Dierks Supplementary Fig. S1

Transcription

1 Dierks Supplementary Fig. S1 ITK SYK PH TH K42R wt K42R (kinase deficient) R29C E42K Y323F R29C E42K Y323F (reduced phospholipid binding) (enhanced phospholipid binding) (reduced Cbl binding) E42K Y323F E42K Y323F Fig. S1 ITK-SYK constructs and point mutants The ITK-SYK fusion contains the pleckstrin homology domain (PH), which mediates its membrane localisation and the Tec-homology domain (TH) of ITK. The SYK fragment comprises the SYK tyrosine kinase (TK) domain. The ITK-SYK mutant K42R contains a point mutation within the SYK kinase domain (lysin > arginine), which induces loss of the SYK kinase activity. The point mutant R29C within the PH domain of ITK demonstrates reduced phospholipid-binding and therefore reduced membrane localization. E42K in contrast demonstrates enhanced phospholipid binding and therefore stronger membrane binding. The ITK-SYK mutant Y323F demonstrates reduced CBL binding due to the loss of the essential tyrosine residue as binding motif.

2 Dierks Supplementary Fig. S2 ITK-SYK sequence and point mutations M N N F I L L E E Q L I K K S Q Q K 18 ATG AAC AAC TTT ATC CTC CTG GAA GAA CAG CTC ATC AAG AAA TCC CAA CAA AAG 54 R R T S P S N F K V R>C F F V L T K A 36 AGA AGA ACT TCT CCC TCG AAC TTT AAA GTC CGC TTC TTC GTG TTA ACC AAA GCC 18 S L A Y F E>K D R H G K K R T L K G S 54 AGC CTG GCA TAC TTT GAA GAT CGT CAT GGG AAG AAG CGC ACG CTG AAG GGG TCC 162 I E L S R I K C V E I V K S D I S I 72 ATT GAG CTC TCC CGA ATC AAA TGT GTT GAG ATT GTG AAA AGT GAC ATC AGC ATC 216 P C H Y K Y P F Q V V H D N Y L L Y 9 CCA TGC CAC TAT AAA TAC CCG TTT CAG GTG GTG CAT GAC AAC TAC CTC CTA TAT 27 V F A P D R E S R Q R W V L A L K E 18 GTG TTT GCT CCA GAT CGT GAG AGC CGG CAG CGC TGG GTG CTG GCC CTT AAA GAA 324 E T R N N N S L V P K Y H P N F W M 126 GAA ACG AGG AAT AAT AAC AGT TTG GTG CCT AAA TAT CAT CCT AAT TTC TGG ATG 378 D G K W R C C S Q L E K L A T G C A 144 GAT GGG AAG TGG AGG TGC TGT TCT CAG CTG GAG AAG CTT GCA ACA GGC TGT GCC 432 Q Y D P T K N A S K K P L P P T P E 162 CAA TAT GAT CCA ACC AAG AAT GCT TCA AAG AAG CCT CTT CCT CCT ACT CCT GAA 486 D N R GAC AAC AGG S 36 TCC 918 S P A Q G N R Q E S T V S F N P Y>F E 324 TCC CCT GCC CAA GGG AAC CGG CAA GAG AGT ACT GTG TCA TTC AAT CCG TAT GAG 972 P E L A P W A A D K G P Q R E A L P 342 CCA GAA CTT GCA CCC TGG GCT GCA GAC AAA GGC CCC CAG AGA GAA GCC CTA CCC 126 M D T E V Y E S P Y A D P E E I R P 36 ATG GAC ACA GAG GTG TAC GAG AGC CCC TAC GCG GAC CCT GAG GAG ATC AGG CCC 18 K E V Y L D R K L L T L E D K E L G 378 AAG GAG GTT TAC CTG GAC CGA AAG CTG CTG ACG CTG GAA GAC AAA GAA CTG GGC 1134 S G N F G T V K K G Y Y Q M K K V V 396 TCT GGT AAT TTT GGA ACT GTG AAA AAG GGC TAC TAC CAA ATG AAA AAA GTT GTG 1188 K T V A V K>R I L K N E A N D P A L K 414 AAA ACC GTG GCT GTG AAA ATA CTG AAA AAC GAG GCC AAT GAC CCC GCT CTT AAA 1242 D E L L A E A N V M Q Q L D N P Y I 432 GAT GAG TTA TTA GCA GAA GCA AAT GTC ATG CAG CAG CTG GAC AAC CCG TAC ATC 1296 V R M I G I C E A E S W M L V M E M 45 GTG CGC ATG ATC GGG ATA TGC GAG GCC GAG TCC TGG ATG CTA GTT ATG GAG ATG 135 A E L G P L N K Y L Q Q N R H V K D 468 GCA GAA CTT GGT CCC CTC AAT AAG TAT TTG CAG CAG AAC AGA CAT GTC AAG GAT 144 K N I I E L V H Q V S M G M K Y L E 486 AAG AAC ATC ATA GAA CTG GTT CAT CAG GTT TCC ATG GGC ATG AAG TAC TTG GAG 1458 E S N F V H R D L A A R N V L L V T 54 GAG AGC AAT TTT GTG CAC AGA GAT CTG GCT GCA AGA AAT GTG TTG CTA GTT ACC 1512 Q H Y A K I S D F G L S K A L R A D 522 CAA CAT TAT GCC AAG ATC AGT GAT TTC GGA CTC TCC AAA GCA CTG CGT GCT GAT 1566

