Lettershttps://doi.org/10.1038/s41590-018-0265-1

The IL-15–AKT–XBP1s signaling pathway contributes to effector functions and survival in human NK cellsYufeng Wang1,2,9, Yibo Zhang1,9, Ping Yi1,3,9, Wenjuan Dong1,4,9, Ansel P. Nalin   1,5, Jianying Zhang6, Zheng Zhu1,4, Lichao Chen1, Don M. Benson1,2, Bethany L. Mundy-Bosse1,2, Aharon G. Freud1,7, Michael A. Caligiuri4,8 and Jianhua Yu   1,2,4,8*

1The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA. 2Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA. 3Third Affiliated Hospital, Third Military Medical University, Chongqing, China. 4Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA, USA. 5Medical Scientist Training Program, The Ohio State University, Columbus, OH, USA. 6Division of Biostatistics, Department of Information Sciences, City of Hope National Medical Center, Duarte, CA, USA. 7Department of Pathology, The Ohio State University, Columbus, OH, USA. 8Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope National Medical Center, Duarte, CA, USA. 9These authors contributed equally: Yufeng Wang, Yibo Zhang, Ping Yi, Wenjuan Dong. *e-mail: jiayu@coh.org

SUPPLEMENTARY INFORMATION

In the format provided by the authors and unedited.

NATure IMMuNoLoGY | www.nature.com/natureimmunology

Supplementary Figure 1

Quantification of IL-15-induced XBP1s and its correlation with NK cell cytotoxicity.

a, Densitometric quantification of the level of XBP1s protein normalized to the level of -Actin protein, assessed by immunoblotting shown in Fig. 1b. NK cells were treated with IL-2 (100 units/ml) or IL-15 (100 units/ml) for 24 h. Immunoblotting was performed with an anti-XBP1s antibody. Bar graphs display mean ± s.d. of n = 8 donors. ***P < 0.001 by linear mixed model. b, NK cells were treated with IL-15 (100 units/ml) for 24 h and co-cultured with MM.1S multiple myeloma cells for 4 h. CD107a surface expression and intracellular level of XBP1s were determined by flow cytometry using anti-CD107a and anti-XBP1s antibodies. n = 4 donors. ***P < 0.001 by two-tailed paired t test. MFI, mean fluorescence intensity.

Supplementary Figure 2

XBP1s positively regulates the expression of GZMB in primary NK cells.

a,b, NK cells were pre-treated with 1 µM of thapsigargin (Thap) for 1 h and then treated with or without IL-15 (100 units ml–1

) for 4 h followed by qPCR (a) or for 24 h followed by immunoblotting to detect XBP1s or GZMB expression (b). Bar graphs display mean ± s.e.m. of n = 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001 by linear mixed model (a). c, Densitometric quantification of the

level of XBP1s or GZMB protein normalized to the level of -Actin protein in (b). Bar graphs display mean ± s.d. of n = 3 donors. **P < 0.01 by linear mixed model. d, NK cells were transfected with XBP1 or a scramble siRNA by electroporation and then were

cultured for 48 h, followed by immunoblotting with XBP1s, GZMB, and -Actin antibodies. e, Densitometric quantification of the

level of XBP1s or GZMB protein normalized to the level of -Actin protein in (d). Bar graphs display mean ± s.d. of n = 3 donors. **P < 0.01, ***P < 0.001 by Student’s two-tailed unpaired t test. f,g,h, NK cells were pretreated with 50 µM of 4µ8C for 1 h and then treated with or without IL-15 (100 units ml

–1) for 4 h followed by qPCR (f) or for 24 h followed by immunoblotting to detect

XBP1s or GZMB expression (g,h). h, Densitometric quantification of the level of XBP1s or GZMB protein normalized to the level

of -Actin protein in (g). Bar graphs display mean ± s.e.m. (f) or mean ± s.d. (h) of n = 3 donors. **P < 0.01, ***P < 0.001 by linear mixed model. The experiment was repeated independently for three donors with similar results (b,d,g). N.S., no significance. Blot images (b,d,g) were cropped, and the full scans are shown in the supplementary figures.

Supplementary Figure 3

Interaction and co-localization of XBP1s with T-BET.

a, Densitometric quantification of the level of T-BET protein normalized to input (i.e., enrichment) in Fig. 3c. 293T cells were co-transfected with T-BET and FLAG-XBP1u, FLAG-XBP1s, or the control (pCDH) lentiviral vector by lentiviral infection, followed by a 48 h culture. Co-IP was performed with an anti-FLAG antibody. Bar graphs display mean ± s.d. of n = 3 independent experiments. **P < 0.01, ***P < 0.001 by linear mixed model. b, NK-92 cells were transduced with FLAG-XBP1s or the control (pCDH) lentiviral vector by lentiviral infection, followed by a 24 h culture. Immunofluorescent staining was performed with anti-FLAG and anti-T-BET antibodies. The nuclei were stained by DAPI. Yellow indicates overlay of red and green. c, NK-92 (upper panel) or primary NK cells (lower panel) were treated with IL-15 (100 units/ml) for 24 h, followed by immunofluorescent staining with an anti-T-BET antibody that was different from the antibody clone used in (b) and Fig. 3d for data validation. An anti-α-tubulin antibody was included to stain the cytoplasm. The nuclei were stained by DAPI. The experiment was repeated

independently for one (b) or three (c) times with similar results. d, Primary human NK cells were treated with or without 50 M of

48c in the presence of IL-15 (100 units/ml) for 16 h. ChIP was performed with a T-BET antibody or control IgG. Precipitated DNA was then analyzed by PCR. Bar graphs display mean ± s.d. of n = 4 donors. **P < 0.01 by Student’s two-tailed unpaired t test.

Supplementary Figure 4

XBP1s positively regulates GZMB gene promoter activity in a STAT5-independent manner.

a,b, 293T cells were co-transfected with the following four plasmids: 1. STAT5A shRNA, STAT5B shRNA, or scramble shRNA; 2. XBP1s or empty vector pCDH (EV); 3. A pGL3 plasmid containing the GZMB promoter; 4. A pRL-TK plasmid (as a control for data normalization). The transfected cells were cultured for 48 h and then harvested and lysed to determine the expression level of XBP1s or STAT5 protein by immunoblotting (a) or GZMB promoter activity by luciferase reporter assays (b). Bar graphs display mean ± s.d. of n = 4 independent experiments. ***P < 0.001 by Student’s two-tailed unpaired t test. The moderate knockdown effect for STAT5A is likely because the antibody used (#25656, Cell Signaling Technology, Inc.) recognizes both STAT5A and STAT5B. N.S., no significance. The experiment in (a) was repeated independently for three times with similar results. Blot images (a) were cropped, and the full scans are shown in the supplementary figures.

Supplementary Figure 5

AKT signaling regulates the expressions of XBP1s and GZMB.

a, Densitometric quantification of the level of XBP1s protein normalized to the level of -actin protein in Fig. 5c. NK cells were pre-treated with or without 10 µM of AKTi-1/2, a potent isozyme selective Akt1/2 kinase inhibitor, followed by stimulation with IL-15 (100 units ml

–1) for 6 h. Cells were then harvested to make lysates for immunoblot analysis. Bar graphs display mean ± s.d. of n =

3 donors. *P < 0.05 by linear mixed model. b,c, AKT1 or scramble shRNA lentiviral construct (pLKO.1) was transduced by lentiviral infection in primary human NK cells for 24 h, followed by fluorescence-activated cell sorting (FACS) for transduced cells based on GFP expression. GZMB expression was analyzed at the protein level by flow cytometry (b) or at the mRNA level by qPCR (c). Bar graphs display mean ± s.d. in (b) or mean ± s.e.m. in (c) of n = 3 donors. **P < 0.01, ***P < 0.001 by Student’s two-

tailed paired t test. d,e, myrAKT4-129 lentiviral construct or empty vector (EV) was transduced by lentiviral infection in human

primary NK cells for 24 h, followed by FACS-based cell sorting for GFP(+) cells. GZMB expression of the sorted cells was analyzed as described in (b, c). Bar graphs display mean ± s.d. in (d) or mean ± s.e.m. in (e) of n = 3 donors. P = 0.07, **P < 0.01, ***P < 0.001 by Student’s two-tailed paired t test. N.S., no significance.

Supplementary Figure 6

AKT signaling regulates XBP1s at the protein level.

a, Densitometric quantification of the level of XBP1s protein normalized to the level of -actin in Fig. 5d. NK cells were pre-treated with IL-15 (100 units/ml) alone or IL-15 plus 10 µM of AKTi-1/2 for 1 h; then cells were cultured with or without 10 µg/ml of cycloheximide (CHX) for 30 min without washing cells. Cells were then harvested to make lysates for immunoblot analysis. Bar graphs display mean ± s.d. of n = 3 donors. P = 0.08, **P < 0.01 by linear mixed model. b, Densitometric quantification of the level

of XBP1s protein normalized to the level of -actin protein in Fig. 5e. 293T cells were co-transfected with 0.04, 0.2, or 1 µg of

pECE-myrAKT4-129 vector or pECE empty vector (EV) and pCDH-FLAG-tagged XBP1s (FLAG-XBP1s) vector or pCDH control

vector. The transfected cells were cultured for 24 h, followed by detecting XBP1s protein expression by immunoblotting with an anti-XBP1s antibody. Bar graphs display mean ± s.d. of n = 3 independent experiments. P = 0.06, *P < 0.05, **P  < 0.01 by linear

mixed model. c, A plasmid containing myrAKT4-129 with constitutively active AKT or a control plasmid was co-transfected with an

XBP1s plasmid, a pGL3 plasmid containing the GZMB promoter, and a pRL-TK plasmid (as a control for data normalization) into 293T cells. The transfected cells were cultured for 48 h. The promoter activity of the GZMB gene was detected by luciferase

reporter assays and the expression of XBP1s and myrAKT4-129 at the protein levels were detected by immunoblot. Bar graphs

display mean ± s.d. of n = 3 independent experiments. ***P < 0.001 by two-way ANOVA model. The blot experiment was repeated independently for three times with similar results. Blot images (c) were cropped, and the full scans are shown in the

supplementary figures. d, Densitometric quantification of the level of XBP1s protein normalized to the level of -actin protein in

Fig. 5g. 293T cells were co-transfected with a pECE-myrAKT4-129 vector or pECE empty vector (EV) and pCDH-FLAG-tagged

XBP1s (FLAG-XBP1s) vector or pCDH control vector, followed by treatment with 10 µg/ml of CHX for 0, 1, 2 and 4 h in the presence or absence of 10 µM of MG132, a protein degradation inhibitor. Bar graphs display mean ± s.d. of n = 3 independent experiments. **P < 0.01, ***P < 0.001 by linear mixed model. N.S., no significance. Blot images (c) were cropped, and the full scans are shown in Supplementary figures.

Fig. 1b

50 kDa

75 kDa 37 kDa XBP1s

Actin

Fig. 1

Fig. 2b

Fig. 2

25 kDa GZMB

50 kDa XBP1s

37 kDa Actin

Fig. 2e

37 kDa Actin

50 kDa XBP1s

Fig. 2

50 kDa α-Tubulin

75 kDa Lamin B

50 kDa XBP1s

Fig. 3 Fig. 3a

50 kDa 50 kDa

T-BET FLAG-XBP1s

Fig. 3b

T-BET

FLAG-XBP1s

50 kDa

50 kDa

75 kDa STAT5

Fig. 3c

200 bp 300 bp

Fig. 3e

37 kDa Actin

15 kDa C-CASP3

50 kDa XBP1s

Fig. 4 Fig. 4a

50 kDa

37 kDa

15 kDa C-CASP3

Actin

XBP1s

Fig. 4b

37 kDa Actin

15 kDa C-CASP3

50 kDa XBP1s

Fig. 4c

Fig. 5

Actin

XBP1u XBP1s

Fig. 5a

50 kDa AKT

50 kDa α-Tubulin

75 kDa Lamin B

50 kDa p-AKT

50 kDa XBP1s

Fig. 5b

50 kDa XBP1s

50 kDa p-AKT

50 kDa AKT

37 kDa Actin

Fig. 5c

Fig. 5

50 kDa

37 kDa

Actin

XBP1s

Fig. 5d

37 kDa Actin

50 kDa

FLAG-XBP1s (IP)

50 kDa myr-AKT

50 kDa FLAG-XBP1s

Fig. 5e

37 kDa Actin

50 kDa FLAG-XBP1s

50 kDa myrAKT

Fig. 5g

50 kDa FLAG-XBP1s

100 kDa

250 kDa

50 kDa

Ub

Fig. 5h

Ub-XBP1s

Ub

GAPDH 37 kDa

100 kDa

250 kDa

100 kDa

250 kDa

Fig. 5i

Fig. 5

Supplementary Fig. 2

37 kDa Actin

GZMB 25 kDa

50 kDa XBP1s

37 kDa Actin

GZMB

25 kDa

50 kDa XBP1s

Supplementary Fig. 2d

Supplementary Fig. 2b

Supplementary Fig. 2g

37 kDa Actin

25 kDa

GZMB

50 kDa XBP1s

Supplementary Fig. 4

50 kDa XBP1s

37 kDa Actin

75 kDa STAT5

Supplementary Fig. 4a

Supplementary Fig. 6

37 kDa Actin

50 kDa myrAKT

50 kDa XBP1s

Supplementary Fig. 6c