DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

TL;DR

DNA-PKcs helps T cells fight cancer and prevent transplant rejection by regulating LAT signaling at the immune synapse.

Contribution

DNA-PKcs is newly identified as a regulator of LAT-dependent T cell signaling and function.

Findings

DNA-PKcs interacts with LAT and localizes to the immune synapse upon T cell activation.

Inhibiting DNA-PKcs impairs LAT localization and reduces T cell function in tumor control and graft rejection.

Phosphorylation of LAT serines S224 and S241 by DNA-PKcs affects T cell signaling and cytokine production.

Abstract

Formation of the immune synapse (IS) following T cell antigen recognition includes recruitment of the linker for activation of T cells (LAT). Once at the IS, LAT tyrosines are phosphorylated, allowing it to serve as a scaffold for the formation of the “signalosome,” a multiprotein complex that drives T cell receptor signaling. Here, we show that upon T cell activation, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interacts with LAT and localizes to the IS. Inhibition of DNA-PKcs diminishes LAT localization at the IS. We identified two LAT serines phosphorylated by DNA-PKcs, S224 and S241, that impact LAT tyrosine phosphorylation, protein binding, and cytokine production. Using our mouse model designed to delete DNA-PKcs expression in mature CD4+ or CD8+ T cells, we show that loss of DNA-PKcs results in T cells that are unable to control tumor growth or induce allogeneic graft rejection. These data highlight DNA-PKcs as a pivotal protein in T cell function.

Rainwater et al. show that DNA-PKcs plays a role in early T cell activation by regulating signaling through LAT at the immune synapse. By influencing how T cells respond to antigens, DNA-PKcs emerges as a promising therapeutic target, and inhibiting DNA-PKcs may offer a powerful approach to preventing transplant rejection.

Figures

A](#F3)). These data indicate that phosphorylation of serines S224 and S241 is crucial for regulating T cell signaling pathways.

A](#F4)). Since LAT indirectly binds to SLP-76 through its interaction with GADS, we also observed that the S241A and S2A mutants consequently prevented the interaction of SLP-76 with the LAT complex ([Figure 4A](#F4)).^[28](#R28)^ These findings indicate that phosphorylation of S241 is critical for the binding of proteins to LAT and regulates the composition of the signalosome.

A](#F5)). To support the hypothesis that DNA-PKcs inhibition affects Golgi-mediated trafficking of LAT, we compared LAT localization patterns in Jurkat T cells treated with brefeldin A, a vesicle transport inhibitor.^[32](#R32)^ Brefeldin A treatment similarly attenuated LAT localization to the plasma membrane as observed with DNA-PKcs inhibitor treatment ([Figures 5B](#F5) and [5C](#F5)). Other TCR-related proteins, such as CD3ζ and Lck, also traffic through the Golgi to localize to the IS.^[33](#R33),[34](#R34)^ If DNA-PKcs influences this process, we would expect defects in the IS localizat

A](#F6)). Reduced cytokine expression was also observed in DNA-PKcs-deficient CD8^+^ T cells activated with αCD3/CD28 ([Figure 6A](#F6)). Loss of DNA-PKcs expression in CD4^+^ T cells did not affect IL-2 production by either alloantigen or αCD3/CD28 stimulation. However, IFN-*γ* was increased in DNA-PKcs-deficient CD4^+^ activated with αCD3/CD28 but decreased when activated with alloantigen ([Figure 6A](#F6)). The effects on cytokine production were not attributable to impaired T cell proliferation or increased apoptosis. Loss of DNA-PKcs expression did not affect the proliferation or enhance

G](#F7) contains representative images of skin grafts. Grafts were considered completely rejected, as seen in the graft from control mice (PKcs^wt/wt^) on day 18, when necrosis consumed 80% of the entire graft. Treatment with the DNA-PKcs inhibitor NU7441 significantly extended graft survival compared to the vehicle-treated controls, supporting our previous findings ([Figure 7H](#F7)).^[39](#R39)^ Although loss of DNA-PKcs in CD4^+^ T cells did not significantly alter the overall rejection time frame, there was an initial delay in rejection that accounts for a minimal increase in survival. For

Full text

INTRODUCTION

Antigen ligation of the T cell receptor (TCR) triggers the formation of a supramolecular structure known as the immune synapse (IS) at the interface between the TCR and the antigen-presenting cell (APC).^1^ This involves polarization of the T cell toward the APC and reorganization of cytoskeletal elements and organelles, including the Golgi complex, beneath the IS. This facilitates the delivery of critical signaling molecules to the IS to initiate molecular pathways impacting T cell response, including transcriptional pathways for differentiation into antigen-specific effector cells. Pharmaceutical targeting of IS proteins led to the development of effective therapies for cancer and infectious diseases, as well as promising approaches for preventing transplant rejection.^2^ It is evident that continuing to explore the IS landscape to identify proteins involved in regulating T cell signaling can yield significant therapeutic benefits.

A critical step in IS signal transduction is recruitment of the transmembrane adaptor molecule linker for activated T cells (LAT) to the synaptic membrane. While many questions remain regarding the trafficking of LAT to the IS, it is well documented that upon TCR/antigen binding, LAT translocates to the IS where it is phosphorylated by the tyrosine kinase ZAP70.^3-5^ The phosphorylated tyrosines serve as binding sites for signaling molecules forming the multimolecular complex known as the “LAT signalosome”.^6^ This complex brings effector molecules into contact to drive proximal TCR signaling and the subsequent T cell response. Loss of LAT expression or mutation of key tyrosine phosphorylation sites results in the misregulation of T cells in animal models.^7-10^

In its canonical role, the phosphoinositide 3-kinase (PI3K)-related kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs), along with family members ATM and ATR, functions in the nucleus as a double-strand DNA damage repair (DDR) kinase to promote non-homologous end-joining.^11,12^ It was originally linked to the developing immune system when mice with a severe combined immunodeficiency (SCID) phenotype, characterized by deficiencies in mature T and B cells, were discovered to harbor DNA-PKcs loss-of-function mutations.^13-16^ This defect is attributed to DNA-PKcs’s role in V(D)J recombination, which promotes B and TCR diversity.

Though less characterized, DNA-PKcs also functions in mature immune cells independent of its role in DDR. For instance, DNA-PKcs is required for Toll-like receptor 9 (TLR9) signaling and interferon (IFN)-α/β production in dendritic cells, functioning as a viral DNA sensor and supporting efficient antigen presentation to drive a T helper (Th)2-mediated response.^17,18^ DNA-PKcs also regulates the differentiation of CD4^+^ T cells into Th1 and Th2 subtypes by altering the expression of Gata3 and T-bet.^19^ Additionally, DNA-PKcs, in conjunction with the KU complex expressed in the cytoplasm of aging CD4^+^ T cells, acts as a sensor for accumulated cytoplasmic DNA, resulting in heightened T cell activation.^20^ Here, we provide further evidence that DNA-PKcs is a critical component of the adaptive immune response and suggest a mechanism by which it impacts CD4^+^ and CD8^+^ T cell function. Here, we demonstrate that upon T cell activation, DNA-PKcs physically interacts with LAT and is required for LAT localization to the IS. We identified two serines within LAT, S224 and S241, that are phosphorylated by DNA-PKcs and are critical for LAT tyrosine phosphorylation and IL-2 and IFN-γ production. Using unique transgenic mouse models designed to delete DNA-PKcs expression specifically within mature CD4^+^ or CD8^+^ T cells, we show that loss of DNA-PKcs results in T cells unable to control tumor growth or induce allogeneic transplant graft rejection. These data highlight a mechanism influencing T cell responses with potential therapeutic application for immune-related diseases.

RESULTS

T cell activation uniquely activates DNA-PKcs in the cytoplasm

The function of DNA-PKcs within the cytoplasm of T cells remains poorly understood. Using cell fractionation and laser confocal microscopy (LSCM), we demonstrate that upon activation of human Jurkat T cells with αCD3/CD28, active DNA-PKcs, indicated by the presence of phosphorylated serine 2056 (pDNA-PKcs), is specifically elevated within the cytoplasm (Figures 1A-1C).^21,22^ Western blot analysis of the cytosolic fractions revealed that when T cells were stimulated with αCD3/CD28 for five minutes and then removed from the stimulus, pDNA-PKcs levels peaked at 15 min and returned to baseline by 45 min (Figures S1A and S1B).

Interestingly, a portion of cytoplasmic pDNA-PKcs localized to the plasma membrane upon αCD3/CD28 activation (Figures 1D and 1E). We investigated whether this phenomenon was unique to DNA-PKcs or if related DDR kinases ATM and ATR exhibited similar expression patterns following T cell activation.^23^ After stimulation of Jurkat T cells, we performed western blot analysis on cytosolic fractions to assess the levels of activated ATM (pS1981) and ATR (pS1989). Unlike DNA-PKcs, TCR stimulation did not induce an increase in pATM or pATR in the cytoplasm (Figures 1F and 1G). This finding was corroborated by LSCM results, which showed that while pATM and pATR levels increased overall, there was no increase in the cytosol as seen with pDNA-PKcs (Figures 1H-1J). These results suggest that DNA-PKcs plays a unique role in T cells, distinct from its well-known function in nuclear DDR, and point to an unexplored step in TCR signaling that involves the localization of DNA-PKcs at the plasma membrane.

LAT is a phosphorylation target of DNA-PKcs

To define the function of DNA-PKcs at the plasma membrane of activated T cells, we evaluated our recently published phosphomass spectrometry study, which analyzed differentially phosphorylated proteins in activated Jurkat T cells treated with or without the DNA-PKcs inhibitor NU7441.^24,25^ We specifically focused on proteins known to localize to the plasma membrane of activated T cells. Notably, we identified the IS-associated protein LAT. Phosphorylation of LAT at serine 241 (S241) was significantly reduced (fold change > 10) upon NU7441 treatment, indicating that DNA-PKcs phosphorylates LAT at this residue. We confirmed that DNA-PKcs phosphorylates LAT using an in vitro DNA-PKcs luminescent kinase assay and a LAT fragment containing the S241 amino acid (Figures S2A and S2B). Analysis of the phosphorylated LAT peptide by mass spectrometry confirmed that DNA-PKcs not only phosphorylates S241 but also serine 224 (S224). Figure S2C illustrates the ratios of phosphorylated S224 and S241 to unphosphorylated LAT in the presence and absence of DNA-PKcs. Sequence alignment of these phosphorylation sites reveals a high degree of conservation from mice to humans. Both serines are located within conserved S/Q motifs, the known DNA-PKcs consensus sites (Figure S2D).

DNA-PKcs inhibition diminishes LAT localization to the immune synapse

To further explore a functional relationship between DNA-PKcs and LAT, we analyzed the expression patterns of LAT and DNA-PKcs by LSCM in Jurkat T cells following activation. As expected, activation resulted in clear localization of LAT to the plasma membrane. Interestingly, we observed colocalization between DNA-PKcs and LAT on the plasma membrane of activated T cells (Figures 2A and 2B). LAT is found to be specifically localized to the IS following antigen recognition. To form a true, “visible” IS, we cocultured Jurkat T cells with Raji B cells preloaded with the Staphylococcal enterotoxin type E (SEE) antigen.^26^ LSCM images in Figures 2C and 2D show LAT and pDNA-PKcs colocalized to the IS at the T-B cell interface. Moreover, we show that DNA-PKcs physically interacts with LAT as LAT co-immunoprecipitated with the pull-down of DNA-PKcs (Figures 2E and 2F).

Surprisingly, DNA-PKcs with NU7441 not only blocked DNA-PKcs localization but also abrogated the expression of LAT at the IS and reduced the interaction between DNA-PKcs and LAT (Figures 2C-2H). To further validate that DNA-PKcs inhibition impacts LAT IS localization, we used short hairpin RNA (shRNA) methods to knock down DNA-PKcs expression in Jurkat T cells and analyzed LAT expression at the plasma membrane. Similar to our previous findings, loss of DNA-PKcs expression significantly reduced LAT expression at the plasma membrane (Figures 2I-2L). This relationship between LAT and DNA-PKcs was further confirmed in primary human CD3^+^ T cells isolated from the commercially available human peripheral blood mononuclear cells. Activation with αCD3/CD28 stimulated the localization of LAT at the plasma membrane, which was reduced with NU7441 treatment (Figures S3A and S3B). These findings indicate a mechanism by which DNA-PKcs is required for the translocation or retention of LAT at the IS

PHOSPHORYLATION AT LAT S224 AND S241 IS NOT REQUIRED FOR PROLIFERATION, BUT IT IS FOR IL-2 PRODUCTION

We next investigated the relevance of the phosphorylation sites S224 and S241 to LAT activity and T cell function. To achieve this, we generated mCherry-tagged lentiviral expression vectors containing serine-to-alanine mutations at S224 and S241, designated as S224A and S241A, respectively, and a double mutant with both S224 and S241 mutated to alanines (S2A). These vectors, along with vectors expressing wild-type (WT) LAT, were transduced into the LAT-deficient Jurkat T cell line J.LAT, previously generated by the Arthur Weiss laboratory.^27^ Following transduction, the expression of WT and mutant LAT was validated using flow cytometry for mCherry expression and western blot for LAT protein levels (Figures S4A and S4B).

