PMC

Glucose and glutamine uptake are regulated during thymocyte β-selection and feed into O-GlcNAcylation. (a) Uptake of ³H-2-deoxyglucose and ¹⁴C-glutamine in thymocytes pooled from 10 wild-type mice sorted into DN3, DN4 and DP subsets. (b) Intracellular flow cytometric analysis of O-GlcNAc and TCRβ was done along with surface markers for the different subsets (n=6 mice). (c) DN3-4 thymocytes were cultured on OP9 or OP9-DL1 in the presence of IL-7 (5 ng/ml) for 2 days. Uptake of ³H-2-deoxyglucose and ¹⁴C-glutamine was measured, as well as (d) intracellular O-GlcNAc levels in either total Thy1⁺ cells on OP9 or OP9-DL1, or (e) cells grown on OP9-DL1 and gated on DP (CD4⁺CD8⁺) and DN (CD4^-CD8^-) thymocytes. ***P < 0.001 (One-way ANOVA). Data are from 1 experiment (a), 2 pooled experiments and representative of 5 similar experiments (b), 1 experiment representative of 3 independent experiments (c,d).

Loss of OGT prevents transformation of PTEN-null thymocytes. Analysis of (a) ³H-2-deoxyglucose uptake and (b) ³H-glutamine uptake in cells isolated from thymi of Pten^fl/flLck-Cre^– and from thymic tumors of Pten^fl/flLck-Cre⁺ mice (n=1). (c) Immunoblot analysis of global O-GlcNAcylation and OGT in equal numbers of thymic lymphoma cells from two representative Pten^fl/flLck-Cre⁺ mice and total thymocytes from two Pten^fl/flLck-Cre^– mice. SMC1 was used as loading control. (d) Kaplan-Meier survival plot comparing kinetics of tumor development in Pten^fl/flLck-Cre⁺ (n=8) mice compared with Pten^fl/flOgt^{fl/fl or fl/Y}Lck-Cre⁺ (n=10) mice. (e) Average thymocytes numbers in old Pten^fl/flOgt^{fl/fl or fl/Y}Lck-Cre⁺ mice (n=8, average age=239 days), as compared to wild-type (WT) mice of similar age (n=3). (f) Representative flow cytometric plots of CD4 and CD8 expression in Thy1⁺ thymocytes derived from Pten^fl/wtOgt^wtLck-Cre⁺ and Pten^fl/flOgt^fl/YLck-Cre⁺ thymi at 6 weeks of age. ***P = 0.000228, ****P < 0.0001 (Log rank (Mantel-Cox) test (d), unpaired t-test (e)). Data are from one experiment, representative of 3 (a-c), cumulative (d,e), and representative of 3 mice (f).

Deletion of OGT at the DP stage of T cell development blocks positive selection. (a) O-GlcNAc flow cytometric analyses in quiescent DPs (TCRβ^loCD69⁻), pre-selected (TCRβ^loCD69^hi), post-selected (TCRβ^hiCD69^hi) thymocytes, and mature CD4SP and CD8SP (TCRβ^hiCD69^loCD24^lo). Populations were electronically gated as depicted in . (b) Quantification of thymic subsets cell numbers in 6- to 12-week old Ogt^wt/wtCd4-Cre⁺ (n=4) and Ogt^fl/flCd4-Cre⁺ mice (n=4). (c) Representative flow cytometry plots of CD4 and CD8 expression on Thy1⁺ thymocytes from Ogt^wt/YCd4-Cre⁺ and Ogt^fl/YCd4-Cre⁺ mice. (d) Total numbers of TCRβ⁺ cells isolated from Ogt^wt/wtCd4-Cre⁺ spleens (n=9) and Ogt^fl/flCd4-Cre⁺ spleens (n=9) are plotted. (e) Immunoblotting analysis of OGT and loading control SMC1 for thymocytes from 3 Ogt^wt/YCd4-Cre⁺ and 3 Ogt^fl/YCd4-Cre⁺ mice. (f) Representative flow cytometry for TCRβ expression and CD69 expression in WT and KO thymocytes (gated on Thy1⁺ living cells). To the right, histogram of TCRβ expression is shown in Ogt^wt/YCd4-Cre⁺ (n=1) and Ogt^fl/YCd4-Cre⁺ (n=3) thymocytes. NS, not significant, ** P=0.00437, ***P < 0.001, **** P < 0.0001 (One-way ANOVA (a), t- tests (b, mean ± s.e.m), Mann-Whitney Rank Sum test (d)). Data are cumulative (a,b,d), representative of 9 mice (c,f), and one experiment (e).

