Nkt1 , the NKT Cell Control Gene Slamf1

Slamf1, the NKT Cell Control Gene Nkt1 Margaret A. Jordan, Julie M. Fletcher, Daniel Pellicci and Alan G. Baxter This information is current as J Immunol 2007; 178:1618-1627; ; of September 24, 2021. doi: 10.4049/jimmunol.178.3.1618 http://www.jimmunol.org/content/178/3/1618 Downloaded from References This article cites 43 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/178/3/1618.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Slamf1, the NKT Cell Control Gene Nkt11

Margaret A. Jordan,2* Julie M. Fletcher,2* Daniel Pellicci,† and Alan G. Baxter3*

Invariant NKT cells play a critical role in controlling the strength and character of adaptive immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. In this study, we report the production and characterization of a NOD.Nkrp1b.Nkt1b congenic mouse strain, apply microarray expression analyses to limit candidate genes within the 95% confidence region, identify Slamf1 (encoding signaling lymphocyte activation molecule) and Slamf6 (encoding Ly108) as potential candidates, and demonstrate retarded signaling lymphocyte activation molecule expression during T cell development of NOD mice, resulting in reduced expression at the CD4؉CD8؉ stage, which is consistent with decreased NKT cell production and deranged tolerance induction in NOD mice. The Journal of Immunology, 2007, 178: 1618–1627. Downloaded from nvariant NKT (iNKT)4 cells are an immunoregulatory pop- mosome 1 that control NKT cell numbers, we produced and char- ulation of lymphocytes that plays a critical role in controlling acterized a NOD mouse line congenic for the C57BL/6 allele at the I the adaptive immune system and contributes to the regulation Nkt1 locus. of autoimmune responses (1–3). We have previously reported deficiencies in the numbers and function of NKT cells in the NOD Materials and Methods Mice

mouse strain (4, 5), which is a well-validated model of type 1 http://www.jimmunol.org/ diabetes (6) and systemic lupus erythematosus (7, 8), and mapped NOD.Nkrp1b, C57BL/6J, and congenic mice were maintained at the Im- genetic control of thymic NKT cell numbers in a first backcross munogenetics Research Facility at the James Cook University in specific (BC1) from C57BL/6 to NOD.Nkrp1b mice (9). The numbers of pathogen-free conditions. The NOD.Nkrp1b strain carries B6-derived alleles at the NK complex on chromosome 6 (from D6mit105 to D6mit135), thymic NKT cells of 320 BC1 mice were determined by fluores- b b ␣ permitting the use of the NK1.1 marker (13, 14). NOD.Nkrp1 .Nkt1 mice cence-activated cell analysis using CD1d/ -galactosylceramide were produced by intercrossing NOD.Nkrp1b and C57BL/6J mice and per- (CD1d/␣-GalCer) tetramer (10). Tail DNA of 138 female BC1 forming serial backcrosses to NOD.Nkrp1b to N10, before intercrossing mice was analyzed for PCR product length polymorphisms at 181 and selection of homozygous congenic founders. These studies have been simple sequence repeats, providing Ͼ90% coverage of the auto- reviewed and approved by the James Cook University Institutional Animal Care and Ethics Committee. somal genome with an average marker separation of 8 cM. Two by guest on September 24, 2021 loci exhibiting significant linkage to NKT cell numbers were iden- DNA preparation tified; the most significant (Nkt1; log-likelihood ratio 6.82) was Extraction of genomic DNA from NOD.Nkrp1b, NOD.Nkrp1b.Nkt1b, mapped near D1mit15 on distal chromosome 1 (9) in the same and C57BL/6 mouse strains was conducted using the CAS-1810 X- region as the NOD mouse lupus susceptibility gene Babs2/Bana3 TractorGene (Corbett Robotics) and the XTR2 X-tractor gene solid sample (11). The second locus (Nkt2; log-likelihood ratio 4.90) was reagent pack (Sigma-Aldrich), which is based on a method developed in mapped between D2mit490 and D2mit280 on chromosome 2 (9) in this laboratory. Briefly, DNA was extracted by digesting an 11-mm tailtip in 400 ␮l of digest buffer (100 mM Tris-HCl (pH 8), 10 mM EDTA, 100 the same region as Idd13, a NOD-derived diabetes susceptibility mM NaCl, 0.5%SDS, 50 mM DTT, and 100 mM proteinase K), O/N, 56°C, gene (12). In an attempt to identify the genetic sequences on chro- 40 rpm in a VORTEMP 56EVC (Labnet). Samples were lysed by addition of 700 ␮l of 5.25 M guanidine thiocyanate lysis buffer (5.25 M guanidine thiocyanate, 10 mM Tris-HCl (pH 6.5), 20 mM EDTA, 4% Triton X-100, *Comparative Genomics Center, James Cook University, Townsville, Queensland, and 64.8 mM DTT), loaded on a glass filter (GF/B) polypropylene micro- Australia; and †University of Melbourne, Department of Microbiology and Immu- plate (Whatman International), and washed twice in propanol wash buffer nology, Parkville, Victoria, Australia and once in 100% ethanol. Samples were eluted in 150 ␮l of elution buffer. Received for publication January 19, 2006. Accepted for publication November The DNA yield was quantified spectrophotometrically. 21, 2006. Genotyping The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Identification of the congenic segment boundaries and the background with 18 U.S.C. Section 1734 solely to indicate this fact. screen were conducted by genotyping the extracted tail DNA using simple 1 A.G.B. is supported by an Australian National Health and Medical Research Coun- sequence repeats chosen from the Whitehead Institute simple sequence cil Senior Research Fellowship, J.M.F. is the recipient of an Australian Postgraduate length polymorphism library, as well as markers designed in-house on the Award, and M.A.J. is the recipient of a James Cook University intramural scholarship. basis of PCR product length polymorphisms between C57BL/6 and This project was funded by the Australian National Health and Medical Research NOD/Lt strains, as described previously (9). Council. RNA preparation and microarray expression analyses 2 M.A.J. and J.M.F. contributed equally to this manuscript. 3 Address correspondence and reprint requests to Dr. Alan G. Baxter, Comparative To minimize activation of the apoptosis cascade, thymi were removed from Genomics Center, Molecular Sciences Building 21, James Cook University, Towns- 4-wk-old female mice and placed in RNA-later (Qiagen) within 120 s of ville, Queensland 4811, Australia. E-mail address: [email protected] the mouse being placed in CO2 for asphyxiation. In our hands, this pro- 4 Abbreviations used in this paper: iNKT, invariant NKT; CD1d/␣-GalCer, CD1/␣- cedure substantially improved the signal to noise ratio of expression anal- galactosylceramide; DP, double positive; SAP, SLAM-associated protein; SH, Src ysis, greatly reducing the numbers of differentially expressed genes homology; SLAM, signaling lymphocyte activation molecule; SP, single positive. identified. The thymi were individually homogenized in the RLT buffer of an Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 RNeasy kit (Qiagen), with contamination minimized by extensive washing www.jimmunol.org The Journal of Immunology 1619

