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1.
Cell ; 186(16): 3333-3349.e27, 2023 08 03.
Article in English | MEDLINE | ID: mdl-37490916

ABSTRACT

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.


Subject(s)
Antigens, Neoplasm , Neoplasms , Proteomics , Receptors, Antigen, T-Cell , Antigens, Neoplasm/metabolism , Epitopes , Immunotherapy , Lymphocytes, Tumor-Infiltrating , Neoplasms/immunology , Neoplasms/therapy , Receptors, Antigen, T-Cell/metabolism
3.
Nat Immunol ; 21(2): 178-185, 2020 02.
Article in English | MEDLINE | ID: mdl-31959982

ABSTRACT

Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.


Subject(s)
Cytotoxicity, Immunologic/immunology , Histocompatibility Antigens Class I/immunology , Minor Histocompatibility Antigens/immunology , Neoplasms/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocyte Subsets/immunology , Animals , CRISPR-Cas Systems , Genome-Wide Association Study , Humans , Immunotherapy/methods , Lymphocyte Activation/immunology , Mice
5.
Nature ; 577(7791): 561-565, 2020 01.
Article in English | MEDLINE | ID: mdl-31942071

ABSTRACT

Checkpoint blockade therapies that reactivate tumour-associated T cells can induce durable tumour control and result in the long-term survival of patients with advanced cancers1. Current predictive biomarkers for therapy response include high levels of intratumour immunological activity, a high tumour mutational burden and specific characteristics of the gut microbiota2,3. Although the role of T cells in antitumour responses has thoroughly been studied, other immune cells remain insufficiently explored. Here we use clinical samples of metastatic melanomas to investigate the role of B cells in antitumour responses, and find that the co-occurrence of tumour-associated CD8+ T cells and CD20+ B cells is associated with improved survival, independently of other clinical variables. Immunofluorescence staining of CXCR5 and CXCL13 in combination with CD20 reveals the formation of tertiary lymphoid structures in these CD8+CD20+ tumours. We derived a gene signature associated with tertiary lymphoid structures, which predicted clinical outcomes in cohorts of patients treated with immune checkpoint blockade. Furthermore, B-cell-rich tumours were accompanied by increased levels of TCF7+ naive and/or memory T cells. This was corroborated by digital spatial-profiling data, in which T cells in tumours without tertiary lymphoid structures had a dysfunctional molecular phenotype. Our results indicate that tertiary lymphoid structures have a key role in the immune microenvironment in melanoma, by conferring distinct T cell phenotypes. Therapeutic strategies to induce the formation of tertiary lymphoid structures should be explored to improve responses to cancer immunotherapy.


Subject(s)
Melanoma/immunology , Melanoma/therapy , Tertiary Lymphoid Structures/immunology , Antigens, CD20/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Chemokine CXCL13/metabolism , Humans , Immunologic Memory/immunology , Melanoma/genetics , Melanoma/pathology , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Phenotype , Prognosis , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Proteomics , RNA-Seq , Receptors, CXCR5/metabolism , Single-Cell Analysis , Survival Rate , T Cell Transcription Factor 1/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tertiary Lymphoid Structures/genetics , Treatment Outcome , Tumor Microenvironment/immunology
6.
Int J Cancer ; 150(11): 1870-1878, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35001363

ABSTRACT

Routine [18F]2-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) may help predict clinical outcomes after response to immunotherapy. With a European Medicines Agency-recommended treatment length until disease progression or unacceptable toxicity, the optimal duration of immunotherapy remains to be defined. In a retrospective study, we retrieved from the Danish Metastatic Melanoma Database (DAMMED), all patients that were annotated as a partial or complete response based on the computed tomography (CT) of serial FDG-PET-CT scans. Patients treated with an anti-Programmed Death (PD)-1-containing regimen for <18 months, and ≥4 months without disease progression after halting anti-PD-1 were included. Cases were divided into an "elective" and a "toxicity" group based on the reason for treatment discontinuation. A total of 140 patients were included. At 29.3 months of median follow-up, a higher proportion of patients remained alive in the "elective" group (93% vs 75%, P = .0031) with an improved melanoma-specific (HR 0.07, 95% CI 0.02-0.32, P = .0041) survival (MSS). Patients without FDG-avid lesions at the time of treatment discontinuation had an improved MSS (HR 0.03, 95% CI 0.01-0.17, P = .0002), and the absence of FDG-avid lesions was the only independent predictive feature of improved MSS in multivariate analysis. In conclusion, patients with metastatic melanoma who obtain an early response and early discontinue immunotherapy have an excellent prognosis, especially in the absence of FDG-PET avid lesions when discontinuing treatment. These data support the option of early discontinuation, limiting possible overtreatment and thereby toxicity, health and economic expenses and improving logistics.


