Supplemental Material

Sphingosine 2 restricts T cell immunopathology but permits viral persistence

Caleb J. Studstill1,#, Curtis J. Pritzl1,#, Young-Jin Seo2,#,*, Dae Young Kim3, Chuan Xia1 ,

Jennifer J. Wolf1, Ravi Nistala4, Madhuvanthi Vijayan1, Yong-Bin Cho2, Kyung Won

Kang5, Sang-Myeong Lee5,6, and Bumsuk Hahm1,*

1Departments of Surgery and Molecular Microbiology & Immunology, University of

Missouri-Columbia, Medical Science Building M331, One Hospital Dr. Columbia, MO

65212, USA

2Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea

3Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of

Missouri-Columbia

4Division of Nephrology, Department of Medicine, M754 Center for Precision Medicine,

University of Missouri, One Hospital Dr. Columbia, MO 65211, USA

5Division of Biotechnology, College of Environmental and Bioresources, Jeonbuk National

University, Iksan 54596, Republic of Korea

6College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644,

Republic of Korea

# These authors equally contributed to this work.

*Corresponding Authors Supplemental Methods

Lymphocyte Isolation

Splenocyte suspensions were obtained as described in the methods section of the main text. Splenic dendritic cells (DCs) were obtained by treating spleens with collagenase D

(1U/mL, Roche) for 10 minutes followed by EDTA treatment (1mM) for 5 minutes. For experiments investigating immune cell infiltration into kidneys, tissues were incubated at

37C in RPMI-1640 (Gibco) containing type 1 collagenase (2mg/mL, Alfa Aesar), DNase I

(5U/mL, Thermo Scientific), FBS (10%, Sigma), HEPES (25mM, Gibco), and penicillin/streptomycin (100U/mL, 100μg/mL, Sigma) for 50 minutes followed by the addition of RPMI-1640 containing FBS (2%). Tissues were then forced through nylon mesh, and cells were used in downstream experiments.

Flow Cytometry Antibodies

Antibodies used were specific for murine CD11b (M1/70), CD11c (N418), MHC-I (H-2Kb,

AF6.88.5), MHC-II (I-Ab, AF6-120.1), 4-1BBL (TKS-1), CD40 (HM40-3), CD8α (53-6.7),

CD3 (145-2C11), CD4 (GK1.5, RM4-5, RM4-4), CD90.1 (Thy1.1, OX-7), CD45.1 (A20),

Ly6C (HK1.4), Ly6G (1A8), B220 (RA3-6B2), IL-2 (JES6-5H4), Ki67 (SolA15), IFN

(XMG1.2), Granzyme B (NGZB), TNFα (MP6-XT22), Tim-3 (RMT3-23), CD160 (CNX46-

3), LAG-3 (C9B7W), and PD-1 (RMP1-30) (BD Pharmingen, eBioscience, Tonbo

Biosciences, BioLegend, and Leinco Technologies).

Derivation of BM-DCs

The femurs and tibias of WT mice were harvested and washed with 70% EtOH. As much excess tissue was removed from the bone as possible. The ends of each bone were then clipped, and a 25-gauge needle and syringe were used to wash the inside of each bone with PBS. The collected cells were passed through a 40μm nylon mesh. Cells were then

7 treated with NH4Cl to remove RBCs and plated in cell dishes at a concentration of 1x10 cells/plate. These cells were grown in complete RPMI-1640 (Gibco) supplemented with

FBS (10%, Sigma), penicillin/streptomycin (100U/mL, 100μg/mL, Sigma), and 2- mercaptoethanol (50μM, Sigma), sodium pyruvate (1%, Gibco), HEPEs (1%, Gibco), and non-essential amino acid (1%, Gibco). Finally, the media was supplemented with

20ng/mL (200U/mL) rmGM-CSF (Peprotech). The cells were grown for 10 days, changing media on days 3, 6, and 8.

In vitro T cell proliferation assay

BM-DCs were treated with the TLR7 agonist loxoribine (0.5mM) in the presence of GP61 peptide (1ug/mL). The other process was performed as described in the Methods section of the main text.

Western Blot Analysis

1x105 GP61-specific CD4+ or GP33-specific CD8+ T cells were adoptively transferred to

C57BL/6 mice. At 8 days post LCMV Arm or LCMV Cl 13 infection, CD4+ or CD8+ T cells were obtained using EasySep CD4+ or CD8+ positive selection reagents (Stem Cell

Technologies) according to the manufacturer’s instructions. Splenocytes or positively selected CD4+ and CD8+ T cells were lysed utilizing a sample buffer containing glycerol

(20%), Tris/HCl pH 6.8 (0.125M), SDS (0.016g/mL), bromophenol blue (0.2mg/mL), and

2-mercaptoethanol (0.358M). Lysates were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membrane. SphK2 (ab264042, Abcam), phospho-SphK2

(pSphK2, SP4631 ECM Biosciences or PA5-39812 Invitrogen), and GAPDH (5174, Cell

Signaling) were identified using specific polyclonal antibodies and detected using a rabbit

IgG-specific, HRP-linked secondary antibody (Thermo Fisher Scientific) and chemiluminescent substrate (Thermo Fisher Scientific) or a rabbit IgG-specific, IRDye

800CW antibody (LI-COR) and imaged with the Odyssey Fc Imaging System (LI-COR).

Antibody-Mediated Neutralization

Sphk2-/- mice were infected with 2x106 PFU LCMV Cl 13 and then given 200μg Armenian hamster IgG (PIP, isotype control), anti-TNFα (TN3-19.12) (Leinco Technologies), or anti-

FasL (MFL3) (Bio X Cell) i.p. on 4, 7, and 10 dpi. Survival was monitored subsequently.

Focus-Forming Assay

Vero cells were infected with serum or homogenized kidney supernatants of mice, followed by overlaying with a 1% methylcellulose (Sigma-Aldrich) in DMDM. Forty-eight hours later, monolayer cells were fixed with 4% paraformaldehyde, permeabilized with

1% Triton X-100, and stained with rat anti-LCMV nucleoprotein antibodies (VL4, Bio X Cell). Following incubation with anti-rat IgG-HRP antibodies (Thermo Fisher Scientific), a colorimetric reaction with DAB (3,3´-diaminodbenzidine) solution (Thermo Fisher

Scientific) was performed to visualize LCMV-infected Foci.

A WT

Sphk2-/-

B WT C Sphk2+/- 100 Sphk2-/- 100 95 90 75 WT +/- 85 Sphk2 50 Sphk2-/- 80 75

25 Weight Change Weight

70 Survival Percent 65 0 0 **** 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Days Post Infection Days Post Infection

Supplemental Figure 1. Sphk2-/- mice experience severe morbidity following LCMV Cl 13 infection, and one genetic copy of Sphk2 is enough to alleviate this morbidity. Groups of WT (●) (n = 5), Sphk2+/- (□) (n = 8), and Sphk2-/- (Δ) (n = 7) mice were infected with LCMV Cl 13 at 2 x 106 PFU. (A) Picture represents the morbidity seen in Sphk2-/- mice compared to WT mice 18dpi. Mice exhibit ascites, lethargy, and become hunched due to fluid retention. For 30 days following LCMV Cl 13 infection, the percent weight change (B) and percent survival (C) were compared between the three mice groups. ****p≤0.0001, Mantel-Cox Logrank test. A B C 200 ** 140 ** 50 **

120 /L)

/L) 40

/L) 150 100 mEq

mEq 30

mEq 80 100 60 20 40

50 10 Sodium ( Sodium

Chloride ( Chloride 20 Potassium ( Potassium 0 0 0 WT Sphk2-/- WT Sphk2-/- WT Sphk2-/-

D n.s. E n.s. F n.s. 3.5 200 400 3 350 300 2.5 150 250 2 100 200 1.5

Alanine Alanine 150

1 50 100 Globulin (g/dL) Globulin 0.5 (mg/dL)Glucose 50

0 0 (U/L) aminotransferase 0 WT Sphk2-/- WT Sphk2-/- WT Sphk2-/-

Supplemental Figure 2. Serum metabolite profile for Sphk2-/- mice indicates an electrolyte imbalance. WT (n = 3) and Sphk2-/- (n = 5) mice were infected with 2x106 PFU LCMV Cl 13 and monitored for morbidity. When Sphk2-/- mice showed signs of severe morbidity at 15-17dpi, they were sacrificed along with infected WT mice, and serum was assessed for a complete clinical chemistry profile analysis, including sodium (A), chloride (B), potassium (C), globulin (D), glucose (E), and alanine aminotransferase (ALT) (F) levels. **p≤0.01, n.s. not significant, bidirectional, unpaired Student’s t-test. A B

9 * 6 * +

8 +

5 D8

7 D8

)

) 5 6 5 4 5

3 GP276/C

4 NP396/C

+

+ 

3  2

T cells Tcells (x10 T cells Tcells (x10 2

1 # of IFN of # 1 IFN of # 0 0 WT Sphk2-/- WT Sphk2-/-

Supplemental Figure 3. Virus-specific CD8+ T cell responses have collectively increased in Sphk2-/- mice during LCMV Cl 13 infection. Groups (n = 4-5 mice per group) of WT and Sphk2-/- were infected with LCMV Cl 13 at 2 x 106 PFU. At 7dpi, the numbers of IFN-positive LCMV GP276 (A) and NP396 (B)-specific CD8+ T cells in the spleen were analyzed following re-stimulation with GP276- and NP396-peptides and intracellular staining. *p≤0.05, bidirectional, unpaired Student’s t- test. A B *

5 ** 6 *

T T T T

+ + *

4 + 5 )

5 4 3 CD4

(x10 3 cells

GP61/CD4 2

GP61/ +

+ 2

 cells

1 1 # IFN # % IFN % 0 0 WT Sphk2-/- WT Sphk2-/-

C D ** 10 n.s. 60 n.s.

T T *** + + T T 50

8 +

) 40 6 5

30

(x10 cells

GP33/CD8 4

+ GP33/CD8

 20

+

 cells cells

2 10

IFN % IFN % 0 # 0 WT Sphk2-/- WT Sphk2-/-

Supplemental Figure 4. SphK2 deficiency affects CD4+, but not CD8+ T cell responses to LCMV Arm infection. Groups of WT and Sphk2-/- mice (n = 5 mice per group) were infected with LCMV Arm. At 7dpi IFN+ GP61/CD4+ (Frequency, A; Number, B) and IFN+ GP33/CD8+ (Frequency, C; Number, D) T cells were determined in the spleens of infected mice by intracellular cytokine staining. ***p≤0.001, **p≤0.01, *p≤0.05, n.s. not significant, one-way ANOVA with Tukey’s post-hoc test. the WT Supplemental spleen and Sphk were 2 - / Figure - measured mice (n 5 . = Analysis 4 by mice plaque LCMV (x105 PFU/spleen) 0.5 1.5 2.5 3.5 per 0 1 2 3 of assay group) virus WT . Bidirectional, titers were 0.27 infected in Sphk Sphk2 unpaired with 2 - / - - / - mice LCMV Student’s upon Cl 13 LCMV . t At - test 7 dpi, . Cl 13 virus infection titers in . A B

1 5 5 * 8 * +

* + +

0.8 4 4 + ) * )

3 6

4

CD4

CD8

CD4

CD8

+

+ + 0.6 3 3 + 4

0.4 2 2

Tcells/Live

Tcells/Live T cells Tcells (x10 T cells Tcells (x10 2

0.2 1 1

# GP66 Tet GP66 #

% GP66 Tet GP66 %

# GP33 Tet GP33 # % GP33 Tet GP33 % 0 0 0 0 WT Sphk2-/- WT Sphk2-/- WT Sphk2-/- WT Sphk2-/-

C D

1 7 n.s. 0.5 10 * ) * 3 6 *

0.8 0.4 + 8

cells )

5 + 3 0.6 0.3 6

4 Ly6C

cells/Live

+

Ly6C

cells (x10 cells

+ + 0.4 + 3 0.2 4

2 cells (x10

0.2 0.1 Ly6G # 2 % B220 %

# B220 # 1 % Ly6G % 0 0 0 0 WT Sphk2-/- WT Sphk2-/- WT Sphk2-/- WT Sphk2-/-

E F

1.2 n.s. 3 * 4 n.s. 10 n.s.

)

)

4 4 1 8

3 cells

0.8 2 cells +

+ 6 cells cells (x10

0.6 2 cells (x10 + + 4 0.4 1

1 % CD11b %

0.2 CD11c % 2

# CD11c # # CD11b # 0 0 0 0 WT Sphk2-/- WT Sphk2-/- WT Sphk2-/- WT Sphk2-/-

Supplemental Figure 6. Virus-specific T cells and neutrophils infiltrate the kidneys of Sphk2- /- mice following LCMV infection. (A-F) WT and Sphk2-/- mice (n = 4-5 mice per group) were infected with 2x106 PFU LCMV Cl 13. At 8dpi, mice were sacrificed, and kidneys were analyzed for

immune cell infiltration via flow cytometry. The percent and number of LCMV GP66 (GP66-77) + + + + + tetramer CD4 T cells (A), LCMV GP33 (GP33-41) tetramer CD8 T cells (B), B220 cells (C), neutrophils [Ly6G(1A8)+ Ly6C+ cells] (D), CD11b+ cells (E), and CD11c+ cells (F) are shown. *p≤0.05, n.s. not significant, bidirectional, unpaired Student’s t-test. A B n.s. 40%40 ** 5 35%35 4.5 Tcells 4 30%+ 30

3.5

Tcells

CD8 + 25%+ 25 3

20%20 /CD4 2.5

+ Thy1.1

+ 2

15%α 25 1.5 10%

/TNF 10 +

%CD45.1 1  5%5 0.5

% IFN % 0%0 0 WT1 Sphk22 -/- WT Sphk2-/-

Supplemental Figure 7. Sphk2+/+ CD8+ T cells function better in a Sphk2-/- environment, whereas Sphk2+/+ CD4+ T cells do not expand differentially based on the environment. (A) 1x104 Sphk2+/+ Thy1.1+ GP33/CD8+ T cells were transferred into WT or Sphk2-/- mice (n = 4-5 mice per group). Mice were infected with 2x106 PFU LCMV Cl 13 one day later. 7dpi, the transferred cells were re-stimulated and analyzed for the percentage of IFN/TNFα-double positive CD8+ T cells via flow cytometric analysis. (B) 1x104 Sphk2+/+ CD45.1+ GP61/CD4+ T cells were transferred into WT or Sphk2-/- mice (n = 4 mice per group). Mice were infected and 7dpi, transferred cells were analyzed for the percentage of CD45.1+ cells out of total CD4+ T cells by flow cytometric analysis. **p≤0.01, n.s. not significant, bidirectional, unpaired Student’s t-test. Loxoribine + GP61

Sphk2+/+

Sphk2-/-

CFSE

Supplemental Figure 8. Sphk2-/- CD4+ T cells proliferate at a higher rate than Sphk2+/+ CD4+ T cells when stimulated by DCs that were activated with a TLR7 ligand. Naïve CD4+ T cells were isolated from Sphk2+/+ (upper panel; open histogram) or Sphk2-/- (lower panel; filled histogram) CD45.1+ GP66/CD4+ mice. Bone marrow-derived dendritic cells (BM-DCs) were incubated with 0.5mM loxoribine and 1μg/mL GP61 peptide for 1 hour. CFSE-stained Sphk2+/+ or Sphk2-/- CD4+ T cells were then co-cultured with the treated BM-DCs and harvested 5 days later. CFSE dilution was checked by flow cytometry analysis and compared to the cells measured at day 0 (dotted histogram). Splenocytes CD4+ T cells

Arm Cl 13 Arm Cl 13

pSphK2

SphK2

GAPDH

Supplemental Figure 9. LCMV infections increase the levels of SphK2 and pSphK2 in splenocytes and CD4+ T cells. WT mice were uninfected or infected with LCMV Arm or LCMV Cl 13. Total splenocytes and CD4+ T cells were harvested from mice at 8dpi. The levels of SphK2 and pSphK2 were detected by western blot analysis. The pictured blot is a representative of at least 3 mice per group. Supplemental Figure 10

DNA Binding Regulatory Response to Intracellular TNFα Signaling Regulation of Transcription Factor Region Nucleic Hypoxia (Hall) Endogenous Receptor Signaling via NFκB (Hall) Cell Cycle (GO) Activity (GO) Acid Binding (GO) Stimulus (GO) Pathway (GO)

Chromatin Binding (GO)

RNA Pol II Specific DNA Transcription Factor Mitotic Cell Negative Regulation of Binding Transcription Binding (GO) Cycle (GO) Apoptotic Signaling Factor Binding (GO) Pathways (GO)

T Cell Differentiation (GO)

z score Molecular Transducer Regulation of Cell Surface (GO) Secretion (GO) Immune Effector Leukocyte Mediated Activity (GO) Immune Process (GO) Immunity (GO) Response (GO)

z score

Supplemental Figure 10. Heatmaps for top relevant sets from GSEA. Following differential expression analysis and GSEA (see Fig. 6), heatmaps were generated for significant, representative sets from (GO) and hallmark (Hall) molecular signature databases. The heatmaps depict colors relating to the z scores for increased (positive values, red) or decreased (negative values, blue) normalized expression values of genes from Sphk2+/+ (WT) or Sphk2-/- (KO) CD4+ T cells (n = 3 samples per group). 100 LCMV Cl 13 αTNFα + LCMV Cl 13 80 αFasL + LCMV Cl 13

60

40

Percent Survival Percent 20 n.s. 0 0 5 10 15 20 25 30 35 Days Post Infection

Supplemental Figure 11. Neutralization of TNFα or FasL does not prevent the death of Sphk2-/- mice following LCMV Cl 13 infection. Sphk2-/- mice were infected with 2x106 PFU LCMV Cl 13. On 4, 7, and 10dpi mice were given i.p. 200ug of hamster IgG control Ab (n = 3), anti- TNFα Ab (n = 5), or anti-FasL Ab (n = 5). Survival was monitored for 32 days. n.s. not significant, Mantel-Cox Logrank test. A 0.3 B 0.8 2.0 n.s. + +

α 1.5 α 0.6 Tcells Tcells + + TNF TNF +

+ 1.0 0.4  

0.5

0.2 % IFN % IFN GP61/CD4 GP61/CD4 0.0 0.0 0 0 50 50 100 100 iSphK2 (mg/kg) iSphK2 (mg/kg) C D 0.11 8000 200 0.10 n.s.

6000 150 Tcells Tcells + +

4000 GP33 on 3 100 - CD8 CD8 + 2000 + 50 Tet Tet MFI Tim MFI CD160 on GP33 GP33 MFI on CD160 0 0 0 0 50 50 100 100 iSphK2 (mg/kg) iSphK2 (mg/kg)

E F 0.19 150 20 n.s. 0.09

15 100 T cells T Tcells + +

1 on GP33 GP33 1 on 10 - CD8 50 CD8 + + 5 Tet Tet MFI PD MFI CD160 on GP33 GP33 MFI on CD160 0 0 0 0 50 50 100 100 iSphK2 (mg/kg) iSphK2 (mg/kg)

Supplemental Figure 12. Inhibition of SphK2 regulates virus-specific T cells. (A-F) LCMV Cl 13-infected mice (n = 3-5 mice per group) were treated with solvent (0mg/kg), 50mg/kg, or 100mg/ kg iSphK2. At 40dpi, spleen (A, C-D) and liver (B, E-F) tissues were analyzed by flow cytometry for IFNγ+TNFα+ GP61/CD4+ T cells (A, B) and the mean fluorescent intensity (MFI) of CD160 (C, E), Tim-3 (D), and PD-1 (F) on GP33 Tet+ CD8+ T cells. n.s. not significant, one-way ANOVA with Tukey’s post-hoc test (A-F). A B MFI MFI Uninfected 42 Uninfected 69 LCMV + Solvent 177 LCMV + Solvent 91

LCMV + iSphK2 199 LCMV + iSphK2 82

MHC-I MHC-II

C D MFI MFI Uninfected 36 Uninfected 34 LCMV + Solvent 53 LCMV + Solvent 99

LCMV + iSphK2 53 LCMV + iSphK2 97

CD40 4-1BBL

Supplemental Figure 13. SphK2 deficiency does not regulate DC activation. (A-D) C57BL/6 mice were infected with LCMV Cl 13 (n = 3 mice per group) and given iSphK2 (100mg/kg) or its solvent starting the day of infection for two days. The expression levels of MHC-I (A), MHC-II (B), CD40 (C), and 4-1BBL (D) were measured on splenic CD11c+ cells of mice at 2dpi. CD11c+ DCs from uninfected mice were used as a control. Mean fluorescence intensities (MFIs) are depicted. A B n.s. n.s. n.s. 7 6 n.s. n.s. * ** 6 5

5

pfu/ml) PFU/ml) 10 4

10 4 3

Solvent 3 LCMV (log 2

LCMV LCMV (log iSphK2 1 2 0

0 10 20 30 40 50 Solvent Days Post Infection iSphK2

Supplemental Figure 14. Inhibition of SphK2 promotes clearance of persistent LCMV infection but does not impair LCMV replication in vitro. (A) C57BL/6 mice (n = 5 mice per group) were infected with LCMV Cl 13 and treated daily for 7 days with 100mg/kg iSphK2 or solvent by oral gavage. At 10, 20, 28, and 42dpi, LCMV titers were determined in the serum of infected mice by plaque assay. (B) BHK cells were infected with LCMV Cl 13 (MOI = 0.1) and treated with 25 or 50μM iSphK2 or solvent control for 48 hours. LCMV Cl 13 titers were determined in the supernatants by plaque assay. **p≤0.01, *p≤0.05, n.s. not significant, bidirectional, unpaired Student’s t-test (A), one-way ANOVA with Tukey’s post-hoc test (B).