The multikinase inhibitor sorafenib is the only standard first-line therapy for hepatocellular carcinoma (HCC). Here, we report the dose-dependent effects of sorafenib on the immune response, which is related to nuclear factor of activated T cells 1 (NFAT1) activity. In vitro and in vivo experiments were performed with low and high doses of sorafenib using human T cells and spontaneous developed woodchuck HCC models. In vitro studies demonstrated that following exposure to a high dose of sorafenib the baseline activity of NFAT1 in T cells was significantly increased. In a parallel event, high dose sorafenib resulted in a significant decrease in T cell proliferation and increased the proportion of PD-1 expressing CD8+ T cells with NFAT1 activation. In the in vivo model, smaller tumors were detected in the low-dose sorafenib treated group compared to the placebo and high-dose treated groups. The low-dose sorafenib group showed a significant tumor growth delay with significantly more CD3+ cells in tumor. This study demonstrates that sorafenib has immunomodulatory effects in a dose- and time-dependent manner. Higher dose of sorafenib treatment was associated with immunosuppressive action. This observed effect of sorafenib should be taken into consideration in the selection of optimum starting dose for future trials.

Background: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. Method: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. Results: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-β1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. Conclusion: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

Vascularized composite tissue allotransplantation (VCA) has transformed patients’ lives by enabling limb, face, abdominal wall, and penile transplants. Despite advancements in screening and immunosuppression, chronic rejection continues to limit the success of VCA. Lack of reliable preclinical models exacerbates this challenge. Here, we report on new mouse models of chronic rejection following heterotopic hind limb VCA. We employed different levels of MHC mismatch using CD8 knockout C57BL/6 mice as recipients along with BALB/c or B6 H2-Ab1^bm12 mice as donors. Transient CD4 T cell depletion was induced to allow graft maturation. Evaluation included gross findings, changes in immune status changes, production of donor-specific antibodies (DSA), C4d levels, and histopathological alterations. Two chronic rejection models displayed common features of clinical chronic graft rejection, such as skin stricture, hair loss, adnexal atrophy, extensive fibrosis and mast cell infiltration without widespread necrotic changes common in acute rejection. Similar to chronic rejection patients, large populations of activated B and plasma cells were detected in the recipient’s immune system as well as increased DSA and C4d production. Collectively, our models closely replicate the immunological and histopathological aspects of chronic graft rejection post-VCA, and could provide a new platform for evaluation of novel therapeutic interventions prior to clinical evaluation.

Background: Surgery, chemotherapy, and radiation often have limited utility for advanced metastatic disease in the liver, and despite its promising activity in select cancers, PD-1 blockade therapy similarly has minimal benefit in this setting. Curaxin, CBL0137, is an experimental anti-cancer drug that disrupts the binding of DNA to histones, destabilizes chromatin, and induces Z-DNA formation which may stimulate anti-tumor immune responses. Methods: Murine cell lines of colon (CT26) and breast (4T1) cancer were interrogated for survival and CBL0137-associated DNA changes in vitro. Immunocompetent models of liver metastases followed by CBL0137 hepatic arterial infusion (HAI) were used to examine in vivo tumor cell DNA alterations, treatment responses, and the immune contexture associated with CBL0137, both alone and in combination with anti-PD-1 therapy. Results: CBL0137 induced immediate changes to favor tumor cell death in vitro and in vivo with an efficient tumor uptake via the HAI route. Toxicity to CBL0137 was minimal and anti-tumor treatment effects were more efficient with HAI compared to intravenous delivery. Immune effects were pronounced with CBL0137 HAI with concurrent depletion of a specific population of myeloid-derived suppressor cells and maintenance of effector T cell populations. Conclusions: Combination of CBL0137 HAI with PD-1 blockade improved survival in 4T1 tumors but not in CT26 tumors, and therapeutic efficacy relies on the finding of simultaneous and targeted depletion of myeloid-derived suppressor cells and skewing of T cell populations to produce synergy with PD-1 blockade therapy.

Background: Vascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non‐life‐threatening conditions. Here we tested whether we could suppress anti‐graft immune activity by using a safe β₂‐adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system‐induced signalling through AR.Methods: A heterotopic hind limb transplantation model was used with C57BL/6 (H‐2b) as recipients and BALB/c (H‐2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of β₂‐AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro‐inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of β₂‐AR signalling in donor and recipient tissue were investigated with β₂‐AR^−/− strains.Results: Treatment with the β₂‐AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. β₂‐AR agonist decreased T‐cell infiltration into the transplanted grafts and decreased memory T‐cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN‐γ, IL‐6, TNF‐α, CXCL‐1/10 and CCL3/4/5/7) were detected following β₂‐AR agonist treatment, and there was a decreased expression of ICAM‐1 and vascular cell adhesion molecule‐1 in donor stromal cells.Conclusions: β₂‐AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress‐signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic.

Vascularized composite tissue allotransplantation (VCA) can replace severely damaged body parts but the unavoidable toxicity of high doses of immunosuppressive drugs, such as tacrolimus, required results in significant morbidity. Here we tested whether we could suppress immune activity in a mouse model of VCA by mimicking the natural immune suppression generated by nervous system-induced signaling of adrenergic receptors (AR) by using a safe and well-studied β-AR agonist (terbutaline). Using wild-type and β2-AR-knockout (KO) mice, we found that increased β2-AR signaling results in delayed rejection in VCA recipients, even with subtherapeutic doses of tacrolimus, and this was associated with changes in immune contexture, expression of pro-inflammatory cytokines and chemokines, and function of endothelial adhesion molecules. We propose that β-AR agonists can be used safely to mimic the natural suppression of immune responses generated by adrenergic stress signaling and thereby reduce the dose needed of other more toxic immunosuppressive drugs.