ROLES FOR BRAF KINASE ACTIVATING MUTATIONS IN MELANOMA: MICROENVIRONMENTAL IMMUNOSUPPRESSION

The BRAF oncogene demonstrates a characteristic mutation (V600E) in a significant fraction of cutaneous melanomas, leading to constitutive activation of the MAP kinase pathway. This genetic lesion endows tumor cells with proliferative and survival advantages, and metastatic melanoma patients treated with the BRAF(V600E)-specific inhibitor, Vemurafenib, have shown dramatic clinical responses. Here, I show that BRAF(V600E) induces transcription of the IL-1α and IL-1β genes in both melanocytes and melanoma cell lines and that this upregulation is specifically abrogated by targeted BRAF(V600E) inhibitors. Furthermore, treatment of melanoma tumor-associated fibroblasts (TAFs) with IL-1α/β significantly enhanced the ability of TAFs to suppress the proliferation and function of melanoma antigen-specific cytotoxic T cells. IL-1α/β treatment of TAFs upregulated multiple immunosuppressive factors, including COX-2 and the PD-1 ligands PD-L1 and PD-L2. Specific BRAF(V600E) inhibitors largely abrogated the ability of melanoma cells to confer T cell-suppressive properties on TAFs. These results support a model in which BRAF(V600E) promotes immune suppression in the melanoma tumor environment through an IL-1-mediated mechanism involving resident stromal fibroblasts. Based on these findings, combination therapies involving targeted BRAF inhibition and T cell-based immunotherapies are warranted

In silico prediction of tumor antigens derived from functional missense mutations of the cancer gene census

Antigen-specific immune responses against peptides derived from missense gene mutations have been identified in multiple cancers. The application of personalized peptide vaccines based on the tumor mutation repertoire of each cancer patient is a near-term clinical reality. These peptides can be identified for pre-validation by leveraging the results of massive gene sequencing efforts in cancer. In this study, we utilized NetMHC 3.2 to predict nanomolar peptide binding affinity to 57 human HLA-A and B alleles. All peptides were derived from 5,685 missense mutations in 312 genes annotated as functionally relevant in the Cancer Genome Project. Of the 26,672,189 potential 8–11 mer peptide-HLA pairs evaluated, 0.4% (127,800) display binding affinities < 50 nM, predicting high affinity interactions. These peptides can be segregated into two groups based on the binding affinity to HLA proteins relative to germline-encoded sequences: peptides for which both the mutant and wild-type forms are high affinity binders, and peptides for which only the mutant form is a high affinity binder. Current evidence directs the attention to mutations that increase HLA binding affinity, as compared with cognate wild-type peptide sequences, as these potentially are more relevant for vaccine development from a clinical perspective. Our analysis generated a database including all predicted HLA binding peptides and the corresponding change in binding affinity as a result of point mutations. Our study constitutes a broad foundation for the development of personalized peptide vaccines that hone-in on functionally relevant targets in multiple cancers in individuals with diverse HLA haplotypes

Screening of previously reported microsatellite markers, associated with panicle characteristics, for maker assisted selection in Malaysian rice (Oryza sativa l.)

Rice (Oryzasativa L.) is an important crop that vital to the lives of billions of people providing almost 23percent of the calories intake. Most of the yield related traits are quantitative in nature. To improve these traits, quantitatively traits loci (QTL) are needed to be identified. Major QTLs have the tendency to be detected across populations and it mainly depends on high phenotypic variations for respective traits. Panicle characteristics are the main determinant of rice yield. Several investigations related to the identification of quantitative trait loci (QTLs) for these traits have been reported. Present studies were carried out to screen the QTLs in Malaysian rice cultivars for the selection of desirable plants through marker assisted breeding. Malaysian rice cultivars were evaluated for three panicle traits; panicle length, number of panicle per plant and number of grains per panicle. Eight microsatellite markers, which have shown association with panicle traits in the literature, were selected to identify the microsatellite loci for panicle traits. Among these microsatellites markers, RM310 distinguished the Malaysian rice varieties based on number of grains per panicle. It is therefore suggested to utilize microsatellite marker RM 310 in molecular screening of rice breeding populations to identify the plant possessing high numbers of grains

Natural Splice Variant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-Kb generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-Db-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-Db DCs were superior to WT-Db DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy

Standardized analysis for the quantification of Vbeta CDR3 T-cell receptor diversity.

International audienceAssessment of the diversity of the T-cell receptor (TCR) repertoire is often determined by measuring the frequency and distribution of individually rearranged TCRs in a population of T cells. Spectratyping is a common method used to measure TCR repertoire diversity, which examines genetic variation in the third complementarity-determining region (CDR3) region of the TCR Vbeta chain using RT-PCR length-distribution analysis. A variety of methods are currently used to analyze spectratype data including subjective visual measures, qualitative counting measures, and semi-quantitative measures that compare the original data to a standard, control data set. Two major limitations exist for most of these approaches: data files become very wieldy and difficult to manage, and current analytic methods generate data which are difficult to compare between laboratories and across different platforms. Here, we introduce a highly efficient method of analysis that is based upon a normal theoretical Gaussian distribution observed in cord blood and recent thymic emigrants. Using this analysis method, we demonstrate that PBMC obtained from patients with various diseases have skewed TCR repertoire profiles. Upon in vitro activation with anti-CD3 and anti-CD28 coated beads (Xcyte Dynabeads) TCR diversity was restored. Moreover, changes in the TCR repertoire were dynamic in vivo. We demonstrate that use of this streamlined method of analysis in concert with a flexible software package makes quantitative assessment of TCR repertoire diversity straightforward and reproducible, enabling reliable comparisons of diversity values between laboratories and over-time to further collaborative efforts. Analysis of TCR repertoire by such an approach may be valuable in the clinical setting, both for prognostic potential and measuring clinical responses to therapy