FIGURE 1. Characterization of the NOD.Nkrp1b.Nkt1b congenic mouse line. The boundaries of the Nkt1 congenic segment on distal chromosome 1 are indicated (A). Proportions (B) and absolute numbers (C) of thymic NKT cell numbers in 5-wk-old mice from the congenic line and the NOD.Nkrp1b parental line as determined by CD1d/␣-GalCer tetramer binding are shown. Values for NOD.Nkrp1b mice are indicated by Ⅺ whereas those for NOD.Nkrp1b.Nkt1b mice by ࡗ. Proportion means and SEM are shown, whereas for numbers, individual values and statistical analysis (Mann-Whitney U test) are given. with RNase-off and RNase-free-DNase-free-water between samples. Ho- 5Ј-ATTTTGCTCTTGTCTCTGC-3Ј, and R primer, 5Ј-GGAATCCCTCTT mogenates were passed through Qiashedder columns (Qiagen) and ex- TAGGTAGACTGC-3Ј; and Gapdh, F primer, 5Ј-ACCACAGTCCATGC tracted (RNeasy; Qiagen). The RNA yield was quantified spectrophoto- CATCACT-3Ј, and R primer, 5Ј-TCCACCACCCTGTTGCTGTA-3Ј. metrically and aliquots electrophoresed for determination of sample Titrated template standards were processed in parallel with unknown Downloaded from concentration and purity. controls. Expression microarray hybridizations were performed by the Australian Genome Research Facility using the one-cycle cDNA synthesis kit (Af- Primer design and sequencing fymetrix) and Affymetrix 430 2.0 mouse gene microarray, which contains Ͼ45,000 probe sets, representing Ͼ34,000 well-substantiated mouse Primers for sequencing were designed using BioTechnix 3d 1.1.0, based on genes. sequence obtained from UCSC Genome Bioinformatics (http://genome. The probed arrays were scanned using the GeneChip Scanner 3000, and ucsc.edu) such that overlapping sequences would be amplified across the the images (.dat files) were processed using GeneChip Operating System promoter, coding, and noncoding mRNA sequences. PCR were performed http://www.jimmunol.org/ (GCOS, Affymetrix) and imported into Avadis Prophetic3.3 (Strand for all mouse strains for all regions on Omn-E thermal cyclers (Hybaid). Each 100-␮l reaction included 10 ␮lof10ϫ PCR buffer with 3 mM MgCl Genomics) for further analysis. The statistical significance threshold was 2 (Roche), 0.4 mM each of dATP, dCTP, dGTP, and dTTP (Astral), 1.6 U of set by permutative analysis (10,000 permutations) and a Kruskal-Wallis ␮ ␮ test. A conservative significance threshold of p Ͻ 0.001 was set; this value Taq Polymerase (Roche), and 2 l of DNA (cDNA). Approximately 20 l coincided with a lack of overlap in signal values between the two groups of mineral oil overlaid the reaction mix. PCR protocol included denatur- (n ϭ 7/group). ation 95°C, 3 min, then 32 (40) cycles (95°C, 1 min; 50–62°C (primer dependant annealing) 1 min, 72°C, 1 min), followed by an extension step First-strand cDNA synthesis of 72°C, 7 min. Reactions were verified by 1% agarose gel electrophoresis. Reactions were then purified using the Qiagen PCR purification kit fol- First-strand cDNA was synthesized from 5 ␮g of total RNA using oli- lowing manufacturers directions. Twenty to 100 ng of PCR product be- go(dT) primers and Superscript II reverse transcriptase following manu- tween 200 and 500 bp/100–160 ng of PCR product between 500 and 1000 by guest on September 24, 2021 facturer’s instructions (Invitrogen Life Technologies). bp were prepared with 6.4 pmol primer and sent to the Australian Genome Research Facility for sequencing (both forward and reverse reactions for Real-time quantitative PCR each). The raw data were retrieved by FTP and analyzed using Sequencher 3.1.1. (Gene Codes). Primers were designed to verify microarray data on independent samples of b b b RNA from NOD.Nkrp1 and NOD.Nkrp1 .Nkt1 mice. All