3 E N Y Y K A Q T H G K W P V K W Y A 54 GAA AAC TAC TAC AAG GCC CAG ACC CAT GGA AAG TGG CCT GTC AAG TGG TAC GCT 162 P E C I N Y Y K F S S K S D V W S F 558 CCG GAA TGC ATC AAC TAC TAC AAG TTC TCC AGC AAA AGC GAT GTC TGG AGC TTT 1674 G V L M W E A F S Y G Q K P Y R G M 576 GGA GTG TTG ATG TGG GAA GCA TTC TCC TAT GGG CAG AAG CCA TAT CGA GGG ATG 1728 K G S E V T A M L E K G E R M G C P 594 AAA GGA AGT GAA GTC ACC GCT ATG TTA GAG AAA GGA GAG CGG ATG GGG TGC CCT 1782 A G C P R E M Y D L M N L C W T Y D 612 GCA GGG TGT CCA AGA GAG ATG TAC GAT CTC ATG AAT CTG TGC TGG ACA TAC GAT 1836 V E N R P G F A A V E L R L R N Y Y 63 GTG GAA AAC AGG CCC GGA TTC GCA GCA GTG GAA CTG CGG CTG CGC AAT TAC TAC 189 Y D V V N * 636 TAT GAC GTG GTG AAC TAA 198 Fig. S2 ITK-SYK sequence and point mutations As described by Streubel et al., the ITK-SYK fusion contains 165 amino acids (495 base pairs) from the ITK gene and 331 amino acids (993 base pairs) from the SYK tyrosine kinase. The sequence in black contains the ITK-part of the sequence and the sequence in red the SYK part of the sequence. The point mutantions inserted to generate the different loss or gain of function constructs are shown within the sequence.

4 Dierks Supplementary Fig. S Percentage of start weight d d 7 d 14 d 21 d 28 d 35 d 42 pmscv GFP pmscv ITK-SYK GFP Fig. S3 Mouse weight in mice transplanted with ITK-SYK-positive bone marrow Average mouse weight of mice transplanted with bone marrow infected with a pmscv/ires/gfp control vector or pmscv/itk-syk/ires/gfp (8 mice per group, ±SD).

5 Dierks Supplementary Fig. S4 GFP ITK-SYK Fig. S4 Enlarged lymph nodes in ITK-SYK-positive mice Representable examples for lymph nodes in mice transplanted with bone marrow either infected with a GFP control virus or an ITK-SYK/GFP virus 4 weeks after transplantation. All ITK-SYK positive mice had enlarged lymph nodes compared to the control group.

6 Dierks, Supplementary Fig. S5 Fig. S5 Spleen infiltration with CD3+ cells in ITK-SYK-positive mice IHC staining for CD3 in spleens from one representative ITK-SYK pos. diseased mouse demonstrates massive infiltration of the spleen with CD3 pos. T-cells with expansion of T- cells into the red pulpa. (photomicrographs with 2-fold and 4-fold magnification)

7 Dierks Supplementary Fig. S6 Fig. S6 Percentage of regulatory T-cells in spleen and bone marrow of transplanted mice Isolation from white blood cells from the spleen and the bone marrow from mice either transplanted with the pmscv/gfp control vector or the pmscv/itk-syk/gfp vector 5 weeks after transplantation. The graphs show the percentage of CD4+CD25+Foxp3+ regulatory T-cells within the GFP-positive or GFP-negative population in control versus ITK-SYK mice in the spleen (left) or bone marrow (right). The percentage of regulatory T-cells in ITK-SYK mice is not significantly enhanced in bone marrow and spleens compared to control mice (p>,5).

