Terrence L. Geiger

Peripheral tolerance depends on limiting conventional T cell responses to self-antigens. To define the contribution of nutritional factors and related epigenetic regulation, we perform in vivo CRISPR screening and identify the ascorbate transporter Slc23a2 as a key regulator of naive T cell (Tn) reactivity. T cell–specific loss of Slc23a2 reduces intracellular ascorbate, induces regional DNA hypermethylation, enhances differentiation of Tn cells into effector and memory populations, and promotes low-grade autoimmune inflammation. These phenotypes mirror those caused by ascorbate deprivation, TET dioxygenase deficiency, and aging. Mechanistically, Slc23a2 maintains TET enzymatic activity through ascorbate, dampens proximal TCR signaling, represses helper T cell lineage programs, and sustains Tcf1 expression and chromatin binding in Tn cells. Deletion of Tcf7 partially phenocopies Slc23a2 deficiency, whereas Tcf1 overexpression mitigates the effects. Together, these findings identify ascorbate and TET-dependent DNA demethylation as a checkpoint that restrains Tn cell reactivity to self-antigens, thereby maintaining peripheral tolerance, a process compromised with aging.

Figure S1 shows that GPR65 is a biomarker of B-ALL immunotherapy response

Figure S8 shows cell-cell communication analysis of tumors, macrophages, and monocytes.

Figure S4 shows CAR-T cell function remains unimpaired in GPR65 KO TME.

Figure S2 shows the strategy to generate GPR65 clones.

Table S1: Differential expression and Activity analysis of human (h.CR vs h.NR) and mouse (m.CR vs m.PR) B-ALL tumor samples. The colum is ranked by combined Z-score of human and mouse DE results. Table S2: Functional enrichment analysis upregulated and downregulated genes in m.CR vs m.PR tumors RNA-seq. Table S3: Differential Expression analysis results of CART T cells treated with No tumor, CR tumors or GPR65 KO tumors. Table S4: Differential Expression analysis of GPR65 KO vs m.CR tumor cells from Day 7 Untreated, Day 11 Untreated and Day 11 CAR-T cell treated tumors. Table S5: NetBID2 Hidden driver analysis of 1035 TF comparing CAR T Treated GPR65 KO and CR tumors.

Figure S3 shows that GPR65 KO mediated CAR-T resistance is independent of antigen expression and CAR-T cell expansion.

Figure S7 shows macrophage depletion and CAR-T cell therapy regimen.

Figure S5 is the scRNA-seq experiment design to evaluate tumors and TME.

Figure S6 shows characterization of mouse and human B-ALL TME.