Subject(s)
Fluorodeoxyglucose F18 , Melanoma , Fluorodeoxyglucose F18/therapeutic use , Glucose , Humans , Immunotherapy/methods , Melanoma/diagnostic imaging , Melanoma/drug therapy , Positron Emission Tomography Computed Tomography/methods , Positron-Emission Tomography , Radiopharmaceuticals/therapeutic use , Retrospective Studies , Treatment Outcome
7.
Cancer Immunol Immunother ; 71(3): 553-563, 2022 Mar.
Article in English | MEDLINE | ID: mdl-34272988

ABSTRACT

BACKGROUND: Studying tumor cell-T cell interactions in the tumor microenvironment (TME) can elucidate tumor immune escape mechanisms and help predict responses to cancer immunotherapy. METHODS: We selected 14 pairs of highly tumor-reactive tumor-infiltrating lymphocytes (TILs) and autologous short-term cultured cell lines, covering four distinct tumor types, and co-cultured TILs and tumors at sub-lethal ratios in vitro to mimic the interactions occurring in the TME. We extracted gene signatures associated with a tumor-directed T cell attack based on transcriptomic data of tumor cells. RESULTS: An autologous T cell attack induced pronounced transcriptomic changes in the attacked tumor cells, partially independent of IFN-γ signaling. Transcriptomic changes were mostly independent of the tumor histological type and allowed identifying common gene expression changes, including a shared gene set of 55 transcripts influenced by T cell recognition (Tumors undergoing T cell attack, or TuTack, focused gene set). TuTack scores, calculated from tumor biopsies, predicted the clinical outcome after anti-PD-1/anti-PD-L1 therapy in multiple tumor histologies. Notably, the TuTack scores did not correlate to the tumor mutational burden, indicating that these two biomarkers measure distinct biological phenomena. CONCLUSIONS: The TuTack scores measure the effects on tumor cells of an anti-tumor immune response and represent a comprehensive method to identify immunologically responsive tumors. Our findings suggest that TuTack may allow patient selection in immunotherapy clinical trials and warrant its application in multimodal biomarker strategies.


Subject(s)
Biomarkers, Tumor , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Neoplasms/etiology , Transcriptome , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Cell Line, Tumor , Coculture Techniques , Computational Biology/methods , DNA Contamination , Gene Expression Profiling/methods , Gene Expression Profiling/standards , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immune Checkpoint Inhibitors , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Organ Specificity , ROC Curve , Tumor Cells, Cultured
8.
Cancer Immunol Immunother ; 70(6): 1771-1776, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33165629

ABSTRACT

BACKGROUND: Immune-related adverse events (IrAEs) are auto-immune reactions associated with immune checkpoint inhibitor-based therapy (ICI). Steroids are currently the first-line option for irAE management; however, recent studies have raised concerns regarding their potential impairment of tumor-specific immune responses. In this study, we investigated the in vitro effects of commonly used irAE treatment drugs on the anti-tumor activity of tumor-infiltrating lymphocytes (TILs). METHODS: Impairment of anti-tumor immune responses by four drugs (antibodies: vedolizumab and tocilizumab; small molecules: mycophenolate mofetil and tacrolimus) reported to be effective in treating irAEs was tested at clinically relevant doses in vitro and compared to a standard moderate dose of corticosteroids (small molecules) or infliximab (antibodies). TIL responses against autologous tumor cell lines, in the presence or absence of irAE drugs, were determined by flow cytometry (short-term tumor-specific T-cell activation) or xCELLigence (T-cell-mediated tumor killing). RESULTS: None of the tested antibodies influenced T-cell activation or T-cell-mediated tumor killing. Low-dose mycophenolate and tacrolimus did not influence T-cell activation, whereas higher doses of tacrolimus (> 1 ng/ml) impaired T-cell activation comparably to dexamethasone. All tested small molecules impaired T-cell-mediated tumor killing, with high-dose tacrolimus reducing killing at levels comparable to dexamethasone-mediated inhibition. In addition, mycophenolate and tacrolimus alone also demonstrated anti-proliferative effects on tumor cells. CONCLUSIONS: These data support clinical testing of targeted immune-regulatory strategies in the initial phase of irAE management, as a potential replacement for corticosteroids.