PCR were con- Cell suspension preparation ducted on the Rotorgene 3000 (Corbett) and PCR mixes set up using a CAS1200 liquid handling platform (Corbett Robotics). Each 25-␮l reaction Thymocyte cell suspensions were prepared by gently grinding the thymus contained 12.5 ␮l of Platinum Sybr Green qPCR Supermix UDG (Invitro- between two frosted microscope slides in MACS buffer (PBS containing 2 gen Life Technologies), 0.5 ␮l each primer (5 ␮M), 1 ␮l of dNTP (10 mM), and 5 ␮l of cDNA. Slamf1 and Slamf6 expression values were nor- malized against Gapdh, as microarray expression analyses had shown that this b Table I. List of genetic markers tested to confirm genetic homogeneity gene was not differentially expressed between NOD.Nkrp1 and of NOD.Nkrp1b.Nkt1b mouse line NOD.Nkrp1b.Nkt1b mice. The primers used for quantitation were as follows: Slamf1 exons 3–5 (microarray probe 1425570_at), F primer, 5Ј-TAATCTTC ATCCTGGTTTTCACGGC-3Ј, and R primer, 5Ј-TTGGGCATAAATAGTA D1mit58, D1mit72, D1mit279, D1mit124, D1mit180, D1mit438, D1mit306, AGGC-3Ј; Slamf1 exon7 (microarray probe 1425569_a_at), F primer, 5Ј-AG D1mit494, D1mit348, D1mit445, D1mit103, D1mit199, D1mit199, ATGAAGAGGGAACAAAGC-3Ј, and R primer, 5Ј-TTGTTTGAAGCATA D1mit102, D1mit449, D1mit288, D1mit 369, D1mit396*, D1mit33,* D1Bax208,* D1Bax15,* D1mit406,* D1mit209,* D1mit155,* D2mit1, AGAGGC-3Ј; Slamf6 S-isoform (microarray probe 1457773_at), F primer, Ј Ј Ј D2mit362, D2mit458, D2mit92, D2mit256, D2mit490, D2mit283, 5 -CCTATTCCTGCTATCACG-3 , and R primer, 5 -AACTTAGAGGAA D2mit412, D2mit528, D2mit265, D3mit60, D3mit203, D3mit25, D3mit187, AATGGGTGC-3Ј; Slamf6 L-isoform (microarray probe 1420659_at), F D3mit13, D3mit84, D3mit147, D3mit19, D4mit264, D4mit139, D4mit301, primer, 5Ј-TGTTTGACCTCTGTGACCTTT-3Ј, and R primer, 5Ј-TACA D4mit9, D4mit11, D4mit204, D4mit226, D4mit59, D5mit48, D5mit387, GGAGGAACCCAACAGGC-3Ј; and Gapdh, F primer, 5Ј-TGCCGCCT D5mit81, D5mit113, D5mit239, D5mit406, D5mit245, D5mit169, D6mit86, GGAGAAACCTGCCAAGTATG-3Ј, and R primer, 5Ј-TGGAAGAGTG D6mit224, D6mit209, D6mit178, D6mit343, D6mit259, D6mit15, D7mit76, GGAGTTGCTGTTG D7mit225, D7mit84, D7mit301, D7mit101, D7mit334, D8mit155, AAGT-3Ј. D8mit281, D8mit191, D8mit54, D8mit81, D8mit211, D8mit166, D9mit90, D9mit285, D9mit26, D9mit335, D9mit165, D9mit269, D9mit136, D9mit17, Analyses of unknown samples were conducted by comparison to a stan- D9mit52, D10mit104, D10Bax30, D10mit15, D10mit198, D10mit42, dard curve for both the gene of interest and the housekeeper. Template D10mit95, D11mit77, D11mit131, D11mit5, D11mit357, D11mit198, standards were prepared by PCR amplification of cDNA from C57BL/6 D11mit61, D12mit190, D12mit156, D12mit259, D12mit141, D13mit158, thymi using primers flanking those used for quantitation: Slamf1 exons 3–5 D13mit221, D13mit7, D13mit202, D13mit230, D13mit78, D14mit207, (microarray probe 1425570_at), F primer, 5Ј-ACCACAGTCCATGCCA D14mit62, D14mit63, D14mit239, D14mit197, D14mit178, D15mit174, TCAC-3Ј, and R primer, 5Ј-TCCACCACCCTGTTGCTGTA-3Ј; Slamf1 D15mit179, D15mit255, D15mit121, D15mit71, D15mit159, D15mit193, exon7 (microarray probe 1425569_a_at), F primer, 5Ј-CTGGACTTTATT D15mit35, D16mit131, D16mit98, D16mit189, D17mit133, D17mit176, CTGGAAGC-3Ј, and R primer, 5Ј-TTGAGGTTCCAGAGTTTTGC-3Ј; D17mit68, D17mit70, D17mit93, D17mit130, D18mit60, D18mit123, Slamf6 Ј D18mit206, D18mit4, D19mit78, D19mit79, D19mit73, D19mit119, S-isoform (microarray probe 1457773_at), F primer, 5 -TGTGTG D19mit91, D19mit35 GTATTACTCCAAGGA-3Ј, and R primer, 5Ј-AGTAACTCCATCCCCA .Presence of C57BL/6-derived alleles ء ,TAGC-3Ј; Slamf6 L-isoform (microarray probe 1420659_at), F primer 1620 GENETIC CONTROL OF NKT CELL NUMBERS

