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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: Transplantation. 2021 Mar 1;105(3):540–549. doi: 10.1097/TP.0000000000003442

DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival

David K Harrison 1,2,, Zachary J Waldrip 1,2,, Lyle Burdine 1,3, Sara C Shalin 4, Marie Schluterman Burdine 1,2,*
PMCID: PMC7902289  NIHMSID: NIHMS1622716  PMID: 32890138

Abstract

Background

Organ transplantation is life-saving and continued investigations into immunological mechanisms that drive organ rejection are needed to improve immunosuppression therapies and prevent graft failure. DNA-dependent protein kinase catalytic subunit, DNA-PKcs, is a critical component of both the cellular and humoral immune responses. In this study, we investigate the contribution of DNA-PKcs to allogeneic skin graft rejection to potentially highlight a novel strategy for inhibiting transplant rejection.

Methods

Fully MHC mismatched murine allogeneic skin graft studies were performed by transplanting skin from BalbC mice to C57bl6 mice and treating with either vehicle or the DNA-PKcs inhibitor NU7441. Graft rejection, cytokine production, immune cell infiltration, and donor-specific antibody (DSA) formation were analyzed.

Results

DNA-PKcs inhibition significantly reduced necrosis and extended graft survival compared to controls (mean survival 14 days vs 9 days respectively). Inhibition reduced the production of the cytokines Interleukin (IL)2, IL4, IL6, IL10, TNFα, and IFNγ and the infiltration of CD3+ lymphocytes into grafts. Furthermore, DNA-PKcs inhibition reduced the number of CD19+ B cells and CD19+ CD138+ plasma cells coinciding with a significant reduction in DSAs. At a molecular level, we determined that the immunosuppressive effects of DNA-PKcs inhibition were mediated, in part, via inhibition of NFκB signaling through reduced expression of the p65 subunit.

Conclusion

Our data confirm that DNA-PKcs contributes to allogeneic graft rejection and highlight a novel immunological function for DNA-PKcs in the regulation of NFκB and concomitant cytokine production.

INTRODUCTION

The canonical function for DNA-dependent protein kinase catalytic subunit or DNA-PKcs is in the sensing and repairing of DNA double-stranded breaks (DSB).1 The protein is recruited to DSB sites where it acts in a complex with the protein KU70/80 to initiate non-homologous end joining (NHEJ).2 Likewise, DNA-PKcs is required for V(D)J recombination, a process that generates DSBs in order to rearrange V, D and J gene segments to promote antibody and T cell receptor diversity.3 Additionally, DNA-PKcs is required for V(D)J recombination-mediated isotype switching in B cells.4 Not surprisingly, all vertebrates harboring loss of function mutations in DNA-PKcs present with a severe immunodeficient phenotype with defects in antibody production and impaired B and T cell maturation.5,6 However, recent investigations highlight functions for DNA-PKcs in both the innate and adaptive immune responses that are distinct from its’ role in V(D)J recombination suggesting novel functions for this DNA repair kinase in the immune response.7

Stimulation of T cells results in DNA-PKcs activation by autophosphorylation of its activation site S2056.8 Once active, DNA-PKcs is a potent regulator of the transcription of pro-inflammatory cytokines via mechanisms that remain unclear.8,9,10 Our laboratory demonstrated that in T cells, DNA-PKcs regulates IL2 production via a NFAT-dependent mechanism whereby DNA-PKcs-mediated phosphorylation of the kinase Chk2 initiates the ubiquitination of CABIN1, an endogenous inhibitor of calcineurin.8,11,12

These findings have prompted investigations into the involvement of DNA-PKcs in immune-related diseases for possible therapeutic solutions. For example, Boulares et al., using the ovalbumin (OVA) allergen or house dust mite (HDM) mouse asthma model determined that DNA-PKcs contributed to the development of asthma by prompting the expression of Th2-cytokines including IL2, IL5, IL13 and IFNγ as well as the production of OVA-specific and HDM-specific IgE antibodies.9 Treatment with the DNA-PKcs inhibitor NU7441 mitigated asthma severity.

