Recent clinical evidence revealed that the use of beta-blockers such as propranolol, prior to diagnosis or concurrently with chemotherapy, could increase relapse-free and overall survival in breast cancer patients. We therefore hypothesized that propranolol may be able to increase the efficacy of chemotherapy either through direct effects on cancer cells or via anti-angiogenic mechanisms. In vitro proliferation assay showed that propranolol (from 50-100 μM) induces dose-dependent anti-proliferative effects in a panel of 9 human cancer and “normal” cell lines. Matrigel assays revealed that propranolol displays potent anti-angiogenic properties at non-toxic concentrations (<50 μM) but exert no vascular-disrupting activity. Combining chemotherapeutic drugs, such as 5-fluorouracil (5-FU) or paclitaxel, with propranolol at the lowest effective concentration resulted in synergistic, additive or antagonistic effects on cell proliferation in vitro depending on the cell type and the dose of chemotherapy used. Interestingly, breast cancer and vascular endothelial cells were among the most responsive to these combinations. Furthermore, Matrigel assays indicated that low concentrations of propranolol (10 – 50 μM) potentiated the anti-angiogenic effects of 5-FU and paclitaxel. Using an orthotopic xenograft model of triple-negative breast cancer, based on injection of luciferase-expressing MDA-MB-231 cells in the mammary fat pad of nude mice, we showed that propranolol, when used alone, induced only transient anti-tumor effects, if at all, and did not increase median survival. However, the combination of propranolol with chemotherapy resulted in more profound and sustained anti-tumor effects and significantly increased the survival benefits induced by chemotherapy alone (+19% and +79% in median survival for the combination as compared with 5-FU alone and paclitaxel alone, respectively; p<0.05). Collectively our results show that propranolol can potentiate the anti-angiogenic effects and anti-tumor efficacy of chemotherapy. The current study, together with retrospective clinical data, strongly suggests that the use of propranolol concurrently with chemotherapy may improve the outcome of breast cancer patients, thus providing a strong rationale for the evaluation of this drug combination in prospective clinical studies.

Background:The use of β-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether β-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma.Methods:Seven β-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma.Results:Three β-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. β-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, β-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, β-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01).Conclusion:β-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.

Key Points• In vivo Treg effect depends on TNFa produced by T cells.• TNF/TNFR2 interaction represents a novel immune checkpoint therapy to modulate alloreactivity after allo-HCT. Therapeutic CD41 Foxp3 1 natural regulatory T cells (Tregs) can control experimental graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) by suppressing conventional T cells (Tconvs). Treg-based therapies are currently tested in clinical trials with promising preliminary results in allo-HCT. Here, we hypothesized that as Tregs are capable of modulating Tconv response, it is likely that the inflammatory environment and particularly donor T cells are also capable of influencing Treg function. Indeed, previous findings in autoimmune diabetes revealed a feedback mechanism that renders Tconvs able to stimulate Tregs by a mechanism that was partially dependent on tumor necrosis factor (TNF). We tested this phenomenon during alloimmune response in our previously described model of GVHD protection using antigen specific Tregs. Using different experimental approaches, we observed that control of GVHD by Tregs was fully abolished by blocking TNF receptor type 2 (TNFR2) or by using TNF-deficient donor T cells or TNFR2-deficient Tregs. Thus, our results show that Tconvs exert a powerful modulatory activity on therapeutic Tregs and clearly demonstrate that the sole defect of TNF production by donor T cells was sufficient to completely abolish the Treg suppressive effect in GVHD. Importantly, our findings expand the understanding of one of the central components of Treg action, the inflammatory context, and support that targeting TNF/TNFR2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD. (Blood. 2016;128(12):1651-1659

CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell–mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-D-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC 50 , 2 mM) but no other conventional CCL2-associated events. We used the classic competitive CCR2

BackgroundIn rheumatoid arthritis (RA), regulatory T cells (Tregs) are defective in their suppressive capacities and fail to control chronic inflammation. TNF-α is involved in inhibition of Treg differentiation and activation, likely via activation of TNF type 1 receptor (TNFR1).1 Conversely, activation of TNFR2 on Tregs is critical for their phenotypic and functional stability in the inflammatory environment.2 Moreover, it has been shown that therapeutic TNF blockade with the anti-TNF monoclonal antibody adalimumab restores the potency of Treg cell suppression in RA by binding to membrane TNF- α on monocytes and promoting Treg cell expansion through enhanced TNFR2 signaling.3 In the present study we aimed to establish the role of TNFR2 on Tregs in control of inflammation at multiple levels, by: 1) studying the action of TNF on Treg function in the presence and absence of TNFR2 in vitro, 2) testing the severity of a model of skin inflammation in TNFR2KO mice, 3) evaluating the evolution of TNFR2-expressing Treg from RA patients during anti-TNF treatment.Materials and methodsMice deficient in the TNFR2 gene (TNFR2 KO) and TNFR2 lox/lox mice to conditionally delete TNFR2 specifically in Tregs were used. CD4+CD25+Treg cells were purified by magnetic sorting. Cell phenotype was evaluated by flow cytometry. ATP concentrations were determined by luminometry. Skin inflammation was induced by applying an imiquimod-containing ointment, to the skin. Peripheral blood Treg where characterised before and after 3 months of anti–TNF treatment in 10 RA patients.ResultsIn vitro, TNF-α enhanced Foxp3 maintenance through TNFR2 signalling in cultured Tregs. In vivo, TNFR2-negative Treg cells, from both TNFR2KO and TNFR2 lox/lox mice, had lower spontaneous suppressive capacities (lower ATP hydrolysis, inhibition of effector T cells proliferation and IFN-γ production). Compared to wt mice, TNFR2KO mice had enhanced skin-inflammation and decreased Treg frequency in lymph nodes. In RA patients, TNF blockade induced an increase in the frequency of TNFR2-expressing Tregs at 3 months of treatment vs. the baselineConclusionsTNFR2 signalling on Tregs may play a major role in controlling inflammation and can be activated both by TNF-α and anti-TNF treatment. Further studies to dissect TNFR2 dependent pathways on Tregs are warranted.ReferencesNie H, Zheng Y, Li R, et al. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nat Med2013;19:322-8.Chen X, Wu X, Zhou Q, et al. TNFR2 is critical for the stabilisation of the CD4+Foxp3+ regulatory T cell phenotype in the inflammatory environment. J Immunol. 2013;190:1076-84.Nguyen DX, Ehrenstein MR. Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis. J Exp Med2016;213:1241-53.