FIGURE 2. Thymic and splenic subsets of NKT cells in the NOD.Nkrp1b. Nkt1b congenic mouse line. Flow cytometric analyses of thymic CD1d/␣- GalCer tetramer-binding NKT cell subset proportions of mice aged 2 and 4 wk (top panels)and6wk(middle left panel) of age from the NOD.Nkrp1b. Nkt1b congenic and the NOD.Nkrp1b parental line as deﬁned by the CD44, CD4, and NK1.1 markers are illustrated. Data for splenic NKT cell subset proportions Downloaded from of mice aged 6 wk are also shown for both inbred lines, as well as for (NOD. b ϫ b b Nkrp1 NOD.Nkrp1 .Nkt1 )F1 mice (middle right panel). Histograms illus- trating thymic (bottom left panel) and splenic (bottom right panel) NKT cell http://www.jimmunol.org/ numbers for 6-wk-old mice are shown in which values from NOD.Nkrp1b mice are indicated by Ⅺ, those for NOD. Nkrp1b.Nkt1b mice are indicated by f, and those for (NOD.Nkrp1b ϫ NOD. b b u Nkrp1 .Nkt1 )F1 mice indicated in (mean Ϯ SEM; n ϭ 4–7). by guest on September 24, 2021

mM EDTA (Amresco) and 0.5% (w/v) BSA (ICN Biomedicals). Spleens tetramer, conjugated to either PE or PE-Cy7 and loaded with ␣-GalCer, were disrupted using a 26-gauge needle and forceps and the resulting cell was produced in house as previously described (10) using recombinant ␤ suspension treated with RBC lysing buffer (Sigma-Aldrich). baculovirus encoding his-tagged mouse CD1d and mouse 2-microglobulin, provided by Prof. M. Kronenberg’s laboratory (La Jolly Institute for Flow cytometric analysis Allergy and Immunology, San Diego, CA). For flow cytometric analyses cells were labeled with anti-␤TCR- For surface staining, Abs were diluted in MACS buffer. Cells were FITC (clone H57-597), anti-CD3-FITC (clone 145-2C11), anti-CD3- preincubated for 15 min with CD16/32 (clone 93; eBioscience), followed allophycocyanin (clone 145-2C11), anti-CD3-allophycocyanin-Cy7 (clone by an additional 20-min incubation with 10% mouse serum to prevent FcR 145-2C11), anti-CD4-allophycocyanin (clone GK1.5), anti-CD4-PerCP- binding, before addition of surface staining Ab mixtures. Viable lympho- Cy5.5 (clone RM 4-5), anti-NK1.1-PE-Cy7 (clone PK136), anti- cytes were identified by the forward and side scatter profile and in some CD8-FITC (clone 53-6.7), anti-CD45R/B220-allophycocyanin (clone cases by propidium iodide exclusion. A forward scatter-area against for- RA3-6B2), anti-CD44-FITC (clone IM7), all from BD Pharmingen, and ward scatter-height gate was used to exclude doublets from analysis. anti-CD150(SLAM)-PE (clone TC15-12F12.2; Biolegend). Mouse CD1d Where possible, an empty fluorescent channel was used to exclude

Table II. Thymic and splenic NKT cell numbers at 6 wk

Total Cell Number NKT Organ Strain n Number (ϫ10Ϫ6) % NKT Cells Cells (ϫ10Ϫ5)

Thymus NOD.Nkrp1 7 113 Ϯ 7 0.09 Ϯ 0.01 1.0 Ϯ 0.2 Ϯ Ϯ Ϯ F1 7 143 9 0.16 0.02 2.3 0.3 NOD.Nkrp1.Nkt1 4 184 Ϯ 7 0.17 Ϯ 0.01 3.1 Ϯ 0.2 Spleen NOD.Nkrp1 575Ϯ 3 0.30 Ϯ 0.02 2.3 Ϯ 0.3 Ϯ Ϯ Ϯ F1 4651 0.44 0.03 2.9 0.3 NOD.Nkrp1.Nkt1 562Ϯ 3 0.55 Ϯ 0.02 3.4 Ϯ 0.2 The Journal of Immunology 1621

FIGURE 3. Averaged log signal intensity of Af- fymetrix Mouse 430 series 2 expression microarray pro- ﬁling of thymi from NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b mice (n ϭ 7/group; A). Results of genes for which a p Ͻ 0.05 was obtained are illustrated. F, Genes that are highly differentially expressed (experiment-wise permutative analysis threshold of p Ͻ 0.001; Kruskal-Wallis test). Di- agonal lines indicate 2-fold differential expression. Num- Downloaded from bers indicate gene identities as listed in Fig. 4. The linkage data from Ref. 9 are presented transformed to physical distances (B), the location of the Nkt1 congenic interval (indicated by the f) presented on the same scale (C), and the locations of the highly differentially expressed genes displayed as a histogram (D). The probability of 21 of the 28 locatable highly differentially expressed genes mapping http://www.jimmunol.org/ to the Nkt1 congenic region by chance is p Ͻ 10Ϫ200 (␹2 one sample test). by guest on September 24, 2021

autofluorescent cells. In general, flow cytometry was performed on a Cytokine measurement FACSVantage SE with FACSDiVa option (BD Biosciences) and data analyzed using either CellQuest Pro or FACSDiVa software (BD Bio- Cytokine levels in cell culture supernatants were determined using Mouse sciences). The data in Fig. 8E were acquired on a CyAn ADP flow Th1/Th2 Cytokine Cytometric Bead Array (BD Biosciences). Capture ␮ ␥ cytometer (DakoCytomation) and analyzed with Summit 4.3 software beads (30 l, specific for IL-2, IL-4, IL-5, IFN- , and TNF) together with ␮ ␮ (DakoCytomation). 30 l of culture supernatant samples and 30 l of PE detection reagent, were incubated for2hin96-well plates. Beads were washed twice with 200 ␮l of wash buffer, resuspended, and data were acquired using a Proliferation assays FACSCalibur (BD Biosciences). Serial dilutions of the provided cytokine standards were prepared and assayed as described above. Standard Single-cell suspensions were cultured in triplicate at 37°C, 5% CO2 for 3–5 curves were generated and samples quantified using the BD CBA soft- days in RPMI 1640 medium with L-glutamine (Invitrogen Life Technolo- ware (BD Biosciences). gies) supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin sul- fate, and 50 ␮M 2-ME. Stimulation was achieved by the addition of Dyna- Results bead Mouse CD3/CD28 T cell expander beads (Dynal Biotech) in varying b b proportions. Some cultures were established in the presence of 100 ␮g/ml NOD.Nkrp1 .Nkt1 congenic mice of the blocking signaling lymphocyte activation molecule (SLAM) peptide A NOD.Nkrp1b.Nkt1b congenic mouse line carrying a C57BL/6- ␮ 132–146 (FCKQLKLYEQVSPPE; Auspep), 100 g/ml of the nonblocking derived chromosomal segment spanning the 95% confidence in- SLAM peptide 83–97 (DLSKGSYPDHLEDGY; Auspep) or inhibiting concentrations (6.25 ␮g/ml) of the TC15 anti-SLAM mAb (Biolegend). Prolif- terval of Nkt1 was produced by serial backcrossing to the b eration was assayed by the addition of 0.25 ␮Ci of 6-3H-labeled thymidine NOD.Nkrp1 strain to N10, followed by intercrossing and selec- per 200-␮l well (GE Healthcare) 8–16 h before harvesting. At termination, tion for Nkt1b homozygotes. The proximal boundary of the con- plates were spun to pellet cells, 100 ␮l of supernatant was removed for genic segment lies between D1mit369 and D1mit396 and the distal cytokine assays, and the cells were harvested with a Tomtec Harvester 96 Mach IIIM, the emission scintillated with MeltiLex A melt-on scintillator boundary is distal to the most telomeric marker available, sheets (Wallac) and detected with a Wallac 1450 Microbeta Jet liquid scin- D1mit155 (Fig. 1A). A background screen of 136 polymorphic loci tillation counter. distributed throughout the rest of the autosomal genome failed to 1622 GENETIC CONTROL OF NKT CELL NUMBERS