Organ transplantation is a highly effective therapy for patients with end-stage organ failure, however; its benefits are often negated when the organ is rejected by the recipient’s own immune system. The aforementioned studies confirming a role for DNA-PKcs in the immune system and the onset of immune-related disorders suggest that DNA-PKcs may be a driving force behind allogeneic graft rejection. If so, DNA-PKcs inhibitors could be a potential option for immunosuppression therapy. We utilized a fully mismatched MHC murine allogeneic skin graft model to determine the effects of DNA-PKcs inhibition on graft rejection. This model is a highly used tool for investigating immunological mechanisms of graft rejection. Our results indicate that DNA-PKcs plays a significant role in the allogeneic immune response.

MATERIALS AND METHODS

Materials

Flow cytometric antibodies - anti-CD3, CD4, CD8, CD19 and CD138 antibodies (cat #100220, 100509, 100711, 115505, 142505 BioLegend). Immunohistochemistry antibodies - anti-CD3 antibody [CD3-12] (ab11089, Abcam) and anti-F4/80 (cat # 70076, Cell Signaling). DSA analysis antibodies - F(ab)2’-IgM-APC (Jackson Immunoresearch Laboratories) and F(ab)2’-IgG-FITC (cat # F11021, Thermo Fisher Scientific). Western blot antibodies - GAPDH antibody (ThermoFisher cat# MA5-15738); IκB and p32/36 IκB (ThermoFisher cat# MA5-15087, Cell Signaling cat# 4814T); p100/p52, p105/p50 and p65/RelA (Cell Signaling cat #55764). MILLIPLEX MAP Mouse High Sensitivity T Cell Magnetic Bead Panel (Millipore, MHSTCMAG-70K). NU7441 (Selleckchem, S2638). PMA (AAJ63916MCR), PHA (NC1293968) and ionomycin (BP25271), Fisher Scientific. Human T-Activator CD3/CD28 (Gibco, Cat # 11161D). Jurkat T cells (ATCC, Clone E6-1, cat # TIB-152)

Mice

Donor ear or tail skin was taken from BalbC mice and transplanted onto the backs of recipient C57bl6 or NOD.CB17-Prkdc scid/J mice purchased from Jackson Laboratories (stock #000664, #001303 respectively). Mice (8 weeks, 25g) were housed in a pathogen-free facility at Arkansas Children’s Research Institute. All protocols were approved by the Institutional Animal Care and Use Committee.

Skin allograft procedure

The skin graft procedure was modified from Pakyari et al.13 In summary, ear or tail skin was dissected from euthanized donor BalbC mice. The back skin of recipient C57bl6 or DNA-PKcs KO mice was tented and cut to prepare the graft bed. Grafts were sutured in place using 6-0 surgical sutures. Mice received daily intraperitoneal injections of either vehicle alone (Control, 40% PEG400/saline/DMSO) or NU7411 (10 mg/kg in 40% PEG400/saline). Grafts were scored visually daily for percent necrosis and considered rejected when necrosis reached 80% of the total graft.

Splenocyte and CD4+ T cell isolation

Harvested spleens were passed through a 70 μm nylon cell strainer into PBS. BD PharM Lyse™ (Fisher Sci.) was added to the cell suspension. CD4+ T cells were isolated using the MidiMACS system (cat# 130-042-301) with the Naïve CD4+ T cell isolation kit (cat# 130-104-453) from Miltenyi Biotec.

Cytokine analysis

Mixed lymphocyte reactions were performed using splenocytes from C57bl6 (responder) and BalbC (stimulator) mice. BalbC splenocytes were treated with 25 μg/mL mitomycin C for 30 minutes at 37°C. Cells were mixed at a ratio of 1:10 responder to stimulator and cultured at 37°C for 48 hours in medium consisting of 0.1mM 2-Mercaptoethanol (2-ME). Supernatants were assayed using the Cytokine 6-Plex Mouse Panel (Invitrogen) for target cytokines following the manufacturer’s protocol. Cytokines were measured with the Luminex 200 platform. The Luminex assay was performed in triplicate and samples in duplicate.

