Efecto de VIH-1 en la desregulación de los linfocitos B. Papel de dendrímeros carbosilano en la respuesta inflamatoria, como agentes transfectantes y en la polarización de macrófagos de tipo M2

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-12-2013Este trabajo ha sido realizado en el Laboratorio de Inmuno-Biología Molecular del Hospital General Universitario Gregorio Marañón de Madrid. El trabajo experimental que se recoge en esta Memoria ha sido financiado por el proyecto europeo EuroNanoMed 2010, Fondos de Investigación Sanitaria (INTRASALUD PI09/02029, PS09/02669, PS09/02523), la Red Temática de Investigación Cooperativa Sanitaria ISCIII (RETIC RD06/0006/0035 y RD12-0017-0037), INDISNET S-2011-BMD2332, FIPSE, y COST action TD0802.This work is divided into two main subjects: “the effect of HIV-1 on the deregulation of B lymphocytes”, and “the role of carbosilane dendrimers in inflammatory response, as transfecting agents and, on the polarization of M2-type macrophages”. PART I: B cells are a critical component of the adaptive immune system, due to its capacity of producing highly specific antibodies. During HIV infection, patients with AIDS could exhibit hyperimmunoglobulinemia, increased expression of cell-activation markers, depletion of memory B cells, polyclonal B-cell hyperactivity, and altered differentiation of naïve B cells that could result in impaired immunoglobulin class switch recombination (CSR), and thus production of nonspecific immunoglobulin (Ig) G, IgE and IgA antibodies. All of these processes finally provoke the exhaustion of B-cell and defective responses against opportunistic pathogens. However, little is know about the molecular mechanism responsible of the B-cell deregulation and whether it is due to a direct effect of HIV on B cells. In this study we have evaluated the effects of a direct exposition of B cells to HIV-1 particles in order to identify and describe the deregulation of B cells by HIV-1 evaluating CSR, AID protein expression, AID-related miRNA expression and Ig production in an environment free of T lymphocytes. We showed that HIV-1 particles deregulate human primary B cells, increasing their survival, proliferation, modifying their phenotype and function on cultured B-cell. Moreover, expression level of AID mRNA in human primary B cells was highly increased and its subsequent IgM/IgE; IgM/IgA and IgM/IgG class switch was detected in vitro. Finally, the results indicate that the mechanism by which HIV-1 deregulates B cells is through the BCR/SYK signaling pathway, promoting the mobilization of BCR in the membrane, which leads to the activation of JNK. In summary, in this study we have demonstrated that direct contact between HIV-1 particles and B-cell was sufficient to induce a deregulation of B-cell. The model in vitro developed in this study, which is independent of CD4 T cells and CD40L, can be useful to study the mechanisms of B-cell deregulation in the context of HIV infection. In addition, these results may highlight a possible relation between HIV-1 infection and B cells hyperactivation, loss of memory B cells or hyperglobulinemia. By all that, these results contribute to the better understanding of the general immune deregulation observed in HIV-1 patients, and allow us to lay the groundwork for development of better anti-HIV vaccines. PART II: New objectives of nanomedicine consist in developing and characterizing nanoparticles as new preventive treatment, therapeutic or diagnostic tools with the aim to improve current treatments. The major advantage of carbosilane dendrimers is based on their regular structure and skeletons and surfaces easy to modify. Moreover, carbosilane dendrimers can be used as molecules that have an effect per se in the treatment of HIV-1, in autoimmune diseases and inflammation, as well as molecules that can shuttle nucleic acids and drugs to the cell interior. We have studied the ability of cationic dendrimers 2G-NN16 and 2G-03NN24 to transfect siRNA-Nef in CD4 T lymphocytes, in the context of HIV-1. The results have shown that both dendrimers are able to form stable complexes with siRNA and protect them against RNase. Both dendrimers facilitate CD4 T lymphocytes transfection with siRNA. The 2G-03NN24 dendrimer is better transfectant than 2G-NN16. Our results indicate that 2G-03NN24 could protect its cargo better than 2G-NN16. We have also studied the effect of 5 carbosilane dendrimers (anionic dendrimer 2G-S16 and cationic dendrimers 2G-NN16, 1G-03NN12, 2G-03NN24 and 3G03NN48) on M1 macrophages. Dendrimers did not induce the release of TNF-α, IL-12p40, CCL3, CCL4, IL-1β and IL-6. Especially, 2G-NN16 decreased the expression of several genes implicated in the pro-inflamnmatory function of M1 macrophages, suppressed the expression of TNF-α and IL-12p40. These data indicate that 2G-NN16 has a slightly non-inflammatory effect, which could be beneficial in HIV therapy because local inflammation allows higher cell activation, which facilitates the HIV-1 infection. An additional effect of the 2G-03NN24 is the decreased expression of the CCR2 co-receptor which is involved in macrophage infection by HIV-1. The results obtained with different dendrimers in M1 open promising lines of research, suggesting that they could be used as safe biological agents without promoting inflammation and that they could be useful for the treatment of several medical conditions. Since most of the tumor-associated macrophages (TAM) are similar to M2, we have also studied the role of carbosilane dendrimers on M2 macrophages to determine if they are able to induce a switch on the macrophages phenotype, to evaluate the potential application of dendrimers in tumor immunotherapy. Tumor microenvironment favours the escape from immunosurveillance, promoting anti-inflammatory responses and inhibiting pro-inflammatory ones. The 2G-03NN24 dendrimer decreases the production of IL-10 by the LPS-stimulated M2 macrophages and also switches the M2 genetic phenotype to a M1 phenotype. Furthermore, this dendrimer decreases the activity of STAT3 by diminishing its phosphorylation through the PDGFR and EGFR receptors pathways. In vivo, dendrimer-treated tumors show that the TAM over-express iNOS, a protein typically expressed by the M1, that possess anti-tumor properties. In addition, other changes induced by the dendrimer would create a more favorable microenvironment within tumors as seen in preliminary in vivo studies. Results indicate that 2G-03NN24 dendrimer might be used for the therapy of tumors through its ability to suppress polarization of M2 macrophages, generally associated with tumor proliferation. These results are very encouraging showing that the 2G-03NN24 dendrimer can be a new anti-tumor compound

