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

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

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

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

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

The Immune System GTPase GIMAP6 Interacts with the Atg8 Homologue GABARAPL2 and Is Recruited to Autophagosomes

<div><p>The GIMAPs (GTPases of the <b><u>i</u><u>m</u></b>munity-<b><u>a</u></b>ssociated <b><u>p</u></b>roteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux. </p> </div

Identification of the domains of GIMAP6 and GABARAPL2 required for their interaction.

<p>Panels A-C) HEK293T cells were transfected with 10µg wild-type GIMAP6 or the indicated mutated derivatives in plasmid pcDNA3Biot1His6iresBirA. Biotinylated and associated proteins were recovered by streptavidin-agarose affinity chromatography 48 h after transfection. Western blots of the recovered proteins were probed with HRP-conjugated streptavidin (to show the GIMAP6 proteins) or rat monoclonal antibody MAC446 to GABARAPL2 followed by an HRP-conjugated goat F(ab’)₂ fragment anti-rat IgG. Panel A) GIMAP6 compared with mutations of the putative AIM motif in GIMAP6. Panel B) GIMAP6 compared with N- and C-terminal mutants of the protein as indicated. In panel B, 1-292 corresponds to full-length GIMAP6. Panel C) Mutations within the GTPase domain of GIMAP6 as indicated. Panel D) HEK293T cells were transfected with a plasmid encoding myc-GIMAP6 together with plasmids encoding biotinylated forms of GABARAPL2 as indicated. Cell lysates were prepared and biotinylated and associated proteins recovered by streptavidin-agarose affinity chromatography. Western blots were probed with HRP-conjugated streptavidin (to show the GABARAPL2 proteins) or an anti-myc monoclonal antibody 9E10 followed by an HRP-conjugated goat anti-mouse IgG to detect myc-tagged GIMAP6. The wild-type protein is represented by 1-117. Results in all four panels are representative of data obtained from three independent experiments.</p