Mechanisms of tolerance induction to foreign proteins

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2013Several immune mediated diseases, such as autoimmunity and allergy or transplant rejection are consequence of inappropriate immune responses towards specific antigens. The therapeutic induction of robust immune tolerance has, therefore been considered the Holy Grail of immunology since the pioneering work of Medawar and colleagues who have demonstrated the feasibility of tolerance induction. Immune tolerance is defined as the unresponsive state to a set of antigens by an immune system that remains immune competent to respond to unrelated antigens. A wealth of mechanisms has evolved to achieve immune tolerance to self as well as many environmental antigens. These mechanisms include negative selection in the thymus together with the production of regulatory T cells dedicated to the prevention of auto reactivity; cytokines like IL-10; immune privileged sites; and peripheral control of effector T cells by apoptosis, anergy, competition, and differentiation of several types of regulatory immune cells. In the last twenty years much emphasis has been given to a powerful regulatory T (Treg) cell population initially characterized by the high expression of CD25 (the alpha chain of the IL-2R), that express the transcription factor Foxp3. Over the years a significant amount of information on Tregs has been acquired which empowered the immunologists to devise new strategies to differentiate and expand these cells and ultimately to use them as cellular therapies. One strategy known to induce immune tolerance takes advantage of biological agents such as monoclonal antibodies (MAbs) that can specifically target certain molecules modulating immune responses. Several studies in different animal models of immune disorders have shown the efficacy of MAbs at inducing long term specific immune tolerance. Non depleting anti-CD4 MAb has been shown able to induce immune tolerance in transplantation and autoimmunity by inducing a specific Treg population. In the work presented in this thesis we have shown that non depleting anti-CD4 can induce robust tolerance to soluble antigens in a mouse model of allergic asthma – a Th2-driven pathology. CD4-blockade at the time of allergen exposure can lead to antigen-specific tolerance therefore sparing the immune response to other antigens. Based on these findings, where the allergen is administered together with an adjuvant, we revisited immune tolerance to therapeutic proteins using a mouse model of hemophilia. It had been reported that tolerance induced with MAbs (such as anti-CD4) was difficult to induce to proteins like factor VIII. We hypothesized that an adjuvant, such as alum, that in the animal models of allergy allows tolerance induction to model antigens and allergens, may be useful to facilitate tolerance to FVIII. We confirmed that indeed tolerance induction to FVIII can be induced when alum is used as an adjuvant – a counter-intuitive finding given the tendency of adjuvants to boost, rather than inhibit, immune responses. We believe that for proper tolerization of antigen-specific cells it is essential to have adequate antigen presentation. Therefore an adjuvant can facilitate the process. This hypothesis will be addressed in future studies. Finally, we investigated the cellular and molecular mechanisms underlying the induction of tolerance to proteins using non-depleting anti- CD4. We anticipated that induction of Foxp3+ T cells would be a major mechanism given prior studies that have clearly established induced Foxp3+ Tregs in tolerance induction in transplantation and autoimmunity. However, we found that tolerance to proteins in alum could be induced in Foxp3-deficient mice, but not in the absence of IL-10. In addition to the dominant tolerance mechanism IL-10-dependent we found evidence for a recessive mechanism based on the activation-cell death and anergy of antigen-specific cells following activation under the cover of anti-CD4. We believe it may be possible to take advantage of both Foxp3- independent and Foxp3-dependent mechanisms to achieve a more robust state of immune tolerance.Várias doenças causadas pelo sistema imunitário, como doenças autoimunes, alergias ou rejeição de transplantes, são causadas por uma resposta inadequada a alguns antigénios. Uma possível forma de tratar estas doenças consiste na indução de tolerância específica e robusta. A tolerância imunitária é definida como a ausência de resposta por parte do sistema imunitário a um dado antigénio, mantendo-se competente para responder a antigénios distintos. Existem vários mecanismos que asseguram tolerância imunitária a antigénios do próprio e vários antigénios ambientais, tais como a selecção negativa no timo de linfócitos autorreactivos juntamente com o desenvolvimento de células T reguladoras (Treg) que suprimem autorreactividade; locais imunoprivilegiados como a placenta; citoquinas imunorreguladoras como a IL-10; e o controlo na periferia de linfócitos maturos através de apoptose, anergia, competição e diferenciação de diferentes populações celulares reguladoras. Nos últimos vinte anos tem sido dada muita ênfase a uma população de células T reguladoras caracterizada pela elevada expressão de CD25 (a cadeia alfa do receptor de IL-2) e de Foxp3 (um factor de transcrição). Com a aquisição de informação sobre as Tregs, os imunologistas têm tido a capacidade de desenvolver novas estratégias para diferenciar e expandir esta população celular, sendo inclusivamente utilizadas Tregs como terapia celular em ensaios clínicos. Uma forma eficaz de induzir tolerância imunitária em modelos animais consiste na utilização de anticorpos monoclonais (MAbs), dirigidos a moléculas envolvidas na activação de linfócitos. Vários estudos em modelos animais de patologias do sistema imunitário demonstram a eficácia destes MAbs na indução de uma tolerância robusta a longo prazo. O anti-CD4 não depletante, em particular, é bastante eficaz na indução de tolerância imunitária em transplantação e autoimunidade induzindo uma população de Tregs. No trabalho descrito nesta tese demonstrámos que o anti-CD4 não depletante é capaz de induzir tolerância num modelo animal de asma alérgica, que é uma patologia dependente de uma resposta imunitária do tipo Th2. O bloqueio do CD4 concomitante com a exposição ao alergénio leva à indução de um estado de tolerância específica para esse antigénio, mantendo-se a competência do sistema imunitário para responder a outros antigénios diferentes. Com base nestes resultados, onde para a indução de tolerância utilizámos antigénios administrados juntamente com um adjuvante (alum), decidimos estudar a indução de tolerância a outras proteínas. Com efeito, estudos prévios mostraram ser particularmente difícil induzir tolerância a factor VIII (FVIII) em modelos animais de hemofilia, utilizando anti-CD4 ou outros anticorpos indutores de tolerância. Decidimos reavaliar a indução de tolerância a FVIII em hemofilia com base na hipótese que um adjuvante poderia facilitar esse processo. Com efeito verificámos que a administração de FVIII com alum permite a obtenção de tolerância robusta ao factor de coagulação com anti-CD4. Estes resultados poderão parecer contra-intuitivos pois os adjuvantes são conhecidos pela sua capacidade de potenciar respostas imunitárias e não de as inibir. No entanto é provável que para indução terapêutica de tolerância seja necessário que o antigénio a tolerar seja apresentado de forma eficaz à maioria de células T efectoras específicas durante o período em que o anti-CD4 se mantém em níveis terapêuticos. Um adjuvante poderá assim facilitar a apresentação de antigénios pelas células dendríticas durante este período. Esta hipótese será avaliada em experiências futuras. Finalmente, procurámos identificar os mecanismos celulares e moleculares associados à indução de tolerância imunitária a proteínas em adjuvante. Como está descrito na literatura que o anti-CD4 promove, em transplantação e autoimunidade, a diferenciação de Tregs Foxp3+, assumimos que o mesmo mecanismo estaria envolvido na indução de tolerância a proteínas em alum. Contudo, verificámos ser possível indução de tolerância em animais deficientes em Foxp3. No entanto o estado de tolerância depende de IL-10, pois não é possível induzir tolerância em animais na ausência desta citoquina. Para além deste mecanismo de tolerância dominante dependente de IL-10 também está envolvido um componente recessivo, que depende da anergia e apoptose de células efectoras específicas É possível que a combinação de estratégias que promovem mecanismos indutores de tolerância independentes de Foxp3 e outras estratégias que promovam tolerância mediada por Treg possam ser explorados de um modo sinergético para alcançar um estado de tolerância mais robusto e em situações mais estringentes.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/49093/2008

