13 research outputs found

    Snail1 transcription factor controls telomere transcription and integrity

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    Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGF-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes

    Cellular Senescence Is Immunogenic and Promotes Antitumor Immunity

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    Senescencia celular; Inmunidad antitumoralSenesc√®ncia cel¬∑lular; Immunitat antitumoralCellular senescence; Antitumor immunityCellular senescence is a stress response that activates innate immune cells, but little is known about its interplay with the adaptive immune system. Here, we show that senescent cells combine several features that render them highly efficient in activating dendritic cells (DC) and antigen-specific CD8 T cells. This includes the release of alarmins, activation of IFN signaling, enhanced MHC class I machinery, and presentation of senescence-associated self-peptides that can activate CD8 T cells. In the context of cancer, immunization with senescent cancer cells elicits strong antitumor protection mediated by DCs and CD8 T cells. Interestingly, this protection is superior to immunization with cancer cells undergoing immunogenic cell death. Finally, the induction of senescence in human primary cancer cells also augments their ability to activate autologous antigen-specific tumor-infiltrating CD8 lymphocytes. Our study indicates that senescent cancer cells can be exploited to develop efficient and protective CD8-dependent antitumor immune responses. Significance: Our study shows that senescent cells are endowed with a high immunogenic potential‚ÄĒsuperior to the gold standard of immunogenic cell death. We harness these properties of senescent cells to trigger efficient and protective CD8-dependent antitumor immune responses.We are grateful to Maria Isabel Mu√Īoz for assistance with the animal protocols; to Kevin Kovalchik for help with data sharing; to Francesca Castoldi for help in total RNA extraction for B16F10 and IMR-90 cells; to Fredrik Fagerstrom-Billai, Susann F√§lt, Anastasios Damdimopoulos, and David Brodin at Bioinformatics and Expression Analysis Core Facility, Karolinska Institute (KI), for assistance in RNA-seq and analysis; to the IRB core facilities (Functional Genomics, Biostatistics/Bioinformatics and Histopathology); and to the PCB (Animal House) for general research support. I. Marin was the recipient of an FPI fellowship from the Spanish Ministry of Science (PRE2018-083381). O. Boix was the recipient of an FPI-AGAUR fellowship from the Generalitat de Catalunya. A. Garcia-Garijo was supported by a PERIS grant (SLT017/20/000131) from the Generalitat de Catalunya. J.A. L√≥pez-Dom√≠nguez and M. Kovatcheva were supported by a fellowship from the Spanish Association Against Cancer (AECC). Work in the laboratory of E. Caron was funded by the Fonds de recherche du Qu√©bec ‚Äď Sant√© (FRQS), the Cole Foundation, CHU Sainte-Justine, the Charles-Bruneau Foundation, the Canada Foundation for Innovation, the National Sciences and Engineering Research Council (#RGPIN-2020-05232), and the Canadian Institutes of Health Research (#174924). E. Garralda received funding from the Comprehensive Program of Cancer Immunotherapy and Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021). The M. Abad lab received funding from the Spanish Ministry of Science and Innovation (RTI2018-102046-B-I00A and RTC-2017-6123-1) and the AECC (PRYCO211023SERR). M. Abad was the recipient of a Ram√≥n y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2013-14747). A. Gros received funding from the Spanish Ministry of Science cofunded by the European Regional Development Fund (ERDF; RTC-2017-6123-1), from the Instituto de Salud Carlos III (MS15/00058), and from CAIMI-II (grant 53/2021) supported by the BBVA Foundation. The work in the laboratory of F. Pietrocola is supported by a KI Starting Grant, a Starting Grant from the Swedish Research Council (2019_02050_3), and grants from the Harald Jeanssons Foundation, the Loo and Hans Osterman Foundation, and Cancerfonden (21 1637 Pj). Work in the laboratory of M. Serrano was funded by the IRB and La Caixa Foundation, and by grants from the Spanish Ministry of Science cofunded by the European Regional Development Fund (SAF-2017-82613-R, RTC-2017-6123-1), the European Research Council (ERC-2014-AdG/669622), Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282), and the AECC (PRYCO211023SERR). The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked ‚Äúadvertisement‚ÄĚ in accordance with 18 USC section 1734

    Exploring the Immunogenicity of Noncanonical HLA-I Tumor Ligands Identified through Proteogenomics

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    Immunogenicity; ProteogenomicsInmunogenicidad; Proteogen√≥micaImmunogenicitat; Proteogen√≤micaPurpose: Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from noncanonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Experimental Design: Peptides presented on HLA-I were identified in 9 patient-derived tumor cell lines from melanoma, gynecologic, and head and neck cancer through proteogenomics. A total of 507 candidate tumor antigens, including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens, were tested for T-cell recognition of preexisting responses in patients with cancer. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell receptors (TCR) and evaluate their therapeutic potential. Results: We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor-reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor PBL against 170 selected nonC-TL, led to the identification of TCRs specific to three nonC-TL, two of which mapped to the 5‚Ä≤ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a noncoding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Conclusions: Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.We thank the patients for their participation in this study, Steven A. Rosenberg for providing valuable reagents and support for NGS studies, R. Pujol for helpful scientific discussion, J. Gonzalez for bioinformatics support, CRG/UPF Flow Cytometry Unit for assistance with cell sorting, and CRG/UPF and IRB Proteomics Units for technical support. A. Gros and this work were funded by the Comprehensive Program of Cancer Immunotherapy & Immunology II (CAIMI-II) supported by the BBVA Foundation (53/2021), Institute Carlos III (MS15/00058 and PI17/01085), AECC (IDEAS197PORT), and La Fundaci√≥ La Marat√≥ de TV3 (201919‚Äď30). We thank CERCA Programme / Generalitat de Catalunya for institutional support. M. Lozano-Rabella was supported by the Ag√®ncia de Gesti√≥ d'Ajuts Universitaris i de Recerca (AGAUR) (2018FI_B 00946). A. Garcia-Garijo was supported by Generalitat PERIS award (SLT017/20/000131). A. Yuste-Estevanez was supported by the Ag√®ncia de Gesti√≥ d'Ajuts Universitaris i de Recerca (AGAUR) (2021 FI_B 00365). J. Palomero was supported by the Beatriu de Pin√≥s programme (BP 2018), cofounded by the Agency for Management of University and Research Grants (AGAUR) and European Union's Horizon 2020

    Cellular senescence is immunogenic and promotes anti-tumor immunity

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    Cellular senescence is a stress response that activates innate immune cells, but little is known about its interplay with the adaptive immune system. Here, we show that senescent cells combine several features that render them highly efficient in activating dendritic cells (DCs) and antigen-specific CD8 T cells. This includes the release of alarmins, activation of interferon signaling, enhanced MHC class I machinery, and presentation of senescence-specific self-peptides that can activate CD8 T cells. In the context of cancer, immunization with senescent cancer cells elicits strong anti-tumor protection mediated by DCs and CD8 T cells. Interestingly, this protection is superior to immunization with cancer cells undergoing immunogenic cell death. Finally, the induction of senescence in human primary cancer cells also augments their ability to activate autologous antigen-specific tumor-infiltrating CD8 lymphocytes. Our study indicates that senescent cancer cells can be exploited to develop efficient and protective CD8-dependent anti-tumor immune responses

    Exploring the Immunogenicity of Noncanonical HLA-I Tumor Ligands Identified through Proteogenomics

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    Purpose: Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from noncanonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Experimental Design: Peptides presented on HLA-I were iden-tified in 9 patient-derived tumor cell lines from melanoma, gyneco-logic, and head and neck cancer through proteogenomics. A total of 507 candidate tumor antigens, including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens, were tested for T-cell recognition of preexisting responses in patients with cancer. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell recep-tors (TCR) and evaluate their therapeutic potential.Rudolf Virchow Center, Center for Integrative and Transla- tional Bioimaging, Julius-Maximilians-University Wueurorzburg, Wueurorzburg, German

    Long-term follow-up of certolizumab pegol in uveitis due to immune-mediated inflammatory diseases : multicentre study of 80 patients

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    Objectives To evaluate effectiveness and safety of certolizumab pegol (CZP) in uveitis due to immune-mediated inflammatory diseases (IMID). Methods Multicentre study of CZP-treated patients with IMID uveitis refractory to conventional immunosuppressant. Effectiveness was assessed through the following ocular parameters: best-corrected visual acuity, anterior chamber cells, vitritis, macular thickness and retinal vasculitis. These variables were compared between the baseline, and first week, first, third, sixth months, first and second year. Results We studied 80 (33 men/47 women) patients (111 affected eyes) with a mean age of 41.6¬Ī11.7 years. The IMID included were: spondyloarthritis (n=43), Beh√ßet's disease (n=10), psoriatic arthritis (n=8), Crohn's disease (n=4), sarcoidosis (n=2), juvenile idiopathic arthritis (n=1), reactive arthritis (n=1), rheumatoid arthritis (n=1), relapsing polychondritis (n=1), Conclusions CZP seems to be effective and safe in uveitis related to different IMID, even in patients refractory to previous biological drugs

    Exploiting circulating lymphocytes and cell-free DNA as a source to develop minimally-invasive personalized T-cell therapies

