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1.
Cancer Immunol Res ; 9(12): 1465-1475, 2021 12.
Article in English | MEDLINE | ID: mdl-34635486

ABSTRACT

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunity/immunology , Immunotherapy/methods , Programmed Cell Death 1 Receptor/therapeutic use , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Humans , Mice , Phosphorylation , Signal Transduction
2.
Sci Transl Med ; 13(581)2021 02 17.
Article in English | MEDLINE | ID: mdl-33597266

ABSTRACT

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.


Subject(s)
Immune Checkpoint Inhibitors , Melanoma , Animals , Antigens, Neoplasm , Epitopes , Humans , Melanocytes , Melanoma/therapy , Mice
3.
Cancer Immunol Res ; 9(2): 184-199, 2021 02.
Article in English | MEDLINE | ID: mdl-33277233

ABSTRACT

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type-specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity-promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell-specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.


Subject(s)
Antineoplastic Agents/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Ferroptosis/drug effects , Tumor Cells, Cultured/drug effects , Animals , Autophagy/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Female , Ferroptosis/genetics , Humans , Mice , Mice, Inbred C57BL , Neoplasms/drug therapy
4.
Cell Rep Methods ; 1(5): 100084, 2021 09 27.
Article in English | MEDLINE | ID: mdl-35474673

ABSTRACT

Oncogenic mutations in KRAS can be recognized by T cells on specific class I human leukocyte antigen (HLA-I) molecules, leading to tumor control. To date, the discovery of T cell targets from KRAS mutations has relied on occasional T cell responses in patient samples or the use of transgenic mice. To overcome these limitations, we have developed a systematic target discovery and validation pipeline. We evaluate the presentation of mutant KRAS peptides on individual HLA-I molecules using targeted mass spectrometry and identify 13 unpublished KRASG12C/D/R/V mutation/HLA-I pairs and nine previously described pairs. We assess immunogenicity, generating T cell responses to nearly all targets. Using cytotoxicity assays, we demonstrate that KRAS-specific T cells and T cell receptors specifically recognize endogenous KRAS mutations. The discovery and validation of T cell targets from KRAS mutations demonstrate the potential for this pipeline to aid the development of immunotherapies for important cancer targets.


Subject(s)
Lung Neoplasms , T-Lymphocytes , Mice , Animals , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Mutation , Receptors, Antigen, T-Cell/genetics , Lung Neoplasms/genetics , Histocompatibility Antigens Class I/genetics
5.
J Exp Med ; 218(1)2021 01 04.
Article in English | MEDLINE | ID: mdl-33045061

ABSTRACT

Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.


Subject(s)
Diabetes Mellitus, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Immune Tolerance , Programmed Cell Death 1 Receptor/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Diabetes Mellitus, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/genetics , Mice , Mice, Inbred NOD , Mice, Neurologic Mutants , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Programmed Cell Death 1 Receptor/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Signal Transduction/genetics
6.
Cell ; 183(7): 1848-1866.e26, 2020 12 23.
Article in English | MEDLINE | ID: mdl-33301708

ABSTRACT

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.


Subject(s)
Immunity , Neoplasms/immunology , Neoplasms/metabolism , Obesity/metabolism , Tumor Microenvironment , Adiposity , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Cell Proliferation , Diet, High-Fat , Fatty Acids/metabolism , HEK293 Cells , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Kinetics , Lymphocytes, Tumor-Infiltrating , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , Principal Component Analysis , Procollagen-Proline Dioxygenase/metabolism , Proteomics
7.
Proc Natl Acad Sci U S A ; 117(38): 23684-23694, 2020 09 22.
Article in English | MEDLINE | ID: mdl-32907939

ABSTRACT

Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.


