Journal Articles

Innovative Methods

Methods with focus on improving cancer immunotherapy approaches

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined gammadeltaTCR

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gamma9delta2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of gamma9delta2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of gamma9delta2T cells, we have developed the concept of T cells engineered to express a defined gammadeltaT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and gammadeltaT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vgamma9Vdelta2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which alphabetaTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous alphabetaTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Author Info: (1) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (2) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht

Author Info: (1) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (2) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (3) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (4) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (5) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (6) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (7) Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands. (8) CRCINA, INSERM 1232, CNRS, Universite d'Angers, Universite de Nantes, Nantes, France. CHU de Nantes, Hotel Dieu, UTCG, Nantes, France. (9) CRCINA, INSERM 1232, CNRS, Universite d'Angers, Universite de Nantes, Nantes, France. CHU de Nantes, Hotel Dieu, UTCG, Nantes, France. (10) Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands. (11) Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands. (12) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. (13) Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands. Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands.

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PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study

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BACKGROUND: Rhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcoma in children and adolescents, defined by skeletal muscle differentiation and the status of FOXO1 fusions. In pediatric malignancies, in particular RMS, scant and controversial observations are reported about PD-L1 expression as a putative biomarker and few immune checkpoint clinical trials are conducted. METHODS: PD-L1 assessment was evaluated by immunohistochemistry (IHC) utilizing two anti-PDL1 antibodies, in a pilot cohort of 25 RMS. Results were confirmed in primary and commercial RMS cell lines by cytofluorimetric analysis and IHC. RESULTS: PD-L1 expression was detectable, by both anti-PD-L1 antibodies, in the immune contexture of immune cells infiltrating and/or surrounding the tumor, in 15/25 (60%) RMS, while absent expression was observed in neoplastic cells. Flow cytometry analysis and PD-L1 IHC of commercial and primary RMS cell lines confirmed a very small percentage of PD-L1 positive-tumor cells, under the detection limits of conventional IHC. Interestingly, increased PD-L1 expression was observed in the immune contexture of 4 RMS cases post chemotherapy compared to their matched pre-treatment samples. CONCLUSION: Here we identify a peculiar pattern of PD-L1 expression in our RMS series with scanty positive-tumor cells detected by flow cytometry, and recurrent expression in the immune cells surrounding or infiltrating the tumor burden.

Author Info: (1) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. (2) Department of Pediatric

Author Info: (1) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. (2) Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy. (3) Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy. (4) Soft tissues and bone, and pediatric pathology unit, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy. (5) Soft tissues and bone, and pediatric pathology unit, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy. (6) Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy. (7) Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. (8) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. Clinical Research Lab (CRAB), Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. (9) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. (10) Unit of Thoracic Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. Clinical Research Lab (CRAB), Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. (11) Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. (12) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. (13) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. (14) Department of Research, Tumor Genomics Unit, Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via venezian 1, 20133, Milan, Italy. patrizia.gasparini@istitutotumori.mi.it.

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TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PD-L1) expression status and tumor-infiltrating lymphocytes in colorectal carcinomas

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Inhibitors targeting the PDCD1 (programmed cell death 1, PD-1) immune checkpoint pathway have revolutionized cancer treatment strategies. The TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PDCD1 ligand 1, PD-L1) expression and tumor-infiltrating lymphocytes (TIL) has been proposed to predict response to immunotherapy. It remains to be determined clinical, pathological, and molecular features of TIME subtypes of colorectal cancer. Using 812 colon and rectal carcinoma cases from the Nurses' Health Study and Health Professionals Follow-up Study, we examined the association of tumor characteristics and survival outcomes with four TIME subtypes (TIME 1, CD274(low)/TIL(absent); TIME 2, CD274(high)/TIL(present); TIME 3, CD274(low)/TIL(present); and TIME 4, CD274(high)/TIL(absent)). In survival analyses, Cox proportional hazards models were adjusted for potential confounders, including microsatellite instability (MSI) status, CpG island methylator phenotype (CIMP) status, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutation status. TIME subtypes 1, 2, 3 and 4 had 218 (27%), 117 (14%), 103 (13%), and 374 (46%) colorectal cancer cases, respectively. Compared with TIL-absent subtypes (TIME 1 and 4), TIL-present subtypes (TIME 2 and 3) were associated with high-level MSI, high-degree CIMP, BRAF mutation, and higher amounts of neoantigens (p < 0.001). TIME subtypes were not significantly associated with colorectal cancer-specific or overall survival. In conclusion, TIL-present TIME subtypes of colorectal cancer are associated with high levels of MSI and neoantigen load, supporting better responsiveness to cancer immunotherapy. Further studies examining tumor molecular alterations and additional factors in the tumor microenvironment may inform development of immunoprevention and immunotherapy strategies.