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    [eng] Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) can medi- ate tumor regression in patients with metastatic melanoma. Retrospective studies of TIL products from responders and non-responders have shown that successful TIL-ACT is associated with an expansion of T cells targeting neoantigens, which are derived from tumor-specific non-synonymous somatic mutations (NSMs) and present exquisite tumor specificity. In addition, ACT with TIL products enriched for neoantigen recognition has demonstrated antitumor activity in selected patients with epithelial tumors other than melanoma, which has fueled the interest in personalized T-cell therapies targeting neo- antigens. The identification and isolation of neoantigen-specific TILs has been possible due to advances in next-generation sequencing that enabled the rapid assessment of the tumor mutational landscape and the development of high-throughput personalized im- munological screenings. However, the need of a tumor biopsy to both identify candidate neoantigens and reactive T cells limits the broad applicability of personalized T-cell ther- apies targeting neoantigens and can underestimate tumor heterogeneity in the advanced metastatic setting. Previous reports have shown that neoantigen-specific T cells can be enriched from peripheral blood by selecting programmed death receptor-1 (PD-1)-ex- pressing lymphocytes in circulation. However, the frequency of neoantigen-specific T cells identified based on PD-1 expression remains very low and a tumor biopsy is still re- quired to identify candidate NSMs. In this thesis, we explored the use of peripheral blood as an alternative minimal- ly-invasive source to identify tumor NSMs and isolate neoantigen-specific or tumor-reac- tive T lymphocytes in patients with metastatic breast, gynecological, colorectal and head and neck cancer or chordoma. We performed whole-exome sequencing (WES) of cell-free DNA (cfDNA) to identify tumor NSMs and compared these to those identified in DNA ex- tracted from tumor biopsies (TuBx DNA). In parallel, we evaluated whether the selection of peripheral blood lymphocytes (PBLs) expressing a combination of cell-surface recep- tors, rather than the expression of PD-1 alone, could further enhance the frequency of neo- antigen-specific T cells and T-cell receptors (TCR) detected or the number of neoantigens recognized. Toidentifycirculating tumor antigen-specific T cells, wesorted CD8+ and CD4+ PBLs of ten patients with different types of epithelial cancers based on the expression of PD-1 into PD-1hi, PD-1dim and PD-1-, and in combination with the cell-surface receptors CD27, CD38, CD39, HLA-DR and 4-1BB, obtaining up to 35 CD8+ and 35 CD4+ T-cell populations for some of the patients studied. In parallel, we also expanded TILs from tumor biopsies and compared the reactivities detected in the two T-cell sources. Ex vivo expanded PBL subsets and TILs were screened using a personalized high-throughput screening strategy, which enabled the detection of T cells targeting neoantigens identified by WES from TuBx DNA and cfDNA as well as T cells recognizing cancer germline antigens. Using this approach, we detected tumor antigen-specific T cells in eight out of 10 patients analyzed. Both CD8+ and CD4+ tumor antigen-reactive T cells were preferentially enriched in T-cell subsets expressing high levels of PD-1, either alone or in combination with other cell- surface markers, being PD-1hiCD39+ the combination of markers that more consistently identified CD4+ and CD8+ tumor reactivities. More importantly, the frequency of tumor antigen-specific T lymphocytes contained in the CD8+ PD-1hiCD39+ population was consistently higher than in any other CD8+ population sorted. This was not observed in CD4+ PBLs since the frequency of neoantigen-specific T cells was highly variable among PBL populations and none of them consistently displayed higher frequencies of neoantigen-reactive lymphocytes. The selection and expansion of CD8+ PD-1hiCD39+ PBLs outperformed the classical non-specific TIL expansion in high-dose interleukin-2 (IL-2) at identifying anti-tumor CD8+ T-cell responses. In contrast, despite the total number of CD4+ reactivities detected considering all populations screened from blood was higher than in TILs, the CD4+ PD-1hiCD39+ T-cell subpopulation identified fewer reactivities than unselected TILs. When comparing