Mouse Hobit is an NKT cell-specific homologue of Blimp-1 that regulates effector differentiation

Klaas P.J.M. van Gisbergen, Natasja A.M. Kragten, Kirsten M.L. Hertoghs, Felix M. Wensveen, Stipan Jonjic,

Jörg Hamann, Martijn A. Nolte, Rene A.W. van Lier.

Supplementary Information

Nature Immunology doi:10.1038/ni.2393

ATG

Hobit gene

stop

exon

1 2 3 4 5

1 kb

a

b c

d e

0

2

4

6

8

PLZ

F m

RN

A (1

02 A

U)

γδ T

δ6.3+ NKT 0

2

4

6

8

10

12

PLZ

F m

RN

A (1

02 A

U)

Total NKT Rest

CD4+ Tem cells

0

10

20

30

40

PLZ

F m

RN

A (A

U)

EM Naive CM Naive CM EM CD8

B NK Myeloid Treg γδ T

ND ND

CD4

δ6.3–

Supplementary Figure 1: Hobit is related to Blimp-1 and shares its expression pattern with PLZF. (a) Schematic representation is shown of the Hobit gene. (b) The neighbor-joining tree shows the phylogenetic relation between the Hobit, Blimp-1, AIM1, and AIM1L amino acid sequences. The scale bar indicates branch length corresponding to 0.1 changes per site. hs, human; mm, mouse; cf, dog; oa, elephant; me, wallaby; md, opossum; oa, platypus; gg, chicken; xt, xenopus; dr, zebrafish. (c-e) Quantitative PCR analyses of PLZF are shown for (c) the indicated subsets of splenocytes, (d) total, NKT enriched and NKT depleted fractions of CD4+ Tem cells and (e) TCR δ6.3- and TCR δ6.3+ γδ T cells and tetramer+ NKT cells. ND denotes non-detectable.

a b –/– 1 –/– 2 WT 2 WT 1

Hobit

Blimp-1

0

4

8

12

Naive CM EM

CD

4+ T

cel

ls (✕

106

)

WT Hobit–/–

0

2

4

6

Naive CM EM

CD

8+ T

cel

ls (✕

106

)

WT Hobit–/–

c

d 0

10

20

30

40

50

60

70

DN DP CD4 SP CD8 SP

Thym

ocyt

es (✕

106

)

WT Hobit–/–

0.0

0.2

0.4

0.6

0.8

1.0

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (✕

106

)

WT Hobit–/–

e

0

0.1

0.2

0.3

0.4

CD27+

CD11b– CD27+

CD11b+ CD27–

CD11b+

NK

cel

ls (✕

106

)

WT Hobit–/–

ATG

Hobit gene

stop

trapping cassette

exon

1 2 3 4 5

1 kb

57.7 0.7

0.1 41.6

18.6 0.3

0.1 81.0

15.1 3.0

1.5 80.5

9.7 8.3

12.9 69.1

45.0 12.0

0.5 42.5

44.1 12.9

4.6 38.4

Ly4

9C/I

WT

Hobit–/–

Ly49A Ly49G2 Ly49H

f g 4.2 32.1

21.1 42.6

13.6 28.4

12.2 45.8

0.7 0.0

0.1 99.3

0.4 0.0

0.0 99.6 IL-4

IFN-γ

Med PMA + iono

WT

Hobit–/–

Supplementary Figure 2: Hobit does not regulate conventional T cells and NK cells, but controls thymic NKT cells. (a) Schematic representation of the Hobit gene is shown and the site that had been targeted by the gene trapping construct is depicted. (b) Hobit and Blimp-1 transcript levels were determined of wild-type and Hobit-/- mice by a Hobit and Blimp-1 specific PCR on cDNA of total splenocytes. (c) Thymocyte differentiation was analyzed in wild-type and Hobit-/- mice using staining for CD4 and CD8 to enumerate double negative (DN), CD4 single positive (SP) CD8 SP and double positive (DP) thymocytes (left panel). DN thymocytes were further analyzed using staining for CD25 and CD44 to separate stage 1-4 (right panel). (d) Effector differentiation of conventional CD4+ (left panel) and CD8+ (right panel) T cells was analyzed in spleen of WT and Hobit-/- mice using staining for CD44 and CD62L that separate CD44loCD62Lhi naive, CD44hiCD62Lhi central memory (CM) and CD44hiCD62Llo effector memory (EM) T cells. (e) NK cell differentiation was analyzed in spleen of wild-type and Hobit-/- mice using staining for CD27 and CD11b. (f) The production of IL-4 and IFN-γ is displayed for representative examples of wild-type and Hobit-/- thymic NKT cell populations with or without PMA and ionomycin stimulation. (g) Dotplots display representative expression of Ly49C/I together with Ly49A (left panels), Ly49G2 (center panels) or Ly49H expression (right panels) on thymic NKT cells of wild-type and Hobit-/- mice.