Subject(s)
Drug-Related Side Effects and Adverse Reactions/prevention & control , Immune Checkpoint Inhibitors/adverse effects , Immunosuppressive Agents/therapeutic use , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/drug therapy , Small Molecule Libraries/therapeutic use , T-Lymphocytes/immunology , Drug-Related Side Effects and Adverse Reactions/etiology , Drug-Related Side Effects and Adverse Reactions/pathology , Humans , Melanoma/immunology , Melanoma/pathology , Tumor Cells, Cultured
9.
Cytotherapy ; 23(8): 724-729, 2021 08.
Article in English | MEDLINE | ID: mdl-33933372

ABSTRACT

BACKGROUND AIMS: Lymphodepletion with non-myeloablative (NMA) chemotherapy is currently a prerequisite for adoptive cell therapy (ACT). ACT based on tumor-infiltrating lymphocytes has long been used in malignant melanoma (MM), but with the advance of ACT into new cancer diagnoses, the patient predisposition will change. The authors here evaluate the bone marrow (BM) toxicity of NMA in combination with checkpoint inhibition and a priori risk factors in a wide range of cancer diagnoses. METHODS: Thirty-one non-MM and MM patients were included from two different clinical trials with ACT. The treatment history was extracted from the medical records, together with the hematology data. Immune monitoring with flow cytometry was performed before and at several time points after therapy. RESULTS: NMA induced reversible myelosuppression in all patients. No significant differences in BM toxicity between MM and non-MM patients were found. The overall hematology counts were reconstituted within 3-6 months but with great individual heterogeneity, including eight patients who developed a second phase of neutropenia after hospital discharge. A performance status >0 was found, and shorter overall survival and sex were statistically associated with longer duration of anemia. By contrast, high expression of co-stimulatory markers CD28+ and CD27+ on T cells at baseline was significantly correlated with shorter duration of neutropenia (P = 0.010 and P = 0.009, respectively), anemia (P = 0.001 and P = 0.001, respectively) and thrombocytopenia (P = 0.017 and P = 0.030, respectively). In addition, following NMA, the authors saw a significant differentiation of T-cell phenotype associated with old age. CONCLUSIONS: ACT with NMA and checkpoint inhibition is tolerable in patients with multiple cancer diagnoses and therapy backgrounds but comes with substantial transient BM toxicity that is comparable in both non-MM and MM patients. Baseline T-cell CD28/CD27 expression level is predictive of duration of BM toxicity. Furthermore, NMA conditioning induces changes in the immune system that may affect a patient's immunocompetence for many months following therapy.


Subject(s)
Immune Reconstitution , Melanoma , Skin Neoplasms , Bone Marrow , Humans , Melanoma/drug therapy , Transplantation Conditioning
10.
J Immunol ; 200(7): 2263-2279, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29483360

ABSTRACT

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , HLA-A2 Antigen/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Antigen, T-Cell/immunology , Staining and Labeling/methods , Cytomegalovirus/immunology , Herpesvirus 4, Human/immunology , Humans , Lymphocyte Activation/immunology , Melanoma/immunology , Orthomyxoviridae/immunology , Protein Binding/immunology , Protein Kinase Inhibitors/metabolism , RNA-Binding Proteins/immunology , Tumor Cells, Cultured
11.
Int J Mol Sci ; 21(15)2020 Jul 23.
Article in English | MEDLINE | ID: mdl-32718045

ABSTRACT

Melanoma of the ocular region (ocular melanoma) comprises about 5% of all patients with melanoma and covers posterior uveal melanoma, iris melanoma, and conjunctival melanoma. The risk of metastasis is much higher in patients with ocular melanoma compared to a primary melanoma of the skin. The subtypes of ocular melanoma have distinct genetic features, which should be taken into consideration when making clinical decisions. Most relevant for current practice is the absence of BRAF mutations in posterior uveal melanoma, although present in some iris melanomas and conjunctival melanomas. In this review, we discuss the genetic biomarkers of the subtypes of ocular melanoma and their impacts on the clinical care of these patients.


