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Purpose:While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1–positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti–4-1BB mAbs.Experimental Design:We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1–expressing murine MC38 cells to assess in vivo antitumoral activity.Results:PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti–PD-L1–resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity.Conclusions:The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB–mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development.See related commentary by Shu et al., p. 3182

Supplementary Table from The PD-L1/4-1BB Bispecific Antibody–Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner

Supplementary Figure from The PD-L1/4-1BB Bispecific Antibody–Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner

Purpose:While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1–positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti–4-1BB mAbs.Experimental Design:We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1–expressing murine MC38 cells to assess in vivo antitumoral activity.Results:PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti–PD-L1–resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity.Conclusions:The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB–mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development.See related commentary by Shu et al., p. 3182

PDF file - 91K, Supplementary Table 1. List of genes analyzed for expression by real-time, quantitative RT-PCR. All the amplifications were performed on a 7000 ABI Prism Instrument (Applied Biosystems), using pre-developed assays ("Mm" code), custom assays (for CCL5 and CXCR4) (Applied Biosystems) or SYBR Green (for Gr-1 and HPRT). The housekeeping genes GAPDH and HPRT were used to normalize the results.

PDF file - 1560K, Quantification of the flow cytometry profiles for the characterization of the four bone marrow cell populations sorted according to the expression of Nrp1 and Gr1. The results (shown as bar charts in the upper part of the figure and as a table in the bottom part) refer to four independent experiments; shown are the mean � s.d. The markers analyzed are the same as in the representative experiment shown in Figure 1D.

PDF file - 3704K, The vessel normalization effect of NEMs is independent of tumor size. B16.F10 tumors were inoculated in mice, which were then injected at days 11 and 13 after tumor inoculation with PBS, Gr1+ cells and NEMs. Mice were sacrificed when tumors reached 1700 mm3 , irrespective of the day. A. Immunofluorescence analysis of the vasculature - representative images. Frozen sections were stained with antibodies against CD31 (green) and α-SMA (red).Nuclei are counterstained with DAPI. Scale bar: 100 μm. B. Quantitative evaluation of the α-SMA/CD31 ratio in vessels of tumors injected with PBS, Gr1+ cells or NEMs. Data are mean�s.e.m. **=P

PDF file - 1471K, Levels of expression of plexins in CD11b+ Gr1+ Nrp1+ cells and in NEMs. Real-time PCR quantifications were normalized to the levels of GAPDH mRNA. Data are mean �s.e.m. **=P

PDF file - 1214K, Levels of expression of HIF-1 target genes in tumors inoculated with bone marrow Gr1+ cells or NEMs. Quantification of the mRNA levels of the indicated factors (VEGF-A, Angiopoietin-2 and Interleukin-6) was obtained by real-time PCR. The results are shown after normalization for the GAPDH gene. Data are mean �s.e.m. **=P

PDF file - 3108K, Flow cytometry analysis of NEMs purified from the bone marrow. Flow cytometry analysis of bone marrow CD11b+/Gr1-/Nrp1+ cells (NEMs) using the indicated antibodies conjugated with phycoerythrin (PE). Percentages of positive cells, referred to the total number of NEMs, are reported. The plots reported in the first row are also shown in Figure 1D, fourth column.

PDF file - 3482K, Proliferative activity of 4T1 tumors in vivo. A. Immunofluorescence staining of frozen tumor sections from mice injected with PBS, Gr1+ cells or NEMs using an antibody against PCNA (red); nuclei are counterstained in blue with DAPI. Analysis was performed after the animal sacrifice at day 18 after tumor inoculation. Scale bar: 100 �m. B. Quantitative analysis showing the percentage of PCNA+ nuclei in the tumor sections. Data are mean�s.e.m.; n=5 per group.