First, we assessed whether the loss of phosphorylation at these sites affects proliferation. J.LAT cells reconstituted with either WT LAT or the mutants (S224A, S241A, or S2A) expectedly proliferated at rates similar to the parental E6.1 Jurkat T cell line (Figures S5A and S5B), as loss of LAT in J.LAT cells was previously reported to not alter proliferation.^27^

Next, we evaluated the impact of the phosphorylation mutations on IL-2 production. After transduction, T cells were activated with αCD3/CD28, and supernatant cytokine levels were quantified using ELISA. While untransduced J.LAT cells were unable to produce measurable levels of the cytokine, J.LAT cells reconstituted with WT LAT produced significant amounts of IL-2 following activation. In contrast, reconstitution with the S224A, S241A, or S2A mutants did not rescue IL-2 production with levels comparable to untransduced J.LAT cells (Figure 3A). These data indicate that phosphorylation of serines S224 and S241 is crucial for regulating T cell signaling pathways.

Phosphorylation at S224 and S241 impacts the LAT signalosome protein composition

Once LAT localizes to the plasma membrane, tyrosines in its cytoplasmic tail, specifically Y132, Y171, and Y191, undergo phosphorylation by the kinase ZAP70.^5^ These residues then serve as docking sites for various signaling proteins that constitute the signalosome. The protein composition of the signalosome dictates downstream signaling pathways and ultimately determines how a T cell responds to an antigen.^6^ Analysis of these tyrosine phosphorylation sites following αCD3/CD28 stimulation revealed that phosphorylation at Y132, Y171, and Y191 was prevented in J.LAT cells reconstituted with either the S241A or S2A mutants, but not with S224A or WT LAT (Figures 3B and 3C). Interestingly, inhibition of DNA-PKcs with NU7441 did not block tyrosine phosphorylation, indicating that other kinases are capable of phosphorylating these serines. This was supported by the observation that treatment of Jurkat T cells with the pan-PI3K inhibitor LY294002 blocked phosphorylation at Y132, Y171, and Y191 (Figures 3D and 3E)

Phosphorylation of these tyrosines is known to impact the binding of key LAT signalosome proteins, such as PLCγ and GADS (GRB2-related adaptor protein).^6,9^ Therefore, we investigated whether mutations at S224 or S241 hindered the binding of these proteins to LAT. Co-immunoprecipitation (coIP) experiments performed in J.LAT cells reconstituted with WT LAT demonstrated binding between LAT and PLCγ and GADS following αCD3/CD28 activation, as expected. However, the S241A and S2A mutants were unable to interact with GADS (Figure 4A). Since LAT indirectly binds to SLP-76 through its interaction with GADS, we also observed that the S241A and S2A mutants consequently prevented the interaction of SLP-76 with the LAT complex (Figure 4A).^28^ These findings indicate that phosphorylation of S241 is critical for the binding of proteins to LAT and regulates the composition of the signalosome.

To further elucidate how these phosphorylation sites affect the signalosome’s composition, we conducted a coIP-mass spectrometry study. This approach allowed us to comprehensively compare the binding patterns between WT LAT and the mutants. J.LAT cells reconstituted with either the mCherry vector alone, WT LAT, S224A LAT, or S241A LAT were activated with αCD3/CD28 for 15 min, followed by mCherry immunoprecipitation. Lysates were subjected to mass spectrometry. After filtering out non-specific binding proteins using the mCherry-only control samples and normalizing to LAT levels, we compared the binding patterns between activated WT and the S224A or S241A mutant cell lines. We identified significant changes in protein binding between WT and both mutants. Notably, the S241A mutant exhibited the most pronounced differences from WT (Figure 4B). This observation is consistent with our findings that the S241A mutant showed defects in the known tyrosine phosphorylation sites. However, further analysis of additional LAT tyrosine residues may reveal alterations in the S224A mutant that account for the observed changes in protein binding compared to WT.

Although we were unable to detect PLCγ and SLP-76 in our mass spectrometry screen, we confirmed a decrease in GADS binding to the S241A mutant, as observed in our coIP experiment. We also observed a significant decrease in the binding of the guanine nucleotide exchange factor Son of Sevenless 1 (Sos1) to S241A. Sos1 promotes LAT signalosome formation and is critical for T cell function.^29^ Interestingly, while some proteins showed decreased binding to the mutants compared to WT, a substantial number of proteins exhibited increased binding to the S224A and S241A mutants. This unexpected increase suggests that phosphorylation of these serines may not only regulate the binding of proteins to LAT but also influence the release of proteins. The exact mechanisms by which phosphorylation at these serines alters protein binding are still under investigation. Nonetheless, these data clearly confirm that phosphorylation of S224 and S241 is a critical step in T cell signaling and may regulate T cell response to antigen.

PHOSPHORYLATION AT S224 AND S241 IS NOT REQUIRED FOR LAT LOCALIZATION TO THE IS

We investigated whether phosphorylation of LAT serines S224 and S241 is required for LAT translocation and retention at the IS. Figures S5C and S5D display LSCM images showing normal LAT translocation to the plasma membrane in J.LAT cells transduced with WT LAT following activation with αCD3/CD28. LAT mutants S224A, S241A, and the double mutant S2A all localized to the membrane similarly to WT LAT. This indicates that although DNA-PKcs is essential for proper LAT localization to the IS, as demonstrated by NU7441 treatment and shRNA knockdown of DNA-PKcs, the phosphorylation of these two serines is not critical. These findings suggest that additional mechanisms mediate DNA-PKcs’s impact on LAT translocation.

Upon TCR activation, current studies suggest that LAT, located in lipid rafts within the plasma membrane and intracellular vesicles, traffics through the Golgi apparatus before being transported to the IS within secretory vesicles.^3,4,30^ Our observation that inhibiting DNA-PKcs prevents LAT accumulation at the IS, combined with previous findings that DNA-PKcs is essential for proper Golgi vesicle development, suggests that DNA-PKcs may play a unique role in regulating Golgi-mediated trafficking.^31^ Therefore, we hypothesized that DNA-PKcs inhibition disrupts the trafficking of LAT-containing vesicles from the trans-Golgi to the IS. To investigate this, we analyzed the localization of DNA-PKcs within the Golgi compartments of T cells. Using a Golgi fractionation kit, we determined that active pDNA-PKcs localizes to both the cis- and trans-Golgi compartments in activated T cells. Notably, pDNA-PKcs was found in secretory vesicles alongside LAT (Figure 5A). To support the hypothesis that DNA-PKcs inhibition affects Golgi-mediated trafficking of LAT, we compared LAT localization patterns in Jurkat T cells treated with brefeldin A, a vesicle transport inhibitor.^32^ Brefeldin A treatment similarly attenuated LAT localization to the plasma membrane as observed with DNA-PKcs inhibitor treatment (Figures 5B and 5C). Other TCR-related proteins, such as CD3ζ and Lck, also traffic through the Golgi to localize to the IS.^33,34^ If DNA-PKcs influences this process, we would expect defects in the IS localization of these proteins as well. Figure 5D shows LSCM images of CD3ζ and Lck (p-Y394) in activated T cells. Consistent with LAT, the signal intensities of CD3ζ and p-Lck at the plasma membrane were also attenuated upon DNA-PKcs inhibition (Figure 5E).

