IAN family critically regulates survival and development of T lymphocytes - PubMed (original) (raw)

IAN family critically regulates survival and development of T lymphocytes

Takeshi Nitta et al. PLoS Biol. 2006 Apr.

Abstract

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell-receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.

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Figures

Figure 1

Figure 1. IAN Family Genes

(A) The cluster of IAN family genes in the genome of indicated species. Mouse IAN genes and their orthologs in human and rat are indicated. For chicken, zebrafish, and thale cress, arrows indicate genes that putatively encode AIG1-domain-containing proteins. In chicken, 19 genes are predicted to encode AIG1 domain–containing proteins, and arrows indicate 15 genes clustered on Chromosome 2. In zebrafish, a cluster of 23 genes was found on Chromosome 16. In thale cress, ten out of 14 predicted genes are clustered on Chromosome 1. (B) Predicted structures of mouse IAN family proteins. Numbers refer to amino acid residues of full-length proteins. (C) A neighbor-joining tree of the AIG1 domain of IAN proteins. A. thaliana AIG1, residues 44–243; N. tabacum NTGP4 (AAD09518), residues 23–222; G. max NTGP4 (BI316235), residues 1–118; O. sativa AIG1 (CAE04223), residues 31–230;Z. mays AIG1 (AW120061), residues 1–200; D. rerio IAN (BC053197), residues 1–200; G. gallus IAN (XP_427942), residues 3–202; M. musculus IAN1, residues 31–230; R. norvegicus IAN1, residues 31–230; H. sapiens IAN1, residues 45–244. No IAN genes were found in the genomes of Drosophila melanogaster (fly), Anopheles gambiae (mosquito), Ciona intestinalis (sea squirt), Caenorhabditis elegans (nematode), Saccharomyces cerevisiae (yeast), and all bacteria and archea.

Figure 2

Figure 2. Expression of Mouse IAN Family Genes

(A) Quantitative RT-PCR analysis of total RNA from C57BL/6 mouse tissues, purified splenocyte subsets, and purified thymocyte subsets. The mRNA levels of IAN family genes were initially normalized to_GAPDH_ levels, and were further normalized to the levels expressed in the thymus. Relative expression of all IAN family genes in the thymus tissue is indicated as 1. (B) Relative mRNA levels of_IAN1, IAN4,_ and_IAN5_ in CD4+CD8+, CD4+CD8+CD5low, and CD4+CD8+CD5high thymocytes from C57BL/6 (wild-type) mice and CD4+CD8+ thymocytes from TCRα-deficient mice [ 42]. (C)IAN1, IAN4, and_IAN5_ mRNA levels in CD4+CD8+ thymocytes from positive selector (AND-TCR Aβ+/+ and 2C-TCRk/b [ 43]) TCR-transgenic mice and null selector (AND-TCR Aβ−/− [ 44] and 2C-TCRk/k) TCR-transgenic mice. (D) Relative mRNA levels of_IAN1, IAN4,_ and_IAN5_ in total, CD4+CD8+CD69low, CD4+CD8+CD69high, CD4+CD8−CD69high and CD4+CD8−CD69low thymocytes from C57BL/6 mice. (E) Thymocytes from TCRα-deficient mice were cultured with or without phorbol 12-myristate 13-acetate (0.2 ng/ml) and ionomycin (0.2 μg/ml) for the indicated periods. (F) Thymocytes from Aβ−/− β2m−/− mice [ 45] were cultured with or without plate-bound anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) antibodies for 24 h. Bar graphs show means ± standard errors.

Figure 3

Figure 3. Overexpression of IAN1, IAN4, and IAN5 in Thymocyte Development

(A) Diagram of MSCV retroviral constructs. IRES, internal ribosomal entry site; LTR, long terminal repeat; Ψ, packaging signal. (B) NIH-3T3 cells infected with retroviruses expressing IAN1-HA, IAN4, IAN5, or EGFP alone were analyzed for EGFP fluorescence by flow cytometry and for IAN protein expression by immunoblotting (IB). The frequency of cells and the mean fluorescence intensity (MFI) in the indicated area are shown. (C) Day 14.5 fetal thymocytes infected with indicated retroviruses were reconstituted in FTOC. EGFP+ cells purified on day 6 were analyzed for mRNA expression. (D) Viable cell numbers of total cells (striped bars) and EGFP+ cells (open bars) in FTOC on day 6. Filled bar indicates a significant reduction (p < 0.01). EGFP,n = 7; IAN1,n = 6; IAN4,n = 6; IAN5,n = 6. (E) EGFP histograms of total cells and CD4/CD8 profiles of EGFP+ cells in FTOC on day 6. Numbers in dot plots show the frequency of cells within boxes. (F) Annexin V and PI staining of indicated EGFP+ thymocyte subpopulations in FTOC on day 6. Filled bar indicates a significant increase (p < 0.05). Bar graphs show means ± standard errors. NS, not significant (p ≥ 0.05); *p < 0.05; **p <0.01. No significant difference in the CD4/CD8 developmental profiles was observed in EGFP− cell populations.