Background: PRS-344/S095012 is a novel 4-1BB (CD137) and programmed death-ligand 1 (PD-L1) bispecific antibody-Anticalin® fusion protein (MabcalinTM protein) designed to cluster 4-1BB on activated T cells exclusively in the presence of PD-L1 expressing cells. We aimed to study PRS-344/S095012 in vivo biodistribution and pharmacokinetics with 89Zr-positron emission tomography (PET) at a dose with antitumoral activity in mice and evaluate the contribution of each targeting arm. Methods: PRS-344/S095012 lacks cross-reactivity to murine 4-1BB and PD-L1. To explore the PRS-344/S095012 biodistribution in a humanized 4-1BB knock-in mouse model, we synthesized the surrogate 89Zr-Atezo-J10 with the same 4-1BB building block and cross-reactivity to murine PD-L1. Humanized 4-1BB knock-in C57BL/6J (h4-1BB KI B6) and C57BL/6J (B6) mice (n=4-6 per group) were subcutaneously engrafted with murine wildtype MC38 colon adenocarcinoma cells. Tumors were grown to a minimum of ≥50 mm3 (average 163 mm3) before tracer injection. Mice received intravenously 30 µg (2.5 MBq) of 89Zr-PRS-344/S095012 or 89Zr-Atezo-J10 supplemented with PRS-344/S095012 or Atezo-J10 up to 10 mg/kg. Four mice groups were formed to distinguish between bispecific (Atezo-J10 in h4-1BB KI B6), monospecific PD-L1 (Atezo-J10 in B6), monospecific 4-1BB (PRS-344/S095012 in h4-1BB KI B6), and isotype (PRS-344/S095012 in B6) binding up to 4 days post-injection (pi). In addition, a fifth group (5 MBq 89Zr-Atezo-J10) was studied to visualize the bispecific biodistribution up to 7 days pi. At days 1, 2, 4, or 2, 4, 7 pi, mice underwent serial PET imaging to obtain mean and maximum standardized uptake (SUVmean/max) and retro-orbital blood sampling, followed by ex vivo biodistribution. Results: PET imaging showed 89Zr-Atezo-J10 specific tumor accumulation with higher tumor-to-blood ratios of respectively 2.2-, 2.6-, and 2.4-fold (p 89Zr-Atezo-J10 spleen uptake was comparable (ns) with monospecific binding of PD-L1 but elevated (p Conclusion: 89Zr-Atezo-J10 specific accumulation in PD-L1 expressing tumors is due to both PD-L1 and 4-1BB binding and is higher than with PD-L1 and 4-1BB mono-targeting. This preclinical study supports the clinical evaluation of 89Zr-PRS-344/S095012’s whole-body distribution and the development of tumor-specific 4-1BB targeting bispecifics. Citation Format: Claudia A. van Winkel, Xiaoyu Fan, Danique Giesen, Glenn Gauderat, Lucia Pattarini, Thomas Jaquin, Anissa Barakat, Anne-Marie De La Bigne, Marleen Richter, Nicole Andersen, Julie Legrand, Helene Lelièvre, Elisabeth G. de Vries, Aizea Morales-Kastresana, Marjolijn N. Lub- de Hooge. Assessment of target-mediated biodistribution of an 89Zr labeled PD-L1/4-1BB bispecific Mabcalin protein. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3579.