tumor and cfDNA, the overlap between NSMs identified in tumor and cfDNA varied notably between patients, with two out of six patients displaying no overlap between TuBx DNA and cfDNA WES. Moreover, WES of cfDNA from six patients preferentially identified clonal somatic mutations in tumor biopsies and enabled the identification of eight of 13 neoantigens in patients displaying some degree of overlap between tumor and cell-free NSMs. Our results underscore peripheral blood as an alternative source to identify cancer-specific neoantigens and CD8+ and CD4+ neoantigen- specific T lymphocytes and TCRs, with important implications for exploiting personalized T-cell responses in advanced cancer patients.[cat] La ter√†pia adoptiva amb c√®l¬∑lules T infiltrants de tumor (TILs) ha demostrat capacitat de mediar regressi√≥ de lesions tumorals en pacients amb melanoma metast√†tic. Estudis retrospectius dels productes cel¬∑lulars utilitzats per tractar pacients que van respondre o no a la ter√†pia han demostrat que la resposta a la ter√†pia adoptiva amb TILs va associada a una major expansi√≥ de c√®l¬∑lules T amb capacitat de recon√®ixer neoant√≠gens, els quals s√≥n p√®ptids que deriven de mutacions som√†tiques no sin√≤nimes (NSMs) espec√≠fiques del tumor. A m√©s, la ter√†pia adoptiva amb productes enriquits amb TILs espec√≠fics contra neoant√≠gens ha demostrat activitat antitumoral en pacients amb diferents tipus de tumors epitelials el que ha promogut l‚Äôinter√®s en el desenvolupament de ter√†pies personalitzades amb limf√≤cits T reactius contra neoant√≠gens. La identificaci√≥ i a√Įllament de limf√≤cits T reactius contra neoant√≠gens ha sigut possible gr√†cies als aven√ßos en les t√®cniques de seq√ľenciaci√≥ el que ha perm√®s una r√†pida avaluaci√≥ del repertori mutacional del tumor aix√≠ com el desenvolupament d‚Äôassajos personalitzats per testar el seu reconeixement. Tot i aix√≤, la necessitat d‚Äôaccedir a una bi√≤psia tumoral tant per identificar els neoant√≠gens candidats com per a√Įllar els limf√≤cits T ha limitat l‚Äôaplicabilitat de les ter√†pies personalitzades basades en c√®l¬∑lules T i a m√©s a m√©s, pot subestimar la heterogene√Įtat tumoral en tumors metast√†tics. Estudis previs han demostrat que els limf√≤cits T reactius contra neoant√≠gens es poden identificar en sang perif√®rica a trav√©s de la selecci√≥ de c√®l¬∑lules T amb expressi√≥ del receptor de mort programada tipus 1 (PD-1). Tanmateix, la freq√ľ√®ncia de c√®l¬∑lules T reactives contra neoant√≠gens despr√©s de seleccionar els limf√≤cits T que expressen PD-1 √©s molt baixa. A m√©s, segueix sent necess√†ria l'obtenci√≥ d'una bi√≤psia tumoral per tal de poder identificar les mutacions no sin√≤nimes del tumor. En aquesta tesi doctoral hem explorat com a ter√†pia m√≠nimament invasiva l‚Äô√ļs de sang perif√®rica per identificar les NSMs del tumor i per a√Įllar els limf√≤cits T espec√≠fics contra neoant√≠gens o contra altres ant√≠gens tumorals en pacients amb c√†ncer de mama, ginecol√≤gic, colorectal, de cap i coll o cordoma, tots en estat metast√†tic. Per aix√≤, hem seq√ľenciat l‚ÄôADN circulant a√Įllat de la sang per tal d‚Äôidentificar mutacions que puguin provenir del tumor i les hem comparat amb aquelles identificades en l‚ÄôADN extret de bi√≤psies tumorals dels mateixos pacients. Paral¬∑lelament, hem seleccionat limf√≤cits de la sang perif√®rica en base a l‚Äôexpressi√≥ simult√†nia de diversos receptors de superf√≠cie amb l‚Äôobjectiu de veure si la co-expressi√≥ de receptors pot millorar la freq√ľ√®ncia de c√®l¬∑lules T espec√≠fiques contra neoant√≠gens o b√© incrementar el repertori de receptors de c√®l¬∑lules T (TCRs) reconeixent un mateix neoantigen comparat amb l‚Äôexpressi√≥ exclusiva de PD-1. Per tal d‚Äôidentificar c√®l¬∑lules T reactives contra neoant√≠gens en la sang perif√®rica, vam separar per citometria de flux limf√≤cits CD8 i CD4 de deu pacients amb diferents tipus de tumors epitelials en base al nivell d‚Äôexpressi√≥ de PD-1 en els grups: PD-1hi, PD- 1dim, PD-1- i en combinaci√≥ amb altres receptors de superf√≠cie com CD27, CD39, CD38, HLA-DR i 4-1BB, obtenint fins a 35 poblacions diferents tant pels limf√≤cits CD8 com pels CD4 per alguns dels pacients inclosos en l‚Äôestudi. Paral¬∑lelament, tamb√© vam expandir els TILs de les bi√≤psies tumorals d‚Äôaquests pacients i vam comparar els neoant√≠gens o ant√≠gens tumorals reconeguts per aquestes dues fonts de limf√≤cits T. Les c√®l¬∑lules T de sang perif√®rica