8 Dierks Supplementary Fig. S7 %ige of CD3 pos. cells within WBCs control Spleen ITK-SYK %ige of CD3 pos. cells within WBCs control ITK-SYK Bone marrow GFP- GFP+ GFP- GFP+ Fig. S7 Number of CD3-positive T-cells in transplanted mice Isolation from white blood cells from the spleen and the bone marrow from mice either transplanted with the pmscv/gfp control vector or the pmscv/itk-syk/gfp vector 5 weeks after transplantation. The graphs show the percentage of CD3 positive T-cells within the GFPpositive or GFP-negative population in control versus ITK-SYK mice in the spleen (left) or bone marrow (right).

9 Dierks Supplementary Fig. S8 Spleen Bone marrow Percentage of T-cell subsets within whole cells control spleen ITK-SYK spleen Percentage of T-cell subsets within whole cells control BM ITK-SYK BM CD4-CD8- CD4+CD8+ CD8+ CD4+ CD4-CD8- CD4+CD8+ CD8+ CD4+ GFP-negative cells GFP-negative cells Fig. S8 T-cell subsets within the GFP-negative T-cell population Isolation from white blood cells from the spleen and the bone marrow from mice either transplanted with the pmscv/gfp control vector or the pmscv/itk-syk/gfp vector 5 weeks after transplantation. The graphs show the average percentage of the different T-cell subsets (CD4+, CD8+, CD4+CD8+, CD4-CD8-) within the GFP-negative population in control versus ITK-SYK mice in the spleen (left) or bone marrow (right). The percentage of CD3-positive cells within the GFP-negative population in ITK-SYK pos. mice is significantly reduced compared to control mice in the spleen (p <,5 unpaired T-Test) and shows a tendency towards reduction in the bone marrow (no statistical significancp >,5).

10 Dierks Supplementary Fig. S9 %ige of B22+ B-cells within WBCs in the spleen control ITK-SYK GFP- GFP+ Fig. S9 Number of B22+ B-cells in the spleen of transplanted mice Isolation from white blood cells from the spleen and the bone marrow from mice either transplanted with the pmscv/gfp control vector or the pmscv/itk-syk/gfp vector 5 weeks after transplantation. Detection of B22 positive cells by flow cytometry. The graphs show the average percentage of the B-cells within spleen cells isolated from control mice or ITK-SYK positive mice.

11 Dierks Supplementary Fig. S1 Retransplantation of ITK-SYK-pos. cells surviving mice survival days bone marrow cells spleen cells Fig. S1 Retransplantation of ITK-SYK-positive cells Retransplantation of bone marrow and spleen cells isolated from ITK-SYK-positive mice into irradiated recipients. Figure shows survival of secondary recipients transplanted with either complete bone marrow or complete spleen cells isolated from diseased primary recipients.

12 Dierks Supplementary Fig. S11 A Vbeta 2 B Vbeta 1 Vbeta 6 Tx1 Mouse 1 Tx1 Mouse 2 Tx2 M1 Mouse A Tx2 M2 Mouse A Tx2 M2 Mouse B Tx2 M2 Mouse C Fig. S11 TCR V spectratyping of ITK-SYK positive T-cells reveals clonal events in secondary recipient mice Spectratyping for the T-cell receptor repertoire using primers for V 1-2 was performed in primary (Transplantation1 = Tx1) and secondary (Transplantation 2 = Tx2) recipients of ITK- SYK/GFP positive bone marrow. Spleen cells were harvested from diseased mice. For mice with a spleen infiltration of below 5 % ITK-SYK/GFP pos. cells, the GFP positive cells were purified by FACS. This was done to avoid overlooking clonal events via cross contamination with ITK-SYK negative T-cells (Tx2 M2 Mouse C, Tx2 M1 Mouse A). All primary recipients developed a polyclonal disease (Tx1 Mouse 1, Tx1 Mouse 2). In contrast, we observed a mono/biclonal disease in 3 out of 4 secondary recipients (Tx2 M2 Mouse A, Tx2 M1 Mouse A, Tx2 M2 Mouse C).A TCR V spectratyping for primary recipient (Tx1 Mouse 1) and for the secondary recipient (Tx2 M1 Mouse A), which received bone marrow from this diseased primary recipient. Clonal event in secondary recipient was found in Vbeta 2. B TCR V spectratyping for primary recipient Tx1 Mouse 2 and its secondary recipients (Tx2 M2 Mouse A/B/C). Clonal event in secondary recipients were found in Vbeta 1 and Vbeta 6. Mouse C had a clonal peak in Vbeta 1 and Vbeta 6, which can be interpreted as either biclonal disease or monoclonal disease with biallelic rearrangement. Tx1 = Transplantation 1 = primary recipients Tx2 = Transplantation 1 = secondary recipients Mouse 1/2 = M1/2 = number of primary recipient mouse Mouse A/B/C = number of secondary recipient mouse