a 3.0 0

0 97.1

93.4 0.8

0 5.8

2.1 95.3

2.5 0.1

2.6 0

0.2 97.3

93.2 1.1

0 5.8

0.1 97.9

1.7 0.3 NK

1.1

WT

Hobit–/–

Ly49C/I

NK1.1+Ly49C/I– NK1.1– NK1.1+Ly49C/I+

Supplementary Figure 3: Ly49C/I+ NKT cells require Hobit to develop from NK1.1lo and NK1.1hi precursors. (a) Thymocytes were enriched for NKT cells through depletion of CD8+ and CD24+ cells and the remaining cells were divided into NK1.1lo, NK1.1hiLy49C/I- and NK1.1hiLy49C/I- fractions. Expression of NK1.1 and Ly49C/I on NKT cells within the indicated fractions of wild-type and Hobit-/- mice is displayed directly after cell sorting.

b

c d

0

20

40

60

80

100

Spleen Liver

NK

1.1+

NK

T ce

lls (%

)

WT Hobit–/–

**

**

WT Hobit–/–

NK1.1

Spleen Liver

0

0.02

0.04

0.06

Total NK1.1– NK1.1+

NK

T ce

lls (✕

106

)

WT Hobit–/–

mLN

**

**

0

0.01

0.02

0.03

Total NK1.1– NK1.1+

NK

T ce

lls (✕

106

)

WT Hobit–/–

pLN

** **

a

e f 1.1 0.0

0.0 98.9

32.3 1.3

0.7 65.8

1.3 0.0

0.3 98.4

10.3 0.2

0.2 89.4 NK

1.1

WT

Hobit–/–

Ly49C/I

NK1.1+Ly49C/I– NK1.1–

NK

1.1

WT

Hobit–/–

Ly49C/I

NK1.1+Ly49C/I– NK1.1–

0.9

0.3 10.3

88.5

0.0

0.1 9.8

90.2

0.0

0.5 84.1

15.3

0.0

0.2 86.8

12.9

g

0

200

400

600

800

1,000

1,200 WT Hobit–/–

NK1.1– NK1.1+ NK1.1+

Ly49C/I– Ly49C/I+

NK

T ce

lls (%

inpu

t)

Input: NK1.1–

0

20

40

60

80

100

120

NK

T ce

lls (%

inpu

t)

*

WT Hobit–/–

NK1.1– NK1.1+ NK1.1+

Ly49C/I– Ly49C/I+

Input: NK1.1+Ly49C/I–

h 27.5 5.3

8.5 58.7

2.7

NK1.1–

6.2 41.9

44.3 7.6 Ly5

.1

37.1

NK1.1+

CD

3

Tetramer NK1.1

NK

T ce

lls (%

of m

ax)

