Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer

Neoplasias genitales; Inmunoterapia; Linfocitos TNeoplàsies genitals; Immunoteràpia; Limfòcits TGenital neoplasms; Immunotherapy; T-LymphocytesBackground Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. Methods To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. Results We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1− and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1− or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. Conclusions Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.AG was funded by the Comprehensive Program of Cancer Immunotherapy and Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021), the La Fundació La Marató de TV3 (201919-30; identification number 488/C/2019), the Spanish Ministry of Science and Innovation (PID2020-118529RB-100) and Instituto de Salud Carlos III (CP15/00058). We thank the CERCA Programme/Generalitat de Catalunya for institutional support. JP was supported by the Beatriu de Pinós programme (BP 2018), cofounded by the Agency for Management of University and Research Grants (AGAUR) and European Union's Horizon 2020. HUB-ICO-IDIBELL Biobank received support from Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC)

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

Immunogenicity; ProteogenomicsInmunogenicidad; ProteogenómicaImmunogenicitat; ProteogenòmicaPurpose: 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′ 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 Fundació La Marató de TV3 (201919–30). We thank CERCA Programme / Generalitat de Catalunya for institutional support. M. Lozano-Rabella was supported by the Agència de Gestió 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 Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) (2021 FI_B 00365). J. Palomero was supported by the Beatriu de Pinós programme (BP 2018), cofounded by the Agency for Management of University and Research Grants (AGAUR) and European Union's Horizon 2020

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

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

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer

Background: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. Methods: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. Results: We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1- and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1- or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. Conclusions: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity

Identifi cation of the personalized repertoire of conventional and non-canonical tumor antigens