Author Info: (1) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (2) Program in MPE Molecular Pathological Epidemiology, Department of Pathology

Author Info: (1) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (2) Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (3) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (4) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (5) Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (6) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (7) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (8) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (9) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (10) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Collaborative Innovation Center of Tianjin for Medical Epigenetics, Key Laboratory of Hormone and Development, Ministry of Health, Metabolic Disease Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, P.R. China. (11) Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Division of Gastroenterology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA. (12) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Department of Epidemiology and Biostatistics, and the Ministry of Education Key Lab of Environment and Health, School of Public Health, Huazhong University of Science and Technology, Hubei, P.R. China. (13) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. College of Pharmacy, Zhejiang Chinese Medical University, Zhejiang, P.R. China. (14) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Department of Medical Oncology, Chinese PLA General Hospital, Beijing, P.R. China. (15) Department of Gastroenterology, Rheumatology, and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan. (16) Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. (17) Division of Pathology, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan. (18) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. (19) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (20) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (21) Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (22) Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA. (23) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. (24) Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Division of Gastroenterology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (25) Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. (26) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (27) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (28) Yale Cancer Center, New Haven, CT, USA. Department of Medicine, Yale School of Medicine, New Haven, CT, USA. Smilow Cancer Hospital, New Haven, CT, USA. (29) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA. (30) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. (31) Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

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Increase in PD-L1 expression after pre-operative radiotherapy for soft tissue sarcoma

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Soft tissue sarcomas (STS) have minimal expression of PD-L1, a biomarker for PD-1 therapy efficacy. Radiotherapy (RT) has been shown to increase PD-L1 expression pre-clinically. We examined the expression of PD-L1, pre- and post-RT, in 46 Stage II-III STS patients treated with pre-operative RT (50-50.4 Gy in 25-28 fractions) followed by resection. Five additional patients who did not receive RT were utilized as controls. PD-L1 expression on biopsy and resection samples was evaluated by immunochemistry using the anti PD-L1 monoclonal antibody (E1L3 N clone; Cell Signaling). Greater than 1% membranous staining was considered positive PD-L1 expression. Changes in PD-L1 expression were analyzed via the Fisher exact test. Kaplan-Meier statistics were used to correlate PD-L1 expression to distant metastases (DM) rate. The majority of STS were T2b (87.0%), high-grade (80.4%), undifferentiated pleomorphic histology (71.7%), and originated from the extremities (84.6%). Zero patients demonstrated PD-L1 tumor expression pre-RT. Post-RT, 5 patients (10.9%) demonstrated PD-L1 tumor expression (p = 0.056). Tumor associated macrophages (TAM) expression of PD-L1 increased after RT: 15.2% to 45.7% (p = 0.003). Samples from controls demonstrated no baseline (0%) or change in tumor PD-L1 expression. Freedom from DM was lower for patients with PD-L1 TAM expression post-RT (3 years: 49.7% vs. 87.8%, log-rank p = 0.006); TAM PD-L1 positivity remained an independent predictor for DM on multivariate analyses (Hazard ratio - 0.16, 95% confidence interval: 0.034-0.721, p = 0.042). PD-L1 expression on human STS tumor and TAM appears to elevate after pre-operative RT. Expression of PD-L1 on TAM after RT was associated with a higher rate of DM.