PRS-344/S095012 is a bispecific antibody-Anticalin® fusion protein targeting PD-L1 and 4-1BB. It is designed to block the PD-1/PD-L1 axis and localize 4-1BB co-stimulation to PD-L1-positive tumor microenvironment (TME) or tumor draining lymph nodes with the aim of maximizing antitumor immunity and increasing the therapeutic window of 4-1BB monoclonal antibodies (mAbs). Multiple in vitro assays have shown that PRS-344/S095012 inhibits PD-1/PD-L1 signaling while simultaneously activating 4-1BB signaling on T cells. This resulted in a synergistic effect on both pathways leading to anti-tumor immune responses. PRS-344/S095012 presents mAb-like pharmacokinetics in vivo and non-clinical mouse models demonstrated dose-dependent efficacy in anti-PD-L1 refractory tumors. In the higher dose range, complete tumor regression was achieved in PD-L1 high and PD-L1 low expressing xenograft mouse models. This first-in-human (FIH), phase 1/2, open-label, multi center, dose escalation and cohort expansion study is designed to determine the safety and tolerability of intravenously administered PRS-344/S095012 in patients with advanced and/or metastatic solid tumors (NCT05159388). Patients with histologically confirmed diagnosis of unresectable, locally advanced, or metastatic solid tumors for which standard treatment options are not available, no longer effective, or not tolerated are eligible. Patients must have Eastern Cooperative Oncology Group (ECOG) status 0 or 1, RECIST 1.1 measurable disease and should have documented progression on prior therapy. Prior therapy with 4-1BB agonists is prohibited. Phase 1 is a dose escalation guided by a Bayesian Logistic Regression Model and a 28-day dose limiting toxicities (DLT) period. Primary objectives are safety and tolerability of PRS-344/S095012; secondary objectives include exploration of antitumor activity and evaluation of pharmacokinetics. Pharmacodynamics and comprehensive biomarker program will be explored. Additional patients may be enrolled to backfill cohort(s)to further explore pharmacokinetic/pharmacodynamic signals. Phase 2 is an expansion with primary objective of anti-tumor activity. First patient was dosed in November 2021 and the study is planned to enroll at sites in Belgium, Spain and Australia. Citation Format: Christiane Jungels, Nuria Kotecki, Emiliano Calvo, Elena Garralda, Timothy Price, Xiaojiang Zahn, Atif Abbas, Lisa Mahnke, Winrich Rauschning, Aizea Morales-Kastresana, Lucia Pattarini, Birgit Bossenmaier, Alix Scholer-Dahirel, Tim Demuth, Julie Legrande. Study of PRS-344/S095012 a PD-L1/4-1BB bispecific antibody-Anticalin®-fusion in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT255.

Background. Preclinical and clinical data suggest that 4-1BB (CD137), a costimulatory immunoreceptor mainly expressed by cytotoxic cells, represents a promising therapeutic target in cancer. A combination of checkpoint blockade with further T-cell activation mediated by 4-1BB co-stimulation may increase response rates and durability of response. However, the efficacy of systemic 4-1BB stimulation is limited by on target peripheral toxicity events. PRS-344/S095012 has been designed to provide the potential of a combinatorial therapy in one molecule and favor the localized stimulation of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. Methods. Anticalin® proteins are 18 kDa protein therapeutics derived from human lipocalins. We utilized phage display technologies to generate an Anticalin protein that binds to 4-1BB with high affinity and specificity. PRS-344/S095012 was generated by recombinant fusion of two 4-1BB-specific Anticalin proteins to a PD-L1-targeting monoclonal antibody with a modified IgG4 backbone that avoids interaction with Fc gamma receptors and restricts 4-1BB agonism to PD-L1 positive tissues. The activity and potency of PRS-344/S095012 were investigated in binding assays, functional in vitro assays with human primary immune cells, as well as in a human 4-1BB knock in mouse model. Results. The bispecific fusion protein PRS-344/S095012 is capable of binding 4-1BB and PD-L1 simultaneously. We show that the bispecific compound retains its ability to block PD-1/PD-L1 receptor-ligand interaction with similar potency to the parental PD-L1 antibody. In relevant in vitro cell-based assays, PRS-344/S095012 enhances T cell effector functions only in the presence of PD-L1 positive cells, in line with the desired mechanism of action. In vitro, we show that PRS-344/S095012 activity is superior to PD-L1 antibodies, and to the combination of clinically relevant 4-1BB and PD-L1 benchmark antibodies. In a human 4-1BB knock in mouse model, subcutaneously implanted with a human PD-L1 expressing tumor, PRS-344/S095012 showed clear superiority to anti-PD-L1 alone and a robust antitumor response that leads to the complete regression of implanted tumors and extension of survival. Conclusion. We report potent costimulatory T cell engagement of the immunoreceptor 4-1BB in a PD-L1-dependent manner, utilizing the PD-L1/4-1BB bispecific compound PRS-344/S095012. This approach has the potential to provide a localized costimulation of the immune system with high efficacy and reduced peripheral toxicity. Furthermore, dual mechanism of action combining PD-L1-dependent 4-1BB agonist activity with simultaneous PD-1/PD-L1 pathway blockade provides an additional therapeutic benefit in preclinical models. Taken together our in vitro and in vivo data outlines proof of concept functionality of PRS-344/S095012 and supports further development of this promising compound. Citation Format: Aizea Morales-Kastresana, Marina Pavlidou, Janet Peper, Lucia Pattarini, Christian Barthels, Eva-Maria Hansbauer, Rachida Bel Aiba, Birgit Bossenmaier, Alix Scholer-Dahirel, Thomas Jaquin, Catherine Gallou, Véronique Blanc, Christine Rothe, Shane Olwill. Simultaneous costimulatory T-cell engagement and checkpoint inhibition by PRS-344/S095012, a PD-L1/4-1BB bispecific compound for tumor localized activation of the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB135.