Loss of OGT at the DN stage of T cell development leads to a strong block in T cell development. (a) Total numbers of cells isolated from thymi of Ogt^wt/wtLck-Cre⁺ mice (n=21) and Ogt^{fl/Y or fl/fl}Lck-Cre⁺ mice (n=21) are plotted. (b) Representative flow cytometry of CD4 and CD8 expression on Thy1⁺ thymocytes from Ogt^wtLck-Cre⁺ and Ogt^fl/YLck-Cre⁺ mice from (a). (c) Quantification of thymic subsets in 6- to 12-week old Ogt^wt/wtLck-Cre⁺ (n=9) and Ogt^{fl/Y or fl/fl}Lck-Cre⁺ mice (n=9). (d) Flow cytometric histogram plots showing intracellular O-GlcNAc levels in Ogt^wt/wtLck-Cre⁺ (WT) and Ogt^fl/YLck-Cre⁺ (KO) DN4 and DP subsets. (e) Intracellular flow cytometry for TCRβ (pre-TCR) in DN3 and DN4 subsets from Ogt^wt/wtLck-Cre⁺ and Ogt^fl/YLck-Cre⁺ thymi. (f-i) OGT^wt/wtLck-Cre⁺ and Ogt^fl/YLck-Cre⁺ DN thymocytes were cultured for 6 days on OP9 or OP9-DL1 feeder cells, in the presence or absence of 5 ng/ml IL-7. 3 days after culture on OP9-DL1 (f) or OP9 (g), Ogt^wt/wtLck-Cre⁺ and Ogt^fl/YLck-Cre⁺ thymocytes were stained for CD4 and CD8. (h,i) Proliferation of Ogt^wt/wtLck-Cre⁺ and Ogt^fl/YLck-Cre⁺ DN thymocytes cultured on OP9-DL1 feeders (h) or OP9 (i) in the presence of 5 ng/ml IL-7. NS, not significant (P=0.251), ***P < 0.001, ****P < 0.0001 (unpaired t-test (a), Mann-Whitney Rank Sum test (c)). Data are cumulative (a,c) or representative of >40 mice (b), 3 mice (d), 3 mice (e) and (f-i) are representative of 3 independent experiments.

T cells utilize glucose and glutamine intake for UDP-GlcNAc biosynthesis through a key nutrient-sensing pathway. (a) Uptake of ³H-2-deoxyglucose or ³H-glutamine by naïve lymph node (LN) T cells maintained in IL-7 (IL-7) (n=2 mice) or TCR stimulated with CD3/CD28 antibodies for 24 hours (n=3 mice). (b) Uptake of ³H-2-deoxyglucose or ³H-glutamine by effector CD8⁺ T cells (CTL) or CD4⁺ T helper cells (T_H1) or naïve T cells maintained in IL-7 for 24 hours (IL-7). (c) Glucose and glutamine intake feed into the hexosamine biosynthesis pathway that produces UDP-N-Acetylglucosamine (UDP-GlcNAc), which is the substrate for OGT. Glucosamine bypasses the rate-limiting step of synthesis of glucosamine-6-phosphate, and the requirement for glucose and glutamine to make UDP-GlcNAc. (d-g) UDP-GlcNAc concentrations measured by LC-MS/MS (n=3 mice each). (d) UDP-GlcNAc in naïve (0 h) CD8⁺ T cells, CD3/CD28 stimulated CD8⁺ T cells (24 h) and CTLs. (e) UDP-GlcNAc in naïve (0 h) CD4⁺ T cells, CD3/CD28 stimulated CD4⁺ T cells (24 h) and T_H1 effectors. (f) UDP-GlcNAc concentrations in OT-I T cells either unstimulated (maintained in IL-7, IL-7) or activated for 24 hours with peptide in full medium or medium lacking either glucose (-Glc) or glutamine (-Gln). (g) UDP-GlcNAc concentrations in OT-I cells stimulated in medium supplemented as indicated. *P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA, n=3 mice each). Data are from one experiment representative of 3 (a, mean and s.e.m. of biological replicates), 4 (b, mean and s.e.m. of technical replicates, n=1), and one experiment each (d-g).

c-Myc regulates protein O-GlcNAcylation through glucose and glutamine flux. (a) O-GlcNAc flow cytometric analyses in day 7 CTLs (n=6 mice) cultured for 20 hours in RPMI containing different doses of glucose or glutamine in the medium. O-GlcNAc MFI was normalized to the MFI of CTLs grown in the highest concentration of glucose or glutamine. (b) O-GlcNAc flow cytometric analyses in 6 hours activated OT-I T cells (n=3 mice) cultured in RPMI containing different doses of glucose or glutamine in the medium, normalized as in (a). (c) O-GlcNAc flow cytometric analysis in total live CD8⁺ T cells from LN of Myc^fl/flCd4-Cre^– (n=3) and Myc^fl/flCd4-Cre⁺ (n=3) mice, stimulated with CD3/CD28 antibodies for 18 hours (+), or fixed directly post-isolation (–). (d-g) GFP-myc^KI LN T cells (n=6 mice) were activated for 6–24 hours in either complete medium, or in medium lacking glucose (-Glc) supplemented or not with glucosamine (+GlcN), or lacking glutamine (-Gln) supplemented or not with glucosamine (+GlcN). (d) O-GlcNAc MFI measured by intracellular flow cytometry after 6 hours and normalized (n=6) to unstimulated cells. (e) GFP-myc fluorescence intensity measured after 6 hours and normalized to unstimulated cells. (f) CD69 MFI measured after 6 hours, and (g) CD71 MFI (n=3 mice) measured after 24 hours, were normalized to cells stimulated in full medium. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). Data are pooled from 2 experiments (a, mean ± s.e.m of n=6 (glucose) n=5 (glutamine)) and representative of 2 experiments (a) or one experiment (b, mean ± s.d. of n=3), one experiment (c) or pooled from 2 experiments (d-g, mean ± s.d.).