FIGURE 5. Microarray profiling of expression levels of SLAM family members of thymi from NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b mice. Af- fymetrix 430 2.0 mouse gene microarray probes and locations were as follows: Downloaded from Slamf1, probe 1 (1425569_a_at) 707–962 bp, probe 2 (1425570_at) 1528– 2038 bp, and probe 3 (1425571_at) 2312–2738 bp; Slamf2 (1427301_at) 522– 705 bp; Slamf3 (1449156_at) 1928–2296 bp; Slamf4, probe 1 (1426120_a_at) 748–978 bp, and probe 2 (1449991_at) 1495–1862 bp; Slamf5, probe 1 (1422875_at) 942–1444 bp, and probe 2 (1446505_at) 68–486 bp; Slamf6, probe 1 (1420659_at) 1877–2323 bp, probe 2 (1425086_a_at) 979–1134 http://www.jimmunol.org/ bp, and probe 3 (1457773_at) 210–667 bp; Slamf7 (1453472_a_at) 1337– 1758 bp; Slamf8 (1425294_at) 997–1541 bp; and Slamf9 (1419315_at) FIGURE 4. Physical locations of highly differentially expressed genes 550–1064 bp. Mann-Whitney U test applied with significance threshold on chromosome 1 in relationship to linkage data from Ref. 9 (A) and lo- corrected for multiple hypothesis testing. cation of the Nkt1 congenic segment (indicated by f; B). The 95% linkage confidence interval is shown (fine lines). C and D, Genes are indicated by histogram. The width of each bar indicates the physical length of the gene NKT cell subsets in NOD.Nkrp1b.Nkt1b congenic mice and the height represents the fold change of differential expression between the NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b mice, with a positive displace- Thymic NKT cell subsets, which are related to each other by a ment indicating higher expression in the NOD.Nkrp1b.Nkt1b congenic line. developmental pathway, can be defined by the cell surface markers by guest on September 24, 2021 D illustrates the 95% confidence interval at higher resolution, and the num- CD4, CD44, and NK1.1 (15, 16). Flow cytometric analyses of bers indicate the identities of individual genes, as indicated: Nr locus de- thymic and splenic NKT cells indicate that the majority of the scription—1) 2010005O13rik, RIKEN cDNA 2010005O13 gene; 2) Rgs5, additional NKT cells found in NOD.Nkrp1b.Nkt1b mice belong to ␤ regulator of G-protein signaling 5; 3) Hsd17b7, hydroxysteroid (17- ) de- the CD4ϩCD44highNK1.1Ϫ population (Fig. 2), which is consid- hydrogenase 7; 4) Nr1i3, nuclear receptor subfamily 1, group I, member 3; ered to be relatively developmentally immature. Similarly, in the 5) Ppox, protoporphyrinogen oxidase; 6) 6030405P05rik, RIKEN cDNA spleen, the majority of the additional NKT cells found in 6030405P05 gene; 7) Slamf1, SLAM; 8) Slamf6, SLAM family member 6; b b ϩ Ϫ 9) Ltap, loop tail-associated protein; 10) Pex19, peroximal biogenesis fac- NOD.Nkrp1 .Nkt1 mice are CD4 NK1.1 . tor 19; 11) Loc623121, similar to IFN-activated gene 203; 12) A1447904, similar to IFN-activated gene 203; 13) Ifi203, IFN-activated gene 203; 14) Microarray gene expression analysis Ifi202b, IFN-activated gene 202B; and 15) Ifi205, IFN-activated gene 205. To identify a subset of candidate genes within the Nkt1 linkage The same key is applied to gene identities in Fig. 3. 95% confidence interval, microarray gene expression analysis was performed on thymi of 4-wk-old NOD.Nkrp1b and NOD.Nkrp1b. Nkt1b mice (n ϭ 7/group; Fig. 3A), following procedures to min- detect any residual C57BL/6-derived genomic contamination (Ta- imize activation of the apoptosis cascade. Thymic RNA was ex- ble I). Flow cytometric analyses of thymic NKT cell numbers and tracted, hybridization and scanning of Affymetrix Mouse 430 proportions, as determined by CD1d/␣-GalCer tetramer binding, series 2 expression microarrays performed by the Australian Ge- confirmed that thymi from the NOD.Nkrp1b.Nkt1b congenic line nome Research Facility, and data imported into Avadis Prophetic have larger proportions (Figs. 1B and 2) and numbers (Fig. 1C, using an RMA summarization algorithm. The statistical signifi- Table II) of iNKT cells than those from the NOD.Nkrp1b parental cance threshold was set by permutative analysis (10,000 permuta- strain controls. The increase in thymic NKT cell numbers in the tions) and a Kruskal-Wallis test applied. A total of only 28 genes congenic line is a product of both a higher proportion of NKT cells were identified as being highly differentially expressed (i.e., those in the thymus and an increase in total thymic cellularity (Table II). with a p Ͻ 0.001), of which 21 mapped to the Nkt1 congenic The proportions of thymic NKT cell numbers in (NOD.Nkrp1b ϫ region (ϳ1.6% of genome; ␹2 ϭ 986; df ϭ 1; p Ͻ 10Ϫ200; ␹2 one b b NOD.Nkrp1 .Nkt1 )F1 mice is intermediate between the congenic sample test; Fig. 3, B–D). This result is indicative of an extremely and parental strains. good signal-to-noise ratio. Splenic NKT cell proportions and numbers are also increased in the Only 15 of the 21 highly differentially expressed genes mapping congenic line compared with the parental line (Fig. 2, Table II; p Ͻ to the Nkt1 congenic region lie within the 95% confidence limits 0.05; Mann-Whitney U test). Again, the proportions and numbers of obtained in the original linkage analysis (Ref. 9; Fig. 4). Their b ϫ b b NKT cells in the spleens of (NOD.Nkrp1 NOD.Nkrp1 .Nkt1 )F1 physical positions and expression fold change are shown in Fig. mice are intermediate between the congenic and parental strains. 4D, and their identities given in the figure legend. Of these genes, The Journal of Immunology 1623

