Nature Immunology: doi:10.1038/ni/-and Cpdm-/-mice were differentially labeled with Cell Trace Far...

Nature Immunology: doi:10.1038/ni.3352

Supplementary Figure 1

Phenotypic analysis of thymocytes.

(a) Expression level of SHARPIN in DN, CD4+, CD8

+ SP and DP thymocytes, CD4

+ naïve T and Treg cells, B cells and macrophages.

(b) Total numbers of CD4+

SP, CD8+

SP and DP thymocytes of Cpdm+/-

and Cpdm-/-

mice (n=8 mice per group). (c) Flow cytometric

analysis (left) or frequencies (right) of CD4+ and CD8

+ T cells in the thymus of 4 wk old Cpdm

+/- and Cpdm

-/- mice (n=10 mice per

group). (d) Percentage of Live/Dead+ DP thymocytes from Cpdm

+/- and Cpdm

-/- mice with or without anti-CD3 and anti-CD28

stimulation (n=8 mice per group). The data are compiled or representative of three to four independent experiments. Error bars indicate

the mean (± s.d.). *P < 0.05 (two-tailed unpaired t-test).



Analysis of T cell development and phenotype in BM chimeric mice.

(a-b) Flow cytometric analysis of CD4+ and CD8

+ in pregated CD45.1

+ or CD45.2

+ cells of Thy or SP from mice reconstituted with

individual (a) or mixed transfers (b) of WT or Cpdm+/-

and Cpdm-/-

BM (n=4-6 mice per group). (c) Sorted naive CD4+CD62L

+CD44

-

CD25- T cells of SP from mice reconstituted with mixed transfers of WT or Cpdm

+/- and Cpdm

-/- BM were cultured with anti-CD3 or anti-

CD3 and anti-CD28. Cell Trace Violet dilution was examined 4 d later by flow cytometry. (d) Percentage of Live/Dead+ CD4

+ T cells of

SP from the mice in (c) with or without anti-CD3 and anti-CD28 stimulation. (e) Flow cytometric analysis (upper) or frequencies (bottom)

of CD62L+ and CD44

+ of CD4

+ T cells in SP or lung from mice reconstituted with individual or mixed transfers of WT or Cpdm

+/- and

Cpdm-/-

BM (n=6-8 mice per group). (f) Cytokine production in CD4+T cells. Isolated lung cells from the mice in (e) were stimulated with

PMA and Ionomycin for 4h (n=6 mice per group). (g) H&E staining (upper) and histology scores (bottom) of the lung tissue section from

the mice in (e). Original magnification, ×100 (n=6 mice per group). (h) Analysis of the phosphorylation status of signaling proteins in

sorted CD4+

T cells from Cpdm+/-

and Cpdm-/-

mice upon TGF-β stimulation. The data are compiled or representative of two to three

independent experiments. Error bars indicate the mean (± s.d.). *P < 0.05 **P < 0.01 and ***P < 0.001 (two-tailed unpaired t-test).



Analysis of Treg cell proliferation and apoptosis.

(a) Flow cytometric analysis of Ki-67 expression in Treg cells of SP from Cpdm+/-

and Cpdm-/-

mice (n=12 mice per group). (b) Sorted

CD4+CD25

+Nrp1

+ Treg cells of SP from Cpdm

+/- and Cpdm

-/- mice were cultured with anti-CD3and anti-CD28. Cell Trace Violet dilution

was examined 2 or 4 d later by flow cytometry. (c) Percentage of Live/Dead+ CD4

+Foxp3

+ Treg cells with or without anti-CD3 and anti-

CD28 in SP from Cpdm+/-

and Cpdm-/-

mice (n=8 mice per group). (d) Flow cytometric analysis of Ki-67 expression in Treg cells of SP

from mixed chimeric mice (n=8 mice per group). (e) Percentage of Live/Dead+ CD4

+Foxp3

+ Treg cells with or without anti-CD3 and anti-

CD28 in SP from mixed chimeric mice (n=6 mice per group). (f-g) Percentage of Live/Dead+ CD4

+Foxp3

+ Treg cells in thymus from

Cpdm+/-

and Cpdm-/-

mice (f) or mixed chimeric mice (g) (n=6 mice per group). The data are compiled or representative of two to four

independent experiments. Error bars indicate the mean (± s.d.). *P < 0.05 **P < 0.01 and ***P < 0.001 (two-tailed unpaired t-test).



SHARPIN-deficient Treg cells have a defect in the suppression of colitis.

Rag1–/–

mice received sorted CD4+CD25

+YFP

+ Treg (CD45.2

+) cells from Foxp3

YFPCreCpdm

+/-or Foxp3

YFPCreCpdm

-/- mice, together with

CD4+CD45RB

hi (CD45.1

+) T cells, or CD4

+CD45RB

hi (CD45.1

+) T cells alone (None). (a-d) Colon length (a), the ratios of

CD4+CD45RB

high (CD45.1

+) to CD4

+YFP

+ (CD45.2

+) Treg cells in the indicated organs (b), absolute numbers of CD4

+YFP

+ (CD45.2

+)

Treg cells in the indicated organs (c), and frequencies of the indicated cytokine-producing CD4+CD45RB

hi (CD45.1

+) T cells in the

indicated organs (d) of the Rag1-/-

recipient mice at 8 wk after transfer (n=6 to 12 mice per group). (e-f) Isolated CD4+

T cells from

Cpdm+/-

and Cpdm-/-

mice were differentially labeled with Cell Trace Far Red DDAO-SE and Violet dye, respectively, or vice versa, and

then co-injected into WT recipient mice. Flow cytometric analysis (left) or percentage (right) of the input population and transferred

CD4+Foxp3

- T cells (e), flow cytometric analysis (left) or percentage (right) of the input population and transferred CD4

+Foxp3

+ T cells

(f), isolated from various tissues of the recipient mice at 2 or 20 hr after injection (n=6 mice per group). The data are compiled or

representative of two to four independent experiments. Error bars indicate the mean (± s.d.). *P < 0.05 **P < 0.01, ***P < 0.001 and

****P < 0.0001 (two-tailed unpaired t-test).



Cytokine production in SHARPIN-deficient memory or naive T cells or Treg cells.