Subject(s)
Breast Neoplasms , Immunotherapy , Mammary Neoplasms, Experimental , Tumor Microenvironment/immunology , Animals , Biomarkers, Tumor/immunology , Breast Neoplasms/immunology , Breast Neoplasms/therapy , CD8-Positive T-Lymphocytes/immunology , Female , Gene Expression Regulation, Neoplastic/immunology , Humans , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/therapy , Mice , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Transcriptome/immunology
8.
Oncoimmunology ; 7(5): e1423172, 2018.
Article in English | MEDLINE | ID: mdl-29721378

ABSTRACT

Background: Approximately 50% of melanomas harbor BRAF mutations. Treatment with BRAF +/- MEK inhibition is associated with favorable changes in the tumor microenvironment thus providing the rationale for combining targeted agents with immunotherapy. Methods: Patients with unresectable Stage III or IV BRAFV600E mutant melanoma were enrolled in a single-center prospective study (n = 6). Patients were eligible to receive two courses of HD-IL-2 and vemurafenib twice daily. The primary endpoint was progression-free survival (PFS) with secondary objectives including overall survival (OS), response rates (RR), and safety of combination therapy as compared to historical controls. Immune profiling was performed in longitudinal tissue samples, when available. Results: Overall RR was 83.3% (95% CI: 36%-99%) and 66.6% at 12 weeks. All patients eventually progressed, with three progressing on treatment and three progressing after the vemurafenib continuation phase ended. Median PFS was 35.8 weeks (95% CI: 16-57 weeks). Median OS was not reached; however, the time at which 75% of patients were still alive was 104.4 weeks. Change in circulating BRAFV600E levels correlated with response. Though combination therapy was associated with enhanced CD8 T cell infiltrate, an increase in regulatory T cell frequency was seen with HD-IL-2 administration, suggesting a potential limitation in this strategy. Conclusion: Combination vemurafenib and HD-IL-2 is well tolerated and associated with treatment responses. However, the HD-IL-2 induced increase in Tregs may abrogate potential synergy. Given the efficacy of regimens targeting the PD-1 pathway, strategies combining these regimens with BRAF-targeted therapy are currently underway, and the role of combination vemurafenib and HD-IL-2 is uncertain. Trial Registration: Clinical trial information: NCT01754376; https://clinicaltrials.gov/show/NCT01754376.

9.
Nature ; 547(7664): 413-418, 2017 07 27.
Article in English | MEDLINE | ID: mdl-28723893

ABSTRACT

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing , Immunotherapy/methods , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Tumor Escape/drug effects , Tumor Escape/immunology , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Genomics , Humans , Interferons/immunology , Loss of Function Mutation , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Mice , NF-kappa B/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/deficiency , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Escape/genetics , Unfolded Protein Response , Xenograft Model Antitumor Assays
10.
J Exp Med ; 214(4): 895-904, 2017 04 03.
Article in English | MEDLINE | ID: mdl-28302645

ABSTRACT

It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1-deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.


Subject(s)
B7-H1 Antigen/physiology , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Tumor Escape , Animals , Cell Line, Tumor , Colorectal Neoplasms/immunology , Lymphocyte Activation , Melanoma/immunology , Mice , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/physiology , Tumor Microenvironment
11.
Nat Immunol ; 17(12): 1436-1446, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27695002

ABSTRACT

Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)-mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell-mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.


Subject(s)
B-Lymphocyte Subsets/immunology , Germinal Center/immunology , Immune Tolerance , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antibody Formation , Cells, Cultured , Epigenesis, Genetic , Forkhead Transcription Factors/metabolism , Interleukin-21 Receptor alpha Subunit/genetics , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout
12.
J Exp Med ; 212(10): 1603-21, 2015 Sep 21.
Article in English | MEDLINE | ID: mdl-26371185

ABSTRACT

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.


Subject(s)
Autoimmunity/immunology , CTLA-4 Antigen/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Age Factors , Amino Acid Sequence , Animals , Autoimmunity/drug effects , Autoimmunity/genetics , CTLA-4 Antigen/immunology , Disease Resistance/genetics , Disease Resistance/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Gene Deletion , Interleukin-10/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , T-Lymphocytes, Regulatory/physiology , Tamoxifen/pharmacology
13.
Cell ; 162(6): 1242-56, 2015 Sep 10.
Article in English | MEDLINE | ID: mdl-26359984

ABSTRACT

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.