BrdU analysis

BalbC splenocytes were treated with 25 μg/mL mitomycin C then mixed with C57bl6 splenocytes at a ratio of 1:10 responder to stimulator and cultured at 37°C in medium containing 0.1mM 2-ME. After 48 hours, cells were assayed using the BrdU Cell Proliferation Assay Kit (Millipore) per the manufacturer’s protocol. The BrdU assay was performed in triplicate.

Immunohistochemistry of skin grafts

Skin grafts (n=3 mice/group) were harvested from randomly picked recipient mice on day 8, fixed in formalin and stained with an anti-CD3 antibody. A derm-pathologist blindly scored the grafts by counting the number of positive cells per high power field of view. 10 fields of view were analyzed from each graft (30 fields/group). Average cell numbers were plotted and a t-test was performed to determine significance.

Flow cytometry

Splenocytes isolated from KO, NU7441, or control treated mice on day 10 were plated in flow cytometry wash buffer (5% FBS, 0.05% sodium azide in PBS). TruStain fcX (1 μg, Biolegend) was added to cells and incubated on ice for 10 minutes. Cells were incubated with anti-CD4, CD8, CD19 and/or CD138 antibodies for 1h at 4°C. 7-AAD (0.25 μg, Biolegend), a viability marker, was added and analyzed using Guava easycyte Flow Cytometer.

Donor-specific antibody detection

Donor splenocytes were isolated from the spleens of BalbC mice and incubated with plasma collected from mice treated with either NU7441 or control (n ≥ 5/group) for 30 minutes. Washed cells were incubated with F(ab)2’-IgM-APC or F(ab)2’-IgG-FITC, and analyzed using a Guava easycyte Flow Cytometer. Mean fluorescence intensity (MFI) was used to determine DSA production.

NFκB luciferase reporter assay

NF-κB reporter Jurkat cells (BPS Bioscience, cat. # 60651) were treated with 5 μM NU7441 prior to activation with 1 μM ionomycin and PMA. Cells were incubated for 6 hours followed by addition of 100 μL One-Step Luciferase Assay Buffer (BPS Biosciences, Cat. # 60690-1). Luminescence measurements were taken on a BioTek Synergy plate reader.

Western blot analysis

Jurkat T cells, primary splenocytes and CD4+ T cells were treated with 5 μM NU7441 (or DMSO) for 30 minutes. Cultures were activated with PMA (50 ng/ml) and PHA (1 μg/ml), PMA (50 ng/ml), Ionomycin (1 μg/ml) or a 1:1 ratio of cells: Dynabeads Mouse T Activator CD3/CD28 (ThermoScientific cat# 11456D) for 3 hours. Western blots were probed with anti-p100/p52, p105/p50, p65, p/IκB overnight then treated with the secondary antibody.

Statistical analysis

Analysis of significance was done using standard t-test and expressed as the mean ± standard deviation. Assays were performed in triplicate. P ≤ 0.05 was considered significant. Kaplan-Meier analysis was used to analyze survival curves. A log-rank test was performed on the Kaplan-Meier survival curves and a t-test and chi-square included.

RESULTS

Inhibition of DNA-PKcs reduces necrosis and extends allogenic skin graft survival