Gram+ infections are worldwide life-threatening diseases in which the pathological role of type I interferon (IFN) has been highlighted. Plasmacytoid predendritic cells (pDCs) produce high amounts of type I IFN following viral sensing. Despite studies suggesting that pDCs respond to bacteria, the mechanisms underlying bacterial sensing in pDCs are unknown. We show here that human primary pDCs express toll-like receptor 1 (TLR1) and 2 (TLR2) and respond to bacterial lipoproteins. We demonstrated that pDCs differentially respond to gram+ bacteria through the TLR1/2 pathway. Notably, up-regulation of costimulatory molecules and pro-inflammatory cytokines was TLR1 dependent, whereas type I IFN secretion was TLR2 dependent. Mechanistically, we demonstrated that these differences relied on diverse signaling pathways activated by TLR1/2. MAPK and NF-κB pathways were engaged by TLR1, whereas the Phosphoinositide 3-kinase (PI3K) pathway was activated by TLR2. This dichotomy was reflected in a different role of TLR2 and TLR1 in pDC priming of naïve cluster of differentiation 4+ (CD4+) T cells, and T helper (Th) cell differentiation. This work provides the rationale to explore and target pDCs in bacterial infection., It is unclear whether and how plasmacytoid pre-dendritic cells (pDCs) respond to bacteria; this study shows that human pDCs sense Gram-positive bacteria through the Toll-like receptors TLR1 and TLR2, and that these receptors have distinct roles in the activation of pDCs.

Introduction Le but de ce travail etait d’etudier les variations des differentes sous-populations de lymphocytes T auxiliaires (LTh) et folliculaires (LTfh) dans la dermatite atopique au cours d’un traitement par dupilumab. Materiel et methodes Nous avons constitue une cohorte de 29 patients atopiques, traites par dupilumab, un anticorps antagoniste monoclonal IgG4 entierement humain ciblant la sous-unite alpha du recepteur a l’IL-4. Le dupilumab inhibe la transduction de signaux via l’IL-4 et l’IL-13 et ainsi les reponses Th2 anormales. Nous avons mesure le pourcentage des sous-populations de LT dans le sang peripherique des 29 patients a 0, 1, 3, 6 et ≥ 12 mois du traitement et les avons compares a 25 controles sains. Grâce a un marquage avec des anticorps fluorescents et une analyse par cytometrie en flux, nous avons mesure et suivi l’evolution du pourcentage des 8 sous-populations principales de LT auxiliaires (Th1, Th2, Th17, Th1/17) et folliculaires (Tfh1, Tfh2, Tfh17, Tfh1/17). Ces mesures ont ete associees a l’evaluation des scores cliniques SCORAD, IGA, EASI et DLQI lors de la collection des echantillons. Resultats Nous avons observe une diminution significative du pourcentage de LTh2 au cours du traitement par dupilumab, variation la plus importante mesuree parmi les populations de LT analysees. Tous les scores cliniques ont diminue de facon significative au cours du traitement. Nous avons ensuite evalue l’association entre les populations de LTh et LTfh avec l’EASI : le pourcentage de LTh2 etait correle de facon significative avec l’EASI a tous les points de mesure excepte M6, ce qui confirme que les LTh2 representent un biomarqueur pharmacodynamique fort du traitement par dupilumab. Nous avons egalement observe une correlation significative entre le pourcentage de LTh17 et le score EASI a M0 et M12. De plus, la difference relative de l’EASI entre M12 et M0 correlait avec la difference relative de pourcentage de LTh17 entre M12 et M0, montrant que la diminution la plus importante du pourcentage de LTh17 entre M12 et M0 avait lieu chez les patients avec la diminution de l’EASI la plus forte. Enfin nous avons separe les patients en fonction de leur reponse au traitement : repondeurs hauts (amelioration de l’EASI a ≥ M12 d’au moins 85 %), bas (amelioration de l’EASI a ≥M12 de moins de 60 %) et intermediaires. Nous avons ainsi observe que les repondeurs bas presentaient des niveaux de LTfh totaux (cellules CXCR5 +) a M12 significativement plus bas compares aux repondeurs hauts et intermediaires. Discussion L’evolution des populations de LTh et LTfh peut etre monitoree dans le sang peripherique des patients atopiques. Les resultats etablissent un lien fort entre les LTh2 et la reponse au dupilumab. Ils suggerent egalement un role important d’autres sous-populations de lymphocytes Th et Tfh dans la physiopathologie de la DA et dans la reponse au traitement.