13 Dierks Supplementary Fig. S12 Percentage of T-cell subsets within GFPpositive nuclear cells control Spleen ITK-SYK K42R Spleen control BM ITK-SYK K42R BM CD4-CD8- CD4+CD8+ CD8+ CD4+ Fig. S11 T-cell subsets within the GFP-positive nuclear cells in control mice versus mice transplanted with kinase-deficient ITK-SYK Isolation from nuclear cells from the spleen and the bone marrow from mice either transplanted with the pmscv/gfp control vector or the pmscv/itk-syk K42R/GFP vector 5 weeks after transplantation. Analysis of T-cell subsets was performed using flow cytometry. The graphs show the average percentage of the different T-cell subsets (CD4+, CD8+, CD4+CD8+, CD4-CD8-) within the GFP-positive population in control versus ITK-SYK K42R mice in the spleen or bone marrow.

14 Dierks Supplementary Fig. S13 A signs of inflammation %ige of mice with inflammation 8.% 6.% 4.% 2.%.% GFP control ITK-Syk wt ITK-Syk R29C ITK-Syk E42K B GFP wt R29C E42K K42R CD11b CD3 ITK-Syk K42R GFP Fig. S13 Increased inflammation in mice carrying the cytoplasmic localized mutant ITK- SYK R29C A Unspecific signs of inflammation 4 weeks after transplantation (swollen tail, swollen and red nose, diarrhea, ear infiltration). B (upper panel) CD3-PECy7/GFP measurement of spleen cells 3 weeks after transplantation. CD3/GFP double positive population appears in all mice transplanted with ITK-SYK pos. bone marrow with intact kinase activity. (lower panel) Unspecific infiltration of CD11b-positive/GFP-negative myeloid cells into the spleens of mice transplanted with ITK-SYK wt and the 2 mutants with intact kinase activity.

15 Dierks Supplementary Fig. S14 Percentage of splenic CD3+/GFP+ T- cells compared to vehicle control vehicle Curcumin 5 mg/kg bid Curcumin 1 mg/kg bid splenic CD3+/GFP+ T-cells Fig. S14 Curcumin induced reduction of ITK-SYK positive T-cells in the spleen Flow cytometry analysis of ITK-SYK/GFP+CD3+ population in the spleens of treated mice. Graph shows percentage of CD3+/ITK-SYK+ T-cells compared to vehicle treated mice. Statistical significance for both treatment groups (unpaired t-test, P <,5).

16 Dierks Supplementary Fig. S15 In-vivo treatment (GFP pos. cells) Vehicle 1 Vehicle 2 Curcumine 5 mg/kg bid 1 Curcumine 5 mg/kg bid 2 Curcumine 1 mg/kg bid 1 Curcumine 1 mg/kg bid 2 p-syk Tyr 525 staining Fig. S15 Reduced ITK-SYK phosphorylation in Curcumin treated mice Detection of phospho-syk Tyr525 by flow cytometry in the ITK-SYK/GFP positive T-cell population after treatment of mice with Curcumin for 14 days in vivo. Graph shows 2 representative examples per treatment group.

17 Dierks Supplementary Fig. S16 11 weight in mg vehicle Curcumin 5 mg/kg bid Spleen weight Curcumin 1 mg/kg bid Percentage of splenic CD3+/GFP+ T cells compared to vehicle control vehicle Curcumin 5 mg/kg bid Curcumin 1 mg/kg bid splenic CD3+ T-cells Fig. S16 Curcumin treatment of healthy Balb/c mice Spleen weight comparison of Balb/c females treated with vehicle or Curcumin for 14 days. No statistical difference was detected in between the different treatment groups (unpaired t- test, P >,5). Flow cytometry analysis of CD3+ population in the spleens of treated mice. Graph shows percentage of CD3+ T-cells compared to vehicle treated mice. Differences are not statistically significant for both treatment groups (unpaired t-test, P >,5).