While the role of DNA-PKcs in the cis- and trans-Golgi of resting and activated T cells remains largely unexplored, our data indicate a potential mechanism by which DNA-PKcs regulates the recruitment of proteins to the IS through the control of Golgi-mediated vesicular trafficking following antigen engagement.

Generation of inducible T cell-specific DNA-PKcs-deleted mice

To thoroughly understand the role of DNA-PKcs in T cells, it is imperative to investigate DNA-PKcs function in mature primary T cells. However, animals with inactivating mutations in DNA-PKcs during embryonic development have a significant reduction in mature B and T cells. This phenotype hinders investigations into the function of DNA-PKcs in mature T cells. To analyze DNA-PKcs activity specifically in mature T cells, we generated two unique mouse models in which DNA-PKcs is genetically deleted specifically in either CD4^+^ or CD8^+^ T cells.

Floxed DNA-PKcs (DNA-PKcs^fl/fl^) mice, obtained from Dr. Jay H. Chung,^35^ were bred with either the tamoxifen-inducible CD4-Cre mouse strain to generate Cre^+^CD4-PKcs^fl/fl^ mice, or with the tamoxifen-inducible E8ICreERT2-CD8 mouse strain, received from Dr. Dario Vignali, to generate Cre^+^CD8-PKcs^fl/fl^ mice (Figure S6A).^35,36^ Control mice in this study expressed Cre recombinase but WT DNA-PKcs (Cre^+^CD4-PKcs^wt/wt^, Cre^+^CD8-PKcs^wt/wt^). Eight-week-old Cre^+^CD4-PKcs^wt/wt^, Cre^+^CD4-PKcs^fl/fl^, Cre^+^CD8-PKcs^wt/wt^, or Cre^+^CD8-PKcs^fl/fl^ mice with a mature immune system were treated with tamoxifen for seven consecutive days. DNA-PKcs expression was then analyzed by qPCR and western blot in CD4^+^ or CD8^+^ T cells isolated from spleens. Tamoxifen treatment resulted in a significant reduction of DNA-PKcs expression (up to 90%), which persisted for a minimum of 30 days (Figures S6B and S6C). Loss of DNA-PKcs expression within CD4^+^ or CD8^+^ T cells did not alter mouse activity or affect splenic size or weight (Figures S6D and S6E). Loss of DNA-PKcs expression did not impact baseline levels of naive CD4^+^ and naive CD8^+^ T cells, B cells, macrophages, or dendritic cells isolated from the spleens of tamoxifen-treated mice (Figures S6F and S6G).

DNA-PKcs-deficient CD4+ and CD8+ T cells have functional deficits

Reduced cytokine production

To evaluate how the loss of DNA-PKcs expression affects T cell function, we first assessed the ability of DNA-PKcs-deficient T cells to produce cytokines in response to antigen presentation using mixed lymphocyte reactions (MLRs). Splenocytes isolated from BALB/c mice and treated with mitomycin C served as stimulators, while WT or DNA-PKcs-deficient CD4^+^ or CD8^+^ T cells isolated from our transgenic mice acted as responders. Loss of DNA-PKcs expression significantly reduced the production of IL-2 and IFN-γ by CD8^+^ T cells following stimulation with allogeneic antigens (Figure 6A). Reduced cytokine expression was also observed in DNA-PKcs-deficient CD8^+^ T cells activated with αCD3/CD28 (Figure 6A). Loss of DNA-PKcs expression in CD4^+^ T cells did not affect IL-2 production by either alloantigen or αCD3/CD28 stimulation. However, IFN-γ was increased in DNA-PKcs-deficient CD4^+^ activated with αCD3/CD28 but decreased when activated with alloantigen (Figure 6A). The effects on cytokine production were not attributable to impaired T cell proliferation or increased apoptosis. Loss of DNA-PKcs expression did not affect the proliferation or enhance apoptosis in either αCD3/CD28-activated CD4^+^ or CD8^+^ T cells, as assessed by CellTrace proliferation assays and annexin V staining (Figures S6H and S6I). This highlights that although chemical inhibition of DNA-PKcs impairs T cell proliferation, the residual levels of DNA-PKcs present in our transgenic T cells may be sufficient to support normal proliferation.^37^ Taken together, these findings suggest that DNA-PKcs is critical for cytotoxic responses of CD8^+^ T cells but less so in CD4^+^ T cells.

Impaired metabolic reprogramming

T cell activation induces metabolic reprogramming characterized by a switch from oxidative phosphorylation to glycolysis. Therefore, we analyzed changes in extracellular acidification rate (ECAR), an indicator of aerobic glycolysis, in our DNA-PKcs-deficient primary T cells.^38^ Basal respiration rates were similar between WT and DNA-PKcs-deficient CD8^+^ T cells; however, upon stimulation with phorbol 12-myristate 13-acetate (PMA)/Ionomycin, DNA-PKcs-deficient CD8^+^ T cells exhibited reduced ECAR and OCR (oxygen consumption rate) rates compared to WT counterparts, indicating impaired metabolic reprogramming (Figure S7A). Interestingly, loss of DNA-PKcs expression in CD4^+^ T cells resulted in a significant increase in ECAR and OCR compared to WT upon PMA/Ionomycin stimulation (Figure S7B). While this effect differs from that observed in CD8^+^ T cells, the higher ECAR in CD4^+^ T cells could indicate a compensatory upregulation of glycolytic pathways or an alternative metabolic adaptation in response to DNA-PKcs deficiency. While research is needed to understand the mechanisms behind this metabolic shift and its impact on CD4^+^ T cell function, these observations further validate a critical role for DNA-PKcs in T cells.

LAT localization to the plasma membrane is reduced in activated DNA-PKcs-deficient primary CD4+ and CD8+ T cells

To further confirm the role of DNA-PKcs in LAT localization to the plasma membrane following T cell activation, as observed in Jurkat T cells, we analyzed LAT expression patterns in splenic CD4^+^ and CD8^+^ T cells isolated from Cre^+^CD4-PKcs^fl/fl^ and Cre^+^CD8-PKcs^fl/fl^ mice using LSCM. As expected, αCD3/CD28 activation in control T cells induced LAT expression at the plasma membrane of mouse T cells. However, LAT expression at the plasma membrane was significantly reduced in both DNA-PKcs-deficient CD4^+^ and CD8^+^ T cells (Figures 6B-6E). This indicates that DNA-PKcs plays a crucial role in LAT trafficking to the plasma membrane following TCR stimulation.

Next, we examined how the loss of DNA-PKcs expression affects LAT tyrosine phosphorylation. In Jurkat T cells, we determined that phosphorylation of LAT at S241 is required for phosphorylation of tyrosines Y132, Y171, and Y191. However, inhibition of DNA-PKcs with the specific inhibitor NU7441 did not impact tyrosine phosphorylation, suggesting that S241 is likely phosphorylated by other kinases. Consistent with this finding, loss of DNA-PKcs expression in CD4^+^ or CD8^+^ T cells did not affect phosphorylation at Y132, Y171, or Y191 (Figures S8A and S8B). These results suggest a dual role for DNA-PKcs in T cells: it is critical for the trafficking of proteins to the IS and influences signaling from the LAT signalosome.