To determine if DNA-PKcs contributes to graft rejection, we performed allogeneic murine skin graft studies using a commercially available DNA-PKcs inhibitor NU7441. NU7441 is a highly specific inhibitor documented to be effective at in both in vitro and in vivo studies.14 Skin from BalbC mice were transplanted onto the backs of C57bl6 mice or DNA-PKcs −/− (KO) mice (Jackson Laboratory, stock #001303) followed by daily intraperitoneal injections of either control (vehicle alone) or NU7441. KO mice develop normally but manifest a compromised immune system with decreased levels of B and T cells. KOs were used as a control to compare the similarity between the genetic loss of DNA-PKcs activity and chemical inhibition of DNA-PKcs on graft rejection. Additionally, syngeneic transplants were performed with BalbC mice. Starting on day 6, skin grafts were visually inspected for signs of rejection. Each day, the percent of total graft necrosis was recorded by an observer blinded to the treatment group. Grafts were considered rejected when 80% of the graft was necrosed. Figure 1A plots the average necrosis rates for each group over a 10-day period. While the average necrosis for control treated grafts was close to 90%, NU7441 treatment significantly lowered the rate to 20% (p < 0.01). As expected, the grafts from KO mice had significantly reduced necrosis. Figure 1B demonstrates progression of necrosis in a graft from each group. Kaplan-Meier survival analysis indicated that DNA-PKcs inhibition significantly extended graft survival. Control treated grafts survived to a mean of 9 days while DNA-PKcs inhibition extended survival to a mean of 14 days (Figure 1C) (control: n=9, NU7441: n=7, KO: n=5, p<0.05). Given the robust response of a fully mismatched allogenic skin graft model, the extension of 5 days is a significant result and confirms that DNA-PKcs is involved in graft rejection. This finding validates further investigations into the use of DNA-PKcs inhibitors to prevent rejection in vascularized transplant models.

Figure 1. Inhibition of DNA-PKcs reduces necrosis and extends survival of allogenic skin grafts.

Figure 1.

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A) Average percent necrosis observed overtime in ear skin grafts (n=10/group, *p ≤ 0.01 compared to control, Error bars = s.d.). B) Images of grafts shows the progression of necrosis. Control treated (40% PEG400/saline/DMSO) graft shows 80% necrosis. C) Kaplan-Meier analysis- inhibiting DNA-PKcs activity significantly extended graft survival (tail skin). Control - mean survival day of 9, NU7441 – 14, p<0.05 compared to control (saline: n=9, NU7441: n=7, KO: n=5). Syn – syngeneic

Loss of DNA-PKcs activity attenuates cytokine production

To further characterize the effects of DNA-PKcs inhibition on the response to disparate MHC alloantigens, we analyzed cytokine production using mixed lymphocyte reactions (MLRs). Splenocytes isolated from BalbC mice and treated with mitomycin C were used as stimulators and splenocytes from C57bl6 mice as responders. Inhibition of DNA-PKcs significantly disrupted production of IL2, which is known to have a significant role in graft rejection. This complements our previously published data indicating DNA-PKcs regulates IL2 production.8 NU7441 treatment also reduced expression of the cytokines IL4, IL6, IL10, TNF-α, and IFNγ (Figure 2). Likewise, analysis of cytokine production in WT or KO splenocytes activated by PMA/ionomycin and treated with or without NU7441, also demonstrated significant defects in production of the above cytokines, although a decrease in IL4 and IFNγ was not observed in KO splenocytes (Figure 3). These cytokines are essential mediators of the immune response and a decrease in expression indicates that DNA-PKcs is critical for a proper allogeneic immune response.

Figure 2. DNA-PKcs inhibition reduces MHC mismatched alloantigen-induced cytokine production.

Figure 2.

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Splenoyctes isolated from C57bl6 mice were mixed with Balbc splenocytes treated with mitomycin C. Cytokine levels were detected in the medium using Multiplex bead-based cytokine assays. NU7441 (5 μM) significantly reduced the production of all investigated cytokines. Error bars = s.d. p ≤ *** 0.001, **0.01 compared to - NU7441.

Figure 3. NU7441 treatment attenuates cytokine production.

Figure 3.

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Cytokine production in isolated splenocytes stimulated with PMA/Iono was measured using Multiplex bead-based cytokine assays. NU7441 (5 μM) significantly reduced the production of all investigated cytokines. Error bars = s.d. p ≤ *** 0.001, **0.01, *0.05 compared to control. n=3 mice/group