Innate immune cells are generated through central and peripheral differentiation pathways, and receive multiple signals from tissue microenvironment. The complex interplay between immune cell state and environmental signals is crucial for the adaptation and efficient response to pathogenic threats. Here, we discuss how systems biology approaches have brought global view and high resolution to the characterization of (1) immune cell diversity, (2) phenotypic, transcriptional and functional changes in response to environmental signals, (3) integration of multiple stimuli. We will mostly focus on systems level studies in dendritic cells and macrophages. Generalization of these approaches should elucidate innate immune cell diversity and plasticity, and may be used in the human to generate hypothesis on cell filiation and novel strategies for immunotherapy.

T follicular helper cells (Tfh) are implicated in various pathological conditions, but how they differentiate in Th2-skewed environments is unknown. Pattarini et al. delineate a pathway for human Tfh differentiation induced by TSLP through OX40L, relevant to atopic dermatitis., T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)–activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC–polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments.

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. Here we show that the chromatin remodeler Mi-2β controls epidermal homeostasis by regulating genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2β depletion caused rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2β in keratinocytes was the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that primes dendritic cells for Th2 induction. It has been implicated in different types of allergic diseases. Recent work suggested that TSLP could play an important role in the tumor microenvironment and influence tumor progression, in particular in breast cancer. In this study we systematically assessed the production of TSLP at the mRNA and protein levels in several human breast cancer cell lines, large-scale public transcriptomics data sets, and primary human breast tumors. We found that TSLP production was marginal, and concerned less than 10% of the tumors, with very low mRNA and protein levels. In most cases TSLP was undetectable and found to be expressed at lower levels in breast cancer as compared to normal breast tissue. Last, we could not detect any functional TSLP receptor (TSLPR) expression neither on hematopoietic cells nor on stromal cells within the primary tumor microenvironment. We conclude that TSLP-TSLPR pathway activity is not significantly detected within human breast cancer. Taken together, these observations do not support TSLP targeting in breast cancer.

