Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

Pigs as Clinically Relevant Models for Synergizing Interventional Oncology and Immunotherapy.

Traditionally, rodent cancer models have driven preclinical oncology research. However, they do not fully recapitulate characteristics of human cancers, and their size poses challenges when evaluating tools in the interventional oncologists' armamentarium. Pig models, however, have been the gold standard for validating surgical procedures. Their size enables the study of image-guided interventions using human ultrasound (US), computed tomography (CT), and magnetic resonance (MR) imaging platforms. Furthermore, pigs have immunologic features that are similar to those of humans, which can potentially be leveraged for studying immunotherapy. Novel pig models of cancer are being developed, but additional research is required to better understand both the pig immune system and malignancy to enhance the potential for pig models in interventional oncology research. This review aims to address the main advantages and disadvantages of using a pig model for interventional oncology and outline the specific characteristics of pig models that make them more suitable for investigation of locoregional therapies.

Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance.

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.

Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma.

BACKGROUND: Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway. METHODS: Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated. RESULTS: The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3ß and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines. CONCLUSIONS: The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.

Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma.

BACKGROUND: The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro. METHODS: Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. RESULTS: More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 µM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. CONCLUSIONS: AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

Antitumor activity of the ERK inhibitor SCH772984 [corrected] against BRAF mutant, NRAS mutant and wild-type melanoma.

BACKGROUND: In melanoma, dysregulation of the MAPK pathway, usually via BRAF(V600) or NRAS(Q61) somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF(V600), NRAS mutant, or wild-type melanoma. METHODS: The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined. RESULTS: Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC50) < 1 µM; intermediately sensitive, IC50 1-2 µM; and resistant, >2 µM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF(V600) mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis. CONCLUSIONS: Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.

Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations.

BACKGROUND: A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. MATERIALS AND METHODS: The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. RESULTS: Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. CONCLUSIONS: The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro.

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs. METHODS AND FINDINGS: Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4(+) and CD8(+) T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1. CONCLUSIONS: Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.

Immunosensitization with a Bcl-2 small molecule inhibitor.

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.

CTLA4 blockade increases Th17 cells in patients with metastatic melanoma.

BACKGROUND: Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature. METHODS: Peripheral blood mononuclear cells (PBMC) were collected before and after receiving tremelimumab within two clinical trials, one with tremelimumab alone (21 patients) and another together with autologous dendritic cells (DC) pulsed with the melanoma epitope MART-126-35 (6 patients). Cytokines were quantified directly in plasma from patients and after in vitro stimulation of PBMC. We also quantified IL-17 cytokine-producing cells by intracellular cytokine staining (ICS). RESULTS: There were no significant changes in 13 assayed cytokines, including IL-17, when analyzing plasma samples obtained from patients before and after administration of tremelimumab. However, when PBMC were activated in vitro, IL-17 cytokine in cell culture supernatant and Th17 cells, detected as IL-17-producing CD4 cells by ICS, significantly increased in post-dosing samples. There were no differences in the levels of Th17 cells between patients with or without an objective tumor response, but samples from patients with inflammatory and autoimmune toxicities during the first cycle of therapy had a significant increase in Th17 cells. CONCLUSION: The anti-CTLA4 blocking antibody tremelimumab increases Th17 cells in peripheral blood of patients with metastatic melanoma. The relation between increases in Th17 cells and severe autoimmune toxicity after CTLA4 blockade may provide insights into the pathogenesis of anti-CTLA4-induced toxicities.

T-cell responses to survivin in cancer patients undergoing radiation therapy.

PURPOSE: The goal of this study was to determine if radiation therapy (RT) of human cancer enhances or diminishes tumor-specific T-cell reactivity. This is important if immunotherapy is to be harnessed to improve the outcome of cancer radiotherapy. EXPERIMENTAL DESIGN: Lymphocytes were isolated from colorectal cancer (CRC) patients before, during, and after presurgical chemoradiotherapy. Similar samples were taken from prostate cancer patients receiving standard RT. The level of CD8(+) T cells capable of binding tetramers for the tumor-associated antigen survivin, which is overexpressed in both cancer types, was enumerated in HLA-A*0201 patient samples. CD4(+), CD25(high), Foxp3(+) cells were also enumerated to evaluate therapy-induced changes in T(regulatory) cells. For CRC patients, most of whom were enrolled in a clinical trial, pathologic response data were available, as well as biopsy and resection specimens, which were stained for cytoplasmic and intranuclear survivin. RESULTS: Survivin-specific CD8(+) T lymphocytes were detected in the peripheral blood of CRC and prostate cancer patients and increased after therapy in some, but not all, patients. Increases were more common in CRC patients whose tumor was downstaged after chemoradiotherapy. Biopsy specimens from this cohort generally had higher nuclear to cytoplasmic survivin expression. T(regulatory) cells generally increased in the circulation following therapy but only in CRC patients. CONCLUSION: This study indicates that RT may increase the likelihood of some cancer patients responding to immunotherapy and lays a basis for future investigations aimed at combining radiation and immunotherapy.

Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy.

Breast cancers (BCs) with expression of estrogen receptor-alpha (ERα) occur in more than 70% of newly-diagnosed patients in the U.S. Endocrine therapy with antiestrogens or aromatase inhibitors is an important intervention for BCs that express ERα, and it remains one of the most effective targeted treatment strategies. However, a substantial proportion of patients with localized disease, and essentially all patients with metastatic BC, become resistant to current endocrine therapies. ERα is present in most resistant BCs, and in many of these its activity continues to regulate BC growth. Fulvestrant represents one class of ERα antagonists termed selective ER downregulators (SERDs). Treatment with fulvestrant causes ERα down-regulation, an event that helps overcome several resistance mechanisms. Unfortunately, full antitumor efficacy of fulvestrant is limited by its poor bioavailability in clinic. We have designed and tested a new generation of steroid-like SERDs. Using ERα-positive BC cells in vitro, we find that these compounds suppress ERα protein levels with efficacy similar to fulvestrant. Moreover, these new SERDs markedly inhibit ERα-positive BC cell transcription and proliferation in vitro even in the presence of estradiol-17ß. In vivo, the SERD termed JD128 significantly inhibited tumor growth in MCF-7 xenograft models in a dose-dependent manner (P < 0.001). Further, our findings indicate that these SERDs also interact with ER-positive immune cells in the tumor microenvironment such as myeloid-derived suppressor cells (MDSC), tumor infiltrating lymphocytes and other selected immune cell subpopulations. SERD-induced inhibition of MDSCs and concurrent actions on CD8+ and CD4 + T-cells promotes interaction of immune checkpoint inhibitors with BC cells in preclinical models, thereby leading to enhanced tumor killing even among highly aggressive BCs such as triple-negative BC that lack ERα expression. Since monotherapy with immune checkpoint inhibitors has not been effective for most BCs, combination therapies with SERDs that enhance immune recognition may increase immunotherapy responses in BC and improve patient survival. Hence, ERα antagonists that also promote ER downregulation may potentially benefit patients who are unresponsive to current endocrine therapies.

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System.

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression.

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.

Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.