Fig 2: Tet2 CD Tet3 KO iNKT cells show expansion and NKT17 cell lineage skewing. (A) Representative flow cytometry plots evaluating aGalactosylseramide (aGalCer) loaded CD1d tetramer binding and TCRβ expression in thymocytes isolated from control, Tet2/3 DKO and Tet2 CD Tet3 KO mice. The aGalCer-CD1d tetramer+ TCRβ+ cells are iNKT cells. (B) Percentage of iNKT cells (identified as described in (A) in the thymus isolated from control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (C) Number of iNKT cells in the thymus of control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (D) Representative flow cytometry plots evaluating the expression of transcription factors PLZF and RORγt in thymus isolated from control, Tet2/3 DKO and Tet2 CD Tet3 KO mice. PLZFlow RORγt negative cells are NKT1, PLZF high RORγt negative are NKT2 and PLZF intermediate RORγt positive cells are NKT17 cells. (E) Percentage of NKT1 cells, (F) NKT2 and (G) NKT17 cells among total iNKT cells in the thymus isolated from control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (H) Percentage of NKT1 cells, (I) NKT2 cells and (J) NKT17 cells among total thymocytes isolated from control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (K) Number of NKT1, (L) NKT2 and (M) NKT17 cells in the thymus isolated from control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. For B, C, E-M: Each dot represents a mouse. 5 independent experiments were performed. Male and female mice for each genotype were analyzed. Control n=10, Tet2/3 DKO n=6, Tet2 CD Tet3 KO n=7 **** (p < 0.0001),*** (p < 0.001), ** (p < 0.01), * (p < 0.05) unpaired t test. Unless otherwise indicated the difference is not statistically significant. Horizontal lines indicate the mean (s.e.m.).

Fig 3: Tet2 and Tet3 are upregulated during T cell lineage specification and are required for optimal Th-POK expression. (A) Gene expression of Tet2 (left) and Tet3 (right) in DP cells, CD4 SP cells and CD8 cells. (Source: Immunological Genome project, Immgen). (B) MA plot depicting log2 fold-change of gene expression in Tet2/3 DKO CD4 SP cells compared to control (wild type) CD4 SP cells. 561 were identified to be upregulated (padj < 0.01, in green) in Tet2/3 DKO SP cells, whereas 255 were downregulated (padj < 0.01, in red). Genes of interest are indicated. n=3 male control (wild type) and 2 male Tet2/3 DKO mice were used. (C) Venn diagram indicating the intersecting genes that are differentially expressed in Tet2/3 DKO iNKT cells compared to wild type iNKT cells (8) and Tet2/3 DKO CD4 SP cells compared to wild type CD4 SP cells. (D) Heatmap indicating the log2 fold-change of the overlapping genes identified in (C). The scale for evaluating differential gene expression is depicted. (E) Representative histogram comparing the protein expression levels of Th-POK in wild type (gray) and Tet2/3 DKO (purple) CD4 SP cells evaluated by flow cytometry. (F) Median Fluorescence Intensity (MFI) of Th-POK expression in CD4 SP cells as evaluated in (E). Each dot represents a mouse. WT n=10 (in black), Tet2/3 DKO n=6 (in purple). For each genotype male and female mice were analyzed with comparable findings. (G) Representative histogram comparing the protein expression levels of Th-POK in wild type (gray) and Tet2/3 DKO (purple) iNKT cells evaluated by flow cytometry. (H) Median Fluorescence Intensity (MFI) of Th-POK expression in iNKT cells as evaluated in (G). Each dot represents a mouse. WT n=7 (in black), Tet2/3 DKO n=7 (in purple). For each genotype male and female mice were analyzed with comparable findings. 5 independent experiments were performed. **** (p < 0.001), unpaired t test. Horizontal lines indicate the mean (s.e.m.).

Fig 4: Characterization of Tet2 Catalytic Dead (Tet2CD) Tet2CD Tet3flx/flx CD4Cre (Tet2CD Tet3 KO) mice. (A) Depicting the point mutations that result in H1795R catalytic dead TET2 mice. The substitution of histidine (H) with arginine (R) compromises the ability to bind Fe2+ that is critical for the catalytic activity of TET2. (B) Sanger sequencing of genomic DNA indicating the mutations at the TET2 catalytic domain. (C) Gene expression evaluation by real time PCR for Tet1, Tet2 and Tet3 in total thymocytes isolated by control (WT), Tet2 CD Tet3 KO, Tet2 CD and Tet2 KO. Data from one representative experiment are shown. The total number of evaluated mice is indicated. (D) Western blot depicting TET2 expression in nuclear extracts from total thymocytes isolated from control (WT), Tet2/3 DKO, Tet2 CD Tet3 KO, Tet2 KO, Tet2 CD and Tet3 KO. Histone 3 (H3) was used as loading control. Data from one representative experiment are shown. (E) Quantification of western blots showing expression of TET2. The number of mice evaluated for each genotype is shown in parenthesis. Control (WT) n=6, Tet2/3 DKO n=6, Tet2 CD Tet3 KO n=3, Tet2 KO n=3, Tet2 CD n=2 and Tet3 KO n=2. (F) Western blot depicting TET3 expression in nuclear extracts from total thymocytes isolated from control (WT), Tet2/3 DKO, Tet2 CD Tet3 KO, Tet3 KO, Tet2 CD and Tet2 KO. Histone 3 (H3) was used as loading control. (G) Quantification of western blots showing expression of TET3. The number of mice evaluated for each genotype is shown in parenthesis. control (WT) n=6, Tet2/3 DKO n=6, Tet2 CD Tet3 KO n=3, Tet3 KO n=3, Tet2 CD n=4 and Tet2 KO n=3.

Fig 5: Tet2 CD Tet3 KO CD8 SP cells exhibit innate like characteristics. (A) Representative flow cytometry plots evaluating CD4 and CD8 expression in the surface of thymocytes (excluding iNKT cells) isolated from control (wild type), Tet2/3 DKO and Tet2 CD Tet3 KO mice. (B) Percentage of CD8 SP cells (determined as described in A) in the thymus isolated from control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (C) Number of CD8 SP cells in the thymus of control (in black), Tet2/3 DKO (in purple), Tet2 CD Tet3 KO (in cyan) mice. (D) Representative histograms evaluating the expression of the transcription factor EOMES in CD8 SP cells. Gray is indicating control cells, purple Tet2/3 DKO and cyan Tet2 CD Tet3 KO. (E) Median fluorescence intensity (MFI) of EOMES expression in control (in black), Tet2/3 DKO (in purple) and Tet2 CD Tet3 KO (in cyan) CD8 SP cells. (F) Representative histograms evaluating the expression of CD44 in CD8 SP cells. Gray is indicating control cells, purple Tet2/3 DKO and cyan Tet2 CD Tet3 KO. (G) Median fluorescence intensity (MFI) of CD44 expression in control (in black), Tet2/3 DKO (in purple) and Tet2 CD Tet3 KO (in cyan) CD8 SP cells. For (B, C, E, G): Each dot represents a mouse. 4 independent experiments were performed. Male and female mice for each genotype were analyzed. Control n=8, Tet2/3 DKO n = 4, Tet2 CD Tet3 KO n = 6 **** (p < 0.0001),*** (p < 0.001), ns, non-significant, unpaired t test. Horizontal lines indicate the mean (s.e.m.).