Author Info: (1) Department of Therapeutic Radiology, Smilow Cancer Center, Yale University School of Medicine, New Haven, CT, USA. (2) Deparment of Pathology, Emory University School of

Author Info: (1) Department of Therapeutic Radiology, Smilow Cancer Center, Yale University School of Medicine, New Haven, CT, USA. (2) Deparment of Pathology, Emory University School of Medicine, Atlanta, GA, USA. (3) Department of Therapeutic Radiology, Smilow Cancer Center, Yale University School of Medicine, New Haven, CT, USA. (4) Deparment of Pathology, Emory University School of Medicine, Atlanta, GA, USA. (5) Deparment of Pathology, Emory University School of Medicine, Atlanta, GA, USA. (6) Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (7) Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (8) Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (9) Division of Surgical Oncology, Department of Surgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (10) Division of Orthopaedic Oncology, Department of Orthopedic Surgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (11) Division of Orthopaedic Oncology, Department of Orthopedic Surgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (12) Division of Orthopaedic Oncology, Department of Orthopedic Surgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (13) Division of Surgical Oncology, Department of Surgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. (14) Deparment of Pathology, Emory University School of Medicine, Atlanta, GA, USA. (15) Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA.

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Prognostic implications of tumor-infiltrating macrophages, M2 macrophages, regulatory T-cells, and indoleamine 2,3-dioxygenase-positive cells in primary diffuse large B-cell lymphoma of the central nervous system

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Primary diffuse large B-cell lymphoma of the central nervous system (CNS-DLBCL) is an aggressive disease with a poor prognosis. The status of the tumor immune microenvironment in CNS-DLBCL remains unclear. We investigated the prognostic implications of tumor-associated macrophages (TAMs), regulatory T-cells (Tregs), and indoleamine 2,3-dioxygenase (IDO)(+) cells in primary CNS-DLBCL (n = 114) by immunohistochemical analysis. The numbers of tumor-infiltrating immune cells, including CD68(+) TAMs, CD163(+) or CD204(+) M2 macrophages, FOXP3(+) Tregs, and IDO(+) cells were all significantly lower in CNS-DLBCL versus systemic DLBCL (n = 165; all P < 0.001), but with little difference in the ratio of CD163(+)/CD68(+) or CD204(+)/CD68(+) cells. An increase in CD68(+) cell numbers was significantly associated with prolonged progression-free survival (PFS) and overall survival in patients with CNS-DLBCL (P = 0.004 and 0.021, respectively). In contrast, an increase in CD204(+) cell numbers or a higher ratio of CD204(+)/CD68(+) cells was related to a shorter PFS (P = 0.020 and 0.063, respectively). An increase in IDO(+) cell numbers was associated with a significantly longer PFS (P = 0.019). In combination, the status of low IDO(+) cell numbers combined with low CD68(+) cell numbers, high CD204(+) cell numbers, or a high CD204(+)/CD68(+) cell ratio all predicted poor PFS in multivariate analyses. This study showed that an increase in CD204(+) cell numbers, suggestive of M2 macrophages, was associated with poor clinical outcome in CNS-DLBCL, whereas increased CD68(+) or IDO(+) cell numbers were related to a favorable prognosis. The analysis of tumor-infiltrating immune cells could help in predicting the prognosis of CNS-DLBCL patients and determining therapeutic strategies targeting tumor microenvironment.

Author Info: (1) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Pathology, Asan Medical Center, University

Author Info: (1) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. (2) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (3) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (4) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (5) Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. (6) Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (7) Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (8) Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (9) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. (10) Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. (11) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. (12) Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. Cancer Research Institute, Seoul National University, Seoul, Republic of Korea.

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Vaccination-induced skin-resident memory CD8(+) T cells mediate strong protection against cutaneous melanoma

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Memory CD8(+) T cell responses have the potential to mediate long-lasting protection against cancers. Resident memory CD8(+) T (Trm) cells stably reside in non-lymphoid tissues and mediate superior innate and adaptive immunity against pathogens. Emerging evidence indicates that Trm cells develop in human solid cancers and play a key role in controlling tumor growth. However, the specific contribution of Trm cells to anti-tumor immunity is incompletely understood. Moreover, clinically applicable vaccination strategies that efficiently establish Trm cell responses remain largely unexplored and are expected to strongly protect against tumors. Here we demonstrated that a single intradermal administration of gene- or protein-based vaccines efficiently induces specific Trm cell responses against models of tumor-specific and self-antigens, which accumulated in vaccinated and distant non-vaccinated skin. Vaccination-induced Trm cells were largely resistant to in vivo intravascular staining and antibody-dependent depletion. Intradermal, but not intraperitoneal vaccination, generated memory precursors expressing skin-homing molecules in circulation and Trm cells in skin. Interestingly, vaccination-induced Trm cell responses strongly suppressed the growth of B16F10 melanoma, independently of circulating memory CD8(+) T cells, and were able to infiltrate tumors. This work highlights the therapeutic potential of vaccination-induced Trm cell responses to achieve potent protection against skin malignancies.