Enhanced antitumor efficacy of oncolytic adenovirus-loaded menstrual blood-derived mesenchymal stem cells in combination with peripheral blood mononuclear cells

Several studies have evaluated the efficacy of using human oncolytic adenovirus-loaded mesenchymal stem cells for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow mesenchymal stem cells infected with the oncolytic adenovirus ICOVIR-5, for treatment of neuroblastoma patients. Results from this clinical trial point out the role of the immune system in the clinical outcome. In this context, a better understanding of the immunophenotypic changes of human mesenchymal stem cells upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMCs) could guide strategies to improve the antitumor efficacy of infected Mesenchymal Stem Cells (MSCs). In this work, we show how infection by an oncolytic adenovirus (OAdv) induces Toll-like receptor 9 overexpression and activation of the NF-κB pathway in menstrual blood-derived mesenchymal stem cells (MenSCs), leading to a specific cytokine secretion profile. Moreover, a pro-inflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T-cells and natural killer cells (NK cells), is generated when OAdv-loaded MenSCs are co-cultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MenSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the oncolytic adenovirus-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy

Safety and Efficacy of an Oncolytic Adenovirus as an Immunotherapy for Canine Cancer Patients

The use of oncolytic virus is an innovative approach that has shown promising results as a treatment in oncology. Epithelial-derived tumors are the most frequent neoplasms in dogs, but gold standard therapies can be highly invasive procedures. Due to the accessible localization of these tumors, the intratumoral administration is feasible. Therefore, we propose to determine the safety and efficacy of intratumoral administration of oncolytic adenovirus ICOCAV15, in canine patients with epithelial-derived tumors. Eight dogs with carcinoma/adenocarcinoma were intratumorally treated with ICOCAV15. No clinically relevant changes were observed in the blood count, biochemistry and coagulation test analyzed during follow-up. The survival time of the 6/8 dogs exceeded the median survival time with chemotherapy, showing a partial response rate of 25% and 75% of stable disease. ICOCAV15 was detected in the target lesion by qPCR and immunohistochemistry. Also, some of the non-treated metastasis showed an infiltration of ICOCAV15 by immunohistochemistry. The immune populations were evaluated, and an increase of CD8+, MAC387+, CD3+ and CD20+ cells was reported in some of the patients after the inoculation. These results show that intratumoral ICOCAV15 is safe and well tolerated by dogs. Also, they suggest ICOCAV15 could be a new tool in veterinary oncology for accessible carcinomas/adenocarcinomas. The use of oncolytic viruses is an innovative approach to lyse tumor cells and induce antitumor immune responses. Eight dogs diagnosed with carcinoma/adenocarcinoma were intratumorally treated with ICOCAV15, an oncolytic canine adenovirus (CAV). To evaluate the treatment's safety, a blood count, biochemistry, and coagulation test were performed before treatment and during follow-up. Immune populations were analyzed by flow cytometry. Anti-adenovirus antibodies were also determined. The immune infiltration, vascularization, and viral presence in the tumor were determined by CD3, CD4, CD20, CD31 and CAV by immunohistochemistry. All the dogs maintained a good quality of life during follow-up, and some had increased median survival time when compared with dogs treated with chemotherapy. No treatment-related adverse effects were detected. The Response Evaluation Criteria In Solid Tumors criteria were also assessed: two patients showed a partial response and the rest showed stable disease at various times during the study. ICOCAV15 was detected inside the tumor during follow-up, and antiviral antibodies were detected in all patients. Furthermore, the tumor-infiltrating immune cells increased after viral administration. Therefore, we suggest that intratumorally administered ICOCAV15 could represent as a new tool for the treatment of canine carcinoma because it is safe, well-tolerated by dogs, and shows promising results