Loss of DNA-PKcs results in dysfunctional T cells unable to control tumor growth

T cells are a central component of eliminating cancerous cells; therefore, to confirm DNA-PKcs as a pivotal player in T cell activity and their ability to mount an effective immune response against tumors, we analyzed the effect of DNA-PKcs-deficient CD4^+^ or CD8^+^ T cells on tumor growth in vivo. Following tamoxifen treatment and injection of MC38 colorectal cancer cells, Cre^+^CD8-PKcs^fl/fl^ mice had significantly accelerated tumor growth, resulting in reduced survival compared to Cre^+^CD8-PKcs^wt/wt^ (control) mice with DNA-PKcs-expressing CD8^+^ T cells (Figures 7A and 7B). This finding was further validated with the B16F10 melanoma tumor, where survival decreased, and tumor growth was accelerated in Cre^+^CD8-PKcs^fl/fl^ mice compared to controls (Figures 7C and 7D). These results demonstrate that the loss of DNA-PKcs in CD8^+^ T cells leads to dysfunctional T cells unable to effectively control tumor growth. Interestingly, MC38 tumors grew at an accelerated pace during the initial response in Cre^+^CD4-PKcs^fl/fl^ mice, where T cell control is most evident; however, tumor growth eventually equalized to control mice, resulting in no significant difference in survival (Figures 7E and 7F). This further suggests that DNA-PKcs plays a different regulatory role in CD4^+^ versus CD8^+^ T cells.

DNA-PKcs-deficient CD8+ T cells do not reject allogeneic skin grafts

Cytotoxic T cells are the primary drivers of graft rejection in transplantation. We recently published that chemical inhibition of DNA-PKcs reduced T cell-mediated rejection and extended graft survival in a murine allogeneic skin graft model.^39^ We used this model to analyze defects in DNA-PKcs-deficient T cell response to alloantigen. Figure 7G contains representative images of skin grafts. Grafts were considered completely rejected, as seen in the graft from control mice (PKcs^wt/wt^) on day 18, when necrosis consumed 80% of the entire graft. Treatment with the DNA-PKcs inhibitor NU7441 significantly extended graft survival compared to the vehicle-treated controls, supporting our previous findings (Figure 7H).^39^ Although loss of DNA-PKcs in CD4^+^ T cells did not significantly alter the overall rejection time frame, there was an initial delay in rejection that accounts for a minimal increase in survival. For instance, average graft necrosis in control mice was 30% by day 8 but less than 10% in Cre^+^CD4-PKcs^fl/fl^. This trend continued until around day 13 when necrosis rates were similar between the control and Cre^+^CD4-PKcs^fl/fl^. However, loss of DNA-PKcs in CD8^+^ T cells extensively attenuated graft necrosis and, in most cases, completely prevented any rejection (Figure 7I). Graft health was preserved for over 30 days with only sparse necrosis observed in Cre^+^CD8-PKcs^fl/fl^ recipients. Furthermore, over 50% of Cre^+^CD8-PKcs^fl/fl^ recipients had complete graft integration with hair growth by day 25 (Figures 7J and 7K). This exciting finding supports the tumor growth data and indicates that DNA-PKcs is required for the cytotoxic response to alloantigens.

DISCUSSION

Our study elucidates the multifaceted role of DNA-PKcs in T cell biology, extending beyond its well-established function in DNA repair. We have uncovered that DNA-PKcs is integral for both trafficking and signaling mechanisms within mature T cells, specifically influencing the localization and function of the immune synapse-associated protein LAT.

We show that DNA-PKcs is localized to the IS following TCR stimulation. This intriguing finding suggests that DNA-PKcs has a critical function in regulating TCR signal transduction induced upon antigen recognition. This corresponds with previous reports indicating that DNA-PKcs, localized to lipid rafts within the plasma membrane of tumor cells, promotes intracellular receptor signal transduction initiated by ionizing radiation.^40^ Similar to our findings, these results were unique to DNA-PKcs and not seen with other DDR kinases. This study, along with our data, supports a function for DNA-PKcs in receptor-mediated signal transduction, specifically TCR signaling in T cells. The mechanism by which DNA-PKcs is activated and recruited to the IS following stimulation is unknown. We are actively investigating these processes, including the involvement of the KU70/ 80 complex in the activation and trafficking of DNA-PKcs to the IS.

One pivotal finding of our research is the identification of DNA-PKcs as a critical regulator of LAT trafficking to the IS. Upon TCR activation, DNA-PKcs is activated and localizes to the IS. Activation of DNA-PKcs is essential for the translocation of LAT to the IS, a process that is impaired upon inhibition or genetic deletion of DNA-PKcs. Interestingly, while phosphorylation of LAT S224 and S241 is crucial for IL-2 production and protein binding, it does not appear to be necessary for LAT’s localization to the plasma membrane. Our Golgi fractionation and brefeldin A experiments support a hypothesis that DNA-PKcs regulates Golgi-mediated vesicular transport of LAT and IS-associated proteins, from the trans-Golgi to the IS. This aligns with previous findings showing that DNA-PKcs also participates in endosome-based inflammatory signaling, such as through its interaction with the endosomal receptor CD158d to activate Akt signaling and promote cytokine production.^41^

We show that phosphorylation of LAT at S224 and S241 is essential for the phosphorylation of LAT Y132, Y171, and Y191, which facilitates the assembly of the LAT signalosome. S224 and S241 reside in the unstructured C terminus tail of LAT distal to the transmembrane domain and helix structure. While tyrosines have been the primary phosphorylation sites studied within LAT, other serine phosphorylation sites have been identified within LAT that are required for protein binding and proximal signaling.^42^ Our mass spectrometry results suggest that phosphorylation of S241 regulates which proteins bind to LAT, as well as the release of proteins during T cell activation. This was evident by the increased binding of several proteins to S241 compared to WT LAT. This is critical given recent findings from the Weiss lab showing temporal LAT protein binding effects and antigen discrimination in T cells.^43^ Future studies should focus on determining the role of LAT serine phosphorylation, specifically S241, in TCR specificity, tolerance to self-antigens, and T cell response outcome.