Protein O-GlcNAcylation regulates c-Myc expression and T cell clonal expansion. (a,b) Total numbers of cells obtained on each day of primary CTLs cultures of splenic T cells from Ogt^wtTamox-Cre⁺ or Ogt^fl/YTamox-Cre⁺ mice. In (a), naïve splenocytes were treated with 4-OHT for 4 hours prior to washing out and activation. In (b) 4-OHT treatment was done on day 5. (c) Cells cultured as in (b) were immunoblotted as indicated. (d) GFP-Myc^KI LN T cells (n=5 mice) were stimulated for 6 hours with CD3/CD28 antibodies in the presence of an OGA or an OGT inhibitor (OGAi, OGTi). GFP-myc MFI normalized to naïve T cells is plotted. (e) sWGA affinity purification (AP) from OT-I CTLs lysates were analyzed by immunoblotting for c-Myc. The cell lysate were either left untreated or treated with CpOGA before the affinity purification and immunoblotting for c-Myc and OGT. Total lysates were probed for total protein O-GlcNAcylation and c-Myc. (f) GFP immune-purification (IP) was done from GFP-Myc^KI CTLs lysate that were either left untreated or CpOGA treated. Immune-purified GFP-Myc was probed with RL2 antibody. The lysates were immunoblotted for total protein O-GlcNAcylation and c-Myc. *P = 0.017, ** P < 0.001 (one-way ANOVA). Data are from one experiment each (a,b, mean ± s.d. n=3 mice for each genotype) and representative of 2 (a) and 5 (b) experiments, data are from 2 pooled experiments (d), and data in (c,e,f) are from one experiment and representative of 2 (c), 2 (e) and 4 (f) experiments.

TCR activation upregulates O-GlcNAcylation in T cells. (a) Immunoblot analysis of O-GlcNAc in naïve (–) and 24 hours CD3/CD28-activated (+) T cells (n=2 mice). (b) Immunoblotting of CTLs lysates either untreated (lanes 1,3,4) or treated with CpOGA (lane 2). Immunoblotting was with the O-GlcNAc antibody (lanes 1,2), or the antibody was pre-incubated with 0.5 M N-Acetylglucosamine (GlcNAc) (lanes 3,4). (c) Intracellular flow cytometric analysis with an O-GlcNAc antibody (RL2) of CTLs and naïve CD8⁺ T cells. (d) Flow cytometric analysis of naïve CD8⁺ T cells with the O-GlcNAc antibody that was either pre-incubated with free N-Acetylglucosamine (+GlcNAc) or not. (e) O-GlcNAc flow cytometric analysis of CTLs treated with CpOGA. (f) Dot plot showing forward scatter (FSC) and O-GlcNAc intensities of cells derived from CTLs (grey) and T_H1 (black) cultures (n=1 mouse). Pearson's correlation=-0.072 (CTL), 0.036 (T_H1). (g) 6 hour activated OT-I T cells were either fixed, permeabilized and analyzed, or live cells were analyzed for O-GlcNAc flow cytometry. (h) Naïve LN T cells (n=3 mice) were stimulated with CD3/CD28 antibodies for 6, 24 or 48 hours, and their O-GlcNAc mean fluorescence intensities (MFI) were normalized as a fractional difference to naïve resting T cells (gated on CD62L^hi, CD44^lo). (i) O-GlcNAc flow cytometry data normalized as in (h) of OT-I CD8⁺ T cells stimulated for 24 hours with OVA-derived peptides. O-GlcNAc MFI of total CD8⁺ T cells was normalized to the MFI of unactivated cells (n=4 mice, pooled from 2 experiments). (j) O-GlcNAc MFI (normalized to naïve cells) of OT-I T cells stimulated with serial dilutions of Q4 peptide for 6 hours (n=3 mice). (k-l) O-GlcNAc flow cytometric analysis in CD69⁺ or CD69^– splenic CD4⁺ (k) and CD8⁺ (l) T cells isolated from mice (n=5) infected i.v. with Listeria. (m) Normalized (to untreated) O-GlcNAc MFI in CTLs treated for the indicated times with an OGA inhibitor (OGAi) or an OGT inhibitor (OGTi). *P < 0.05, **P < 0.01, ***P < 0.001 (h, One-way ANOVA, compared to 0 h time point, k,l, paired t-test). Data are representative of 2 similar experiments (a) 1 experiment (b), 3 experiments (h,i,j, mean ±s.d. shown), experiments shown in c-g are representative of at least 5 experiments, (m) left, mean (±s.d.) of n=5 cultures from 2 experiments shown, right, n=1, representative of 2 experiments.