FIGURE 6. Validation of expression microarray pro- ﬁling of Slamf1 expression in thymi from NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b mice. Gene structure and exon structure of the three SLAM isoforms are shown together with the relative locations of microarray probe targets and RT-PCR primer sites (A). The microarray Downloaded from results (B; n ϭ 7) and RT-PCR validation (C; n ϭ 6–9) performed on an independent sample are shown. http://www.jimmunol.org/ by guest on September 24, 2021

the most prominent candidates for control of NKT cell numbers strains. While the NOD.Nkrp1b.Nkt1b mice and C57BL/6 mice are Slamf1 and Slamf6 because signaling through SLAM-associ- (data not shown) express high levels of SLAM on DP thymocytes, ated protein (SAP) appears to be essential for thymic positive se- with a relatively lower level expressed on mature SP cells, expres- lection of NKT cells (reviewed in Ref. 17; see Discussion). sion of SLAM on the developing T cells of NOD.Nkrp1b mice is The expression data from the thymi of NOD.Nkrp1b and retarded, reaching its peak of expression only at the mature SP NOD.Nkrp1b.Nkt1b mice for Slam family members 1–9 are shown stage (Fig. 8C, D). At each developmental stage, the levels of in Fig. 5. Only Slamf1 and Slamf6 were significantly differentially expression of SLAM on the thymocytes of (NOD.Nkrp1b ϫ b b expressed in the congenic line compared with the parental line NOD.Nkrp1 .Nkt1 )F1 mice were intermediate between the two ( p Ͻ 0.001; Mann-Whitney U test). parental strains (data not shown). Levels of expression of SLAM on mature thymic NKT cells are similar (Fig. 8E). Characterization of SLAM expression in thymus and spleen Consistent with the levels of SLAM expression on mature SP Validation of Slamf1 and Slamf6 microarray data was obtained by thymocytes, splenic expression was relatively similar on both T quantitative RT-PCR of the sequences probed by the array on an and B cells of both strains and the (NOD.Nkrp1b ϫ NOD.Nkrp1b. b independent sample set (Fig. 6, n ϭ 6–9; Fig. 7, n ϭ 5–9). Val- Nkt1 )F1 mice (Fig. 8, F–I; data for F1 mice not shown). idation of SLAM expression on thymic and splenic lymphocytes was also performed by flow cytometry (Fig. 8). Consistent with Functional consequences of differences in SLAM expression microarray and RT-PCR quantitation of thymic SLAM expression, To determine whether the difference in SLAM expression on DP thymocytes from NOD.Nkrp1b.Nkt1b congenic mice expressed thymocytes between the NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b significantly more SLAM on their surfaces than those of the pa- strains was sufficient to have functional effects, an assay of SLAM rental strain (Fig. 8A). The cell surface markers CD3, CD4, and function was established. As SLAM acts as a costimulator through CD8 can be used to define the developmental pathway of T cells homotypic interactions, and the difference in levels of expression from the least mature CD4ϪCD8Ϫ (double-negative) CD3Ϫ, was largely restricted to the DP population of thymocytes, SLAM through a double-positive (DP) intermediate stage, to the most function was assessed by measuring TCR-stimulated proliferation mature CD4 or CD8 single-positive (SP) subsets immediately be- of whole thymocytes. Whole thymocytes or purified CD4ϩ spleno- fore thymic export (Fig. 8B). Flow cytometric analysis of SLAM cytes were stimulated in vitro with anti-CD3/anti-CD28 coated expression on thymocytes from each developmental stage revealed beads and the proliferative response detected by thymidine incor- major differences in the developmental program of thymic SLAM poration five or three days later, respectively. The validity of this expression between the NOD.Nkrp1b and NOD.Nkrp1b.Nkt1b system as a surrogate measure of SLAM function was confirmed 1624 GENETIC CONTROL OF NKT CELL NUMBERS

FIGURE 7. Validation of expression microarray proﬁling of Slamf6 expression in thymi from NOD. Nkrp1b and NOD.Nkrp1b.Nkt1b mice. Gene structure and exon structure of the two Slamf6 isoforms are shown together with the relative locations of microarray probe targets and RT-PCR primer sites (A). The microarray results (B; n ϭ 7) and RT-PCR validation (C; Downloaded from n ϭ 5–9) performed on an independent sample are shown. http://www.jimmunol.org/ by guest on September 24, 2021

by the inhibition of proliferation by the addition of 100 ␮g/ml of marker, the NOD.Nkrp1b parental line used in these studies is con- the blocking SLAM peptide 132–146 or inhibiting concentrations genic for Nkrp1b, the allele encoding NK1.1, and was specifically (6.25 ␮g/ml) of the TC15 anti-SLAM mAb (Fig. 9A). developed by us to allow analysis of the major NKT cell subsets Consistent with varying expression levels of SLAM on DP thy- (13, 14). The presence of C57BL/6-derived alleles at the Nkrp1 mocytes modulating the signal threshold of the responding mature locus on chromosome 6 does not affect either the numbers of NKT SP thymocytes, greater proliferation was seen in cultures of thy- cells, nor the strain’s susceptibility to autoimmune disease (14). mocytes from NOD.Nkrp1b.Nkt1b mice than from those of Flow cytometric analyses of thymic and splenic NKT cell subsets NOD.Nkrp1b mice (Fig. 9B; n ϭ 6; p Ͻ 0.05; Mann-Whitney U defined by the cell surface markers CD44, CD4, and NK1.1 (15, test). The addition of exogenous IL-2 eliminated this difference 16, 21) indicated that the majority of the additional NKT cells between the strains, which is consistent with SLAM’s role as a found in NOD.Nkrp1b.Nkt1b mice belonged to the CD4ϩ NK1.1Ϫ costimulator. No significant difference in TCR-stimulated prolif- population, which is considered to be developmentally relatively eration of splenic CD4ϩ T cells was observed, which is consistent immature (15, 16). This finding suggests that the addition of the with the absence of a difference in the levels of SLAM expression C57BL/6-derived allele is sufficient to increase the number of cells on this population. entering the NKT cell developmental pathway, but insufficient to The supernatants from the cultures were then assayed for cyto- push those cells through to maturity. The functional characteristics kines. Thymocytes from NOD.Nkrp1b mice produced significantly of these cells, as well as the additional effects of Nkt2, the other less IL-4 and IL-5, and slightly more IFN-␥, in a manner analo- NKT cell control locus, are issues of great interest. Ϫ Ϫ Ϫ Ϫ gous to the cytokine phenotypes of Slamf1 / and Sap / targeted Two strategies were applied to reduce the number of Nkt1 can- mutant mice (18–20). A similar deviation in IL-4 production was didate genes under consideration. The first was the use of gene ϩ seen in cultures of CD4 splenocytes (Fig. 9, C–E). expression microarrays. As a generalization, this has not been a particularly helpful strategy in the past, and reports of hundreds or Discussion thousands of differentially expressed genes in congenic mice have The production and characterization of the NOD.Nkrp1b.Nkt1b been published (e.g., Ref. 22). In our experience, a dramatic im- mouse strain described here formally confirmed the location of a provement in signal-to-noise ratio could be attained by avoiding major NKT cell control gene on distal chromosome 1, as the con- engagement of the activation and apoptosis cascades. In this spe- genic mice had a 2-fold increase in proportions, and a three-fold cific case, thymi were removed from mice within 120 s of the increase in absolute numbers, of thymic NKT cells at six weeks of induction of anoxia and placed immediately in RNAlater. The sec- age. While conventional NOD lines lack the NK1.1 developmental ond strategy applied was the use of a stringent statistical threshold, The Journal of Immunology 1625