DNA-PKcs inhibition stunts T lymphocyte migration into the grafts

To better understand the effect of DNA-PKcs inhibition on T cells, we analyzed proliferation in MLRs. BalbC splenocytes, treated with mitomycin C, were co-cultured with splenocytes from C57bl6 mice. NU7441 significantly reduced the proliferation of activated T cells (Figure 4). We evaluated the level of T lymphocytes from the spleens of mice treated with control or NU7441. Splenocytes isolated on Day 10 were labeled with either anti-CD4 or anti-CD8 antibodies and measured by flow cytometry. As anticipated, both types of T cells were drastically reduced in the control KO mice. (Figure 5A). Although NU7441 treatment tended to reduce the absolute number of CD4+ T helper and CD8+ cytotoxic cells (Figure 5A) there were no statistical differences compared to control. We next analyzed the effect of DNA-PKcs inhibition on T cell infiltration into the grafts. Grafts isolated on Day 10, when rejection was at its peak in control grafts, were stained with anti-CD3 antibody to visualize infiltrating T cells. T cell infiltration was highest in control grafts but NU7441 treatment significantly reduced infiltration (Figure 5B). The KO grafts had the lowest level of infiltration as expected. Additionally, we analyzed the level of macrophages in KO and NU7441 treated grafts, but saw no difference (Figure S1).

Figure 4. DNA-PKcs inhibition reduces T cell proliferation in response to alloantigens.

Figure 4.

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Splenocytes isolated from C57bl6 mice were mixed with Balbc splenocytes treated with mitomycin C at a 1:10 ratio. Proliferation was measured by BrdU incorporation. NU7441 (5 μM) significantly reduced the proliferation of activated immune cells. Error bars = s.d. p ≤ **0.01 compared to - NU7441.

Figure 5. Immune cell infiltration into transplant skin grafts is stunted in NU7441 treated animals.

Figure 5.

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A) Flow cytometry analysis of splenocytic immune cells isolated from control or NU7441 treated or KO mice. CD4+ and CD8+ T cells were lower in NU7441 mice but not significantly. B) Histological images of grafts stained with anti-CD3 antibody. Graph is the average number of immune cells (brown, punctate staining) per high power field (HPF)-10, 20x field of views. Immune cell infiltration was decreased in KO and NU7441 treated animals compared to control. Error bars = s.d. p ≤ * 0.05, **0.01 n=3 mice/group (30 HPFs/group)

Reduced levels of CD19+ B cells and CD19+ CD138+ plasma cells with NU7441 treatment

We analyzed the level of B cells by flow cytometry using anti-CD19 antibodies. As anticipated, B cells were drastically reduced in KO mice. (Figure 6). Interestingly, NU7441 treatment significantly lowered the level of CD19+ B cells in mice compared to controls. IL6 is known to play a critical role in the proliferation of B cells and their maturation into plasma cells which secrete high levels of antibodies.15 Given that DNA-PKcs inhibition reduced IL6 production, we analyzed the level of CD19+ CD138+ plasma cells in our recipient mice (Figure 6). Statistical analysis revealed a significant drop in the level of plasma cells in NU7441 treated mice. As anticipated, both B cell populations were drastically reduced in KO mice. (Figure 6).

Figure 6. Reduced levels of CD19+ B cells and CD19+ CD138+ plasma cells with DNA-PKcs inhibition.

Figure 6.

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Flow cytometry analysis of splenocytic immune cells isolated from control, NU7441 treated or KO mice. CD19+ B cells as well as CD19+ CD138+ plasma cells were significantly reduced with NU7441 treatment compared to levels in control treated mice. Error bars = s.d. *p ≤ 0.01 compared to control. n= 5 mice/group.

Treatment with DNA-PKcs inhibitors reduces donor-specific antibody production

Donor-specific antibodies are a main driver of antibody-mediated chronic graft rejection (AMR) and there are currently no drugs used in the clinic that effectively prevent DSA formation in transplant patients. To determine if the drop in B cells and plasma cells observed in NU7441 treated mice correlated to a decrease in DSA production, we analyzed the level of DSAs in the serum of mice treated with control or NU7441. Donor splenocytes were incubated with serum harvested from recipient mice on Day 10 and analyzed for bound IgM and IgG by flow cytometry. Detected bound antibodies are those developed in response to donor tissue and specifically recognize donor cells. Donor-specific IgM and IgG antibodies were elevated in control treated mice (Figure 7). Most importantly, inhibiting DNA-PKcs activity with NU7441 drastically reduced DSAs to levels similar to those observed in DNA-PKcs KO mice. This data suggests a mechanism by which DNA-PKcs inhibition mitigates the production of antibodies generated in response to donor tissue by reducing IL6 production. This mechanism is supported by published data showing that treatment with monoclonal anti-IL6 antibodies can reduce DSA formation in a mouse skin graft model.16 DSAs are associated with chronic AMR, therefore, they likely have little to do with the delayed acute skin graft rejection observed in our non-vascularized model. However, these are notable findings given the great need for new therapeutic targets to prevent AMR of solid organs and should be further evaluated in vascularized transplant models.

