TNF-Like Weak Inducer of Apoptosis (TWEAK) Promotes Beta Cell Neogenesis from Pancreatic Ductal Epithelium in Adult Mice

Aim/Hypothesis The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice. Methods: C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreERTM: R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px. Results: TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion. Conclusions/Interpretation We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury

Proposed Model for TWEAK’s effect on pancreatic regeneration.

<p>TWEAK signals through Fn14, which is normally expressed at low levels in adult pancreatic ducts and upregulated after injury, resulting in proliferation of duct epithelium followed by differentiation to mature pancreatic cell types, including β-cells. In addition, TWEAK/Fn14 signaling induces a localized leukocyte infiltrate that may be beneficial to the tissue regeneration, thereby creating a positive feedback loop. Although not addressed in the current study, this likely occurs through induction of chemokines or cytokines that promote inflammatory cell recruitment. We speculate that the proliferating duct epithelial cells regress to a less differentiated state with progenitor potential, giving rise to Ngn3+ endocrine progenitors that then differentiate to new endocrine cells.</p

TWEAK treatment promotes proliferation of pancreatic duct and duct adjacent cells through its receptor Fn14.

<p>Pancreatic tissue of normal adult mice stained for Ki-67 (black) on day 3 after injection of (A) control Ig or (B) Fc-TWEAK, and costained with Ki-67 (red) and ductal epithelial marker CK (CK) (green) on day 4 after (C) control Ig or (D) Fc-TWEAK acute treatment. Scale bar  = 10 µm. (E–F) Quantification of the % Ki-67⁺ pancreatic (E) duct and (F) duct adjacent cells in mice treated with control Ig or Fc-TWEAK twice weekly. Data are shown as mean±SEM (n = 4). * p<0.05 for Fc-TWEAK vs control Ig treatment. (G–H) Quantification of the % Ki-67⁺ (G) duct and (H) duct adjacent cells in normal adult WT or Fn14 KO mice treated with control Ig or Fc-TWEAK twice per week. Data are shown as mean±SEM (n = 4) for percentage of Ki-67+ duct cells (E&G) or Ki-67+ duct adjacent cells per total duct cells (F&H); * p<0.05 for Fc-TWEAK treatment vs control Ig.</p

Chronic TWEAK treatment mimics the response to the partial pancreatectomy.

<p>Ductal structures with characteristics of regenerative foci were identified by H&E staining (A) at day 18 after TWEAK treatment twice weekly in all 4 treated animals. Inset of (A) is shown on serial sections stained with Ki-67 (B), CK (C), insulin (D) and glucagon (E). (F–H) Serial sections of area from another representative animal were stained with H&E (F), CD3 (G) F4/80 (H); isotype control (I) and Fn14 (J). The islets were labeled as “i”. Scale bar  = 50 µm in all panels.</p

Supplementary Figures 1–20 from Lymph Node–Targeted Vaccine Boosting of TCR T-cell Therapy Enhances Antitumor Function and Eradicates Solid Tumors

Supplementary Fig. S1: Design of a lymph node-targeted TCR-T cell therapy vaccine combination. Supplementary Fig. S2: Representative flowcytometric gating strategy. Supplementary Fig. S3: TCR-T therapy in irradiation pre-conditioned hosts does not improve activity against established B16F10 tumors. Supplementary Fig. S4: AMP-vaccination enhances TCR-T cell therapy and eradicates established B16F10 solid tumors. Supplementary Fig. S5: Vaccination alone is not effective to treat established B16F10 solid tumors. Supplementary Fig. S6: AMP-vaccination enhances TCR-T cell therapy against B16F10 solid tumors at multiple TCR-T dose levels. Supplementary Fig. S7: AMP-vaccination enhances TCR-T cell therapy and eradicates established solid tumors. Supplementary Fig. S8: Exposure to lymph nodes from AMP-vaccinated mice enhances TCR-T cell cytokine production ex vivo. Supplementary Fig. S9: Exposure to lymph nodes from AMP-vaccinated mice enhance antigen-specific naïve TCR-T cell cytokine production ex vivo. Supplementary Fig. S10: Gene ontology pathway classification for Nanostring analysis. Supplementary Fig. S11: AMP-vaccination induces differential mRNA levels across a broad range of transcripts present in lymph node and tumor. Supplementary Fig. S12: Tumor infiltrating TCR-T cells induced by AMP-vaccination do not exhibit exhausted phenotype. Supplementary Fig. S13: AMP-vaccine combination with TCR-T results in tumor-specific endogenous T cell responses in lymph nodes which correlate to tumor infiltrating responses. Supplementary Fig. S14: Depletion of TCR-T cells from long-term survivors. Supplementary Fig. S15: Schematic of vaccination protocol with fluorescently labelled components. Supplementary Fig. S16: AMP-vaccination induces DC activation within lymph nodes of HLA A*11:01 transgenic mice. Supplementary Fig. S17: NY-ESO-1-specific human TCR-T cells are specifically activated by AMP-peptide pulsed autologous DCs in vitro. Supplementary Fig. S18: mKRAS- and HPV16 E7-specific human TCR-T cells are specifically activated by AMP-peptide-pulsed autologous DCs in vitro. Supplementary Fig. S19: mKRAS-specific human TCR-T cells are specifically activated in vitro by autologous DCs pulsed with AMP-peptides incubated in mock in vivo conditions. Supplementary Fig. S20: mKRAS-specific human TCR-T cells co-cultured with AMP-peptide-pulsed autologous DCs in vitro enhance in vivo anti-tumor efficacy.</p

Supplementary Table 1 from Lymph Node–Targeted Vaccine Boosting of TCR T-cell Therapy Enhances Antitumor Function and Eradicates Solid Tumors

Flow Cytometry Antibodies</p

The Inflammatory Cytokines TWEAK and TNFα Reduce Renal Klotho Expression through NFκB

Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNFα, and siRNA-mediated knockdown of IκBα all activated NFκB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFκB with parthenolide prevented TWEAK- or TNFα-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAK-induced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNFα, downregulate Klotho expression through an NFκB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD