Mary Maas

Bi-directional activation between mesenchymal stem cells and CLL B-cells: Implication for CLL disease progression

It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B‐cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long‐term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co‐culture of MSC with CLL B‐cells protected the latter from both spontaneous apoptosis and drug‐induced apoptosis. The CD38 expression in previously CD38 positive CLL B‐cells was up‐regulated with MSC co‐culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B‐cells was found in the co‐culture. CD71 upregulation was more significantly associated with high‐risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B‐cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi‐directional activation between bone marrow stromal cells and CLL B‐cells.

A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

BackgroundDendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines.MethodsWe prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma.ResultsThree preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent) in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections.ConclusionStandardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Skeletal muscle and bone marrow derived stromal cells: A comparison of tenocyte differentiation capabilities

This study investigated the comparative ability of bone marrow and skeletal muscle derived stromal cells (BMSCs and SMSCs) to express a tenocyte phenotype, and whether this expression could be augmented by growth and differentiation factor‐5 (GDF‐5). Tissue harvest was performed on the hind limbs of seven dogs. Stromal cells were isolated via serial expansion in culture. After four passages, tenogenesis was induced using either ascorbic acid alone or in conjunction with GDF‐5. CD44, tenomodulin, collagen I, and collagen III expression levels were compared for each culture condition at 7 and 14 days following induction. Immunohistochemistry (IHC) was performed to evaluate cell morphology and production of tenomodulin and collagen I. SMSCs and BMSCs were successfully isolated in culture. Following tenocytic induction, SMSCs demonstrated an increased mean relative expression of tenomodulin, collagen I, and collagen III at 14 days. BMSCs only showed increased mean relative expression of collagen I, and collagen III at 14 days. IHC revealed positive staining for tenomodulin and collagen I at 14 days for both cell types. The morphology of skeletal muscle derived stromal cells at 14 days had an organized appearance in contrast to the haphazard arrangement of the bone marrow derived cells. GDF‐5 did not affect gene expression, cell staining, or cell morphology significantly. Stromal cells from either bone marrow or skeletal muscle can be induced to increase expression of matrix genes; however, based on expression of tenomodulin and cell culture morphology SMSCs may be a more ideal candidate for tenocytic differentiation.

Comprehensive immune profiling reveals substantial immune system alterations in a subset of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a median lifespan of 2–3 years after diagnosis. There are few meaningful treatments that alter progression in this disease. Preclinical and clinical studies have demonstrated that neuroinflammation may play a key role in the progression rate of ALS. Despite this, there are no validated biomarkers of neuroinflammation for use in clinical practice or clinical trials. Biomarkers of neuroinflammation could improve patient management, provide new therapeutic targets, and possibly help stratify clinical trial selection and monitoring. However, attempts to identify a singular cause of neuroinflammation have not been successful. Here, we performed multi-parameter flow cytometry to comprehensively assess 116 leukocyte populations and phenotypes from lymphocytes, monocytes, and granulocytes in a cohort of 80 ALS patients. We identified 32 leukocyte phenotypes that were altered in ALS patients compared to age and gender matched healthy volunteers (HV) that included phenotypes of both inflammation and immune suppression. Unsupervised hierarchical clustering and principle component analysis of ALS and HV immunophenotypes revealed two distinct immune profiles of ALS patients. ALS patients were clustered into a profile distinct from HVs primarily due to differences in a multiple T cell phenotypes, CD3+CD56+ T cells and HLA-DR on monocytes. Patients clustered into an abnormal immune profile were younger, more likely to have a familial form of the disease, and survived longer than those patients who clustered similarly with healthy volunteers (344 weeks versus 184 weeks; p = 0.012). The data set generated from this study establishes an extensive accounting of immunophenotypic changes readily suitable for biomarker validation studies. The extensive immune system changes measured in this study indicate that normal immune homeostatic mechanisms are disrupted in ALS patients, and that multiple immune states likely exist within a population of patients with ALS.

The CD4+/CD14+HLA-DRlo/neg ratio as a prognostic biomarker in cancer patients

The immune status of an individual is made up of the absolute number (cells/μl) and relative ratios of each category of immune cells. To determine the extent of similarity of the immune status between individuals across malignancies, we described the immune status using quantitative whole blood flow cytometry of ten immune markers and generated immune phenotypes from 40 healthy volunteers and 120 patients with glioblastoma, renal cell carcinoma, non-Hodgkin’s lymphoma, ovarian cancer or with a non-malignant condition (acute lung injury). After normalization, we used unsupervised hierarchical clustering and principal component analysis to sort individuals by similarity of immune status into discreet groups of immune profiles. Immune profiles sort not only patients by immune similarity, but they also identify independently regulated immune markers. We noticed an inverse relationship between the number of CD14+HLA-DRlo/neg monocytes and CD4+ T cells. We found that the combination of these markers acted as a potent novel biomarker for assessing the patients’ immune status. Using survival and immunophenotype data from glioblastoma, renal cell carcinoma, non-Hodgkin’s lymphoma patients, we calculated the ratio of the number of CD4+ T cells to the number of CD14+HLADRlo/neg monocytes (cells/μ l) and subgrouped those with a high or low ratio, with a cut-point ratio of 2.0. The 40 healthy volunteers had a mean CD4+/CD14 +HLA-DRlo/neg ratio of 39.8 (median 22.5) with a minimum of 3.9. We analyzed the overall survival of GBM, NHL, and RCC patients with high and low ratio using multivariate analysis to control for age and disease type. The median overall survival for patients with a ratio above 2.0 was 30 months (n=68) compared to 9 months for patients with a low ratio (n=39; p=0.006 by multivariate analysis). Thus, the CD4+/CD14+HLA-DRlo/neg ratio has the potential to be a powerful biomarker for risk stratification and prognosis for a broad array of malignancies.

Ten-color, whole blood flow cytometric analysis of human myeloid subsets; implications for immune monitoring in cancer patients

Myeloid cells comprise a heterogeneous population of cells that include granulocytes and monocytes. Recent data reveals a complex role of myeloid cells as mediators of tumor progression and tumor induced immunosuppression. Myeloid-derived suppressor cells (MDSCs) have been extensively studied in mouse models and are thought to be actively recruited to the tumor and suppress immunity through multiple mechanisms. Characterizations of human MDSCs have been much more problematic due to the lack of equivalent surface markers in mice and the variation and inconsistent use of human cell surface markers. The use of these populations as biomarkers for immunity requires standardization in sample prep and reporting. Typically, blood is prepared for analysis by collection of the mononuclear cell fraction. However, we found that this method can alter the cell populations of interest. For example, paired samples from healthy ten volunteers showed an increase of CD16 expression on monocytes purified by density gradient centrifugation versus those stained directly from whole blood. In addition, whole blood flow cytometry allows universal reporting terms as results can be reported in cells/μL. In effort to begin to standardization human myeloid subsets, we developed a ten color flow panel to analyze unfractionated peripheral blood. As part of this development, we felt it was very important to use surface markers and non-overlapping gating strategies that would enable us to define distinct myeloid populations. We used a combination of ten markers that allows us to measure over 15 myeloid phenotypes including granulocytes (using CD15, CD16 and CD66b), monocyte sub-populations (identified by CD14 and CD16), suppressive monocytes (CD14+HLA-DRlo/neg), myeloid derived suppressor cell populations (LINnegCD33 +HLA-DR+), and circulating dendritic cell populations (LINnegHLA-DR+ subgrouped by CD11c and CD123). In addition to typical bivariate plotting strategies, we also used radar plot analyses to look at multiple markers simultaneously. Data from healthy volunteer controls and cancer patients of the non-overlapping myeloid populations will be shown. This approach will allow standardization of the collection and reporting of these critically important populations.

Immunotherapy of malignant melanoma using a dendritic cell-amplified tumor RNA vaccine: A phase I study

2597 Background: We prepared a dendritic cell (DC) vaccine specific for malignant melanoma and tested its safety in a phase 1 clinical trial of immunotherapy of stage IV malignant melanoma. Methods: Immature DCs were prepared from immunomagnetically isolated patients’ CD14+ cells. Messenger RNA was isolated from fresh tumor tissue obtained at surgery and was reversely transcribed, amplified, and in vitro transcribed. Amplified RNA was tested for the presence of melanoma-associated antigens gp100, tyrosinase and Mart1 and introduced into immature DCs by electroporation. Then the DCs were matured and cryopreserved. Five million cells were injected at three-week intervals for up to four injections. Results: We studied six patients (median age 52, range 40–66) suffering from predominantly non-visceral disease (four patients). After surgery, the tumor was measurable in three patients, evaluable in two and non-evaluable (completely resected) in one patient. Prior to DC therapy, two patients were treated by radi...