La identificació d'antígens tumorals és necessària per tal d'entendre millor la resposta immunològica contra els tumors i poder desenvolupar immunoteràpies més efectives. Els cribatges immunològics en pacients amb càncer identifiquen freqüentment respostes naturals dels limfòcits T contra antígens tumorals convencionals com els neoantigens, els antígens cancerosos de línia germinal i els antígens de diferenciació de teixits. Recentment, noves evidències suggereixen que els pèptids derivats de proteïnes no canòniques són presentats de forma específica en les molècules HLA de classe-I de les cèl·lules tumorals, el que diversifica el repertori d'antígens tumorals que es poden usar com a diana en tractaments. Actualment, els reptes principals en aquest camp són determinar si aquests antígens no-canònics es presenten de forma restringida a les cèl·lules tumorals i si els limfòcits T poden generar respostes de forma natural contra ells, dos fets importants per desenvolupar noves opcions terapèutiques. En aquesta tesi, hem estudiat en detall el repertori personalitzat d'antígens tumorals reconeguts per limfòcits T reactius contra la línia tumoral derivada de 9 pacients afectats per diferents tipus de càncer. Hem estudiat tan antígens tumorals convencionals com diferents fonts d'antígens tumorals no-canònics. En la primera part, ens hem centrat en determinar si els limfòcits T reactius reconeixien antígens tumorals convencionals per mitjà de tres metodologies diferents: cribatge immunològic personalitzat de neoantigens amb TMG, predictors in-silico i immunopeptidòmica d'HLA-I. Malgrat els esforços, la majoria de dianes antigèniques dels limfòcits T reactius contra el tumor no es van poder determinar. A la segona part, hem utilitzat un mètode proteogenomic per identificar pèptids derivats de proteïnes no-canòniques presentats en HLA-I a les línies tumorals dels pacients (en anglès, nonC-TL). Vem identificar nonC-TL en tots els pacients en la majoria de casos derivats de regions 5'UTR. Tot i representar la font d'antígens tumoral més abundant, no es van poder detectar respostes de limfòcits T contra nonC-TL en cap dels pacients estudiats. Per contra, els limfòcits T reactius contra el tumor dels pacients reconeixien preferencialment neoantigens, antígens cancerosos de línia germinal o antígens de diferenciació de teixits. Per mitjà de l'estimulació in-vitro de limfòcits de sang perifèrica de donants amb nonC-TL, vam demostrar que poden induir una resposta immunològica de novo. Concretament, vam trobar TCR específics contra 3 nonC-TL, dos dels quals deriven de la regió 5'UTR dels gens HOXC13 i ZKSCAN1 i el tercer d'ells deriva d'una variant d'splicing no-codificant del gen C5orf22C.Cal remarcar, el potencial terapèutic d'aquests tres nonC-TL degut a que s'expressen en diferents tipus tumorals però no es detecten en cèl·lules sanes. En conjunt, els nostres resultats demostren que els nonC-TL tenen un gran potencial terapèutic ja que poden ser immunogènics, freqüentment presentats en HLA-I de cèl·lules tumorals, compartits entre pacients, i poc expressats en cèl·lules sanes. Malgrat l'estudi exhaustiu de totes les possibles fonts antigèniques en pacients amb càncer, incloent nonC-TL, neoantigens, els antígens cancerosos de línia germinal, antígens de diferenciació de teixits, la diana de la majoria de limfòcits T reactius contra el tumor continua per determinar, el que deixa espai a futures investigacions.La identificación de antígenos tumorales es critica para un mejor entendimiento de la respuesta inmune contra los tumores y para el desarrollo de inmunoterapias más efectivas. Los cribados inmunológicos en pacientes con cáncer permiten frecuentemente identificar respuestas naturales de los linfocitos T contra antígenos tumorales convencionales como los neoantigenos, antígenos cancerosos de línea germinal y antígenos de diferenciación de tejidos. Recientemente, nuevas evidencias sugieren que los péptidos derivados de proteínas no-canónicas son presentados de forma específica en las moléculas HLA de clase-I de las células tumorales, lo que diversifica el repertorio de antígenos tumorales que se pueden usar como dianas en los tratamientos. Actualmente, los retos principales en este campo son determinar si estos antígenos no-canónicos se presentan de forma restringida en las células tumorales y si los linfocitos T pueden generar respuestas de forma natural contra ellos, dos hechos vitales desarrollar nuevas opciones terapéuticas. En esta tesis doctoral, hemos estudiado con detalle el repertorio personalizado de antígenos tumorales reconocidos por los linfocitos T reactivos contra la línea tumoral derivada de 9 pacientes afectados por diferentes tipos de cáncer. Concretamente, se han estudiado antígenos tumorales convencionales así como diversas fuentes de antígenos no-canónicos. En la primera parte, nos hemos centrado en determinar si los linfocitos T reactivos contra el tumor reconocían específicamente antígenos tumorales convencionales por medio de tres metodologías diferentes: cribaje inmunológico personalizado de neoantigens con TMG, predictores in-silico y immunopeptidómica de HLA-I. A pesar de los esfuerzos, la mayoría de dianas antigénicas de los linfocitos T reactivos contra el tumor no se pudieron determinar. En la segunda parte, hemos usado un método proteogenómico para identificar péptidos derivados de proteínas no-canónicas presentados en HLA-I en las líneas tumorales de los pacientes (en inglés, nonC-TL). Estos nonC-TL fueron identificados en todos los pacientes, derivando en la mayoría de los casos de regiones 5'UTR. A pesar de representar la fuente de antígenos tumorales más abundante, no se pudieron detectar respuestas de linfocitos T contra nonC-TL en ninguno de los pacientes estudiados. Contrariamente, en pacientes con cáncer, los linfocitos T reactivos contra el tumor reconocían preferencialmente neoantigenos, antígenos cancerosos de línea germinal y antígenos de diferenciación de tejidos. A través de la estimulación in vitro de linfocitos de sangre periférica de donantes con nonC-TL, demostramos que éstos pueden inducir una respuesta inmunológica de novo. Concretamente, encontramos TCR específicos contra 3 nonC-TL, dos de los cuales derivan de la región 5'UTR de los genes HOXC13 y ZKSCAN1 y el tercero de ellos deriva de una variante de splicing no-codificante del gen C5orf22C. Cabe remarcar el potencial terapéutico de estos tres nonC-TL debido a que se expresan en diferentes tipos tumorales pero no se detectan en células sanas. En conjunto, nuestros resultados demuestran que ellos nonC-TL tiene un gran potencial terapéutico ya que pueden ser inmunogénicos, frecuentemente presentados en HLA-I en células tumorales, compartidos entre pacientes y poco expresados en células sanas. A pesar del estudio exhaustivo de todas las posibles fuentes antigénicas en pacientes con cáncer, incluyendo nonC-TL, neoantigenos, antígenos cancerosos de línea germinal y antígenos de diferenciación de tejidos, la diana especifica de la mayoría de linfocitos reactivos contra el tumor continua por determinar, lo que deja espacio a futuras investigaciones.The identification of tumor antigens is critical to better understand the immune response to tumors and to develop more effective cancer immunotherapies. Immunological screenings have frequently identified natural T-cell responses to conventional tumor antigens such as neoantigens, cancer-germline, and differentiation antigens in cancer patients. Recent evidence suggests that peptides derived from nonC proteins are specifically presented on HLA-I from tumor cells, thus expanding the repertoire of targetable tumor antigens. One of the main concerns is whether those nonC HLA-I ligands can naturally elicit T-cell responses and whether they are specifically presented by tumor cells, with important implications for designing therapeutic interventions. In this thesis, we have studied in detail the personalized repertoire of tumor antigens recognized by tumor-reactive T cells in 9 cancer patients with distinct tumor histologies including conventional tumor antigens, but also expanding the scope to non-canonical tumor antigen sources. In the first part, we investigated whether tumor-reactive T cells recognize conventional tumor antigens through three different methods including personalized neoantigen screening with TMG, in silico prediction and pHLA-I immunopeptidomics. However, the specific antigen/s targeted by the majority of tumor-reactive lymphocytes could not be determined. In the second part, we used a proteogenomics approach to identify peptides derived from non-canonical proteins presented on HLA-I of patient-derived TCL (nonC-TL). We frequently identified nonC-TL which were mainly derived from 5'UTR regions and represented the most abundant source of candidate tumor antigens. However, pre-existing T-cell responses targeting nonC-TL were not detected in any patient studied. In contrast, tumor-reactive lymphocytes consistently displayed preferential recognition of neoantigens and also recognized cancer-germline and tissue differentiation antigens. Nonetheless, we showed that nonC-TL can elicit de novo T-cell responses via in vitro sensitization of donor PBL. We identified TCR specific to three nonC-TL peptides, two of which mapped to the 5' UTR regions of HOXC13 and ZKSCAN1 genes, and one mapping to a non-coding spliced variant of C5orf22C gene. Importantly, we found that these immunogenic nonC-TL display therapeutic potential, as they were expressed across diverse tumor types, but were barely detected in healthy cells. Altogether, our results show that nonC-TL antigens hold great promise as new therapeutic targets as they can be immunogenic, are frequently presented on HLA-I of tumor cells, shared across patients, and barely expressed on healthy cells. However, despite the extensive immunological screening performed including nonC-TL, neoantigens, cancer-germline, and melanoma-associated antigens, the specificity of many tumor-reactive lymphocytes remains unknown and warrants further investigation