o infiltrants de tumor es van expandir ex vivo i es van testar en assajos funcionals personalitzats el que ens va permetre detectar c√®l¬∑lules T amb capacitat de recon√®ixer neoant√≠gens identificats per seq√ľenciaci√≥ de l‚Äôexoma tant de l‚ÄôADN extret de bi√≤psies tumorals com de l‚ÄôADN circulant i ant√≠gens tumorals de l√≠nia germinal expressats en la bi√≤psia tumoral. Utilitzant aquesta aproximaci√≥, es van detectar c√®l¬∑lules T reactives contra neoant√≠gens en vuit dels deu pacients estudiats. En la majoria dels casos, els limf√≤cits CD8 i CD4 reactius es van identificar en poblacions que expressaven alts nivells de PD-1 (PD-1hi), sol o en combinaci√≥ amb altres marcadors, sent PD-1hiCD39+ la combinaci√≥ que identificava limf√≤cits T reactius contra ant√≠gens tumorals de manera m√©s consistent. A m√©s, en els limf√≤cits CD8+, la poblaci√≥ PD-1hiCD39+ sempre contenia la freq√ľ√®ncia m√©s elevada de limf√≤cits reactius en el cas que aquests s‚Äôhaguessin identificat en diferents poblacions separades de la sang. Aquesta darrera observaci√≥ no aplica pels limf√≤cits CD4+ ja que en aquest cas cap de les poblacions contenia la freq√ľ√®ncia m√©s alta de limf√≤cits T reactius contra neoant√≠gens de manera consistent. La selecci√≥ i expansi√≥ de limf√≤cits CD8+ PD-1hiCD39+ de la sang perif√®rica va permetre capturar un major nombre de reactivitats contra ant√≠gens tumorals que l‚Äôestablert sistema d‚Äôexpansi√≥ inespec√≠fica de TILs en pres√®ncia d‚Äôaltes dosis d‚Äôinterleuquina-2 (IL-2). Per altra banda, tot i que el nombre total de reactivitats identificades en les poblacions de limf√≤cits CD4+ a√Įllades de la sang perif√®rica √©s superior a les identificades en els TILs, la combinaci√≥ CD4+ PD- 1hiCD39+ identifica menys reactivitats que les detectades en els TILs. Pel que fa a les NSMs identificades en l‚ÄôADN tumoral i en l‚ÄôADN circulant, vam observar que el nombre de mutacions compartides entre les dues fonts d‚ÄôADN era altament variable, essent nul en dos dels sis pacients estudiats. La seq√ľenciaci√≥ d‚Äôexoma de l‚ÄôADN circulant dels sis pacients va identificar preferencialment mutacions som√†tiques clonals en les bi√≤psies tumorals. Tamb√© va permetre la identificaci√≥ de vuit dels 13 neoant√≠gens detectats en pacients que mostraven un cert grau de solapament entre les NSMs detectades en l‚ÄôADN extret de la bi√≤psia tumoral i l‚ÄôADN circulant. En resum, els nostres resultats demostren que l'√ļs exclusiu de sang perif√®rica permet detectar neoant√≠gens i els limf√≤cits CD4+ i CD8+ capa√ßos de recon√®ixer aquest tipus d'ant√≠gens tumorals el que t√© implicacions importants pel desenvolupament de ter√†pies cel¬∑lulars personalitzades en pacients amb tumors avan√ßats.[spa] En esta tesis, el objetivo principal era explorar la sangre perif√©rica para poder identificar neoant√≠genos tumorales y los linfocitos capaces de reconocerlos. Para conseguir este objetivo, se realiz√≥ la secuenciaci√≥n del exoma del ADN circulante extra√≠do de muestras de plasma as√≠ como la secuenciaci√≥n del ADN extra√≠do de biopsias tumorales. Paralelamente, se seleccionaron diferentes poblaciones de linfocitos T CD4+ y CD8+ de la sangre en base a la expresi√≥n de ciertos marcadores de co-estimulaci√≥n o inhibici√≥n y se testaron usando ensayos funcionales inmunol√≥gicos personalizados para ver su capacidad de reconocer neoant√≠genos y otros ant√≠genos tumorales. Mediante estos an√°lisis, se detectaron un total de 15 reactividades en la sangre, 8 CD8+ y 7 CD4+. En el caso de los CD8+, se observ√≥ que la poblaci√≥n CD8+ PD-1hi CD39+ era la poblaci√≥n que conten√≠a m√°s reactividades contra ant√≠genos tumorales y que conten√≠a la frecuencia m√°s alta de linfocitos reactivos. En el caso de los CD4+, la poblaci√≥n PD-1hiCD39+ tambi√©n fue la poblaci√≥n que identific√≥ m√°s reactividades aunque no detect√≥ una proporci√≥n alta de reactividades indicando que esta puede no ser la mejor combinaci√≥n de marcadores para CD4+. Por otro lado, la secuenciaci√≥n del ADN circulante permiti√≥ identificar 8 de los 13 neoant√≠genos detectados en los 6 pacientes en los que se secuenci√≥n el ADN circulante. Por tanto, estos resultados demuestran que es posible utilizar la sangre perif√©rica para identificar neoant√≠genos y linfocitos espec√≠ficos contra neoant√≠genos lo que puede permitir el desarrollo de ter√°pias celulares personalizadas m√≠nimamente invasivas √ļnicamente utilitzando la sangre perif√©rica