Blood represents the most accessible source of human dendritic cells (DCs). We present here a method to isolate three DC subtypes, as identified until now, from peripheral blood: plasmacytoid dendritic cells (pDCs), CD141(+) myeloid DCs, and CD1c(+) myeloid DCs. The method is based on the sequential depletion of non-DCs. First, depletion of granulocytes, erythrocytes, and platelets is obtained by blood centrifugation over a Ficoll gradient. Then, antibodies recognizing non-DCs, combined with magnetic beads, allow enrichment of DCs from peripheral blood mononuclear cells (PBMCs). Finally, enriched DCs are purified and separated into the different subtypes by immunolabeling and fluorescence-activated cell sorting (FACS) using DC-specific surface markers.DC studies might contribute to the comprehension of human immune processes in physiological and pathological conditions. Human blood DCs targeting might be a useful tool to ameliorate inflammatory diseases and improve vaccination strategies.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine expressed by epithelial cells during allergic inflammation, and activating dendritic cells (DC). Its expression and functional role in cancer remain controversial. We conducted retrospective (n = 89), and prospective studies including patients with untreated primary head and neck squamous cell carcinoma (HNSCC). We found that TSLP was overexpressed by HNSCC tumor cells, and associated with a highly differentiated status. However, no significant difference in overall and recurrence-free survival was found between patients bearing a tumor with high and low TSLP levels, respectively. Surprisingly, there was no significant association between the levels of TSLP expression, and the number of tumor-infiltrating mature DCLAMP(+) DC. In order to explain the apparent lack of TSLP-induced DC activation, we performed phenotypic and functional experiments on freshly resected tumors. Tumor-infiltrating immune cells, including DC, did not express the TSLP receptor heterodimer (TSLPR chain, IL-7Ralpha chain). Furthermore, freshly sorted blood CD11c(+) DC from healthy donors cultured with tumor-conditioned supernatant exhibited an activated profile, but this was not affected by an anti-TSLP blocking antibody, suggesting a DC activation pathway independent of tumor-derived TSLP. Overall, our results demonstrate that TSLP is overexpressed in HNSCC but its function is hampered by the lack of TSLPR-expressing cells in the tumor microenvironment. Such a dissociated ligand-receptor expression may impact intercellular communication in other immune activation pathways, and tumor types.

Blood represents the most accessible source of human dendritic cells (DCs). We present here a method to isolate three DC subtypes, as identified until now, from peripheral blood: plasmacytoid dendritic cells (pDCs), CD141(+) myeloid DCs, and CD1c(+) myeloid DCs. The method is based on the sequential depletion of non-DCs. First, depletion of granulocytes, erythrocytes, and platelets is obtained by blood centrifugation over a Ficoll gradient. Then, antibodies recognizing non-DCs, combined with magnetic beads, allow enrichment of DCs from peripheral blood mononuclear cells (PBMCs). Finally, enriched DCs are purified and separated into the different subtypes by immunolabeling and fluorescence-activated cell sorting (FACS) using DC-specific surface markers.DC studies might contribute to the comprehension of human immune processes in physiological and pathological conditions. Human blood DCs targeting might be a useful tool to ameliorate inflammatory diseases and improve vaccination strategies.

Background: Microsurgical tissue engineering is an emerging topic in regenerative medicine. Here we describe a new microsurgical model of bioengineering in rats based on the use of an arterovenous loop (AV) implanted into a commercially available crosslinked collagen/glycosaminoglycan template. Methods: The microvascular loop was created between the femoral artery and vein and covered by the template folded onto itself. The chamber was isolated from the outside tissue by an outer silicon layer to impede tissue ingrowth. Results :A t 1-month postimplantation, the tissue chamber was found heavily vascularized, as assessed by laser Doppler perfusion analysis. Histological examination showed that the AV loop was integrated into the collagen matrix of the template and that the whole template was filled with a newly formed soft connective tissue. Most interestingly, the whole scaffold was found heavily vascularized, including the formation of a large number of a-SMA-positive arterioles. Conclusions: The developed microsurgical chamber provides a highly vascular, isolated tool for in vivo tissue engineering. V C 2007 Wiley-Liss, Inc. Microsurgery 27:623‐629, 2007. One of the central requirements in tissue engineering is the possibility to induce neoangiogenesis or neovasculogenesis to provide adequate blood supply to the engineered tissue and to sustain viability and growth of the cells supporting regeneration. 1 This requirement appears particularly stringent for all in vivo applications of tissue engineering, including the formation of new specialized tissues (dermis, bone, cartilage, liver) or in plastic reconstructive surgery. Over the last few years, a number of interesting surgi

In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen–glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine.

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetylases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In particular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phosphorylation and intracellular signaling upon prolonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetylation is a critical mechanism that directly affects VEGFR2 function.