Author Info: (1) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (2) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (3) Laboratory of

Author Info: (1) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (2) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (3) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (4) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (5) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (6) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (7) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (8) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. (9) Department of Microbiology and Immunology, Stanford University, CA, USA. (10) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile. Facultad de Ciencias Medicas, Escuela de Medicina, Universidad de Santiago de Chile, Santiago, Chile. (11) Centro de Investigacion Biomedica, Facultad de Medicina, Universidad de los Andes, Santiago, Chile. (12) Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile. Millennium Institute on Immunology and Immunotherapy, Universidad de Chile, Santiago, Chile. (13) Department of Microbiology and Immunology, Stanford University, CA, USA. (14) Laboratory of Gene Immunotherapy, Fundacion Ciencia & Vida, Santiago, Chile.

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Immunosuppressive activity of tumor-infiltrating myeloid cells in patients with meningioma

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Meningiomas WHO grade I and II are common intracranial tumors in adults that normally display a benign outcome, but are characterized by a great clinical heterogeneity and frequent recurrence of the disease. Although the presence of an immune cell infiltrate has been documented in these tumors, a clear phenotypical and functional characterization of the immune web is missing. Here, we performed an extensive immunophenotyping of peripheral blood and fresh tumor tissue at surgery by multiparametric flow cytometry in 34 meningioma patients, along with immunosuppressive activity of sorted cells of myeloid origin. Four subsets of myeloid cells, phenotypically corresponding to myeloid-derived suppressor cells (MDSCs) are detectable in the blood and in the tumor tissue of patients and three of them are significantly expanded in the blood of patients, but show no evidence of suppressive activity. At the tumor site, a large leukocyte infiltrate is present, predominantly constituted by CD33(+) myeloid cells, largely composed of macrophages endowed with suppressive activity and significantly expanded in grade II meningioma patients as compared to grade I.

Author Info: (1) IOV-IRCCS, Via Gattamelata, Padova, Italy. (2) Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. (3) IOV-IRCCS, Via Gattamelata, Padova, Italy. (4)

Author Info: (1) IOV-IRCCS, Via Gattamelata, Padova, Italy. (2) Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. (3) IOV-IRCCS, Via Gattamelata, Padova, Italy. (4) Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. (5) Department of Medicine, Verona University Hospital, Verona, Italy. (6) Department of Neurosurgery, Azienda Ospedaliera di Padova, Padova, Italy. (7) IOV-IRCCS, Via Gattamelata, Padova, Italy. Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy.

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Anti-CTLA-4 based therapy elicits humoral immunity to galectin-3 in patients with metastatic melanoma

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The combination of CTLA-4 blockade ipilimumab (Ipi) with VEGF-A blocking antibody bevacizumab (Bev) has demonstrated favorable clinical outcomes in patients with advanced melanoma. Galectin-3 (Gal-3) plays a prominent role in tumor growth, metastasis, angiogenesis, and immune evasion. Here we report that Ipi plus Bev (Ipi-Bev) therapy increased Gal-3 antibody titers by 50% or more in approximately one third of treated patients. Antibody responses to Gal-3 were associated with higher complete and partial responses and better overall survival. Ipi alone also elicited antibody responses to Gal-3 at a frequency comparable to the Ipi-Bev combination. However, an association of elicited antibody responses to Gal-3 with clinical outcomes was not observed in Ipi alone treated patients. In contrast to being neutralized in Ipi-Bev treated patients, circulating VEGF-A increased by 100% or more in a subset of patients after Ipi treatment, with most having progressive disease. Among the Ipi treated patients with therapy-induced Gal-3 antibody increases, circulating VEGF-A was increased in 3 of 6 nonresponders but in none of 4 responders as a result of treatment. Gal-3 antibody responses occurred significantly less frequently (3.2%) in a cohort of patients receiving PD-1 blockade where high pre-treatment serum Gal-3 was associated with reduced OS and response rates. Our findings suggest that anti-CTLA-4 elicited humoral immune responses to Gal-3 in melanoma patients which may contribute to the antitumor effect in the presence of an anti-VEGF-A combination. Furthermore, pre-treatment circulating Gal-3 may potentially have prognostic and predictive value for immune checkpoint therapy.