Our in vivo studies using Cre^+^CD4-PKcs^fl/fl^ and Cre^+^CD8-PKcs^fl/fl^ mouse models reveal a differential impact of DNA-PKcs deficiency on CD4^+^ versus CD8^+^ T cells, although similar deletion of DNA-PKcs was observed in both models. While CD8^+^ T cells lacking DNA-PKcs displayed significant defects in controlling tumor growth and rejecting skin grafts, DNA-PKcs-deficient CD4^+^ T cells did not exhibit similar levels of dysfunction, although they did display alterations in LAT IS localization. This disparity suggests that DNA-PKcs plays a more critical role in the cytotoxic functions of CD8^+^ T cells compared to the helper functions of CD4^+^ T cells. Previous studies showed reduced expression of DNA-PKcs prevents Th1 or Th2 CD4^+^ T cell differentiation, suggesting that additional evaluation of the CD4 model is needed to determine the effects of DNA-PKcs on the CD4^+^ T cell population.^19^ It should be noted that LAT is more highly expressed in human T cells than mouse T cells and could have more of an impact on human T cell function.^44^ It is also important to note that loss of DNA-PKcs expression in mice has varying effects on ATM signaling.^45^ Although these studies have not focused specifically on T cells, it is critical that we analyze how loss of DNA-PKcs expression in T cells from our transgenic mice alters ATM signaling to determine if this is a driving mechanism for T cell dysfunction. This is a future direction for the lab, and with our transgenic model fully developed and validated, we can continue to explore this and the other functions of DNA-PKcs in T cell signaling, potentially identifying changes in T cell subpopulations that account for the attenuated response to antigen.

DNA-PKcs inhibitors have been widely studied for the ability to sensitize tumor cells to chemotherapy and radiation therapy.^46,47^ The impaired signaling in DNA-PKcs-deficient T cells translates to diminished effector functions such as cytokine production and cytotoxic activity, which are crucial for anti-tumor immunity. These findings indicate that DNA-PKcs inhibitors are a double-edged sword for cancer therapy: inhibition weakens tumor cells but also diminishes T cell immunity. This accounts for the mild benefits of DNA-PKcs inhibitors in cancer clinical trials, where the best response is stable disease despite significant results in rodent models.^48-50^ Alternatively, this study supports our previous publication highlighting the potential benefit of DNA-PKcs inhibitors as anti-rejection therapy for transplant patients, where loss of T cell immunity improves graft survival.^39^

In summary, our findings position DNA-PKcs as a pivotal regulator of T cell function, bridging the processes of protein trafficking and signalosome assembly. By modulating both the localization of key signaling proteins and the downstream signaling pathways, DNA-PKcs orchestrates effective T cell responses essential for immune surveillance and pathogen defense. These insights not only advance our understanding of T cell biology but also open avenues for therapeutic interventions targeting DNA-PKcs in immune-related diseases.

Limitations of the study

Several limitations should be considered when interpreting these findings. Although this study highlights previously unrecognized roles for DNA-PKcs in T cell signaling and trafficking, the mechanisms governing its activation and recruitment to the immune synapse remain undefined. The contribution of KU70/80 or other DNA damage response components is only partially explored and requires further mechanistic study. Our identification of DNA-PKcs as a regulator of LAT trafficking is supported by pharmacologic inhibition, genetic deletion, and localization assays; however, the specific molecular machinery linking DNA-PKcs to Golgi-mediated vesicular transport is still unknown. While Golgi fractionation and brefeldin A experiments implicate DNA-PKcs in regulating LAT export from the trans-Golgi network, these assays cannot fully resolve the sequence or intermediates of the trafficking pathway. Additionally, although phosphorylation of serine residues S224 and S241 influences LAT signalosome assembly, the downstream consequences for antigen specificity, TCR sensitivity, and self-tolerance remain to be determined. The in vivo models used to assess DNA-PKcs deficiency in CD4^+^ and CD8^+^ T cells also have limitations. Despite comparable gene deletion efficiency, DNA-PKcs loss produced distinct functional effects in each lineage, and further work is needed to determine whether alterations in T cell subset composition contribute to these differences. The implications of therapeutic DNA-PKcs inhibition must be interpreted cautiously. Because this study relies on T cell-specific genetic deletion, it does not fully replicate the pharmacologic context of systemic inhibitor treatment. Selective chemical inhibition in CD8^+^ T cells has not yet been performed and will be essential to determine whether pharmacologic blockade produces effects similar to genetic loss.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell lines

Jurkat E6.1 human T cell leukemia (male) and Raji B cells (male) were obtained from American Type Culture Collection (ATCC) with certification of analysis. LAT deficient Jurkat T cells, J. LATs, were developed from the Jurkat cell line and were kindly provided by Arthur Weiss, MD, PhD (UCSF)^27^ and validated by western blot and fluorescence microscopy to not express LAT. Cells were maintained in ATCC-modified RPMI 1640 with 10% FBS (Invitrogen cat: A5256701) and 1% Penicillin and streptomycin. Tumor cells lines, B16-F10 and MC-38, were provided by Brian Koss, PhD (UAMS). B16-F10 cells were maintained in DMEM with 10% FBS and 1% P/S. MC-38 cells were cultured with 10% FBS, 1% P/S, 0.1mM non-essential amino acids (NEAA), 1mM sodium pyruvate and 10mM HEPES. All cell lines were identified as mycoplasma free from manufacturer.

Primary cell culture

Single-cell suspensions enriched for naive CD4^+^ or CD8^+^ T cells from mouse spleens were isolated. Briefly, spleens were minced and passed through a 70-μm-nylon cell strainer into serum containing PBS. After washing in PBS and lysing red blood cells, T cells were isolated from mouse splenocytes using EasySep Naive CD4^+^ T or CD8^+^ T cell isolation kits (StemCell: Catalog #19765 and 19858 respectively) according to the manufacturer’s protocol. Human PBMCs were treated similarly, but with EasySep Human Naive Pan T cell Isolation Kit (StemCell: Catalog # 17961). Mouse primary T cells were maintained in RPMI 1640 with 1x NEAA, 1x sodium pyruvate and 55 μM Beta-Mercaptoethanol. Human PBMCs were ordered from Stem cell Technologies and maintained in RPMI 1640 with 10% FBS and 1% Penicillin and streptomycin.

Sex as a biological variable

Our study examined male and female animals, and similar findings are reported for both sexes.

Animals

Mice used in the study were housed in the Arkansas Children’s Research Institute Animal Facility. All animal studies were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences/Arkansas Children’s Research Institute (Animal Welfare assurance number: A3063-01). Mice were tamoxifen treated starting at 8 weeks of life. All mice were between 8 and 12 weeks and weighed between 20 and 30 grams at time of skin transplant or tumor cell injection. For surgeries, mice were anesthetized by inhalation of 1–5% isoflurane. Following tumor cell injection or skin transplants, mice were housed individually. Animals were randomly assigned to control or experimental group with equal numbers of males and females represented in each study. Euthanasia was performed with CO_2_ inhalation followed by cervical dislocation. Animals are housed at 72°F, with monthly sentinel testing conducted in each room to monitor for potential pathogens.