Background Thymic stromal lymphopoietin (TSLP) is a major proallergic cytokine that promotes T H 2 responses through dendritic cell (DC) activation. Whether it also plays a role in human autoimmune inflammation and associated pathways is not known. Objective In this study we investigated the potential role of several epithelium-derived factors, including TSLP, in inducing IL-23 production by human DCs. We further dissected the role of TSLP in patients with psoriasis, an IL-23–associated skin autoimmune disease. Methods The study was performed in human subjects using primary cells and tissue samples from patients with psoriasis and healthy donors. We analyzed the production of IL-23 in vitro by blood and skin DCs. We studied the function for TSLP and its interaction with other components of the inflammatory microenvironment in situ and ex vivo . Results We found that TSLP synergized with CD40 ligand to promote DC activation and pathogenic IL-23 production by primary blood and skin DCs. In situ TSLP was strongly expressed by keratinocytes of untreated psoriatic lesions but not in normal skin. Moreover, we could demonstrate that IL-4, an important component of the T H 2 inflammation seen in patients with atopic dermatitis, inhibited IL-23 production induced by TSLP and CD40 ligand in a signal transducer and activator of transcription 6–independent manner. Conclusion Our results identify TSLP as a novel player within the complex psoriasis cytokine network. Blocking TSLP in patients with psoriasis might contribute to decreasing DC activation and shutting down the production of pathogenic IL-23.

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1− resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth. Cancer Res; 72(24); 6371–81. ©2012 AACR.

Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A(165) and VEGF-B(167) on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.

Experimental and clinical evidence indicate that bone marrow cells participate in the process of new blood vessel formation. However, the molecular mechanisms underlying their recruitment and their exact role are still elusive. Here, we show that bone marrow cells are recruited to the sites of neoangiogenesis through the neuropilin-1 (NP-1) receptor and that they are essential for the maturation of the activated endothelium and the formation of arteries in mice. By exploiting adeno-associated virus vector-mediated, long-term in vivo gene expression, we show that the 165-aa isoform of VEGF, which both activates the endothelium and recruits NP-1+ myeloid cells, is a powerful arteriogenic agent. In contrast, neither the shortest VEGF121 isoform, which does not bind NP-1 and thus does not recruit bone marrow cells, nor semaphorin 3A, which attracts cells but inhibits endothelial activation, are capable of sustaining arterial formation. Bone marrow myeloid cells are not arteriogenic per se nor are they directly incorporated in the newly formed vasculature, but they contribute to arterial formation through a paracrine effect ensuing in the activation and proliferation of tissue-resident smooth muscle cells.

Microsurgical tissue engineering is an emerging topic in regenerative medicine. Here we describe a new microsurgical model of bioengineering in rats based on the use of an arterovenous loop (AV) implanted into a commercially available crosslinked collagen/glycosaminoglycan template.The microvascular loop was created between the femoral artery and vein and covered by the template folded onto itself. The chamber was isolated from the outside tissue by an outer silicon layer to impede tissue ingrowth.At 1-month postimplantation, the tissue chamber was found heavily vascularized, as assessed by laser Doppler perfusion analysis. Histological examination showed that the AV loop was integrated into the collagen matrix of the template and that the whole template was filled with a newly formed soft connective tissue. Most interestingly, the whole scaffold was found heavily vascularized, including the formation of a large number of alpha-SMA-positive arterioles.The developed microsurgical chamber provides a highly vascular, isolated tool for in vivo tissue engineering.

Skeletal myogenesis is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes, typical of the differentiated muscle tissue. While the molecular players involved in early myogenesis have been extensively characterized, information about the later steps of the process is scanty. Here, we describe a novel myogenic cell line (MYOP7), composed of highly proliferating Sca-1+ muscle precursor cells, which can be induced to terminally differentiate into spontaneously contracting multinucleated myotubes. By performing high-density microarray analysis on these cells, we identified a series of genes, differentially expressed in proliferating vs differentiating conditions, which are candidates to play a major role in the later phase of myogenesis. In addition, we confirmed that the late stages of muscle differentiation are characterized by a marked upregulation of the cellular receptors for the vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel formation during both vasculogenesis and angiogenesis. The prolonged expression of VEGF in the normoperfused skeletal muscles of adult rodents after gene transfer using AAV vectors induces the formation of a large set of new capillaries and small arteries. Notably, this process is accompanied by the massive infiltration by mononuclear cells. This observation raises the possibility that these cells might represent circulating progenitors that are eventually incorporated in the newly formed vessels. Here we explore this possibility by exploiting 4 different experimental models based on (a) the transplantation of male bone marrow into female recipients; (b) the transplantation of Tie2-GFP transgenic bone marrow; (c) the transplantation of bone marrow in the presence of erythropoietin (EPO), a mobilizer of endothelial progenitor cells (EPCs); and (d) the reimplantation of ex vivo–expanded EPCs. In all 4 models, VEGF acted as a powerful attractor of bone marrow–derived mononuclear cells, bearing different myeloid and endothelial markers proper of the EPCs to the sites of neovascularization. In no case, however, were the attracted cells incorporated in the newly formed vasculature. These observations indicate that new blood vessel formation induced by VEGF occurs through bona fide sprouting angiogenesis; the contribution of the infiltrating bone marrow–derived cells to this process still remains enigmatic.