(a) The methylation pattern of CNS2 of the Foxp3 locus in sorted CD4+YFP

+CD25

+ Treg cells and CD4

+YFP

-CD25

- conventional T cells

from Foxp3YFPCre

Cpdm+/-

or Foxp3YFPCre

Cpdm-/-

mice. Individual CpG motif within CNS2 is vertically arranged and color-coded according

to the degree of methylation at that site (n=6 mice per group). (b) Sorted CD45.1+CD4

+CD25

+Nrp1

+ or CD45.2

+CD4

+CD25

+Nrp1

+ Treg

cells from mixed chimeric mice were cultured with anti-CD3 and anti-CD28 for 36 h. Cytokine production was measured by bio-plex

multi-cytokine assay (n=6 mice per group). (c) Flow cytometric analysis of Foxp3 (upper) or RORγt (bottom) expression in CD4+

T cells

from Cpdm+/-

and Cpdm-/-

mice upon stimulation with the indicated concentration of anti-CD3 or anti-CD3 and anti-CD28 in the

presence of TGF-β (1ng/ml) and IL-2. (d-e) Cytokine production was measured by bio-plex multi-cytokine assay in sorted memory

CD4+CD44

+CD62L

- T cells (d) or naïve CD4

+CD62L

+CD44

-CD25

- T cells (e) from Cpdm

+/- and Cpdm

-/- mice upon TCR and CD28

stimulation for 36 h. f, Sorted naïve CD4+CD62L

+CD44

-CD25

- T cells from Cpdm

+/- and Cpdm

-/- mice were cultured under TH0, TH1, TH2

and TH17 polarizing conditions for 5 days. IFN-γ, IL-4 or IL-17A producing cells were analyzed by intracellular cytokine staining and flow

cytometry 6 h after restimulation with anti-CD3 and anti-CD28 (n=6 mice per group). The data are compiled or representative of two to

four independent experiments. Error bars indicate the mean (± s.d.). *P < 0.05 **P < 0.01, ***P < 0.001 and ****P < 0.0001 (two-tailed

unpaired t-test).



Phosphorylation status of signaling proteins upon stimulation with various stimuli.

(a) Analysis of the phosphorylation status of signaling proteins in sorted CD4+


and Cpdm-/-

mice upon TCR and

CD28 stimulation. (b) Analysis of the phosphorylation status of signaling proteins in sorted DP thymocytes from Cpdm+/-

and Cpdm-/-

mice upon TCR stimulation. (c) Analysis of the phosphorylation status of signaling proteins in sorted CD4+


and

Cpdm-/-

mice upon TNF stimulation. (d) Splenocytes from Cpdm+/-

and Cpdm-/-

mice were stimulated with PMA and Ionomycin for 10

min. Phosphorylation status of ERK1/2 in CD4+Foxp3

- T or CD4

+Foxp3

+ Treg cells were analyzed by intracellular phosphor-protein

staining. The data are representative of three independent experiments.



Linkage-specific ubiquitination and the mapping of ubiquitination sites on SHARPIN.

(a-c) Jurkat T cells were transiently expressed with the indicated plasmids and analyzed for ubiquitination. (a) HA-Ub WT and

SHARPIN WT or mutants (F354V, K42R, K168/169R and K312R); (b) HA-Ub WT or mutants (K11, K48 and K63) and SHARPIN WT or

mutants (K42R, K168/169R and K312R); and (c) HA-Ub WT or mutants (K11 and K48) and SHARPIN WT or mutants (F354V and

K312R). Cells were stimulated with OKT3 for 5 min, and lysed by adding SDS lysis buffer (2% SDS) and immunoprecipitated with anti-

Myc antibody. The data are representative of three independent experiments.



Knockdown of SHARPIN reduces the generation of Treg cells, but knockdown of HOIP does not.

(a) Analysis of mAmetrine expression in peripheral blood (left) and SHARPIN or HOIP protein expression (right) from BM chimeric mice

8 wks after reconstitution with retrovirus vector expressing control, HOIP or SHARPIN shRNA. (b) Splenocytes from WT mice were

stimulated with concanavalin A, followed by retroviral transduction of retrovirus vector expressing control, HOIP or SHARPIN shRNA.

Four days after transduction, the phosphorylation status of signaling proteins was analyzed in sorted mAmetrine+CD4

+T cells upon TCR

stimulation. (c-d) Flow cytometric analysis of Foxp3 expression in pregated mAmetrine+CD45.2

+CD4

+ T cells in thymus (c) or SP (d) of

the mice in (a) (n=6 mice per group). The data are compiled or representative of three independent experiments with two mice each.

Error bars indicate the mean (± s.d.). *P < 0.05 **P < 0.01, ***P < 0.001 and ****P < 0.0001 (two-tailed unpaired t-test).


Supplementary Table 1


Supplementary Table 2


Nature Immunology: doi:10.1038/ni/-and Cpdm-/-mice were differentially labeled with Cell Trace Far...

Documents

Transcript of Nature Immunology: doi:10.1038/ni/-and Cpdm-/-mice were differentially labeled with Cell Trace Far...