    Exploiting circulating lymphocytes and cell-free DNA as a source to develop minimally-invasive personalized T-cell therapies

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    Tesi realitzada al Vall d'Hebron Institut d'Oncologia (VHIO) / Programa de Doctorat en Biomedicina[eng] Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) can medi- ate tumor regression in patients with metastatic melanoma. Retrospective studies of TIL products from responders and non-responders have shown that successful TIL-ACT is associated with an expansion of T cells targeting neoantigens, which are derived from tumor-specific non-synonymous somatic mutations (NSMs) and present exquisite tumor specificity. In addition, ACT with TIL products enriched for neoantigen recognition has demonstrated antitumor activity in selected patients with epithelial tumors other than melanoma, which has fueled the interest in personalized T-cell therapies targeting neo- antigens. The identification and isolation of neoantigen-specific TILs has been possible due to advances in next-generation sequencing that enabled the rapid assessment of the tumor mutational landscape and the development of high-throughput personalized im- munological screenings. However, the need of a tumor biopsy to both identify candidate neoantigens and reactive T cells limits the broad applicability of personalized T-cell ther- apies targeting neoantigens and can underestimate tumor heterogeneity in the advanced metastatic setting. Previous reports have shown that neoantigen-specific T cells can be enriched from peripheral blood by selecting programmed death receptor-1 (PD-1)-ex- pressing lymphocytes in circulation. However, the frequency of neoantigen-specific T cells identified based on PD-1 expression remains very low and a tumor biopsy is still re- quired to identify candidate NSMs. In this thesis, we explored the use of peripheral blood as an alternative minimal- ly-invasive source to identify tumor NSMs and isolate neoantigen-specific or tumor-reac- tive T lymphocytes in patients with metastatic breast, gynecological, colorectal and head and neck cancer or chordoma. We performed whole-exome sequencing (WES) of cell-free DNA (cfDNA) to identify tumor NSMs and compared these to those identified in DNA ex- tracted from tumor biopsies (TuBx DNA). In parallel, we evaluated whether the selection of peripheral blood lymphocytes (PBLs) expressing a combination of cell-surface recep- tors, rather than the expression of PD-1 alone, could further enhance the frequency of neo- antigen-specific T cells and T-cell receptors (TCR) detected or the number of neoantigens recognized. Toidentifycirculating tumor antigen-specific T cells, wesorted CD8+ and CD4+ PBLs of ten patients with different types of epithelial cancers based on the expression of PD-1 into PD-1hi, PD-1dim and PD-1-, and in combination with the cell-surface receptors CD27, CD38, CD39, HLA-DR and 4-1BB, obtaining up to 35 CD8+ and 35 CD4+ T-cell populations for some of the patients studied. In parallel, we also expanded TILs from tumor biopsies and compared the reactivities detected in the two T-cell sources. Ex vivo expanded PBL subsets and TILs were screened using a personalized high-throughput screening strategy, which enabled the detection of T cells targeting neoantigens identified by WES from TuBx DNA and cfDNA as well as T cells recognizing cancer germline antigens. Using this approach, we detected tumor antigen-specific T cells in eight out of 10 patients analyzed. Both CD8+ and CD4+ tumor antigen-reactive T cells were preferentially enriched in T-cell subsets expressing high levels of PD-1, either alone or in combination with other cell- surface markers, being PD-1hiCD39+ the combination of markers that more consistently identified CD4+ and CD8+ tumor reactivities. More importantly, the frequency of tumor antigen-specific T lymphocytes contained in the CD8+ PD-1hiCD39+ population was consistently higher than in any other CD8+ population sorted. This was not observed in CD4+ PBLs since the frequency of neoantigen-specific T cells was highly variable among PBL populations and none of them consistently displayed higher frequencies of neoantigen-reactive lymphocytes. The selection and expansion of CD8+ PD-1hiCD39+ PBLs outperformed the classical non-specific TIL expansion in high-dose interleukin-2 (IL-2) at identifying anti-tumor CD8+ T-cell responses. In contrast, despite the total number of CD4+ reactivities detected considering all populations screened from blood was higher than in TILs, the CD4+ PD-1hiCD39+ T-cell