Fig 2: DNA-PKcs inhibition reduces LAT localization at the immune synapse(A and B) (A) LSCM imaging at 63× magnification and (B) histogram MFI analysis of 2-min αCD3/CD28 TCR-stimulated Jurkat T cells identify areas of colocalization where pDNA-PKcs and LAT peaks overlap (*) and areas where peaks do not overlap (arrow).(C) LSCM imaging at 63× magnification of SEE-pulsed Raji B cells (blue) cocultured and conjugated with E6.1 Jurkat T cells shows pDNA-PKcs (green) colocalized with LAT (red) at the immune synapse (arrow).(D) LSCM immune synapses were quantified by MFI of pDNA-PKcs and LAT by drawing a quantifying line along the interface of Raji B cell and Jurkat T cell, each dot representing an area of immune synapse. Data are represented as mean ± SEM.(E) Co-immunoprecipitation (coIP) in Jurkat T cells shows that DNA-PKcs interacts with LAT following TCR stimulation.(F) TCR stimulation increases LAT pull-down by DNA-PKcs 7.5-fold, which is reduced by DNA-PKcs inhibitor NU7441 to 2.5-fold when quantified on ImageJ with one dot representing one coIP experiment. Data are represented as mean ± SEM.(G) LSCM imaging at 63× magnification of E6.1 Jurkat T cells demonstrates LAT (red) localization at the plasma membrane with pDNA-PKcs (green) upon TCR stimulation, which decreases with NU7441 (5 μM). Arrows identify areas of colocalization.(H) LSCM quantification reveals significant increases in MFI for pDNA-PKcs and LAT after TCR stimulation, along with colocalization events, which decrease upon DNA-PKcs inhibition with NU7441. Each dot represents a single quantified cell. Data are represented as mean ± SEM.(I and J) shRNA-mediated knockdown of DNA-PKcs (>70% reduction) reduces total DNA-PKcs expression on western blot. Data are represented as mean ± SEM.(K) LSCM imaging at 63× magnification shows that shRNA inhibition of DNA-PKcs attenuates LAT (red) localization at the plasma membrane after 2 min of 5 μg/mL αCD3/CD28 TCR stimulation.(L) Quantification of LSCM with each dot representing one shRNA-transfected cell. Data are represented as mean ± SEM.Scale bars, 5 μm. Representative images and western blots from n = 3 independent experiments with all cells quantified within the field of view on ICC. Statistical significance determined using one-factor ANOVA plus Tukey’s multiple comparisons (α = 0.05, **p < 0.01, **p < 0.005, and ****p < 0.0001).

Fig 3: Loss of phosphorylation at S224 and S241 prevents LAT tyrosine phosphorylation, binding to the signalosome, and cytokine production(A) JLAT transductants with DNA-PKcs phosphorylation sites 224 or 241 mutated, stimulated with 5 μg/mL αCD3/CD28 for 24 h, are unable to produce IL-2, as assessed by ELISA. Data are represented as mean ± SEM.(B) Western blot analysis of ZAP70 tyrosine phosphorylation events on LAT (Y132, 171, and 191) in resting or 2-min αCD3/CD28 (5 μg/mL) TCR-stimulated JLAT transductants. Following 2-min αCD3/CD28 stimulation, tyrosine’s 132, 171, and 191 are phosphorylated in WT and S224A JLAT transductants but not in S241A and S2A JLAT transductants.(C) ImageJ quantification of band intensity shows that WT and S224A LAT have tyrosine phosphorylation (132, 171, and 191) 9-fold higher than the S241 and S2A transductants, with one dot representing one experiment. Data are represented as mean ± SEM.(D) ZAP70 tyrosine phosphorylation of residues 132, 171, and 191 on LAT is blocked with pan-PI3K inhibitor LY294002 (20 μM) but not with selective PI3K inhibitors in WT LAT transduced JLAT T cells.(E) ImageJ quantification of band intensity shows tyrosine phosphorylation (132, 171, and 191) 2.5-fold lower in WT LAT-transduced JLAT T cells treated with LY294002 compared to other inhibitors tested, with one dot representing one experiment. Data are represented as mean ± SEM.Representative blots from n = 3 independent experiments. One-factor ANOVA plus Tukey’s multiple comparisons to determine statistical significance (α = 0.05, *p < 0.05, **p < 0.01, ***p < 0.0005, and ****p < 0.0001).

Fig 4: Loss of phosphorylation at S224 and S241 alters LAT signalosome formation(A) JLAT transductants were stimulated with 5 μg/mL αCD3/CD28 for 15 min. mCherry coIP western blot shows that phosphorylation of LAT by DNA-PKcs at S241 is required for signalosome formation, given altered pull-down of GADS and SLP-76 in JLAT cells expressing S241A LAT and S2A LAT. Representative blots from n = 3 independent experiments.(B) CoIP coupled mass spectrometry was completed in JLAT transductants expressing WT, S224A, or S241A LAT stimulated with 5 μg/mL αCD3/CD28 for 15 min. Pull-down was normalized to mCherry-expressing JLAT cells, and protein pull-down was further normalized to relative LAT pull-down with log-fold differences shown between the groups, n = 5 replicates/transductant. The top statistically different proteins pulled down with mCherry-tagged LAT are represented in WT, S224A, and S241A LAT transductants.

Fig 5: DNA-PKcs-deficient CD4+ and CD8+ T cells have functional deficits and impaired LAT localization at the plasma membrane(A) Isolated CD8+ T cells from Cre+CD8-PKcswt/wt and Cre+CD8-PKcsfl/fl mice and isolated CD4+ T cells from Cre+CD4-PKcswt/wt and Cre+CD4-PKcsfl/fl mice were stimulated for 48 h by either 5 μg/mL αCD3/CD28 or by coculture with splenocytes from BALB/c mice (MLR, 4:1 donor to responder). Supernatant was analyzed for IL-2 and IFN-γ by ELISA. n = 3 mice per group, with each dot representing a technical replicate from an individual mouse. Data are represented as mean ± SEM.(B and C) LSCM at 100× magnification of isolated (B) CD4+ T cells and (C) CD8+ T cells were stimulated for 2 min with plate-bound αCD3/CD28 at 5 μg/mL. Scale bars, 5 μm. Representative images from n = 3 mice per group.(D and E) Quantification of LAT signal at the plasma membrane (F-Actin: green) with each dot representing MFI from an area of the plasma membrane. Data are represented as mean ± SEM.(A) Unpaired Student’s t test and (D and E) one-factor ANOVA plus Tukey’s multiple comparisons were used to determine statistical significance (α = 0.05, *p < 0.05, **p < 0.01, ***p < 0.0005, and ****p < 0.0001), n = at least 3 mice/group.