Supplementary Information A Slfn mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence Michael Berger, Philippe Kres, Karine Crozat, Xiaohong Li, Ben A. Croker, Owen M. Siggs, Daniel Popkin, Xin Du, Brian R. Lawson, Argyrios N. Theofilopoulos, Yu Xia, Kevin Khovananth, Eva Marie Y. Moresco, Takashi Satoh, Osamu Takeuchi, Shizuo Akira & Bruce Beutler Supplementary Figures 1-7 Nature Immunology: doi:1.13/ni.17
3 5 3 1 5 1 c 3 1 Ly 9 D NK p Ig G IL1 /1 IL1 3 1 95 75 55 35 5 Killing of C3H cells (%) m ed iu m IFN-γ+ NK cells (%) 5 Killing of Tap1 / cells (%) IL-1p7 (pg/ml) 5 TNF (pg/ml) IFN-γ (pg/ml) a Tap1 / 1 Tap1 / d Myd poc/poc Supplementary Figure 1. Homozygous mice display intact cytokine production and NK cell activity ut are susceptile to Listeria monocytogenes. (a) IFN-γ, TNF, and IL-1p7 concentrations in serum measured 3 h after MCMV infection. Results are representative of 3 independent experiments. () Negatively isolated NK cells from spleens of (n=) or mice (n=) were stimulated ex vivo with the indicated stimuli for h in the presence of Brefeldin A. NK cells were then fixed, permeailized, and stained for intracellular IFN-γ expression, and then analyzed y flow cytometry. Results are representative of 3 independent experiments. (c) In vivo NK killing assay. Control, TAP deficient, and C3H splenocytes were laeled with different intensities of CFSE. Control and either TAP deficient (left) or C3H splenocytes (right) were mixed at a 1:1 ratio and injected i.v. into recipient mice of the indicated genotype (n= recipients per genotype). One day after injection, PBMCs were collected from recipient mice and analyzed y flow cytometry for the numer of control and NK target cells remaining. % killing reflects numer of NK targets relative to control cells. Results are representative of independent experiments. (d) Bacterial load was visualized in homozygotes, Mydpoc/poc and mice days after challenge with 5 x cfu luminescent L. monocytogenes. Results are representative of independent experiments. Nature Immunology: doi:1.13/ni.17
Relative Ca + flux..3.. CD hi.1. 1 3 1 3..5..3 CD 1 3 Kinetics Time (sec)..5..3 CD 1 3 Time (sec) CD lo Time (min) 5 P-NFAT1 NFAT1 P-NFAT1 NFAT1 Anti-CD3, CD & CD 3 PMA+ iono c Time (min) N-p5 C-p5 Anti-CD3, CD & CD 1 5 1 5 d Time (min) P-Erk Anti-CD3, CD & CD 1 5 3 1 5 3 e Untreated Erk f Untreated P-AKT (T3) CD9 CD5 P-AKT (S73) Supplementary Figure. Intact TCR signaling in homozygous T cells. (a) Pooled purified splenic T cells from homozygous or WT mice, preloaded with Indo-1 AM, were surface stained with iotinylated anti-cd3, iotinylated anti-cd, and fluorescent-conjugated antiodies to CD, CD, and CD. Ca + flux, as measured y Indo-1 fluorescence, was monitored y flow cytometry after the addition of avidin to crosslink CD3 and CD (TCR engagement). Representative plots of Ca + flux in CD susets of CD + and CD + T cells are shown. A s period was used to estalish aseline levels efore the addition of avidin. Results are representative of independent experiments each using 3 mice per genotype. () Purified splenic T cells were activated with CD3/CD/CD antiody-coated eads for the indicated times. Cell extracts were then sujected to immunolot analysis using anti-nfat1. (c) Cells were activated and collected as in. Cells then were separated into cytoplsmic and nuclear fractions followed y immunolot analysis using anti-p5. (d) Cells were activated and collected as in, and sujected to immunolot analysis using anti-phospho-erk or anti-erk. (e) Cells were activated as in and collected after 1 h and sujected to flow cytometry analysis using either anti-phospho-akt (Thr3) or anti-phospho-akt (Ser73). For -e, results are representative of independent experiments. (f) Splenic CD + T cells from WT or homozygous mice were left untreated, or stimulated with a comination of anti-cd3ε and anti-cd. Cells were analyzed y flow cytometry for CD5 or CD9 expression h later. Results are representative of 3 independent experiments each using mice per genotype. Nature Immunology: doi:1.13/ni.17
CD + cells CD + cells 3% % CD5.1.1% CD5.1.3% CD CFSE CD CFSE c B cells % CD5.1 5% CD CFSE Supplementary Figure 3. Impaired homeostatic expansion of homozygous T cells. 5x1 CFSE laeled splenocytes from either WT or homozygous mice (Ly5. + ) were adoptively transferred into irradiated ( rad) WT recipient mice (Ly5.1 + ) (n=3 mice). Seven days after the transfer, spleen cells were sujected to flow cytometric analysis for CD5.1 versus CD (a), CD (), or B (c) staining. Circles indicate donor cells. Histograms show CFSE staining of the donor population. Results are representative of 3 independent experiments. Nature Immunology: doi:1.13/ni.17
% 1% % % CD CD1 CD lo CD hi CDL Supplementary Figure. Homozygous CD + T cells are semi-activated. (a) Flow cytometric analysis of CD versus CD1 (IL-Rβ) staining of splenic CD + cells from WT or homozygous mice. () Flow cytometric analysis of CDL staining of CD lo CD + (left) or CD hi CD + (right) cells from WT or homozygous mice. For a and, results are representative of 3 independent experiments each using mice per genotype. Nature Immunology: doi:1.13/ni.17
3 Apoptosis (%) 5 1 5 Untreated CpG CD Untreated Supplementary Figure 5. Normal activation of DCs. Pooled splenic DCs from WT (n=3) or homozygous mice (n=3) were stimulated in vitro with (.1 μm) of CpG oligonucleotides for h. (a) The percentage of apoptotic cells was determined y Annexin V staining followed y flow cytometric analysis. () Flow cytometric analysis of CD staining. Results are representative of independent experiments. Nature Immunology: doi:1.13/ni.17
CD + lymphocytes (%) 1 11 1 9 7 5 3 1 Lod score (Z) 1 13 1 111 1 9 7 5 3 1 D1Mit D1Mit11 D1Mit13 D1Mit1 DMit35 DMit75 DMit5 D3Mit3 D3Mit9 DMit35 DMit17 DMit3 D5Mit3 D5Mit39 D5Mit9 DMit7 DMit3 D7Mit7 D11Mit35 D7Mit35 DMit1 DMit5 DMit 13 D11Mit D11Mit D9Mit D9Mit355 D1Mit D1Mit3 D11Mit D11Mit D11Mit D11Mit D1Mit D1Mit1 D13Mit D13Mit1 D1Mit1 D1Mit1 DMit DMit D1Mit1 D17Mit57 D17Mit7 D1Mit D19Mit9 DXMit5 DXMit DXMit11 c 79 M 3.5 M Mouse # Marker D11Mit3 D11Mit3 D11Mit119 D11Mit35 D11Mit3 Mutant phenotype px D D Wild-type phenotype 33px 39px 5px 3px D D e d Chr. 11 11 Genes f COG5 divergent AAA 1 5 359 37 I135N Supplementary Figure. Positional cloning of the mutation. (a) Plot shows the percentage of CD + lymphocytes in WT and homozygous mice. CD + T cell deficiency in the lood was used to map the mutation. () Strongest linkage of the phenotype was oserved with a marker on Chromosome 11, D11Mit. (c) Fine mapping of the mutation narrowed the critical interval to a.5 M region (79 M- 3.5 M) on Chr. 11. (d) Schematic representation of Chr. 11. The red rectangle represents the critical region, which contains 11 genes. (e) Genomic sequence from a part of the Slfn gene reveals a T A transversion. (f) Diagram of the Slfn protein. The COG5 domain partially overlaps the divergent AAA domain. The position of the mutation (I135N) is indicated. Nature Immunology: doi:1.13/ni.17
Cells (%) 3 1 CD + T cells CD + T cells B cells NK cells Monocytes 1 1 Slfn1 +/ Slfn1 / Cells (%) 3 1 1 5 1 Slfn3 +/ Slfn3 / Supplementary Figure 7. Normal immune cell populations in Slfn1 / and Slfn3 / mice. Flow cytometric analysis of CD + T cells, CD + T cells, B + B cells, NK cells (CD3 negative), and CD11 + LyC hi LyG monocytes from (a) Slfn1 +/ (n=) and Slfn1 / mice (n=) or () Slfn3 +/ (n=3) and Slfn3 / mice (n=3). Results are representative of independent experiments. Nature Immunology: doi:1.13/ni.17