METHOD DETAILS

Western blot, cell fractionation and co-immunoprecipitation

Cells were harvested with ice-cold PBS and placed on ice. Cells were spun down at 500xg for 10 min, washed with ice-cold PBS (phosphate-buffered saline) prior to lysis. Whole cell lysate was collected using RIPA lysis buffer (150 mM NaCl, 0.1% Triton X-100, 0.1% SDS. 50 mM Tris-HCl, 0.5% Deoxycholate, 1 mM EDTA and EGTA plus protease and phosphatase inhibitors). For cytosolic extraction, cells were lysed with 0.1% NP-40 (plus protease and phosphatase inhibitors) on ice for 10 min. Cells were then centrifuged at 10,000xg for 20 min. Supernatant was then used for western blot analysis. Co-immunoprecipitation was completed using CO-IP lysis buffer (20 mM Tris-Hcl, 300 mM NaCl, 2 mM EDTA and EGTA, 10% NP40 plus protease and phosphatase inhibitors) followed by primary antibody incubation and protein A/G bead pulldown. Input sample was collected from the cytosolic extract.

Laser scanning confocal microscopy (LSCM)

Micro-slide 8-well glass-bottom coverslips (Ibidi, Cat: 80807) were coated with 0.01% poly-L-lysine (Poly-Lys, Sigma-Aldrich, P8920) in PBS ± CD3 for 2 h at 37°C. T cells were seeded at a concentration of 250,000 cells/ml onto the coated coverslips with or without soluble CD28 for the specified duration. After incubation, cells were fixed with 4% paraformaldehyde for 10 min, followed by a single wash with PBS. The cells were then permeabilized using 0.1% Triton X-100 in PBS for 20 min and washed once more with PBS. To block nonspecific binding, cells were incubated in 2.5% BSA in TBST for 1 h. Cells were incubated with primary and secondary antibodies of interest, washed three times with TBST and stained with Hoechst. Imaging was performed using a Zeiss LSM scanning confocal microscope. Analysis of images was completed using the ZenBlue software.

Immune conjugate formation

Raji B cells were washed, resuspended at a concentration of 1 × 10^6^ cells/mL in RPMI without FCS and labeled with Hoechst dye (Biorad Cat#639) for 20 min at 37°C. Labeling was stopped with RPMI 10% FCS and cells were washed once and resuspended at 1 × 10^6^ cells/mL. Cells were pulsed with Recombinant Staphyloccocus Enterotoxin Type E (SEE, Cellgenetech, MBS1112600) (1 μg/mL) or left untreated for 30 min at 37°C before being washed once and resuspended at a concentration of 1 × 10^6^ cells/mL. 100,000 Raji cells were incubated on coverslips for 30 min, washed once with warmed PBS and 150,000 Jurkat cells were added for 30 min. Cells were fixed, permeabilized and blocked similar to aforementioned imaging.

Cellfree kinase assay

The DNA-PK Kinase Enzyme System (Cat #V4106) and ADP-Glo Assay (Cat #V6930) from Promega were used as previously described (Waldrip et al., 2021). Briefly, DNA-PK enzyme was incubated with control peptide or LAT peptide in a 96-well plate format with 150 μM ATP, 1 μg of peptide. DNA-PK enzyme was used at 50 units per reaction, in a reaction volume of 25 μL. Enzyme, peptide, and ATP were incubated for 1 h at room temperature prior to ADP-Glo protocol. Luminescence was measured on synergy HTX multiplate reader (BioTek) using an integration time of 0.25 s per well. Enzyme plus ATP samples, no peptide, were used to determine baseline luminescence since DNA-PKcs is known to phosphorylate itself.

Development of stable LAT phosphomutant cell lines

The CRISPR knockout LAT cell line, J.LAT, was developed, validated and kindly supplied by the Arthur Weiss laboratory (UCSF). Transfer plasmids containing mCherry fluorescent probe and mCherry tagged WT LAT, S224A LAT, S241A LAT or S224/241A LAT were developed, validated and purchased from vectorbuilder. Envelope and packing plasmids from Addgene were transfected into HEK293T cells with transfer plasmids to produce lentiviral particles which were then used to transduce J.LAT cells. J.LAT transductants were screened by their mCherry expression by flow cytometry and cells were grown in the presence of puromycin (2 μg/mL). Transduction of LAT version was validated by PCR (plasmidosaurus). LAT expression was validated across mutants by western blot.

Non-replicative VSV-g pseudotyped lentiviral particles were produced by transfecting HEK-293T cells with Gag, Pol, rev, encoding plasmid (psPAX2), envelop encoding plasmid (pMD2.G) and either the WT LAT or Mutant LAT construct. Mutant and WT LAT lentiviral transfer plasmids were validated through vectorbuilder quality control prior to use in vitro. Mutant and WT LAT were expressed using the pcDNA3 lentiviral expression system which also encodes a puromycin-resistance gene for production of stable lines. LAT transfer plasmids were combined with packaging and envelope plasmids and transfected into HEK293T cells in antibiotic free DMEM (10% FBS) according to published methods. 48 h later, viral supernatant was collected from HEK293T cell culture and passed through a 0.2 μM filter. Viral media was incubated with J.LAT cells via spinfection (2000 rpm for 1 h) with polybrene (5 μg/mL) and incubated at 37°C for 24 h. Viral media was removed and transduced cells were replenished with fresh RPMI and allow to grow for 2 days prior to puromycin addition (2 μg/mL). MCherry expression (tagged to the c-terminus of LAT) was measured weekly.

Transfection

Jurkat T cells were transfected with shRNA to DNA-PKcs using Lonzo electroporation system. Following pulse, Jurkat T cells were plated in fresh, supplemented RPMI and incubated at 37°C for 36 h prior to analysis. Knockdown of protein expression was validated on western blot.

Golgi analysis in T cells

E6.1 Jurkat T cells were cultured under normal conditions and plated at 1 x10^6^ cells/mL. Cells were harvested and washed with icecold PBS and pellets were used in the Golgi apparatus enrichment kit (cat: GO-037, Invent Biotechnologies). Following initial lysis with kit lysis buffer, 15% of sample volume was collected for input. This kit separates cis Golgi from the secretory trans Golgi and with kit recommended controls of GM130 and CLINT1, respectively. Following protocol from manufacture, these fractions were collected and run on western blot. For LSCM analysis of the Golgi, cells were treated with Brefeldin (10 μg/mL) for 4 h before activation.

Co-immunoprecipitation mass spectrometry

Following the co-immunoprecipitation protocol as outlined above, lysates were submitted to the UAMS proteomics core for analysis via the following.

FASP methods – Orbitrap Exploris DIA

Protein samples were reduced, alkylated, and digested on-bead using filter-aided sample preparation with sequencing grade modified porcine trypsin (Promega).^51^ Tryptic peptides were then separated by reverse phase XSelect CSH C18 2.5 μm resin (Waters) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 60 min gradient from 98:2 to 65:35 buffer A:B ratio. Eluted peptides were ionized by electrospray (2.4 kV) through a heated capillary (275°C) followed by data collection on an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).