Supplementary Figure 4: Peripheral differentiation of NKT cells depends on Hobit. (a,b) The absolute number of total, NK1.1lo and NK1.1hi NKT cells was determined in (a) peripheral (pLN) and (b) mesenteric LN (mLN) of wild-type and Hobit-/- mice. (c) Representative histograms display expression of NK1.1 on wild-type and Hobit-/- NKT cells of spleen and liver. (d) Quantification of the percentage of wild-type and Hobit-/- NKT cells that express NK1.1 in spleen and liver. (e-g) Dotplots display expression of NK1.1 and Ly49C/I on the indicated NKT enriched splenocyte fractions at (e) day 0 and (f) day 4 of culture on OP9-Delta-Like-1 cells in the presence of IL-7 and IL-15. (g) The differentiation potential of splenic NK1.1lo (left panel) and NK1.1hiLy49C/I- (right panel) was analyzed and the NK1.1lo, NK1.1hiLy49C/I- and NK1.1hiLy49C/I+ NKT cells that had developed after 4 days of culture were represented as a percentage of input NKT cells. (h) Dotplots display staining for CD3 and tetramer on NK1.1lo and NK1.1hi NKT enriched splenocyte fractions (left panels) and expression of Ly5.1 and NK1.1 on NKT cells within these fractions (right panels) before adoptive transfer into recipients. Ly5.1 expression separates Ly5.1+ wild-type and Ly5.2+ Hobit-/- donor NKT cells.

a

0

0.2

0.4

0.6

0.8

1

NK

T ce

lls (✕

106

)

WT Hobit–/–

Rest Div Rest Div NK1.1– NK1.1+

*

b

3.7 2.7

6.0 2.6 T

etra

mer

BrdU

NK1.1– NK1.1+

WT

Hobit–/–

c

0

1

2

3

4

5

NK1.1- NK1.1+

WT Hobit–/–

NK

T ce

lls (✕

106

)

*

*

NK1.1– NK1.1+

Supplementary Figure 5: Hobit does not impair proliferation of peripheral NKT cells. (a) Representative dotplots show tetramer binding and BrdU incorporation in NK1.1lo and NK1.1hi fractions of wild-type and Hobit-/- NKT cells of spleen. (b) Using nuclear staining for Ki67, absolute numbers of resting (rest, Ki67-) and dividing (div, Ki67+) NKT cell subsets of wild-type and Hobit-/- spleen were determined. (c) The numbers of NK1.1lo and NK1.1hi NKT cells were quantified in the liver of wild-type and Hobit-/- mice after 3 days of antigenic stimulation with α-GalCer.

a b

1

10

100

1000

10000

100000

Ctrl α-GalCer

IFN

-γ m

RN

A (A

U)

*

WT Hobit–/–

c

0

5

10

15

IL-4 IFN-γ

Cyt

okin

e+ N

KT

cells

(%)

WT Hobit–/–

*

4.6

7.1 72.5

15.9 9.2

34.4 47.2

9.2

16.4

12.6 48.0

22.9 32.3

28.7 22.4

16.6 NK

1.1

IFN-γ

Med PMA + iono

WT

Hobit–/–

h

e d

0

5

10

15

20

Ctrl α-GalCer

CD

69 (

geo

MFI

✕ 1

02)

WT Hobit–/–

0

1

2

3

4

Cyt

okin

e (g

eo M

FI ✕

104

) WT Hobit–/–

Total NK1.1– NK1.1+ IL-4 IFN-γ

**

**

**

f

g 393 598

596 362

1417 2173

2430 1397

CD

3

Tetramer

α-GalCer Ctrl

WT

Hobit–/– 0

5

10

15

Ctrl α-GalCer

WT Hobit–/–

IFN

-γ+

NK

cel

ls (%

)

T-bet GATA3 0

20

40

60

80

100

0

1

2

3

4 WT Hobit–/–

T-be

t / G

ATA

3 (A

U)

Supplementary Figure 6: Hobit modulates IFN-γ production after antigen-dependent activation of NKT cells. (a) Representative dotplots display expression of NK1.1 and intracellular production of IFN-γ of wild-type and Hobit-/- NKT cells with and without brief stimulation using PMA and ionomycin. (b) The percentage of IL-4 and IFN-γ producing NKT cells was quantified under steady state conditions in the liver after brief in vitro culture in the presence of Brefeldin A. (c) The expression of IFN-γ at the mRNA level was determined in NKT cells isolated from wild-type and Hobit-/- mice that were non-immunized (ctrl) or that were immunized with α-GalCer 2 hrs previously. (d) The expression of CD69 was determined on liver NKT cells of wild-type and Hobit-/- mice that had not been immunized (ctrl) or that had been immunized with 2 µg of α-GalCer. (e) Preparations of liver lymphocytes of wild-type and Hobit-/- mice were stimulated with PMA and ionomycin during 4 hrs and the production of IL-4 and IFN-γ was analyzed using intracellular cytokine staining. The mean fluorescence intensity of expression of IL-4 or IFN-γ was examined in IL-4 or IFN-γ producing total, NK1.1lo and NK1.1hi NKT cells derived of liver. (f) The expression of T-bet (left panel) and GATA3 (right panel) was examined in liver-derived NKT cells of wild-type and Hobit-/- mice using quantitative PCR. (g) The production of IFN-γ was determined in NK cells of wild-type and Hobit-/- mice using intracellular staining at steady state (ctrl) and after 2 hr stimulation with α-GalCer in vivo. (h) Dotplots display expression of CD3 and binding of tetramer on liver lymphocytes of wild-type and Hobit-/- mice that were not immunized (ctrl) or that were immunized for 2 hrs with α-GalCer. Insets represent geo MFI of CD3 expression (top) and tetramer binding (bottom) on NKT cells.

PMA + iono

NK cells

0

10

20

30

Ctrl

WT Hobit–/–

IFN

-γ+

NK

cel

ls (%

)

poly(I:C)

*

d

f

poly(I:C)

NK cells

0

20

40

60

80

Ctrl

WT Hobit–/–

GzB

+ N

K c

ells

(%)

*

PMA + iono

*

0

200

400

600

800

WT Hobit–/–

GzB

mR

NA

(AU

) *

a

e

0

10

20

30

Med poly(I:C)

GzB

+ N

KT

cells

(%)

WT Hobit–/–

**

**

b c

GzB

8.0

Med

28.4

IL-15

31.7

IFN-β

23.5

poly(I:C)

7.6

poly(I:C) + anti-IFNR

23.6

poly(I:C) + anti-CD122

20.5

poly(I:C) + anti-CD1d

Tet

ram

er

0

10

20

30

40

50

Ctrl poly(I:C) PMA + iono Ctrl poly(I:C) PMA + iono

NK1.1– NK1.1+

GzB

+ N

KT

cells

(%)

*

* WT Hobit–/–

g

0

20

40

60

80

Ctrl PMA + iono

IFN

-γ+

NK

T ce

lls (%

)

WT Hobit–/–

poly(I:C)

*

NKT cells

NKT cells

Supplementary Figure 7: NKT but not NK cells produce granzyme B in a Hobit dependent manner. (a) Expression of granzyme B (GzB) was analyzed at the mRNA level using quantitative PCR within mature thymic NKT cells of wild-type and Hobit-/- mice. (b) Granzyme B expression was analyzed in wild-type and Hobit-/- splenocytes after culture with medium (med) and poly(I:C) for 24 hrs. (c) Representative dotplots display binding of tetramer and intracellular expression of granzyme B in wild-type NKT cells of spleen after 24 hr stimulation with medium (med), IL-15, IFN-β, and poly(I:C) in the absence or presence of the indicated blocking antibodies. (d) The production of IFN-γ was analyzed in liver NKT cells of wild-type and Hobit-/- mice without stimulation (ctrl), after stimulation in vitro with PMA and ionomycin for 4 hrs or after stimulation in vivo with poly(I:C) for 24 hrs. (e,f) The production of (e) IFN-γ and (f) granzyme B was analyzed in liver NK cells of wild-type and Hobit-/- mice without stimulation (ctrl), after stimulation in vitro with PMA and ionomycin for 4 hrs or after stimulation in vivo with poly(I:C) for 24 hrs. (g) Granzyme B expression was analyzed in NK1.1lo and NK1.1hi subsets of liver NKT cells of wild-type and Hobit-/- mice without stimulation (ctrl), after stimulation in vitro with PMA and ionomycin for 4 hrs or after stimulation in vivo with poly(I:C) for 24 hrs.

a

1

10

100

1000

10000

100000

1000000

WT Hobit–/–

Spleen Liver

Vira

l loa

ds (P

FU/o

rgan

)

Supplementary Figure 8: Hobit does not have an impact on viral load after infection with mCMV. (a) The viral load was determined by plaque assay in spleen and liver of wild-type and Hobit-/- mice that had been infected with mCMV Δ m157 for 3 days.