subpopulation identified fewer reactivities than unselected TILs. When comparing tumor and cfDNA, the overlap between NSMs identified in tumor and cfDNA varied notably between patients, with two out of six patients displaying no overlap between TuBx DNA and cfDNA WES. Moreover, WES of cfDNA from six patients preferentially identified clonal somatic mutations in tumor biopsies and enabled the identification of eight of 13 neoantigens in patients displaying some degree of overlap between tumor and cell-free NSMs. Our results underscore peripheral blood as an alternative source to identify cancer-specific neoantigens and CD8+ and CD4+ neoantigen- specific T lymphocytes and TCRs, with important implications for exploiting personalized T-cell responses in advanced cancer patients.[cat] La ter√†pia adoptiva amb c√®l¬∑lules T infiltrants de tumor (TILs) ha demostrat capacitat de mediar regressi√≥ de lesions tumorals en pacients amb melanoma metast√†tic. Estudis retrospectius dels productes cel¬∑lulars utilitzats per tractar pacients que van respondre o no a la ter√†pia han demostrat que la resposta a la ter√†pia adoptiva amb TILs va associada a una major expansi√≥ de c√®l¬∑lules T amb capacitat de recon√®ixer neoant√≠gens, els quals s√≥n p√®ptids que deriven de mutacions som√†tiques no sin√≤nimes (NSMs) espec√≠fiques del tumor. A m√©s, la ter√†pia adoptiva amb productes enriquits amb TILs espec√≠fics contra neoant√≠gens ha demostrat activitat antitumoral en pacients amb diferents tipus de tumors epitelials el que ha promogut l‚Äôinter√®s en el desenvolupament de ter√†pies personalitzades amb limf√≤cits T reactius contra neoant√≠gens. La identificaci√≥ i a√Įllament de limf√≤cits T reactius contra neoant√≠gens ha sigut possible gr√†cies als aven√ßos en les t√®cniques de seq√ľenciaci√≥ el que ha perm√®s una r√†pida avaluaci√≥ del repertori mutacional del tumor aix√≠ com el desenvolupament d‚Äôassajos personalitzats per testar el seu reconeixement. Tot i aix√≤, la necessitat d‚Äôaccedir a una bi√≤psia tumoral tant per identificar els neoant√≠gens candidats com per a√Įllar els limf√≤cits T ha limitat l‚Äôaplicabilitat de les ter√†pies personalitzades basades en c√®l¬∑lules T i a m√©s a m√©s, pot subestimar la heterogene√Įtat tumoral en tumors metast√†tics. Estudis previs han demostrat que els limf√≤cits T reactius contra neoant√≠gens es poden identificar en sang perif√®rica a trav√©s de la selecci√≥ de c√®l¬∑lules T amb expressi√≥ del receptor de mort programada tipus 1 (PD-1). Tanmateix, la freq√ľ√®ncia de c√®l¬∑lules T reactives contra neoant√≠gens despr√©s de seleccionar els limf√≤cits T que expressen PD-1 √©s molt baixa. A m√©s, segueix sent necess√†ria l'obtenci√≥ d'una bi√≤psia tumoral per tal de poder identificar les mutacions no sin√≤nimes del tumor. En aquesta tesi doctoral hem explorat com a ter√†pia m√≠nimament invasiva l‚Äô√ļs de sang perif√®rica per identificar les NSMs del tumor i per a√Įllar els limf√≤cits T espec√≠fics contra neoant√≠gens o contra altres ant√≠gens tumorals en pacients amb c√†ncer de mama, ginecol√≤gic, colorectal, de cap i coll o cordoma, tots en estat metast√†tic. Per aix√≤, hem seq√ľenciat l‚ÄôADN circulant a√Įllat de la sang per tal d‚Äôidentificar mutacions que puguin provenir del tumor i les hem comparat amb aquelles identificades en l‚ÄôADN extret de bi√≤psies tumorals dels mateixos pacients. Paral¬∑lelament, hem seleccionat limf√≤cits de la sang perif√®rica en base a l‚Äôexpressi√≥ simult√†nia de diversos receptors de superf√≠cie amb l‚Äôobjectiu de veure si la co-expressi√≥ de receptors pot millorar la freq√ľ√®ncia de c√®l¬∑lules T espec√≠fiques contra neoant√≠gens o b√© incrementar el repertori de receptors de c√®l¬∑lules T (TCRs) reconeixent un mateix neoantigen comparat amb l‚Äôexpressi√≥ exclusiva de PD-1. Per tal d‚Äôidentificar c√®l¬∑lules T reactives contra neoant√≠gens en la sang perif√®rica, vam separar per citometria de flux limf√≤cits CD8 i CD4 de deu pacients amb diferents tipus de tumors epitelials en base al nivell d‚Äôexpressi√≥ de PD-1 en els grups: PD-1hi, PD- 1dim, PD-1- i en combinaci√≥ amb altres receptors de superf√≠cie com CD27, CD39, CD38, HLA-DR i 4-1BB, obtenint fins a 35 poblacions diferents tant pels limf√≤cits CD8 com pels CD4 per alguns dels pacients inclosos en l‚Äôestudi. Paral¬∑lelament, tamb√© vam expandir els TILs de les bi√≤psies tumorals d‚Äôaquests pacients i vam comparar els neoant√≠gens o ant√≠gens tumorals reconeguts per aquestes dues fonts de limf√≤cits T. Les c√®l¬∑lules T de sang perif√®rica o infiltrants de tumor es van expandir ex vivo i es van testar en