Author Info: (1) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (2) Center for Immuno-oncology, Dana-Farber Cancer Institute and Harvard Medical School

Author Info: (1) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (2) Center for Immuno-oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. Department of Biostatistics & Computational Biology, Dana-Farber Cancer Institute, Boston, MA. (3) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (4) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (5) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (6) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. Center for Immuno-oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (7) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. (8) Department of Pathology Brigham and Women's Hospital, Dana-Farber Cancer Institute, Boston, MA. (9) Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. Melanoma Disease Center, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. Center for Immuno-oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA.

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CD133-directed CAR T cells for advanced metastasis malignancies: A phase I trial

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Expressed by cancer stem cells of various epithelial cell origins, CD133 is an attractive therapeutic target for cancers. Autologous chimeric antigen receptor-modified T-cell directed CD133 (CART-133) was first tested in this trial. The anti-tumor specificity and the postulated toxicities of CART-133 were first assessed. Then, we conducted a phase I clinical study in which patients with advanced and CD133-positive tumors received CART-133 cell-infusion. We enrolled 23 patients (14 with hepatocellular carcinoma [HCC], 7 with pancreatic carcinomas, and 2 with colorectal carcinomas). The 8 initially enrolled patients with HCC were treated by a CART-133 cell dose escalation scheme (0.05-2 x 10(6)/kg). The higher CAR-copy numbers and its reverse relationship with the count of CD133+ cells in peripheral blood led to the determination of an acceptable cell dose is 0.5-2 x 10(6)/kg and reinfusion cycle in 23 patients. The primary toxicity is a decrease in hemoglobin/platelet (

Author Info: (1) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (2) Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China. (3) Department of

Author Info: (1) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (2) Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China. (3) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (4) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (5) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (6) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (7) Department of 3Geriatric Hematology, Chinese PLA General Hospital, Beijing, China. (8) Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China. (9) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (10) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (11) Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China. (12) Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China. (13) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. (14) Department of Molecular & Immunology, Chinese PLA General Hospital, Beijing, China. Department of Bio-therapeutic, Chinese PLA General Hospital, Beijing, China.

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Differential Regulation of T-cell mediated anti-tumor memory and cross-protection against the same tumor in lungs versus skin

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A major advantage of immunotherapy of cancer is that effector cells induced at one site should be able to kill metastatic cancer cells in other sites or tissues. However, different tissues have unique immune components, and very little is known about whether effector T cells induced against tumors in one tissue can work against the same tumors in other tissues. Here, we used CT26 murine tumor models to investigate anti-tumor immune responses in the skin and lungs and characterized cross-protection between the two tissues. Blockade of the function of Treg cells with anti-CD25 allowed for T cell-dependent rejection of s.c. tumors. When these mice were simultaneously inoculated i.v. with CT26, they also rejected tumors in the lung. Interestingly, in the absence of s.c. tumors, anti-CD25 treatment alone had no effect on lung tumor growth. These observations suggested that T cell-mediated anti-tumor protective immunity induced against s.c. tumors can also protect against lung metastases of the same tumors. In contrast, NKT cell-deficiency in CD1d(-/-) mice conferred significant protection against lung tumors but had no effect on the growth of tumors in the skin, and tumor rejection induced against the CT26 in the lung did not confer protection for the same tumor cells in the skin. Thus, effector cells against the same tumor do not work in all tissues, and the induction site of the effector T cells is critical to control metastasis. Further, the regulation of tumor immunity may be different for the same tumor in different anatomical locations.

Author Info: (1) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. Mary H. Weiser Food Allergy Center, University of Michigan, Ann Arbor, Michigan, USA. (2) Vaccine Branch

Author Info: (1) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. Mary H. Weiser Food Allergy Center, University of Michigan, Ann Arbor, Michigan, USA. (2) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. Institute for Public Health Genomics, Department of Genetics & Cell Biology, School for Oncology & Developmental Biology (GROW), FHML, Maastricht University, The Netherlands. (3) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. (4) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. (5) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. (6) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. (7) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA. (8) Vaccine Branch, CCR, NCI, NIH Bethesda, MD USA.

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