Subject(s)
Melanoma/genetics , Programmed Cell Death 1 Receptor/metabolism , Signal Transduction , Animals , Antineoplastic Agents/administration & dosage , B7-H1 Antigen/genetics , Cell Line, Tumor , Cells, Cultured , Gene Knockdown Techniques , Heterografts , Humans , Mice , Mice, Inbred C57BL , Neoplasm Transplantation
14.
Cancer Immunol Res ; 2(7): 643-54, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24903021

ABSTRACT

BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8(+) T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten(-/-) syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8(+) T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8(+) T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Melanoma/drug therapy , Melanoma/immunology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Antigens, Neoplasm/metabolism , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/antagonists & inhibitors , Cell Line, Tumor , Cytokines/biosynthesis , Dose-Response Relationship, Drug , Humans , Immunocompetence , Indoles/administration & dosage , Ipilimumab , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Male , Melanoma/pathology , Melanoma/secondary , Mice, Inbred C57BL , Middle Aged , Sulfonamides/administration & dosage , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Tumor Escape/drug effects , Tumor Escape/immunology , Vemurafenib , Xenograft Model Antitumor Assays/methods
15.
Oncoimmunology ; 2(10): e26615, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24251082

ABSTRACT

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) ß chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10-14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones.

16.
Nano Lett ; 12(8): 4131-9, 2012 Aug 08.
Article in English | MEDLINE | ID: mdl-22769232

ABSTRACT

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4-3 µm) in MSCs enhances MR parameters such as the r(2) relaxivity (5-fold), residence time inside the cells (3-fold) and R(2) signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation.


Subject(s)
Ferric Compounds/chemistry , Magnetic Resonance Imaging/methods , Mesenchymal Stem Cells/chemistry , Nanoparticles/chemistry , Polyglactin 910/chemistry , Animals , Cell Proliferation , Cells, Cultured , Humans , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred BALB C , Particle Size , Surface Properties
17.
J Invest Dermatol ; 132(4): 1084-93, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22189784

ABSTRACT

In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2-, and CD133-Sox2- (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2- spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2- cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function.


Subject(s)
Cell Proliferation , Dermis/cytology , Dermis/metabolism , Hydrogels , SOXB1 Transcription Factors/metabolism , AC133 Antigen , Alkaline Phosphatase/metabolism , Animals , Antigens, CD/metabolism , Cell Culture Techniques/methods , Cells, Cultured , Glycoproteins/metabolism , Hair/anatomy & histology , Hair Follicle/growth & development , Integrin alpha1beta1/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Peptides/metabolism
18.
Arch Otolaryngol Head Neck Surg ; 135(7): 662-9, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19620587

ABSTRACT

OBJECTIVES: To determine the expression of the p53 family member p73 in vestibular schwannoma (VS) and to determine the potential role of this tumor suppressor in regulating the proliferation of HEI193, a human papillomavirus E6-E7 immortalized VS cell line. METHODS: Immunohistochemical staining was used to investigate the expression of p73 in 34 cases of archived VS tissue, while Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of p73 in HEI193. After transfection of a full-length p73 plasmid (TAp73alpha), flow cytometry analysis was performed to determine the effect of p73 expression on cell cycle distribution, while annexin V-FITC (fluorescein isothiocyanate) analysis and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay were used to measure apoptosis. The effect of p73 expression on ionizing radiation-induced cell death was also investigated with annexin V staining, TUNEL assay, and flow cytometry analysis. RESULTS: Of the 34 vestibular schwannoma tissues examined, p73 was expressed in 14 (41%) but was not expressed in HEI193. Transfection of p73 alone resulted in increased apoptosis and necrosis, and G(1) accumulation with concomitant induction of p21. The presence of p73 also significantly increased early apoptosis (P = .046), late apoptosis (P < .001), and necrosis (P = .009) on exposure of the HEI193 cells to ionizing radiation. CONCLUSION: Forced expression of p73, perhaps by gene therapy, to induce apoptosis directly or to sensitize VS tumors to ionizing radiation may have relevant therapeutic applications.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic/physiology , Neuroma, Acoustic/metabolism , Nuclear Proteins/physiology , Oncogene Proteins, Viral/genetics , Protein-Tyrosine Kinases/genetics , Repressor Proteins/genetics , Tumor Suppressor Proteins/physiology , Adult , Aged , Annexin A5/analysis , Apoptosis/physiology , Blotting, Western , Cell Cycle/physiology , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Genetic Therapy , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Male , Middle Aged , Neuroma, Acoustic/therapy , Nuclear Proteins/metabolism , Papillomavirus E7 Proteins , Retrospective Studies , Transfection , Tumor Protein p73 , Tumor Suppressor Proteins/metabolism
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