assajos funcionals personalitzats el que ens va permetre detectar c√®l¬∑lules T amb capacitat de recon√®ixer neoant√≠gens identificats per seq√ľenciaci√≥ de l‚Äôexoma tant de l‚ÄôADN extret de bi√≤psies tumorals com de l‚ÄôADN circulant i ant√≠gens tumorals de l√≠nia germinal expressats en la bi√≤psia tumoral. Utilitzant aquesta aproximaci√≥, es van detectar c√®l¬∑lules T reactives contra neoant√≠gens en vuit dels deu pacients estudiats. En la majoria dels casos, els limf√≤cits CD8 i CD4 reactius es van identificar en poblacions que expressaven alts nivells de PD-1 (PD-1hi), sol o en combinaci√≥ amb altres marcadors, sent PD-1hiCD39+ la combinaci√≥ que identificava limf√≤cits T reactius contra ant√≠gens tumorals de manera m√©s consistent. A m√©s, en els limf√≤cits CD8+, la poblaci√≥ PD-1hiCD39+ sempre contenia la freq√ľ√®ncia m√©s elevada de limf√≤cits reactius en el cas que aquests s‚Äôhaguessin identificat en diferents poblacions separades de la sang. Aquesta darrera observaci√≥ no aplica pels limf√≤cits CD4+ ja que en aquest cas cap de les poblacions contenia la freq√ľ√®ncia m√©s alta de limf√≤cits T reactius contra neoant√≠gens de manera consistent. La selecci√≥ i expansi√≥ de limf√≤cits CD8+ PD-1hiCD39+ de la sang perif√®rica va permetre capturar un major nombre de reactivitats contra ant√≠gens tumorals que l‚Äôestablert sistema d‚Äôexpansi√≥ inespec√≠fica de TILs en pres√®ncia d‚Äôaltes dosis d‚Äôinterleuquina-2 (IL-2). Per altra banda, tot i que el nombre total de reactivitats identificades en les poblacions de limf√≤cits CD4+ a√Įllades de la sang perif√®rica √©s superior a les identificades en els TILs, la combinaci√≥ CD4+ PD- 1hiCD39+ identifica menys reactivitats que les detectades en els TILs. Pel que fa a les NSMs identificades en l‚ÄôADN tumoral i en l‚ÄôADN circulant, vam observar que el nombre de mutacions compartides entre les dues fonts d‚ÄôADN era altament variable, essent nul en dos dels sis pacients estudiats. La seq√ľenciaci√≥ d‚Äôexoma de l‚ÄôADN circulant dels sis pacients va identificar preferencialment mutacions som√†tiques clonals en les bi√≤psies tumorals. Tamb√© va permetre la identificaci√≥ de vuit dels 13 neoant√≠gens detectats en pacients que mostraven un cert grau de solapament entre les NSMs detectades en l‚ÄôADN extret de la bi√≤psia tumoral i l‚ÄôADN circulant. En resum, els nostres resultats demostren que l'√ļs exclusiu de sang perif√®rica permet detectar neoant√≠gens i els limf√≤cits CD4+ i CD8+ capa√ßos de recon√®ixer aquest tipus d'ant√≠gens tumorals el que t√© implicacions importants pel desenvolupament de ter√†pies cel¬∑lulars personalitzades en pacients amb tumors avan√ßats.[spa] En esta tesis, el objetivo principal era explorar la sangre perif√©rica para poder identificar neoant√≠genos tumorales y los linfocitos capaces de reconocerlos. Para conseguir este objetivo, se realiz√≥ la secuenciaci√≥n del exoma del ADN circulante extra√≠do de muestras de plasma as√≠ como la secuenciaci√≥n del ADN extra√≠do de biopsias tumorales. Paralelamente, se seleccionaron diferentes poblaciones de linfocitos T CD4+ y CD8+ de la sangre en base a la expresi√≥n de ciertos marcadores de co-estimulaci√≥n o inhibici√≥n y se testaron usando ensayos funcionales inmunol√≥gicos personalizados para ver su capacidad de reconocer neoant√≠genos y otros ant√≠genos tumorales. Mediante estos an√°lisis, se detectaron un total de 15 reactividades en la sangre, 8 CD8+ y 7 CD4+. En el caso de los CD8+, se observ√≥ que la poblaci√≥n CD8+ PD-1hi CD39+ era la poblaci√≥n que conten√≠a m√°s reactividades contra ant√≠genos tumorales y que conten√≠a la frecuencia m√°s alta de linfocitos reactivos. En el caso de los CD4+, la poblaci√≥n PD-1hiCD39+ tambi√©n fue la poblaci√≥n que identific√≥ m√°s reactividades aunque no detect√≥ una proporci√≥n alta de reactividades indicando que esta puede no ser la mejor combinaci√≥n de marcadores para CD4+. Por otro lado, la secuenciaci√≥n del ADN circulante permiti√≥ identificar 8 de los 13 neoant√≠genos detectados en los 6 pacientes en los que se secuenci√≥n el ADN circulante. Por tanto, estos resultados demuestran que es posible utilizar la sangre perif√©rica para identificar neoant√≠genos y linfocitos espec√≠ficos contra neoant√≠genos lo que puede permitir el desarrollo de ter√°pias celulares personalizadas m√≠nimamente invasivas √ļnicamente utilitzando la sangre perif√©rica

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    Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFő≤-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes
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