Educated T-Cells Mount Stronger Anti-Tumor Response in Myeloma

In efforts to educate the body to fight off cancer, researchers have found that some immune cells are "smarter" than others. Working with collections of human cells, Johns Hopkins Kimmel Cancer Center scientists tested kill-rates of two kinds of T-cells "primed" to home in on myeloma, a cancer of the bone marrow. Those that live in the bone marrow outperformed their counterparts circulating in the blood by more than 90 percent. "It is very difficult to design cancer therapies that get the body's immune system to recognize and kill cancer cells that the system has ignored for a long time," says Ivan Borrello, M.D., assistant professor of oncology and director of the research, which is published in the March 1 issue of Cancer Research. "Now, we have evidence that 'educating' T-cells in the bone marrow may be the most effective way to get an anti-tumor response." In nature, T-cells are responsible for identifying cells that are foreign to the body, including genetically altered cancer cells, and marking them for destruction. In the Hopkins study of both kinds of T-cells, those from the blood and bone marrow, scientists mixed them with magnetic beads coated with tumor antibodies, a sort of "artificial intelligence" that activated and expanded the T-cells' cancer-killing mode. The marrow T-cells identified not only mature myeloma cells but the primitive cells responsible for the disease. Activated bone marrow T-cells stopped the growth of 86 percent of myeloma stem cell colonies compared to 47 percent for activated t-cells taken from circulating blood. The researchers' next step is to determine whether the cells' ability to limit cancer growth in culture dishes ultimately may do the same in patients. Kimmel Cancer Center researchers are planning studies in a small number of myeloma patients to test the activated marrow T-cells alone and in combination with a myeloma vaccine. "While T-cells from circulating blood traditionally are used in immunotherapy strategies because they are easy to obtain and grow, they often don't recognize the tumor," says Borrello. "In the case of myeloma, we believe the marrow T-cells have certain surface markers that may help them migrate back to the site of the tumor," he says. "Moreover, the marrow itself contains some type of stimulant to attract the cells," says Kimberly Noonan, researcher and first author of the paper. To treat patients, the scientists will collect a small amount of bone marrow from patients and with relative ease, grow and activate large numbers of T-cells from that source. These would then be given intravenously back to patients. However, according to Borrello, they may find that an additional cancer vaccine may increase the overall anti-tumor effect of the marrow T-cells. They also believe that patients with other blood, bone marrow and solid tumors such as breast cancer may benefit from this type of immunotherapy. Evidence from other research groups indicates that breast cancer patients have T-cells in their bone marrow that are specific to their tumor. Myeloma strikes close to 16,000 Americans annually and kills 11,300. Other participants of this research include William Matsui, Paolo Serafini, Rebecca Carbley, Gladys Tan, Hyam Levitsky, and Katherine Whartenby from Johns Hopkins; and Jahan Khalili and Mark Bonyhadi from Xcyte Therapies

Human late memory CD8+ T cells have a distinct cytokine signature characterized by CC chemokine production without IL-2 production

Late memory T cell skewing is observed in the setting of immune recovery after cord blood transplantation, and may be associated with inferior control of viral reactivation and cancers. Therefore, we sought to understand how late memory cells differ functionally from earlier stage memory T cells, and whether surface phenotypes associated with differentiation stages were predictably associated with functional signatures. Higher order cytokine flow cytometry allows characterization of human T cells based on complex phenotypic markers and their differential capacity to simultaneously secrete effector proteins, including cytokines and chemokines. We used 8-color, 10-parameter cytokine flow cytometry to characterize the functional activation of human late memory CD8(+) T cells defined by CD45RA and CD27 expression (CD27(-)CD45RA(+)). We assessed the 15 possible functional signatures of cells defined by production of IL-2, IFN-gamma, TNF-alpha, and MIP-1beta alone or in combination, following activation with Ags stimulating bypassing surface proteins (PMA:ionomycin) or through the TCR (e.g., viral Ags). Late memory CD8(+) T cells produced abundant amounts of CC chemokines (MIP-1beta, MIP-1alpha, and RANTES) but not IL-2. IL-2/IFN-gamma coproduction, characteristic of protective immune responses to viral infections, was absent in late memory CD8(+) T cells. These data demonstrate that functional cytokine signatures are predictably associated with CD8(+) maturation stages, and that the polarization of late memory CD8(+) T cells toward CC chemokine production and away from IL-2 production suggests a unique functional role for this subset

Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p<0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p<0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease