Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8+ T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness

Abstract

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8⁺ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8⁺ T cell death. Protective CD8⁺ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.

Keywords: CD8+ T cells; ER stress; HSAN-I; SPTLC2; neurological diseases.

Figure 1.. The SPTLC2 mutations affect HSAN-I patient CD8⁺ T cell effector cytokine production, proliferation, and survival.

(A) FACS dot plots and bar graphs show the percentages of CD4⁺ and CD8⁺ T cells in PBMCs from HSAN-I patients and healthy subjects. (B) PBMCs from HSAN-I patients and healthy subjects were stimulated with PMA and ionomycin for 6 hours in the presence of brefeldin A before FACS analysis of IFNγ- and TNFα-producing CD8⁺ T cells. (C-D) PBMCs from HSAN-I patients and healthy subjects were labeled with Celltrace Violet (CTV) and stimulated with anti-CD2, anti-CD3 and anti-CD28 for 3 days before FACS analysis. FACS plots and bar graphs show the percentages of proliferating (C) and apoptotic (D) CD8⁺ T cells. Data are expressed as mean ± SD and cumulative of three independent experiments (ten pairs of samples for each experiment described in A-D). **p<0.01; n.s., not significant. See also Figure S1.

Figure 2.. Sptlc2-deficiency impairs antiviral effector CD8⁺ T cell formation.

(A) Antigen-specific P14 CD8⁺ T cells were purified on day 6 (D6) after LCMV-Armstrong infection from the “P14 chimeric mice”. Naïve P14 T cells were also purified before LCMV infection (D0) to analyze the basal level of SPTLC2 protein by western blot. Sptlc2-deficient P14 CD8⁺ T cells were included as negative controls. Bar graphs show the densitometry quantification of the SPTLC2 immunoblot bands. GRP94 was used as a loading control. (B) P14 TCR transgenic mouse splenocytes were stimulated with GP_33–41 peptide or various cytokines as indicated for 3 days for immunoblot. Each lane represents an individual mouse sample (A-B). (C-F) Sptlc2^Flox/FloxCd4-cre (Fl/Fl) mice and wildtype littermates (+/+) were infected with LCMV-Armstrong and sacrificed 8 days later. Representative FACS plots and bar graphs (C-D) show the percentages and numbers of D^bGP_33–41 and D^bNP_396–404 tetramer-positive splenic Sptlc2-deficient or wildtype CD8⁺ T cells (C), the effector cytokine-producing CD8⁺ T cells after restimulation with or without the LCMV peptide _GP33–41 for 6 hours (D) and viral titers in mouse serum (E) and spleens (F). Data are expressed as mean ± SD (error bars) and are representative of two (six (A) or three (B) pairs of mice in total) or three (C-F, eight pairs of mice in total) independent experiments (three in each experiment). *p<0.05; **p<0.01. See also Figure S2.

Figure 3.. CD8⁺ T cells require cell-intrinsic SPTLC2 expression for robust effector T cell responses.

(A-D) Congenically mismatched Sptlc2^Flox/FloxCd4-cre and Sptlc2^+/+Cd4-cre P14 CD8⁺ T cells (10⁴ cells each) were mixed and adoptively transferred into B6 recipient mice, which were subsequently infected with LCMV-Armstrong (A). The percentages (B-C) and numbers (D, day 8) of Sptlc2-sufficient (WT) and -deficient donor CD8⁺ T cells are shown. (E-H) FACS plots and bar graphs show the percentages or numbers of apoptotic CD8⁺ T cells (E and F), SLECs and MPECs (E and G), and cytokine-producing CD8⁺ T cells (E and H). Data (C, D, F-H) are expressed as mean ± SD (error bars) and cumulative of three independent experiments (six to eight mice in total). *p<0.05; **p<0.01; n.s., not significant.

Figure 4.. Sptlc2-deficiency causes aberrant mTORC1 activation and ER stress in antiviral CD8⁺ T cells.

(A) Volcano plots show the distribution of significantly up- and down-regulated genes in the Sptlc2-deficient CD8⁺ T cells before and 8 days after LCMV-Armstrong infection. (B) A heat map shows the z-score of the mRNA levels of the indicated genes in CD8⁺ T cells purified from three pairs of Sptlc2^Flox/FloxCd4-cre (Fl/Fl) mice and wildtype littermates (+/+) at day 8 after LCMV-Armstrong infection. “Effector” block contains hallmark genes of effector CD8⁺ T cells. Genes regulating the sphingoid base salvage pathways and ER-stress are shown in the “SB” and “ER stress” blocks. Sptlc2 is included as a control. (C) FACS plots show the percentages of phosphorylated S6 (pS6) in Sptlc2-sufficient (+/+) and -deficient (Fl/Fl) CD8⁺ T cells after in vitro stimulation with anti-CD3 and anti-CD28. pS6 MFI is expressed as mean ± SD (error bars) and cumulative of three independent experiments (one pair of mice in each experiment). (D) The ER stress marker expression in CD8⁺ T cells of Sptlc2^Flox/FloxCd4-cre (Fl/Fl) and Sptlc2^+/+Cd4-cre (+/+) littermate mice infected with LCMV-Armstrong 8 days earlier was analyzed by western blot. Rapamycin was administered every other day (100 μg/kg body weight) as indicated. T cells of three mice in each group were collected separately from two independent infection experiments and analyzed together. Each lane represents an individual sample. Bar graphs show the densitometry quantification of the immunoblot bands. Data are expressed as mean ± SD (error bars). *p<0.05; **p<0.01. See also Figure S3.