Precursor spectra were acquired with a scan from 385 to 1015 Th at a resolution set to 60,000 with 100% AGC, max time of 50 msec, and an RF parameter at 40%. DIA was configured on the Orbitrap 480 to acquire 40 x 15 Th isolation windows, normalized AGC target 500%, maximum injection time 40 ms). A second DIA was acquired in a staggered window (15 Th) pattern with optimized window placements from 390 to 990 Th.

Buffer A = 0.1% formic acid, 0.5% acetonitrile.

Buffer B = 0.1% formic acid, 99.9% acetonitrile.

Following data acquisition, data were searched using Spectronaut (Biognosys version 19.1) against the UniProt Homo sapien database (May 2024) using the direct DIA method with an identification precursor and protein q-value cutoff of 1%, generate decoys set to true, the protein inference workflow set to maxLFQ, inference algorithm set to IDPicker, quantity level set to MS2, cross-run normalization set to false, and the protein grouping quantification set to median peptide and precursor quantity. Protein MS2 intensity values were assessed for quality using ProteiNorm.^52^ The data was normalized using Log_2_ Median Intensities and analyzed using proteoDA to perform statistical analysis using Linear Models for Microarray Data (limma) with empirical Bayes (eBayes) smoothing to the standard errors. Proteins with an FDR adjusted p-value <0.05 and a fold change >2 were considered significant. Heatmaps were constructed using R version 4.3.0 and the following packages: tidyverse, ggsci, and cowplot.

Flow cytometry

Isolated CD4^+^ and CD8^+^ T cells were stained and checked for isolation specific purity prior to assay. Cells were stained for cell surface markers and resuspended in eBioscience flow cytometry staining buffer (Invitrogen, cat #00-4222-26). Viability was assessed by staining with 7-AAD or propidium iodide Viability Staining Solution. For proliferation analysis, isolated T cells were stained with cell trace violet (Invitrogen catalog#: C34557). Cell Trace violet fluorescent intensity was measured at day 0 and day 5. For apoptosis analysis, cells were stained with Annexin V and propidium iodine according to manufacturer protocol. Double-positive cells were quantified for comparison.

Metabolic analysis

Metabolic analysis was completed as described previously^37^; specifically extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) was measured using the Seahorse XFe bioanalyzer. 2 × 10^5^ CD4^+^ or CD8^+^ T cells per well (≥8 wells per sample) were spun onto Cell-Tak (Corning)–coated seahorse 96-well plates and preincubated at 37°C for approximately 20 min in the absence of CO2. ECAR was measured under basal conditions and in response to activation (PMA/Ionomycin), 2 μmol L–1 oligomycin and 10 mmol L–1 2-Deoxy-D-glucose (2-DG).

Mixed lymphocyte reaction

Donor splenocytes were collected from 8 week-old BALB/c mice (Jackson laboratories). Spleens were meshed through 70 μm filter, red blood cells were lysed and whole splenocytes were resuspended in supplemented RPMI. Donor splenocytes were treated with Mitomycin C (25 μg/mL) for 30 min prior to coculture with CD4^+^ or CD8^+^ T cells. Donor splenocytes were plated at a 4:1 ratio to responder splenocytes and incubated for 48 h. Following incubation, cells were spun down, and supernatant was collected for ELISA analysis of IL2 (Biolegend) and IFNγ (R&D systems) according to manufacturer protocol.

Tumor study

Tumor growth studies were conducted in C57BL/6 Cre^+^-PKcs^wt/wt^, Cre^+^CD4-PKcs^fl/fl^, or Cre^+^CD8-PKcs^fl/fl^ mice. All mice were between 8 and 12 weeks and weighed between 20 and 30 grams at time of tumor cell injection. Animals were randomly assigned to control or experimental group with equal numbers of males and females represented. Mice were treated with tamoxifen for 7 days to induce recombination. MC-38 or B16-F10 cells were harvested during the logarithmic growth phase, washed in PBS, and implanted subcutaneously into the flank of each mouse using a 27-gauge needle (1x10^6^ cells in 100 μL) 2 days post tamoxifen treatment. Following tumor cell injection, mice were housed individually. Mice were monitored daily for signs of distress and tumor growth. Tumor growth was measured on alternate days using digital calipers. Tumor endpoint was established at a diameter of 20mm or earlier if mice exhibited signs of discomfort. Tumor volume was calculated using the formula: Tumor Volume=(Length×Width^2^)/2.

Skin transplants

Donor BALB/c tail skin was transplanted onto the backs of BALB/c mice or tamoxifen-treated C57BL/6 Cre^+^-PKcs^wt/wt^, Cre^+^CD4-PKcs^fl/fl^, or Cre^+^CD8-PKcs^fl/fl^ mice, and graft rejection monitored over time. All mice were between 8 and 12 weeks and weighed between 20 and 30 grams at time of skin transplant. Animals were randomly assigned to control or experimental group with equal numbers of males and females represented. Prior to surgery, mice were treated with tamoxifen for 7 days, rested for one day, then underwent skin transplant. Briefly, skin grafts were harvested from the tails of 8-week-old BALB/c mice from Jackson laboratories. Tails were removed, longitudinally cut and peeled from underlying tissue. Tail skin was placed in PBS on ice and cut to size, ~200mm.^3^ Recipient C57BL/6 mice were anesthetized to prepare graft bed on the dorsal surface between front paw shoulder blades about 1 cm caudal to neck fold. Graft bed was shaved and cleaned with ethanol and iodine. Graft bed was marked with surgical pen and incised with scissors. Full thickness native skin was removed down to panniculus carnosus, a thin layer of striated muscle deep to the dermis. Grafts were then placed on graft bed and attached with 6-0 prolene monofilament non-absorbable suture (Ethicon, cat: 8706). Following attachment of graft, mice were given meloxicam for pain and removed from anesthesia. Mice were woken up on heating pad and then placed in single housed cages. Animals were treated with NU7441 (10 mg/kg in 40% PEG400/saline) or vehicle (40% PEG400/saline/DMSO) daily via intraperitoneal injection. Graft necrosis was tracked daily for 30 days after surgery. Animals were euthanized upon graft death (>80% necrosis).

Reagents

A complete list of antibodies, cell lines, animal models, and reagents are provided in the key resources table.

Study approval

All animal studies completed in this study were approved by the Institutional Animal Care and Use Committee (Animal Welfare assurance number: A3063-01).

QUANTIFICATION AND STATISTICAL ANALYSIS

The data in this manuscript were analyzed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. A significance threshold of α = 0.05 was applied, with p ≤ 0.05 considered statistically significant. Details on the number of experimental replicates, animals per experiment, and statistical tests used are provided in the respective figure legends. For proteomic data, the data was normalized using Log_2_ Median Intensities and analyzed using proteoDA to perform statistical analysis using Linear Models for Microarray Data (limma) with empirical Bayes (eBayes) smoothing to the standard errors. Proteins with an FDR adjusted p-value <0.05 and a fold change >2 were considered significant. Heatmaps were constructed using R version 4.3.0 and the following packages: tidyverse, ggsci, and cowplot.

Supplementary Material

1

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116796.