Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Download Full-text

Functional Analysis and Gene Expression Profile of Umbilical Cord Blood Regulatory T Cells.

Blood ◽

10.1182/blood.v114.22.4085.4085 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4085-4085

Author(s):

Giovanni Fernando Torelli ◽

Roberta Maggio ◽

Nadia Peragine ◽

Sabina Chiaretti ◽

Maria Stefania De Propris ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Gene Expression Profile ◽

Expression Profile ◽

Production Capacity ◽

T Cell Response ◽

Foxp3 Gene ◽

Before And After

Abstract Abstract 4085 Poster Board III-1020 Introduction Umbilical cord blood (CB) stem cells are now broadly used in the unrelated stem cell transplant setting and comparative studies with different stem cell sources have shown that CB transplant is characterized by a lower risk of graft-versus-host disease (GVHD). The immaturity of CB T cells has been generally regarded as the main contributing factor accounting for this phenomenon; the possible role played by CB regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is now under investigation, but very scare data are so far available. Aim of this study was to analyze and compare the functional properties and the gene expression profile of Tregs expanded from CB units with those expanded from the peripheral blood (PB) of adult normal donors. Methods Tregs were purified from mononuclear cells obtained from 23 CB units and from the PB of 13 adult normal donors using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with the anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml) MoAbs in the presence of IL-2 (100 U/ml). Immunophenotypic analyses were performed before and after expansion. To assess their suppressive functions, expanded Tregs were seeded with autologous effector T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The IL-10 production capacity of expanded Tregs was tested using an ELISA assay. The two-sided student t test was used to evaluate the significance of differences between groups. Gene expression profile experiments were performed using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analyses were carried out using the dChip software; a t test was used to evaluate the presence of specifically expressed classes of genes. Functional annotation analysis was performed using the DAVID software. Results CB and PB Tregs presented similar immunophenotypic appearances before and after expansion. Im particular, after expansion they presented a comparable expression of surface CD4, CD25, CD62L, CCR5 and CD45RO, and of cytoplasmic CTLA-4 and Foxp3, while they both were negative for the CD45RA antigen, thus indicating the loss of their naïve features. On the contrary, Tregs obtained from CB (n=23) presented a much higher expansion capacity compared to those obtained from PB (n=13): mean fold increase (range), CB 10.3 (1.6-24), PB 3.9 (1.5-10), p 0.003. CB expanded Tregs (n=6) exerted a potent suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC, that resulted inferior even though not significantly compared to that exerted by PB expanded Tregs (n=5): mean fold reduction (range), CB 7.8 (2.5-15.1), PB 14.3 (1.5-23.7), p 0.14. Tregs expanded from CB (n=4) and PB (n=1) presented a high and comparable in vitro IL-10 production capacity: mean pg/ml (range), CB 326.5 (226-426), PB 382. Gene expression profile analysis showed a higher number of upregulated genes in Tregs expanded from CB (n=2) compared to Tregs expanded from PB (n=3); among them, a significant enrichment of genes involved in cell proliferation, cell cycle checkpoints, signal transduction, cell differentiation, apoptosis, TGF-β receptor pathway and the GrNH pathway was observed. This suggests that CB Tregs retain a more undifferentiated program and are characterized by the high expression of genes which might provide an advantage in cell expansion. Finally, when looking at the Foxp3 gene expression levels, no difference was observed between the two populations. Conclusions These results demonstrate that Tregs contained in CB retain an expansion potential superior to that of Tregs isolated from the PB of normal donors, as confirmed by functional analyses and gene profile. Tregs expanded from CB and PB seem to exert a potent and comparable suppressive function of the proliferative effect in mixed lymphocyte reaction assays. The maintaining of the modulatory properties after expansion is confirmed by the expression of the Foxp3 gene and protein, and by the production of IL-10. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T-cell response. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Early T-Cell Precursor-ALL in Adult T-ALL.

Blood ◽

10.1182/blood.v114.22.911.911 ◽

2009 ◽

Vol 114 (22) ◽

pp. 911-911 ◽

Cited By ~ 1

Author(s):

Martin Neumann ◽

Sandra Heesch ◽

Stefan Schwartz ◽

Nicola Gökbuget ◽

Dieter Hoelzer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Real Time ◽

Gene Expression Profile ◽

Expression Profile ◽

Real Time Pcr ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Study Group

Abstract Abstract 911 Introduction: Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods: We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results: Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion: In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

Download Full-text