Figure 4

Figure 4. Knockdown of IAN1, IAN4, and IAN5 in Thymocyte Development

(A) Diagram of retroviral shRNA constructs. Puro_r,_ puromycin resistance gene. SIN-LTR, self-inactivating long terminal repeat. (B) BW5147 cells expressing IAN1-HA, IAN4-HA, or IAN5-HA were infected with shRNA retroviruses, and the infected cells were enriched by puromycin selection. Protein expression levels were analyzed by anti-HA IB.Luciferase (Luc) shRNA was used as control. (C) Day 14.5 fetal thymocytes infected with shRNA retroviruses were reconstituted in FTOC. EGFP+ cells purified on day 6 were analyzed for mRNA expression. (D) Viable cell numbers of total cells (striped bars) and EGFP+ cells (open bars) in FTOC on day 6. (E) EGFP histograms of total cells and CD4/CD8 profiles of EGFP+ cells in FTOC on day 6. The frequency of EGFP+ cells and the mean fluorescence intensity (MFI) in the indicated area are shown in the histograms. Numbers in dot plots show the frequency of EGFP+ cells within boxes. (F) Frequencies of indicated cell populations on day 6. Filled bars indicate significant difference from the values in the control group (Luc shRNA) (p < 0.05). Bar graphs show means ± standard errors. NS, not significant (p ≥ 0.05);* p < 0.05;** p < 0.01. In (D) and (F),Luc shRNA,n = 11;IAN1 shRNA,n = 5;IAN4 shRNA,n = 11;IAN5 shRNA,n = 8. No significant difference in the CD4/CD8 developmental profiles was observed in EGFP− cell populations.

Figure 5

Figure 5. Differential Roles of IAN4 and IAN5 in Thymocyte Development

(A) Day 14.5 fetal thymocytes infected with shRNA retroviruses were reconstituted in FTOC. Frequencies of indicated cell populations on day 10 are shown.Luc shRNA,n = 4;IAN4 shRNA,n = 5. (B) Day 14.5 fetal thymocytes from AND-TCR-transgenic or 2C-TCR-transgenic mice were infected and reconstituted in C57BL/6 fetal thymus lobes for 6–8 d. For 2C-TCR–transgenic thymocytes, mature CD8SP cells were analyzed by gating CD8SP CD5high population. AND-Luc shRNA,n = 11; AND-IAN4 shRNA,n = 12; 2C-Luc shRNA,n = 5; 2C-IAN4 shRNA,n = 5. (C) Day 14.5 fetal thymocytes infected with shRNA retroviruses were reconstituted in FTOC (as shown in Figure 4C– 4F). EGFP+ thymocytes on day 6 were analyzed for DN subpopulations by staining with anti-CD25 and anti-CD44 antibodies.Luc shRNA,n = 6;IAN5 shRNA,n = 8. (D) CD4−CD8−CD25−CD44− DN4 cells were purified from day 17 fetal thymocytes by depleting the cells expressing CD4, CD8, CD25, or CD44, infected with indicated retroviruses, and reconstituted in FTOC for 2 d;n = 4. Bar graphs show means ± standard errors. NS, not significant (p ≥ 0.05);* p < 0.05;** p < 0.01.

Figure 6

Figure 6. Interaction of IAN Family Proteins with Bcl-2 Family Proteins

(A) 293T cells were co-transfected with FLAG-tagged IAN molecules together with Bcl-2, Bcl-xL, HA-tagged Bax, HA-tagged Bak, HA-tagged Bad, BimEL, HA-tagged IκBα, or EGFP. Cell lysates were IP with anti-FLAG M2 antibody and IB with indicated antibodies. (B) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were IP with anti-FLAG M2 antibody and IB with anti-Bcl-2 or anti-Bcl-xL antibody. (C) 23–1–8 T cells expressing FLAG-tagged IAN4 or FLAG-tagged IAN5 were IP with normal IgG or anti-Bcl-2 or anti-Bcl-xL antibody and IB with anti-FLAG M2 antibody. Arrows indicate FLAG-tagged IAN4 or FLAG-tagged IAN5. (D) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were cultured in the presence or absence of IL-2 for 36 h. Cell lysates were IP with anti-FLAG M2 antibody and IB with anti-Bax antibody. (E) Nuclear and heavy membrane fractions prepared from 23–1–8 T cells were lysed in buffer containing 1% CHAPS. The lysates were IP with normal rabbit IgG or anti-IAN4 antibody and IB with anti-Bcl-2 antibody. Means and standard errors (n = 4) of relative intensities of the bands were analyzed by using NIH Image software. * p < 0.05;** p < 0.01.

Figure 7

Figure 7. Knockdown of IAN5 in 23–1–8 T Lymphocytes

(A) 23–1–8 T lymphocyte clones expressing shRNAs were analyzed for_IAN5_ mRNA expression and cultured in the presence or absence of IL-2. Cell viability was quantified by PI staining and flow cytometry analysis. (B and C) Cells in 48-h culture were analyzed for apoptosis induction. Frequencies of Annexin-V-positive cells (B) or mitochondrial membrane potential (Δψm)-negative cells (C) are shown. (D) 23–1–8 T cells expressing shRNAs with or without human_Bcl-xL_ were analyzed for_IAN5_ expression by quantitative RT-PCR and for human_Bcl-xL_ expression by conventional RT-PCR (left panel). Cells were cultured in the presence or absence of IL-2 for 72 h or in the presence of IL-2 and 5 μM helenalin for 48 h, and cell viability was quantified by PI staining (right panel). Graphs show means ± standard errors. NS, not significant (p ≥ 0.05);** p < 0.01.

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References

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