Stefan Tomiuk

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Absolute quantification of microRNAs by using a universal reference

MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(-) hematopoietic progenitor cells.

Purification of neuronal precursors from the adult mouse brain: comprehensive gene expression analysis provides new insights into the control of cell migration, differentiation, and homeostasis.

The progeny of neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain consists in polysialylated NCAM-expressing immature neurons (PSA(+) cells), which migrate to the olfactory bulb (OB) to differentiate into GABAergic interneurons. We purified murine PSA(+) cells directly from the adult brain by FACS and analyzed their gene expression profile by SAGE. Comparative analyses led to the identification of precursor-enriched genes, including Survivin, Sox-4, Meis2, Dishevelled-2, C3aR1 and Riken 3110003A17, and many so far uncharacterized transcripts. Cluster analysis showed that groups of genes involved in axon guidance and gene clusters implicated in chemotaxis are strongly upregulated, indicating a role of both cues in the control of cell migration in the adult brain. Furthermore, genes involved in apoptosis and cell proliferation are co-expressed, suggesting that the amount of precursors that is present in the adult brain is a result of an equilibrium of these processes.

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor‐matched CD133+ cells with the more differentiated CD34+CD133− and CD34−CD133− cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing‐based profiling, we found that between 109 (CD133+) and 216 (CD34−CD133−) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR‐142‐3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34+CD133− cells. Eighteen miRNAs were significantly differentially expressed between CD133+ and CD34+CD133− cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase‐based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR‐142‐3p as well as tropomyosin 1 and frizzled homolog 5 by miR‐29a. Overexpression of miRNAs in CD133+ cells demonstrated that miR‐142‐3p has a negative influence on the overall colony‐forming ability. In conclusion, the miRNAs expressed differentially between the CD133+ and CD34+CD133− cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell‐based therapies with CD133+ cells and delineate for the first time how the stem cell character of CD133+ cells is defined by the expression of specific miRNAs. STEM CELLS 2011;29:847–857

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Molecular and functional characterization of allogantigen specific anergic T cells suitable for cell therapy

Background CD4+ regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. Design and Methods The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Results Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. Conclusions Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.

High Weight Differences between Donor and Recipient Affect Early Kidney Graft Function—A Role for Enhanced IL-6 Signaling

The frequency of delayed function of kidney transplants varies greatly and is associated with quality of graft, donor age and the duration of cold ischemia time. Furthermore, body weight differences between donor and recipient can affect primary graft function, but the underlying mechanism is poorly understood. We transplanted kidney grafts from commensurate body weight (L‐WD) or reduced body weight (H‐WD) donor rats into syngeneic or allogeneic recipients. Twenty‐four hours posttransplantation, serum creatinine levels in H‐WD recipients were significantly higher compared to L‐WD recipients indicating impaired primary graft function. This was accompanied by upregulation of IL‐6 transcription and increased tubular destruction in grafts from H‐WD recipients. Using DNA microarray analysis, we detected decreased expression of genes associated with kidney function and an upregulation of other genes such as Cyp3a13, FosL and Trib3. A single application of IL‐6 into L‐WD recipients is sufficient to impair primary graft function and cause tubular damage, whereas immediate neutralization of IL‐6 receptor signaling in H‐WD recipients rescued primary graft function with well‐preserved kidney graft architecture and a normalized gene expression profile. These findings have strong clinical implication as anti‐IL6R treatment of patients receiving grafts from lower‐weight donors could be used to improve primary graft function.

Transcriptional profiling identifies an interferon‐associated host immune response in invasive squamous cell carcinoma of the skin

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) represent the 2 most common types of nonmelanoma skin cancer. Both derive from keratinocytes but show a distinct biological behavior. Here we present transcriptional profiling data of a large cohort of tumor patients (SCC, n = 42; BCC, n = 114). Differentially expressed genes reflect known features of SCC and BCC including the typical cytokeratin pattern as well as upregulation of characteristic cell proliferation genes. Additionally, we found increased expression of interferon (IFN)‐regulated genes (including IFI27, IFI30, Mx1, IRF1 and CXCL9) in SCC, and to a lower extent in BCC. The expression of IFN‐regulated genes correlated with the extent of the lesional immune‐cell infiltrate. Immunohistological examinations confirmed the expression of IFN‐regulated genes in association with a CXCR3+ cytotoxic inflammatory infiltrate on the protein level. Of note, a small subset of SCC samples with low expression of IFN‐regulated genes included most organ transplant recipients receiving immunosuppressive medication. Collectively, our findings support the concept that IFN‐associated host responses play an important role in tumor immunosurveillance in the skin.

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

IntroductionChemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells.MethodsWe performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations.ResultsHigh expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy.ConclusionsIn this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.