Figure 7. Treatment with DNA-PKcs inhibitor reduces donor-specific antibody formation.

Figure 7.

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Flow cytometry was used to detect IgM and IgG DSAs in serum from recipient mice. Levels were decreased in KO and NU7441 compared to control treated mice. X = average, MFI = mean fluorescent intensity, * p ≤ 0.01 compared to control, n ≥ 4.

DNA-PKcs is required for expression of the p65 subunit of the transcription factor complex NFκB.

The transcription factor NFκB is a complex of proteins including the precursors p105 and p100 and the subunits p50, p52 and p65. Given its well published role in driving expression of critical cytokines, including IL2 and IL6, we evaluated the effect of DNA-PKcs inhibition on NFκB.17,18

To determine if NFκB activity was altered with loss of DNA-PKcs activity, we treated T cells modified to stably express a NFκB luciferase reporter construct with NU7441. Activation of T cells caused induction of NFκB activity which was significantly inhibited by the loss of DNA-PKcs activity (Figure 8). We next determined if the loss of NFκB activity resulted from a change in expression of the NFκB inhibitor protein, IκB. Treatment with NU7441 reduced the phosphorylation of serines 32 and 36 of IκB which correlates with a decrease in NFκB transcriptional activity and suggests that DNA-PKcs may play a role in regulating IκB degradation (Figure 9). Our data also indicated that DNA-PKcs inhibition had a significant effect on the expression of the p65 subunit. A decrease in protein expression of p65 was detected in Jurkat T cells treated with PMA/PHA, primary splenocytes activated with PMA/iono as well as in isolated CD4+ T cells stimulated with antibodies to CD3 and CD28. To confirm that loss of p65 expression was directly due to a decrease in DNA-PKcs activity, we analyzed levels in splenocytes harvested from DNA-PKcs KO mice. While stimulation of splenocytes induced expression of p65 in WT splenocytes, we did not observe an increase in p65 expression in KO cells indicating a direct link between DNA-PKcs and p65 expression (Figure 9). We did not detect a change in p65 mRNA levels with DNA-PKcs inhibition (data not shown) indicating a post-transcriptional mechanism of regulation. DNA-PKcs inhibition did not alter expression of p100, p105, p50 or p52 (Figure 9). These findings suggest that DNA-PKcs modulates the immune system and expression of cytokines such as IL2 and IL6, in part, by enhancing NFκB activity through regulation of p65 expression.

Figure 8. DNA-PKcs inhibition significantly reduces NFκB activity in T cells.

Figure 8.

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PMA/PHA stimulated T cells were treated with the DNA-PKcs inhibitor NU7441 and analyzed for NFκB activity using a luciferase reporter assay. error bars = s.d. **p< 0.05 compared to untreated. DMSO = vehicle control

Figure 9. DNA-PKcs is required for expression of the p65 subunit of NFκB.

Figure 9.

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A) Western blot analysis and Image J quantitation indicated that loss of DNA-PKcs activity with NU7441 significantly reduced expression of the p65 subunit in Jurkat cells stimulated with PMA/PHA, primary splenocytes stimulated with PMA/ionomycin and isolated splenocytic CD4+ T cells activated with antibodies to CD3/CD28. Expression of p65 was also lost in splenocytes harvested from DNA-PKcs KO mice. B) NU7441 treatment did not alter expression of the NFκB subunits p100, p105, p50, p52, or IκB in Jurkat T cells stimulated with PMA/PHA but did reduce IκB phosphorylation. WT – wild type