Direct Phenotypical and Functional Dysregulation of Primary Human B Cells by Human Immunodeficiency Virus (HIV) Type 1 In Vitro

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) induces a general dysregulation of immune system. Dysregulation of B cell compartment is generally thought to be induced by HIV-related immune activation and lymphopenia. However, a direct influence of HIV-1 particles on B cells was recently proposed as the third pathway of B cells dysregulation. METHODS/PRINCIPAL FINDINGS: We evaluated the direct and specific consequences of HIV-1 contact on activation, survival, proliferation and phenotype of primary B cells in vitro. Moreover, we examined expression of activation-induced cytidine deaminase (AID) mRNA that is responsible for class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that changes observed in cellular proliferation, phenotypes and activation of B cells could be caused by direct contact between HIV-1 particles and primary B cells in vitro. Finally, direct HIV-1-derived B cells activation led to the increase of AID mRNA expression and its subsequent CSR function was detected in vitro. CONCLUSION/SIGNIFICANCE: We showed that HIV-1 could directly induce primary B cells dysregulation triggering phenotypical and functional abilities of B cells in vitro that could explain in some extent early B-cell abnormalities in HIV disease

Cellular Virotherapy Increases Tumor-Infiltrating Lymphocytes (TIL) and Decreases their PD-1+ Subsets in Mouse Immunocompetent Models.

Factor de impacto: 6,639 Q1Oncolytic virotherapy uses viruses designed to selectively replicate in cancer cells. An alternative to intratumoral administration is to use mesenchymal stem cells (MSCs) to transport the oncolytic viruses to the tumor site. Following this strategy, our group has already applied this treatment to children and adults in a human clinical trial and a veterinary trial, with good clinical responses and excellent safety profiles. However, the development of immunocompetent cancer mouse models is still necessary for the study and improvement of oncolytic viroimmunotherapies. Here we have studied the antitumor efficacy, immune response, and mechanism of action of a complete murine version of our cellular virotherapy in mouse models of renal adenocarcinoma and melanoma. We used mouse MSCs infected with the mouse oncolytic adenovirus dlE102 (OAd-MSCs). In both models, treatment with OAd-MSCs significantly reduced tumor volumes by 50% and induced a pro-inflammatory tumor microenvironment. Furthermore, treated mice harboring renal adenocarcinoma and melanoma tumors presented increased infiltration of tumor-associated macrophages (TAMs), natural killer cells, and tumor-infiltrating lymphocytes (TILs). Treated mice also presented lower percentage of TILs expressing programmed cell death protein 1 (PD-1)-the major regulator of T cell exhaustion. In conclusion, treatment with OAd-MSCs significantly reduced tumor volume and induced changes in tumor-infiltrating populations of melanoma and renal cancer.PI14CIII/00005/Ministerio de Economía y Competitividad PI17CIII/00013/Ministerio de Economía y Competitividad P2017/BMD-3692/Consejería de Educación, Juventud y Deporte of Comunidad de Madrid General grant/Fundación Oncohematología Infantil General grant/AFANIONS

Biodistribution Analysis of Oncolytic Adenoviruses in Canine Patient Necropsy Samples Treated with Cellular Virotherapy.

Oncolytic immunotherapy with competent viruses is an emerging approach in cancer treatment. The clinical safety of many types of oncolytic viruses (OVs) has been demonstrated. However, there is a lack of information about viral biodistribution in patients. The available data about oncolytic adenovirus biodistribution in human subjects treated intravenously consists of virus detection in body fluids, a few tumor biopsies, and a single report of patient necropsy samples. There is no information about adenoviral biodistribution in patients treated intravenously with cellular vehicles carrying an oncolytic adenovirus. We previously published reports regarding the efficacy and clinical safety of infusing mesenchymal stem cells (MSCs) infected with an OV in human and canine patients. In this study, we performed necropsies on 12 canine patients treated with dCelyvir, canine MSCs infected with ICOCAV17, a canine oncolytic adenovirus. The prevalence of microscopic lesions, especially chronic inflammatory responses in different organs, was higher than expected. Concomitantly, we found a positive immunoreaction to ICOCAV17 in analyzed samples. These findings support a possible role of the virus in development of histopathological alterations and ongoing systemic viral replication of ICOCAV17 in the period after therapy administration.This study was funded by Fundación Universidad Alfonso X el Sabio , Madrid, Spain ( 1.010.909 to A.J.P.-B.); Instituto de Salud Carlos III , Spain ( PI14CIII/00005 and PI17CIII/00013 to J.G.-C.); Consejería de Educación, Juventud y Deporte, Comunidad de Madrid , Spain ( P2017/BMD-3692 to J.G.-C.); Fundación Oncohematología Infantil ; AFANION ; and Asociación Pablo Ugarte , whose support we gratefully acknowledge. The graphical abstract was created with BioRender.S

Tumor-Homing of Mesenchymal Stem Cells Infected with Oncolytic Virus in a Canine Patient

Intravenous administration of oncolytic adenovirus (OAds) can be challenging, although various vehicles for the delivery of the virus to the tumor have been described. The efficacy of mesenchymal stem cells (MSCs) as a virus vehicle has been reported in mouse models and canine and human patients, but the actual action mechanism has never been described in patients. It is of importance to determine whether MSCs infected with OAds can reach the tumor and release the virus in a clinical setting. For this purpose, GFP-labeled MSCs were infected with an OAd and inoculated into a companion dog diagnosed with spontaneous lung carcinoma. Forty-eight hours later, the tumor was excised and analyzed microscopically by flow cytometry for GFP fluorescence detection, and a cellular culture was established. Peripheral blood samples were taken to quantify the oncolytic adenovirus by qRT-PCR. Green fluorescence cells detected in the cellular culture by microscopy and flow cytometry revealed 0.69% GFP-positive cells in the tumor. OAd in peripheral blood was confirmed by qRT-PCR during follow-up. For the first time, the tumoral-homing capacity of OAds infected-MSC has been confirmed in a clinical setting, helping to explain the clinical response mechanism, whose efficacy was previously reported in canine and human patients

AID mRNA expression in B cells and Igs production.

<p>(A)AID mRNA expression was quantified at 24 h post treatment in B cells. NT or treated B cells were cultured <i>in vitro</i> and mRNA was extracted and AID mRNA expression was quantified by real-time PCR. Fold increase was calculated as ratio of AID mRNA expression in comparison to NT condition. Each grey dot represents one experiment. Black bar represent average fold increase. (* = p<0.05). IgG (B), IgA (C) and IgE (D) production were quantified in cell culture supernatant by ELISA kit. (B) Mean of 10 experiments was represented (+SD). (C, D) Only experiments with detectable level of IgA or IgE was shown where each bar represent the result obtained for one individual donor. (ND: not determined).</p

Expression of activation markers on B cells.

<p>(A) Histogram plots of CD71 (left panel) and CD69 (right panel) expression markers at the surface of B-cell. (B) B cells were NT or treated with 25 ng and 125 ng of p24^gag of HIV_NL4-3, with 125 ng of p24^gag of boiled-HIV_NL4-3, with LPS/IL-4, mock-treated or treated with CD40L/IL-4. At day 1 (grey bars) or day 4 (black bars) post-treatment, CD71 and CD69 surface markers were followed by flow cytometry. Mean of 7 individuals donors excepted for mock and CD40L/IL-4 conditions (3 individuals donors) (+SD; * = p<0.05 in comparison to NT for day 1 or 4 post-treatment). Mock corresponded to the supernatant of MT2 non-infected cells. (C) B cells were NT or treated with 125 ng of p24^gag of HIV_NL4-3, with 125 ng of p24^gag of HIV_NL4-3 treated with anti-HIV serum (Vol/Vol), mock-treated/anti-HIV serum or treated with LPS/IL-4. At day 1 post-treatment percentage of CD71 and CD69 were followed by flow cytometry. Mean of 3 individuals donors is represented (± SD; * = p<0.05).</p

Class switch detected by intracellular labeling in in vitro B cells.

<p>(A) NT or treated B cells were cultured during 5 days. Intracellular CD19, IgM, IgD (A), IgG, IgA or IgE (B) markers were followed by flow cytometry. (A) IgD and IgM expression levels were followed in CD19+ population by percentage of positive cells for these markers *MFI of the same both markers ( = iMFI; integrated MFI). Mean of 5 individual donors (+SEM; * = p<0.05). (B) IgG, IgA and IgE expression levels were followed in CD19+ population by percentage of positive cells for each marker. Mean of 5 individual donors for IgG and IgA and 3 individual donors for IgE (+SEM; * = p<0.05).</p