁺ DAPI^-; Q4 –annexin V^- DAPI^-. The right-hand panel summarises the percentage of annexin V⁺ cells as a percentage of total DAPI^- CD4⁺ T cells for five animals in each group. ns P>0.05; ** P<0.01. C) CD4⁺ enriched naive splenocytes from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre mice were activated by maintenance on anti-CD3 antibody-coated plates in medium containing anti-CD28 and IL2 or were maintained in medium without activation as described in the Materials and Methods section. After 24 h cells were harvested and stained as in (B). The percentage of CD4⁺ T cells in each quadrant is indicated. n = 3 for each group; ns P>0.05; ** P<0.01.</p