DISCUSSION

Recent studies have demonstrated that DNA-PKcs, a well-known DNA double strand break repair kinase, is involved in both the humoral and cellular immune responses and that these effects are independent of its role in DNA repair.7 This has spurred investigations into the role of DNA-PKcs in the onset of immune-related disorders such as asthma, arthritis and in this study, the rejection of allogeneic grafts.19-21 Here, we seek to determine if DNA-PKcs is involved in immunological mechanisms that trigger rejection of allogeneic transplants. Identifying a role for DNA-PKcs in graft rejection would support a novel therapeutic application of DNA-PKcs inhibitors (which are currently in Phase I oncological clinical trials) in transplant immunosuppression.22

We performed allogeneic skin graft studies in mice treated with the DNA-PKcs inhibitor NU7441 which significantly reduced necrosis and extended survival by a mean of 5 days; a significant extension given the robustness of rejection in a fully mismatched MHC skin graft model. This result suggests a critical function for DNA-PKcs in mechanisms that promote graft rejection.

In an effort to further define how loss of DNA-PKcs activity impedes graft rejection, we analyzed the effects of NU7441 on cytokine production in MLRs. NU7441 treatment blocked IL2 production in response to an allogeneic antigen. This is an important observation given the connection between IL2 and cell-mediated transplant rejection. IL2 is typically elevated in patients actively undergoing graft rejection and our most effective immunosuppressants in clinical use, such as FK506, prevent cell-mediated rejection by inhibiting IL2 production. Therefore, this effect is most likely a main contributor to the delayed rejection of NU7441 treated grafts. DNA-PKcs inhibition also reduced TNFα, IFNγ and IL6, all cytokines that have previously been reported to promote graft rejection. Furthermore, there was reduced expression of IL4 and IL10, anti-inflammatory cytokines believed to dampen the immune response, however, both IL4 and IL10 can act as pro-inflammatory molecules under certain environmental stressors, so the effects of these cytokines on transplant rejection are unclear.23,24 Of note, immune cells isolated from DNA-PKcs KO mice generated normal levels of both IFNγ and IL4 when stimulated with PMA/ionomycin but NU7441 treatment significantly reduced production of both. These differences may be a result of off-target effects of NU7441 but may also indicate a function of DNA-PKcs in adult immune cells that are not apparent when DNA-PKcs activity is absent during embryonic development and have a dysfunctional immune system. In summary, this data suggests that inhibition of DNA-PKcs activity impedes graft rejection through erroneous cytokine production, particularly IL2, resulting in an immunosuppressed environment.

We next evaluated how DNA-PKcs deficiency altered T cell proliferation when stimulated with a disparate MHC molecule. Treatment with NU7441 significantly reduced proliferation of T cells. Given, the critical role of IL2 in T cell proliferation, this result is, in part, due to a drop in IL2 production. We also analyzed the effects of DNA-PKcs inhibition on immune cells in vivo. Although the levels of CD4+ and CD8+ T cells in NU7441-treated mice were decreased, the results were not as significant as expected from our proliferation data in MLRs. This result is mostly likely due to the difference between NU7441 concentration in in vitro and in vivo experiments. Zhao et al., has reported that NU7441 at 10mg/kg (as used in our study) is significantly cleared from organs, including the spleen, 4 hours after injection.14 The limited solubility and short half-life preclude complete DNA-PKcs inhibition in a mature immune system. We surmise using DNA-PKcs inhibitors with improved solubility, such as those used in phase I clinical trials, may result in more pronounced T cell effects and further prolongation of graft survival.22 Despite these limitations, we do see a significant drop in the total number of T cells (CD3+) that have infiltrated into the graft. The decrease in T cell infiltration and cytokine production suggests that in the mature immune system DNA-PKcs is required for the function of T cells and their ability to respond to immune stimuli.