Figure 5.. Sptlc2-deficiency reduces sphingolipid biosynthetic flux and prolongs mTORC1 activation through protein phosphatases in CD8⁺ T cells.

(A) Sptlc2^Flox/FloxCd4-cre (Fl/Fl) or Sptlc2^+/+Cd4-cre (+/+) P14 CD8⁺ T cells were cultured with the cognate peptide GP_33–41 for 3 days. The primed CD8⁺ T cells were then expanded using IL-2 for another 2 days before lipid extraction and mass spectrometry analysis of sphingolipids. The heat map shows the z-score of the indicated metabolites. (B) The sphingolipid de novo synthetic pathway is depicted with tracers highlighted in red. (C) P14 CD8⁺ T cells as described in (A) were expanded using IL-2. Then T cells were washed and cultured in HBSS plus 1% FBS in the presence of isotope-labeled L-serine for 0~120 minutes for mass spectrometry analysis of the tracer incorporation into various sphingolipids. (D) Sptlc2^Flox/FloxCd4-cre (Fl/Fl) or Sptlc2^+/+Cd4-cre (+/+) CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 for 3 days in the presence or absence of sphingolipids (5 μM 3-KDS, 5 μM sphinganine, 50 nM C16 ceramide, 50 nM C18 dihydroceramide, 4 μg/ml palmitoyl sphingomyelin, and 1 μM sphingosine) or microcystin-LR (it inhibits both PP1 and PP2A at 25 nM) as indicated before FACS analysis of pS6. Data (A and C, two pairs of mice in each experiment) are cumulative of two independent experiments or representative (D, one pair of mice in each experiment) of three experiments. Data are expressed as mean ± SD (error bars) (C). *p<0.05; **p<0.01. See also Figure S4, S5 and S6.

Figure 6.. Sphingolipid supplementation and inhibition of ER stress-induced cell death partially restore Sptlc2-deficient antiviral T cell survival and expansion.

(A-B) Sptlc2^Flox/FloxCd4-cre (Fl/Fl) or Sptlc2^+/+Cd4-cre (+/+) CD8⁺ T cells were labeled with (B) or without (A) CTV and stimulated with anti-CD3 and anti-CD28 for 3 days in the presence or absence of 3-keto-sphinganine (3-KDS, 5 μM), sphinganine (Spha, 5 μM), sphingomyelin (Sphm, 4 μg/ml), rapamycin (Rapa, 10 nM), and salubrinal (Salu, 5 μM) before FACS analysis of apoptosis (A) and proliferation (B). (C-E) Sptlc2^Flox/FloxCd4-cre (Fl/Fl) and wildtype P14 CD8⁺ T cells from littermates were mixed (1×10⁴ cells from either group) and adoptively transferred into C57BL/6 mice, which were infected with LCMV and sacrificed 8 days later. Sphinganine (100 μg/kg), rapamycin (100 μg/kg), and salubrinal (1 mg/kg) were peritoneally injected every other day (day 0~8 after LCMV infection). Bar graphs show the ratio (D) and total numbers (E) of Sptlc2-deficient and -sufficient donor CD8⁺ splenic T cells at day 8 after LCMV infection. (F) FACS plots and bar graphs show the percentages or numbers of apoptotic CD8⁺ T cells, SLECs and MPECs, and cytokine-producing CD8⁺ T cells. Data are representative (A-B, three pairs of mice in total) and cumulative (D-F, six pairs of mice in total) of three independent experiments. Data are expressed as mean ± SD (error bars) (D-F). *p<0.05; **p<0.01; n.s., not significant. See also Figure S4 and S5.

Figure 7.. Supplementing with sphinganine and inhibiting ER stress-induced cell death corrects the immunodeficiency in SPTLC2-mutated HSAN-I patient PBMCs.

(A) PBMCs from HSAN-I patients and healthy subjects were stimulated with PMA and ionomycin for 6 hours in the presence or absence of the indicated compounds (upper panel). FACS plots show the production of IFNγ and TNFα by CD8⁺ T cells. Alternatively, PBMCs were labeled with CTV (middle panel only) and stimulated with anti-CD2, anti-CD3 and anti-CD28 for 3 days (both middle and lower panels). FACS plots show the percentages of proliferating (middle panel) and apoptotic (lower panel) CD8⁺ T cells. (B) Bar graphs show the cumulative results obtained from ten pairs of samples. Data are expressed as mean ± SD. **p<0.01. See also Figure S7.