RESUMEN
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3-5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of ß2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy.
Asunto(s)
Neoplasias del Colon , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T , Humanos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Reparación de la Incompatibilidad de ADN/genética , Receptores KIR , Línea Celular Tumoral , Organoides , Presentación de Antígeno , Genes MHC Clase I/genéticaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of cancer immunotherapies, a comprehensive understanding of local and systemic immune profiles in patients with PDAC is required. Here, our goal was to decipher the interplay between local and systemic immune profiles in treatment-naïve patients with PDAC. METHODS: The immune composition of PDAC, matched non-malignant pancreatic tissue, regional lymph nodes, spleen, portal vein blood, and peripheral blood samples (collected before and after surgery) from 11 patients with PDAC was assessed by measuring 41 immune cell markers by single-cell mass cytometry. Furthermore, the activation potential of tumor-infiltrating lymphocytes as determined by their ability to produce cytokines was investigated by flow cytometry. In addition, the spatial localization of tumor-infiltrating innate lymphocytes in the tumor microenvironment was confirmed by multispectral immunofluorescence. RESULTS: We found that CD103+CD8+ T cells with cytotoxic potential are infrequent in the PDAC immune microenvironment and lack the expression of activation markers and checkpoint blockade molecule programmed cell death protein-1 (PD-1). In contrast, PDAC tissues showed a remarkable increased relative frequency of B cells and regulatory T cells as compared with non-malignant pancreatic tissues. Besides, a previously unappreciated innate lymphocyte cell (ILC) population (CD127-CD103+CD39+CD45RO+ ILC1-like) was discovered in PDAC tissues. Strikingly, the increased relative frequency of B cells and regulatory T cells in pancreatic cancer samples was reflected in matched portal vein blood samples but not in peripheral blood, suggesting a regional enrichment of immune cells that infiltrate the PDAC microenvironment. After surgery, decreased frequencies of myeloid dendritic cells were found in peripheral blood. CONCLUSIONS: Our work demonstrates an immunosuppressive landscape in PDAC tissues, generally deprived of cytotoxic T cells and enriched in regulatory T cells and B cells. The antitumor potential of ILC1-like cells in PDAC may be exploited in a therapeutic setting. Importantly, immune profiles detected in blood isolated from the portal vein reflected the immune cell composition of the PDAC microenvironment, suggesting that this anatomical location could be a source of tumor-associated immune cell subsets.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Linfocitos T CD8-positivos/metabolismo , Carcinoma Ductal Pancreático/patología , Humanos , Inmunoterapia , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.
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Antígenos CD/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , Monocitos/metabolismo , Receptores de Superficie Celular/sangre , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Macrófagos Asociados a Tumores/metabolismoRESUMEN
Cancers are characterized by extensive heterogeneity that occurs intratumorally, between lesions, and across patients. To study cancer as a complex biological system, multidimensional analyses of the tumor microenvironment are paramount. Single-cell technologies such as flow cytometry, mass cytometry, or single-cell RNA-sequencing have revolutionized our ability to characterize individual cells in great detail and, with that, shed light on the complexity of cancer microenvironments. However, a key limitation of these single-cell technologies is the lack of information on spatial context and multicellular interactions. Investigating spatial contexts of cells requires the incorporation of tissue-based techniques such as multiparameter immunofluorescence, imaging mass cytometry, or in situ detection of transcripts. In this Review, we describe the rise of multidimensional single-cell technologies and provide an overview of their strengths and weaknesses. In addition, we discuss the integration of transcriptomic, genomic, epigenomic, proteomic, and spatially-resolved data in the context of human cancers. Lastly, we will deliberate on how the integration of multi-omics data will help to shed light on the complex role of cell types present within the human tumor microenvironment, and how such system-wide approaches may pave the way toward more effective therapies for the treatment of cancer.
RESUMEN
OBJECTIVE: The subset distribution and immunophenotype of circulating immune cells ("peripheral blood immune cell profile") may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). METHODS: The subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. RESULTS: The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. CONCLUSIONS: Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores/sangre , Neoplasias del Colon/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Complejo CD3 , Estudios de Casos y Controles , Quimioterapia Adyuvante , Neoplasias del Colon/sangre , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Femenino , Estudios de Seguimiento , Humanos , Inmunofenotipificación , Células Asesinas Naturales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Pronóstico , Linfocitos T/efectos de los fármacosRESUMEN
OBJECTIVE: A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC. DESIGN: Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence. RESULTS: We discovered that a previously unappreciated innate lymphocyte population (Lin-CD7+CD127-CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes. CONCLUSION: This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.
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Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Antígenos CD/metabolismo , Antígenos CD8/metabolismo , Estudios de Casos y Controles , Neoplasias del Colon/metabolismo , Citometría de Flujo , Humanos , Cadenas alfa de Integrinas/metabolismo , Recuento de Linfocitos , Linfocitos Infiltrantes de TumorRESUMEN
OBJECTIVE: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. METHODS: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. RESULTS: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. CONCLUSION: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.
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Neoplasias Colorrectales/inmunología , Células Asesinas Naturales/inmunología , Células T Asesinas Naturales/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Complejo CD3/inmunología , Complejo CD3/metabolismo , Antígeno CD56/inmunología , Antígeno CD56/metabolismo , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/metabolismo , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Masculino , Persona de Mediana Edad , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Receptor 2 Gatillante de la Citotoxidad Natural/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Células T Asesinas Naturales/metabolismo , Análisis de Supervivencia , Linfocitos T/metabolismo , Adulto JovenRESUMEN
BACKGROUND: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers. METHODS: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material. RESULTS: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39+CD103+ T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-ß pathway activation and worse clinical outcome. CONCLUSIONS: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-ß pathway to reinforce T cell reactivity in this patient group.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias Colorrectales/patología , Antígenos de Neoplasias/química , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/genética , Epítopos/química , Epítopos/inmunología , Epítopos/farmacología , Humanos , Inmunoterapia , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/metabolismo , Proteínas de Microfilamentos/genética , Mutación , Péptidos/síntesis química , Péptidos/farmacología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Although most cancer types have been viewed as immunologically silent until recently, it has become increasingly clear that the immune system plays key roles in the course of tumor development. Remarkable progress towards understanding cancer immunogenicity and tumor-immune system interactions has revealed important implications for the design of novel immune-based therapies. Natural immune responses, but also therapeutic interventions, can modulate the tumor phenotype due to selective outgrowth of resistant subtypes. This is the result of heterogeneity of tumors, with genetic instability as a driving force, and obviously changes the immunogenicity of tumors. In this review, we discuss the immunogenicity of colorectal cancer (CRC) in relation to tumor development and treatment. As most tumors, CRC activates the immune system in various ways, and is also capable of escaping recognition and elimination by the immune system. Tumor-immune system interactions underlie the balance between immune control and immune escape, and may differ in primary tumors, in the circulation, and in liver metastases of CRC. Since CRC immunogenicity varies between tumors and individuals, novel immune-based therapeutic strategies should not only anticipate the molecular profile, but also the immunological profile of a specific tumor.