Subject(s)
Melanoma , Mutation , Proto-Oncogene Proteins B-raf , Uveal Neoplasms , Humans , Melanoma/enzymology , Melanoma/genetics , Melanoma/therapy , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Uveal Neoplasms/enzymology , Uveal Neoplasms/genetics , Uveal Neoplasms/therapy
12.
Int J Cancer ; 145(5): 1408-1413, 2019 09 01.
Article in English | MEDLINE | ID: mdl-30575963

ABSTRACT

Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high dose corticosteroids, or tumor necrosis factor (TNF) antagonists in refractory cases, is the mainstay of treatment for irAEs. It is currently unknown what impact corticosteroids and anti-TNF have on the activity of antitumor T cells. In our study, the influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10-125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing ability of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high doses impaired the activation (-46 and -62%, respectively) and tumor-killing ability (-48 and -53%, respectively) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (-20%) and tumor killing (-10%). A 72-hr resting period after withdrawal of dexamethasone was sufficient to rescue the in vitro activity of TILs, while a short withdrawal did not result in a full rescue. In conclusion, clinically relevant doses of infliximab only had a minor influence on the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. However, the activity of TILs could be restored after withdrawal of steroids. These data indirectly support steroid-sparing strategies and early initiation of anti-TNF therapy for the treatment of irAEs in immuno-oncology.


Subject(s)
Dexamethasone/adverse effects , Infliximab/adverse effects , Lymphocytes, Tumor-Infiltrating/drug effects , T-Lymphocytes/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Antineoplastic Agents, Hormonal/adverse effects , Humans , Immune Tolerance/drug effects , Lymphocyte Activation/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , Melanoma/pathology , T-Lymphocytes/immunology
13.
Br J Cancer ; 120(8): 870, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30890776

ABSTRACT

Since the publication of this paper, the authors noticed that the funding information was not complete. The correct funding information is now shown in the Acknowledgements section. Acknowledgements The studies were supported by grants from the OvaCure Foundation, the Danish Cancer Society Research Foundation, the Anticancer Fund, Aase og Ejnar Danielsens Foundation and the Independent Research Fund Denmark (grant number DFF-4183-00597).

14.
Br J Cancer ; 120(4): 424-434, 2019 02.
Article in English | MEDLINE | ID: mdl-30718808

ABSTRACT

BACKGROUND: Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens. METHODS: Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality. RESULTS: Minimally expanded TILs (Young TILs) were successfully established from all patients. Young TILs contained a high frequency of CD3+ cells with a variable CD4/CD8 ratio. TILs could be expanded to clinical numbers. Importantly, recognition of autologous tumour cells was demonstrated in TILs in >50% of the patients. We confirmed with mass spectrometry the presentation of multiple tumour antigens, including peptides derived from the cancer-testis antigen GAGE, which could be recognised by antigen-specific TILs. Antigen-specific TILs could be isolated and further expanded in vitro. CONCLUSION: These findings support the hypothesis that patients with OC can benefit from ACT with TILs and led to the initiation of a pilot clinical trial at our institution . TRIAL REGISTRATION: clinicaltrials.gov: NCT02482090.


Subject(s)
Lymphocytes, Tumor-Infiltrating/immunology , Ovarian Neoplasms/immunology , T-Lymphocyte Subsets/immunology , Tumor Microenvironment , Adoptive Transfer , CD4-CD8 Ratio , Female , Humans , Immunophenotyping , Interferon-gamma/pharmacology , Ovarian Neoplasms/therapy
15.
Cancer Immunol Immunother ; 68(10): 1689-1700, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31375885

ABSTRACT

Immunotherapy aims to activate the immune system to fight cancer in a very specific and targeted manner. Despite the success of different immunotherapeutic strategies, in particular antibodies directed against checkpoints as well as adoptive T-cell therapy, the response of patients is limited in different types of cancers. This attributes to escape of the tumor from immune surveillance and development of acquired resistances during therapy. In this review, the different evasion and resistance mechanisms that limit the efficacy of immunotherapies targeting tumor-associated antigens presented by major histocompatibility complex molecules on the surface of the malignant cells are summarized. Overcoming these escape mechanisms is a great challenge, but might lead to a better clinical outcome of patients and is therefore currently a major focus of research.