In response to donor antigen, B cells proliferate and differentiate into plasma cells which secrete high levels of donor-specific antibodies that are a critical component of AMR.19 Given that loss of DNA-PKcs activity in KO mice results in reduced B cells and a corresponding decrease in antibody production, we evaluated the level of CD19+ B cells and observed a significant decrease in the number of B cells in mice treated with NU7441 compared to controls. A key point with respect to B cell biology and AMR is our observation that DNA-PKcs is required for IL6 production. This was observed in both DNA-PKcs KO splenoctyes and cells treated with NU7441. IL6 drives the proliferation and maturation of CD19+ B cells into CD19+ CD138+ plasma cells.25 Jordan et al. have indicated that IL6 plays a critical role in the formation of DSAs and AMR, and elevated serum levels of IL6 in transplant patients correlate with an increased risk for AMR.26,15 In clinical studies, treatment of patients undergoing AMR with anti-IL6 receptor monoclonal antibodies significantly reduced DSA levels and promoted graft survival.27 In the current study, DNA-PKcs inhibition decreased the level of CD19+ and CD19+ CD138+ B cells which corresponded to a decrease in both IgM and IgG DSA production in NU7441 treated mice. However, due to the low frequency detected of both CD19+ CD138+ B cells and DSAs, the impact of NU7441 on humoral immunity is inconclusive in this study. This finding does, however, correlate with previous studies showing that loss of DNA-PKcs activity hindered the production of OVA or HDM specific IgE antibodies in asthma models and that treatment with anti-IL6 antibodies reduced both IgM and IgG DSAs in mouse models of skin allografts.12,14,16 A more appropriate model such as the murine vascularized kidney transplant model needs to be used to properly investigate the effect of NU7441 on humoral immunity during transplant rejection. It is also important to note that DNA-PKcs is required for V(D)J recombination, a process of antibody diversification that is required for proper antibody production.3 The decrease in DSA formation in this study can also be partially attributed to a defect in V(D)J recombination. While DSAs and the humoral immune response have little to do with the acute rejection of skin grafts in this model, the potential of DNA-PKcs inhibitors to prevent DSAs is important to the development of effective therapies that can protect against chronic AMR and a topic of future investigation.

IL2 and IL6 expression are highly regulated by the transcription factor NFκB.17,18 NFκB is a complex of proteins including p65 which contains a DNA-binding region and is critical for the transcriptional activity of NFκB. It is involved in numerous immune responses including lymphocyte maturation and the expression of multiple pro-inflammatory cytokines.28 DNA-PKcs has been previously shown to alter NFκB via phosphorylation of the p50 subunit which enhances the DNA binding ability of the complex.29 In this study, we show that DNA-PKcs inhibition significantly attenuates NFκB activity in T cells through regulation of p65 expression. Inhibition of DNA-PKcs with NU7441 in activated T cells or in DNA-PKcs KO splenocytes resulted in loss of p65 expression but not the p50 or p52 subunits. Additionally, we did not see a drop in p65 mRNA indicating that DNA-PKcs is affecting the post-transcriptional status of p65. Investigations concerning mechanisms behind p65 regulation by DNA-PKcs are ongoing. We have previously demonstrated that DNA-PKcs regulates NFAT activity.8 Those findings combined with our current data indicating a role for DNA-PKcs in the regulation of NFκB, explain the significant drop in cytokine production upon DNA-PKcs inhibition.

In conclusion, we confirm that DNA-PKcs has a significant function in the immune mechanisms that drive rejection of allogeneic skin grafts. This study provides proof of concept that DNA-PKcs inhibitors should be evaluated for their potential utility as solid organ transplant therapy with current calcineurin inhibitors or as stand-alone therapy.

Supplementary Material

Supplemental Digital Content to Be Published (cited in text)

Acknowledgments

Funding

Center for Pediatric Translational Research NIH COBRE P20GM121293.

Abbreviations

AMR

antibody-mediated rejection

DNA-PKcs

DNA dependent-protein kinase catalytic subunit

DSA

donor specific antibody

DSB

double stranded break

HDM

House dust mite

IL

interleukin

MLR

mixed lymphocyte reaction

NHEJ

non-homologous end joining

NFAT

Nuclear factor of activated T-cells

NFκB

Nuclear factor kappa-light-chain-enhancer of activated B cells

OVA

Ovalbumin allergen

Footnotes

Disclosure

The authors declare no conflicts of interest.

References

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