Vascular endothelial growth factor (VEGF) is a major regulator of blood vessel formation during development and in the adult organism. Recent evidence indicates that this factor also plays an important role in sustaining the proliferation and differentiation of different cell types, including progenitor cells of different tissues, including bone marrow, bone, and the central nervous system. Here we show that the delivery of the 165-aa isoform of VEGF-A cDNA using an adeno-associated virus (AAV) vector exerts a powerful effect on skeletal muscle regeneration in vivo. Following ischemia-, glycerol-, or cardiotoxin-induced damage in mouse skeletal muscle, the delivery of AAV-VEGF markedly improved muscle fiber reconstitution with a dose-dependent effect. The expression of both VEGF receptor-1 (VEGFR-1) and VEGFR-2 was upregulated both in the satellite cells of the damaged muscles and during myotube formation in vitro; the VEGF effect was mediated by the VEGFR-2, since the transfer of PlGF, a VEGF family member interacting with the VEGFR-1, was ineffective. These results are consistent with the observation that VEGF promotes the growth of myogenic fibers and protects the myogenic cells from apoptosis in vitro and prompt a therapeutic use for VEGF gene transfer in a variety of muscular disorders.

Top of pageAbstract While the ability of VEGF to drive a powerful angiogenic response is widely recognized, the central question as to whether the newly formed vessels support a physiological increase in tissue perfusion and meet the increased flow demand during exercise remains, as does the question whether other factors, such as the angiopoietins, might be required to promote further vessel maturation. To explore these issues, we exploited AAV vectors for the prolonged expression of VEGF165 and Ang1 in the skeletal muscle. Eighty-four rats were injected in their right hindlimb with AAV-LacZ, AAV-VEGF, AAV-Ang1, or AAV-VEGF+AAV-Ang1 (n=21 for each group, 4|[times]|1011 particles for each vector). Muscular blood flow (MBF) and vascular permeability were assessed at 1, 2 and 4 months using PET and 13N-ammonia (3.7 Mbq iv), under both resting conditions and after 15 min of muscle activity, induced by electrical stimulation at 180 spikes/min. Resting perfusion did not vary significantly in the animals injected with AAV-VEGF alone; surprisingly, in the same animals, perfusion after exercise markedly decreased over time (0.70|[plusmn]|0.13, 0.66|[plusmn]|0.02, 0.50|[plusmn]|0.02 ml/min/g at 1, 2 and 4 months respectively, vs. 0.89|[plusmn]|0.03 control; p

The potential use of bone marrow-derived cells (BMCs) to promote new blood vessel formation, recapitulating vasculogenesis in the adult, has recently aroused much excitement, and a rapid transposition to the clinic. However, new experimental evidence has challenged this notion, demonstrating that BMCs transplanted into ischemic tissues still adopt mature hematopoietic fates and do not transdifferentiate into vascular structures. To better define the actual role of bone marrow-derived cells in the formation of new blood vessels, we developed a series of AAV vectors to drive the persistent expression of several factors, differing in their capacity to promote angiogenesis and to recruit BMCs.

Bone marrow-derived cells have been recently proposed to participate in the development of new blood vessels during physiological and pathological angiogenesis in adult organisms.

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.