Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity Ben G. Wen, Mathew T. Pletcher, Masaki Warashina, Sun Hui Choe, Niusha Ziaee, Tim Wiltshire, Karsten Sauer*, and Michael P. Cooke* Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121 Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved January 27, 2004 (received for review October 24, 2003) The mechanisms governing positive selection of T cells in the were unable to detect expression of Itpkb in thymocytes from thymus are still incompletely understood. Here, we describe a mutant mice. N-ethyl-N-nitrosourea induced recessive mouse mutant, Ms. T-less, Itpkb, also known as inositol (1,4,5) 3 kinase B, converts which lacks T cells in the peripheral blood because of a complete inositol (1,4,5) trisphosphate (IP3) to inositol (1,3,4,5) tetrakis- ؉ ؉ block of thymocyte development at the CD4 CD8 stage. Single phosphate (IP4) (6). IP3 is a critical mediator of TCR induced nucleotide polymorphism mapping and candidate gene sequenc- Ca2ϩ release from internal stores (7). Several studies suggest ing revealed a nonsense mutation in the inositol (1,4,5) trisphos- roles for IP4 in calcium signaling in nonlymphoid cells, possibly phate 3 kinase B (Itpkb) gene in Ms. T-less mice. Accordingly, Ms. by modulating the levels of IP3 (8–10). T-less thymocytes do not show detectable expression of Itpkb Mammals express three Itpk isoforms: Itpka, Itpkb, and Itpkc protein and have drastically reduced basal inositol (1,4,5) trisphos- (6, 11, 12). Itpka and Itpkb are regulated through the binding of phate kinase activity. Itpkb converts inositol (1,4,5) trisphosphate Ca2ϩ͞calmodulin. Disruption of the brain-enriched Itpka gene to inositol (1,3,4,5) tetrakisphosphate, soluble second messengers results in minor enhancements of long-term potentiation in the 2؉ 2؉ that have been implicated in Ca signaling. Surprisingly, Ca CA1 region of the hippocampus; yet no other major defects have responses show no significant differences between wild type (WT) been noted in these mice (13). This mild phenotype may reflect and mutant thymocytes. However, extracellular signal-regulated functional redundancy with Itpkb, which shows an overlapping kinase (Erk) activation in response to suboptimal antigen receptor expression pattern (Fig. 5, which is published as supporting stimulation is attenuated in Ms. T-less thymocytes, suggesting a information on the PNAS web site). However, Itpkb is also role for Itpkb in linking T cell receptor signaling to efficient and enriched in lymphoid tissues. Itpkc shows a broader tissue sustained Erk activation. expression pattern. Surprisingly, we did not detect any significant effects on he development of mature T cells is a tightly regulated calcium responses in TCR-stimulated CD4ϩCD8ϩ T cells from Tprocess that has been studied extensively at the cellular and Ms. T-less mice. Instead, we found a specific defect in the molecular level. Lymphoid precursors destined to become T cells activation of extracellular signal-regulated kinase (Erk), a crit- arrive in the thymus from the bone marrow, where they face a ical mediator of positive selection (1). This result identifies Itpkb gauntlet of checkpoints to determine their ultimate fate (re- as a unique link between the TCR and the Ras mitogen-activated viewed in ref. 1). Briefly, T cell development in the thymus can protein kinase (MAPK) pathway, which is essential for T cell be followed by the expression of the two T cell receptor (TCR) development. coreceptors CD4 and CD8. Thymocytes at the earliest develop- Ϫ Ϫ mental stage are CD4 CD8 double negative cells. After suc- Materials and Methods ␤ cessful rearrangement of the TCR chain, they undergo rapid Mice. All mice used in this study were between 6 and 12 weeks proliferation and begin expressing both coreceptors simulta- ϩ ϩ of age. ENU mutagenized C57BL͞6 mice were generated as neously, thereby entering the CD4 CD8 double positive (DP) described (14). Mice were maintained by backcrossing affected stage. At this stage, the TCR ␣ chain is rearranged and expressed animals to C57BL͞6 and housed in the Genomics Institute of the on the cell surface to form a functional receptor. In addition, DP ϩ ϩ Novartis Research Foundation Specific Pathogen Free animal cells face a fate decision to either become mature CD4 or CD8 facility. All procedures were approved by the Genomics Institute single positive T cells or to die. This fate decision is directed by of the Novartis Research Foundation Institutional Animal Care the avidity and affinity of the TCR on DP thymocytes to self peptides presented by MHC class I or class II molecules (re- and Use Committee. viewed in ref. 2). Cells that recognize the peptide–MHC complex Flow Cytometry. with high or no affinity die by apoptosis during negative selection Single cell suspensions of thymus, lymph node, or by neglect, respectively. Cells that recognize the peptide– or spleen were stained with FITC-, phycoerythrin-, peridinin chlorophyll protein-, and allophycocyanin-conjugated antibod- MHC complex with intermediate affinity are positively selected ␤ to mature into CD4ϩ or CD8ϩ T cells. The molecular events ies against B220, TCR , CD4, CD8, CD3, CD69, CD44, CD45.1, dictating this differentiation process are an area of intense and CD45.2 (Pharmingen and eBioscience, San Diego). Cells investigation (reviewed in refs. 3 and 4). were analyzed by flow cytometry on a FACSCalibur flow In an attempt to find mediators of immune function, we are conducting a forward genetics screen in mice by using N-ethyl- This paper was submitted directly (Track II) to the PNAS office. N-nitrosourea (ENU) (5). Here, we describe Ms. T-less,a Abbreviations: ENU, N-ethyl-N-nitrosourea; DP, double positive; TCR, T cell receptor; Itpkb, recessive mouse mutant that lacks peripheral T cells because of inositol (1,4,5) trisphosphate 3 kinase B; IP3, inositol (1,4,5) trisphosphate; IP4, inositol a nearly complete block of T cell development at the DP stage. (1,3,4,5) tetrakisphosphate; Erk, extracellular signal-regulated kinase; PIP2, phosphatidyl- Single-nucleotide polymorphism mapping and candidate gene inositol (4,5) bisphosphate; GAP, GTPase-activating protein; MAPK, mitogen-activated sequencing revealed a nonsense mutation in the Itpkb gene. The protein kinase; GRP, guanine nucleotide-releasing protein. mutant allele encodes a N-terminally truncated protein, which *To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. lacks the lipid kinase domain. We used various antibodies but © 2004 by The National Academy of Sciences of the USA 5604–5609 ͉ PNAS ͉ April 13, 2004 ͉ vol. 101 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306907101 Downloaded by guest on September 27, 2021 Fig. 1. Ms. T-less mice display a paucity of peripheral T cells and a CD4ϩCD8ϩ block in T cell development. (A) Peripheral blood lymphocytes from mutant and control mice on a C57BL͞6J background were stained with antibodies to CD3, B220, CD4, and CD8. The scatter plots show lymphocyte subpopulations as % of total lymphocytes. Thymocytes from WT (wt) and mutant (mut) mice were stained with antibodies against CD4 and CD8 to follow T cell development (B) and with antibodies to activation markers, including CD69, CD3, and TCR␤ (C). The histograms in C are gated on CD4ϩCD8ϩ thymocytes. (D) Spleens from WT and mutant mice were stained with antibodies against CD4 and CD8 to analyze the peripheral T cell compartment. cytometer (Becton Dickinson). Acquisition and analysis were Immunoblotting and Northern Analysis. Itpkb was immunoprecipi- performed with CELLQUEST (Becton Dickinson) and FLOWJO tated from whole thymocyte extracts with an antibody against (TreeStar, Ashland, OR) software. the N-terminal region of rat Itpkb (Santa Cruz Biotechnology). Precipitate eluates or whole cell lysates from sorted DP thymo- -Analysis of Ca2؉ Responses. The protocol for measuring intracel- cytes were separated by SDS͞PAGE, transferred to nitrocellu lular calcium levels by flow cytometry was derived from L. B. lose, probed with the N-terminal antibody, and developed by Dustin (15). Thymocytes at 10 ϫ 106͞ml were labeled in DMEM enhanced chemiluminescence (ECL, Amersham Pharmacia). ϩ 10 mM Hepes with 2 ␮M Fura red (Molecular Probes), 1 ␮M For Northern blot analysis, RNA was isolated from whole Fluo-4 (Molecular Probes), and 0.2% Pluronic (Molecular thymocytes or sorted DP thymocytes, separated on a denaturing Probes) for 30 min at room temperature. Cells were washed formaldehyde-agarose gel, transferred to nylon, and hybridized twice in DMEM ϩ 10 mM Hepes with 1% FCS and rested for with a radioactive probe against an internal portion of the Itpkb 20 min in the dark. For stimulation, labeled cells were incubated transcript. with biotinylated ␣CD3 and ␣CD4 (Pharmingen and eBio- science) on ice for 15 min, washed, then resuspended with Erk Activation. Thymocytes were incubated with biotinylated prewarmed streptavidin in Hanks’ balanced salt solution (HBSS) ␣CD3 and͞or ␣CD4 for 30 min at 4°C with rotation, followed by 2ϩ with EGTA or CaCl2, and analyzed by flow cytometry. Ca stimulation with prewarmed streptavidin or phorbol 12- mobilization was determined ratiometrically as described in ref. myristate 13-acetate at the indicated time points. Stimulation 15. For single-cell calcium imaging, we adapted a protocol was stopped by adding ice-cold PBS. Cells were then washed and described in ref. 16. Briefly, thymocytes were isolated as above, lysed. Protein lysates were subjected to SDS͞PAGE, transferred labeled with 1 ␮M Fura-2 (Molecular Probes) and 0.2% Plu- to nitrocellulose, probed with antibodies to Erk1͞Erk2 or phos- ronic, stained with biotinylated ␣CD3 and ␣CD4, and adhered pho-Erk1͞Erk2 (Cell Signaling Technology), and detected by to poly(L-lysine)-treated coverslips. Cells were stimulated by enhanced chemiluminescence. perfusion of streptavidin and imaged on an inverted microscope (Nikon) under 40ϫ magnification with an UV light source.