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Molecular Epidemiology of Hepatitis A Virus in a Group of Portuguese Citizens Living in Lisbon Area

Hepatitis A virus (HAV) is the most important cause of acute infectious hepatitis worldwide. In Portugal, due to improvements in sanitation epidemic outbreaks of HAV infection have become less frequent. This report is the first, to our knowledge that characterized HAV in Portugal. For the detection and molecular characterization of HAV cases in a group of Portuguese individuals in the Lisbon area, 31 serum samples were tested: 8 from symptomatic children from an acute hepatitis A outbreak in a Roma (Gipsies) community (2004–2005), and 22 from patients with acuteHAV from sporadic cases (2005–2006). A sample of CSF involved in a case of meningitis was also included. IgM anti-HAV detection and nested reverse transcription (RT-PCR), with primers located at the VP1-P2a region, was undertaken to detect HAV genome. In positive samples, molecular characterization was followed by phylogenetic analysis. All samples (n¼31) were positive for IgM anti-HAV. HAV RNA was found in 96.7% of cases. All isolates were classified as genotype I: 22 belonged to sub-genotype IA (73.3%), and 8 to sub-genotype IB (26.7%). All strains obtained from an acute HAV outbreak had sub-genotype IA, in which seven isolates (87.5%) had identical sequences. In HAV sporadic cases sub-genotypes IA and IB were identified, and this may reflect the co-circulation of these two subgenotypes in Portugal. Molecular epidemiology of HAV infection in this group of Portuguese appears to be similar to other European countries. HAV phylogenetic studies can provide important information for the design of appropriate public health measures

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data

Abstract Introduction Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). Methods The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. Results The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. Conclusions We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis

<p>Abstract</p> <p>Background</p> <p>ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis.</p> <p>Methods</p> <p>UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand <it>in vitro</it> and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed <it>in vitro.</it> Furthermore, UCX® cells were administered <it>in vivo</it> in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time.</p> <p>Results</p> <p>UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations.</p> <p>Conclusion</p> <p>The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.</p