FIGURE 8. SLAM expression on thymocytes (A–E) and splenocytes (F–I) of NOD.Nkrp1b and NOD. Nkrp1b.Nkt1b mice (n ϭ 5/group). Profiles from NOD. Nkrp1b mice are indicated with the fine lines, whereas those from NOD.Nkrp1b.Nkt1b are indicated with the heavy lines. The T cell developmental pathway is indicated (B) and example profiles of SLAM expression on thymocyte subsets gated for CD3, CD4, and CD8 expression presented (C). Means and SEM of mean fluo- rescence intensities (MFI) are shown for each stage (n ϭ Downloaded from 5; D). Values for NOD.Nkrp1b mice are indicated by Ⅺ, whereas those for NOD.Nkrp1b.Nkt1b mice by ࡗ. SLAM levels of thymic CD1d/␣-GalCer tetramer-binding NKT cell CD4ϩ and double-negative subsets were determined by flow cytometry (n ϭ 3; E). Splenocytes were gated by ␤TCR (T cells) and B220 (B cells; G). Example profiles are shown (H) as well as individual values (n ϭ 5; I). http://www.jimmunol.org/ by guest on September 24, 2021

rather than ad hoc fold difference thresholds, which have no ob- tion, SapϪ/Ϫ targeted mutant mice showed a similar defect in T vious biological validity. As a consequence of these procedures, 21 cell-mediated IL-4 production and slightly increased IFN-␥ pro- of the 28 locatable highly differentially expressed genes mapped to duction, compared with SAP-sufficient wild-type CD4ϩ T cells the Nkt1 congenic region and only fifteen of these genes lay within (19, 20, 32, 33). Significantly, SAP-deficient X-linked lymphopro- the Nkt1 95% confidence interval. To our knowledge, no microar- liferative patients as well as mice bearing targeted deletions of ray expression analysis of congenic mice has produced a better SAP (34–36) or FynT (37, 38) lack NKT cells, indicating a critical signal to noise ratio. Of the 15 highly differentially expressed role for the SAP/FynT signaling pathway, presumably activated genes lying within the Nkt1 95% confidence interval, the most following recruitment to one or more members of the SLAM family prominent candidates for control of NKT cell numbers are Slamf1 of cell surface receptors. As NKT cells are positively selected on DP and Slamf6, as signaling through SAP appears to be essential for thymocytes (39, 40), selection is dependent on the SAP/FynT sig- thymic positive selection of NKT cells (reviewed in Ref. 17). naling pathway (34–38), and SLAM is known to be expressed on Slamf1 encodes the Ig-like receptor termed SLAM or CD150, the surfaces of DP thymocytes (41), SLAM-SLAM interactions which forms homotypic interactions and modulates immune re- may be responsible (42). Slamf1 lies within a haplotype block sponses (17, 23). It associates with, and signals through, the Src containing genes encoding nine SLAM family members, many of homology (SH)2-domain containing adaptor protein SAP, which is which contain multiple polymorphism between the minority hap- mutated in the human inherited immunodeficiency X-linked lym- lotype, expressed in C57BL/6, C57L, C57BR, C57BL/10, and RF phoproliferative disease (24–26), and FynT, a Src-related protein (haplotype 1) and the majority haplotype, which is expressed in tyrosine kinase, which is recruited to SAP through a unique inter- 129/SvJ, A/J, AKR/J, BALB/cJ, C3H/HeJ, CBA/J, DBA/2J, MRL/ action involving the SH2 domain of SAP and the SH3 domain of MpJ, NOD/Lt, NZB/B1WJ, NZW, SJL/J, and others (haplotype 2; FynT (27). Ligation of SLAM with mAbs enhanced TCR-stimu- Ref. 43). The lupus susceptibility gene Sle1b has been localized to lated proliferation and cytokine production by human and mouse T this region by congenic mapping and is expressed in haplotype 2 cells (28–30), which is consistent with a role as a costimulator (43). (31). T cells from Slamf1Ϫ/Ϫ targeted mutant mice deficient in RT-PCR and flow cytometry confirmed a major difference in SLAM expression have a severe defect in TCR-activated produc- SLAM expression on the thymocytes of NOD.Nkrp1b.Nkt1b and tion of IL-4 in vitro but produce slightly more IFN-␥, consistent NOD.Nkrp1b mice. Comparison of SLAM levels on thymic sub- with an important role in modulating the character of immune sets revealed variation in the developmentally regulated pattern of responses (18). Consistent with its role in SLAM signal transduc- expression between the strains. While the NOD.Nkrp1b.Nkt1b mice 1626 GENETIC CONTROL OF NKT CELL NUMBERS