Subject(s)
Immunotherapy/methods , Neoplasms/therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor Escape , Antigen Presentation , HLA-G Antigens/physiology , Histocompatibility Antigens Class I/immunology , Humans
16.
Acta Oncol ; 58(11): 1603-1611, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31271119

ABSTRACT

Background: Chemotherapeutic agents are often mutagenic. Induction of mutation associated neo-epitopes is one of the mechanisms by which chemotherapy is thought to increase the number of tumor-infiltrating lymphocytes. It is not known, however, whether treatment with various chemotherapeutic agents with different mutagenic capacity induce a significantly different number of stromal tumor-infiltrating lymphocytes (StrTIL) in residual cancer.Methods: One hundred and twenty breast carcinoma cases with residual disease that were treated with one of three types of pre-operative chemotherapy regimens were selected for the study. The percentage of StrTIL was evaluated in pretreatment core biopsies (pre-StrTIL) and post-treatment surgical tumor samples (post-StrTIL). TIL changes (ΔStrTIL) were calculated from the difference between post-StrTIL and pre-StrTIL.Results: When analyzing the pre-StrTIL and post-StrTIL among the three treatment groups, we detected significant StrTIL increase independently of the treatment applied. Based on distant metastases-free survival analysis, both post-StrTIL and ΔStrTIL was found to be independent prognostic factor in HR negative cases. Conclusions: Significant increase of StrTIL in the residual disease was observed in patients treated with the highly (platinum), moderately (cyclophosphamide) and marginally mutagenic chemotherapeutic agents (taxane, anthracycline). Increase in StrTIL in residual cancer compared to pretreatment tumor tissue is associated with improved distant metastasis-free survival in cases with HR negative breast carcinoma.


Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Lymphocytes, Tumor-Infiltrating/drug effects , Neoplasm, Residual/drug therapy , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/immunology , Female , Humans , Middle Aged , Mutagens , Neoplasm, Residual/immunology , Preoperative Care
17.
Int J Mol Sci ; 20(18)2019 Sep 11.
Article in English | MEDLINE | ID: mdl-31514305

ABSTRACT

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.


Subject(s)
Cell Movement , ErbB Receptors/metabolism , Melanoma/drug therapy , Melanoma/pathology , Vemurafenib/therapeutic use , Adult , Aged , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Erlotinib Hydrochloride/pharmacology , Erlotinib Hydrochloride/therapeutic use , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitory Concentration 50 , Male , Melanoma/genetics , Middle Aged , Mutation/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phenotype , Proto-Oncogene Proteins B-raf/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Time Factors , Vemurafenib/pharmacology
18.
Cancer Immunol Immunother ; 67(5): 703-711, 2018 May.
Article in English | MEDLINE | ID: mdl-29520474

ABSTRACT

The exclusion of "real-world" patients from registration clinical trials of cancer immunotherapy represents a significant emerging issue. For instance, a large fraction of cancer patients develops brain metastases during the course of the disease, but results from large prospective clinical trials investigating this considerable proportion of the cancer patient population are currently lacking. To provide a useful tool for the clinician in a "real-world" setting, we have reviewed the available literature regarding the safety and efficacy of immune check-point inhibitors in patients with cancer metastatic to the brain. Overall, these data provide encouraging evidence that these therapeutic agents can induce intracranial objective responses, particularly in patients with asymptomatic and previously untreated brain metastases. Larger prospective studies are needed to confirm these initial results.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Brain Neoplasms/drug therapy , Immunotherapy , Brain Neoplasms/immunology , Brain Neoplasms/secondary , Humans , Prognosis
19.
J Immunol ; 194(1): 463-74, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25452566

ABSTRACT

Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.


Subject(s)
Flow Cytometry/methods , Fluorescent Antibody Technique/methods , Receptors, Antigen, T-Cell/immunology , Staining and Labeling/methods , T-Lymphocytes/immunology , Antibodies/chemistry , Antibodies/immunology , Cells, Cultured , Fluorescent Dyes/chemistry , Humans , Phycocyanin/chemistry , Protein Binding/immunology , Protein Kinase Inhibitors/pharmacology , T-Lymphocytes/cytology
20.
Immunology ; 146(1): 11-22, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26076649

ABSTRACT

Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Major Histocompatibility Complex/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Staining and Labeling/methods , Antibodies/immunology , CD8-Positive T-Lymphocytes/cytology , Flow Cytometry/methods , Fluorescent Dyes , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Peptides/immunology , Protein Multimerization
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