Figure 4. Mitochondrial Respiration Compensates for Gpi1 Deficiency in the Homeostatic SFB Th17 Cells.

(A) Lactate secretion of in vitro-cultured Olfr2 and Gpi1 KO np/p Th17 cells. Cells were cultured as in Figure 3A. At 96 h, cells were re-plated in fresh RPMI medium with 10% dialyzed FCS, and supernatants were collected 12 h later for lactate quantification by GC-MS.

(B) Seahorse experiment showing the oxygen consumption rate (OCR) of in vitro-cultured Th17 cells at baseline and in response to oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin (R + A). 10 replicates were used for the npTh17 Olfr2 and Gpi1-targeted cells, 7 replicates for the pTh17 Olfr2, and 6 replicates for the pTh17 Gpi1 sets of targeted cells.

(C) ATP linked respiration rate quantified based on experiments in (B). Representative data from three independent experiments are shown.

(D) Relative number of Th17 cells cultured for 120 h in vitro (as in Figure 3A), in the presence or absence of 10 nM antimycin A. Cell number was normalized to control (Olfr2-targeted cell number was set as 100). n = 3; data are representative of two independent experiments.

(E) 7B8 Cas9-naive CD4⁺ T cells were electroporated with a mixture of two guide amplicons (for double KO) before transfer into recipient mice. Left, representative FACS plots showing the frequencies of co-transferred Olfr2⁺Olfr2 control and indicated gene KO CD4⁺ T cells in the SILP of the SFB model at day 15. Right, compilation of cell-number ratios of glycolysis gene KO group to the co-transferred Olfr2⁺Olfr2 control. The ratio was normalized to Olfr2⁺Olfr2/Olfr2⁺Olfr2 co-transfer. Three independent experiments were performed with the same conclusion.

p values were determined using t tests.