FIGURE 9. The costimulation effect of SLAM expression by DP thymocytes on anti-CD3/CD28-induced proliferation of whole thymocytes from NOD.Nkrp1b b b

and NOD.Nkrp1 .Nkt1 mice is illustrated in A in the Downloaded from absence or presence of blocking SLAM peptide 132– 146 or inhibiting concentrations of the TC15 anti- SLAM mAb. Proliferation of control cultures containing no added inhibitors or the nonblocking SLAM peptide 83–97 is also shown. Functional effects of allelic SLAM expression on TCR signaling in vitro of thymocytes and b b splenocytes from NOD.Nkrp1 (Ⅺ) and NOD.Nkrp1 . http://www.jimmunol.org/ Nkt1b mice (f; n ϭ 6/group; B–E). Cultures of whole thymocytes (3 days) and puriﬁed CD4ϩ splenocytes (5 days) were stimulated with anti-CD3/anti-CD28-coated beads in the presence or absence of IL-2, and proliferation assayed by thymidine incorporation (B). Levels of IL-4 (C), IL-5 (D), and IFN-␥ (E) in culture supernatants were measured by cytokine bead array. by guest on September 24, 2021

express increasing levels of SLAM through T cell development to ing of differential gene expression between the strains in whole peak on DP thymocytes and then decline to relatively lower levels thymic RNA preparations. Second, because NKT cells are positively on mature SP cells, expression of SLAM on developing T cells of selected on DP thymocytes via a mechanism dependent on the SAP/ NOD.Nkrp1b is retarded, reaching its peak of expression only at FynT signaling pathway, decreased SLAM expression at this de- the mature SP stage. Consistent with the levels of SLAM expres- velopmental stage may provide an explanation for the reduced sion on mature SP thymocytes, splenic expression was relatively numbers of NKT cells in NOD mice. Third, as SLAM also acts as similar between the strains on both T and B cells. This difference a costimulator for conventional T cells, it is possible that the rel- in SLAM expression was of functional importance, as it affected atively lower levels of SLAM expression at the stage of negative both TCR-stimulated proliferation as well as cytokine production. selection (late DP stage) compared with those at maturity (SP thy- Significantly, thymocytes and CD4ϩ splenocytes from NOD. mocytes and in the periphery) result in a lowering of the signaling Nkrp1b mice produced less IL-4, and slightly more IFN-␥,ina threshold of conventional T cells in the periphery. If true, this may manner analogous to the cytokine phenotypes of Slamf1Ϫ/Ϫ and result in an increased proportion of peripheral T cells capable of SapϪ/Ϫ targeted mutant mice (18–20). responding to self-Ags. The retardation of developmentally programmed SLAM expres- In conclusion, the data presented make a strong case for the sion in NOD mice has three significant implications. First, as DP hypothesis that the control of NKT cell numbers attributed to the thymocytes account for Ͼ80% of the thymus, it explains the find- Nkt1 gene is mediated by differential expression of Slamf1 and are The Journal of Immunology 1627 consistent with an additional contribution by Slamf6. In addition, it 21. Gadue, P., and P. L. Stein. 2002. NK T cell precursors exhibit differential cyto- is possible that the retarded programmed expression of SLAM on kine regulation and require Itk for efficient maturation. J. Immunol. 169: 2397–2406. developing conventional T cells of NOD mice may contribute to 22. Eaves, I. A., L. S. Wicker, G. Ghandour, P. A. Lyons, L. B. Peterson, J. A. Todd, lowering their TCR signaling threshold in the periphery thereby and R. J. Glynne. 2002. Combining mouse congenic strains and microarray gene expression analyses to study a complex trait: the NOD model of type 1 diabetes. contributing to autoimmune disease in this strain. Genome Res. 12: 232–243. 23. Veillette, A., and S. Latour. 2003. The SLAM family of immune-cell receptors. Acknowledgments Curr. Opin. Immunol. 15: 277–285. We acknowledge the invaluable advice and suggestions of Dale Godfrey 24. Sayos, J., C. Wu, M. Morra, N. Wang, X. Zhang, D. Allen, S. van Schaik, L. Notarangelo, R. Geha, M. G. Roncarolo, et al. 1998. The X-linked lympho- and Stuart Tangye, technical assistance by Tatiana Tsoutsman, Tim Butler, proliferative-disease gene product SAP regulates signals induced through the and Rhiana Magee, and the help and support of our animal attendants co-receptor SLAM. Nature 395: 462–469. Nicole Fraser and Rohan Henderson. 25. Nichols, K. E., D. P. Harkin, S. Levitz, M. Krainer, K. A. Kolquist, C. Genovese, A. Bernard, M. Ferguson, L. Zuo, E. Snyder, et al. 1998. Inactivating mutations References in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proc. Natl. Acad. Sci. USA 95: 13765–11370. 1. Godfrey, D. I., and M. Kronenberg. 2004. Going both ways: immune regulation 26. Coffey, A. J., R. A. Brooksbank, O. Brandau, T. Oohashi, G. R. Howell, J. M. via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388. Bye, A. P. Cahn, J. Durham, P. Heath, P. Wray, et al. 1998. Host response to EBV 2. Van Kaer, L. 2005. ␣-Galactosylceramide therapy for autoimmune diseases: infection in X-linked lymphoproliferative disease results from mutations in an prospects and obstacles. Nat. Rev. Immunol. 5: 31–42. SH2-domain encoding gene. Nat. Genet. 20: 129–135. 3. Brigl, M., and M. B. Brenner. 2004. CD1: antigen presentation and T cell func- 27. Latour, S., R. Roncagalli, R. Chen, M. Bakinowski, X. Shi, P. L. Schwartzberg, tion. Ann. Rev. Immunol. 22: 817–890. D. Davidson, and A. Veillette. 2003. Binding of SAP SH2 domain to FynT SH3 4. Baxter, A. G., S. J. Kinder, K. J. Hammond, R. Scollay, and D. I. Godfrey. 1997. ϩ Ϫ Ϫ domain reveals a novel mechanism of receptor signalling in immune regulation. Association between ␣␤TCR CD4 CD8 T cell deficiency and IDDM in

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