The establishment of a clinical immune-monitoring platform at the CHUV has as its principal goals to identify the immunological profile associated with different infectious agents, cancers, auto-immune related and hematologic diseases. Profiling will consist of phenotypic, functional and genetic characterization of immune cells and tissues. This characterization will allow for associations to be made between the immunological profile of a patient and the emergence of their disease. Immune and/or cancerous cell profiling can also permit the predicted evolution of a disease and can be compared in clinical cases of immunological control versus disease recurrence. The immune-monitoring platform will perform valuable studies that can identify the specific cells targeted and mechanism of action associated with a specific therapy. With the orthogonal screening approaches in place and under development, a prime objective is also to identify immune related biological markers that are associated with a response to therapy. These goals will contribute to research discoveries, improved therapy and the innovative advancement of patient care.
The clinical immune-monitoring platform will be established for the characterization of cells and tissues from patients with: infectious diseases, cancers, systemic inflammatory diseases, organ transplantation or an immunocompromised condition. This platform will also be applied to evaluate the immune response to seasonal flu vaccines in at risk populations such as organ transplant patients, immunocompromised individuals and the elderly population. In addition, immune-monitoring in phase I/II clinical trials will be performed for experimental vaccines directed against infectious diseases such as malaria, tuberculosis and HIV. With these objectives and range of applications, the clinical immune-monitoring platform will extend its services and expertise to the CHUV departments of laboratory, medicine, oncology, neurosciences and surgery. This platform is equally pertinent for the Biobanque Institutionnelle Lausannoise (BIL) project to support the discovery and development of new therapies and to position Lausanne as a focal point of medicinal genomics.
The clinical immune-monitoring platform will achieve its goals through the implementation of established and emerging analysis technologies. These technologies include the use of flow cytometry for phenotypic and functional profiling of live cells, gene expression analysis at the single cell levels using the Fluidigm system and mass cytometry for an in depth characterization of cellular phenotype and function using the CyTOF instrument.
The flow cytometry analysis facility at the Division of Immunology and Allergy (IAL, Departments of Laboratories and Medicine) conforms to ISO/CEI 17025 standards and is accredited for diagnostic purposes. A large flow cytometry facility is also available at the Department of Oncology. These services are widely used for patients in need of immune-monitoring for conditions such as viral infection, immunodeficiency, cancer, HLA typing and transplantation. Through the use of validated panels of fluorochrome conjugated antibodies, multi-parametric analysis up to eighteen different biological targets can be quantitatively analyzed to evaluate phenotypic and functional aspects of the immune response at the single cell level. Flow cytometry has the additional advantage of being able to sort live cells which allows for culturing and further characterization of discrete immune related populations. Numerous reagents are available and immune profiling is established for many different diseases.
Analysis using the Fluidigm system combines the multi-parameter separation and cell sorting capabilities of the flow cytometer with a microfluidic chip technology for multiplexed analysis of messenger RNA levels. In a standard experiment, a sample is stained with fluorochrome- conjugated monoclonal antibodies and specific phenotypic cell populations are sorted into individual wells of a 96-well PCR plate based on the protein expression of cell surface markers. In a 96×96 TaqMan PCR reaction, 96 samples can be analyzed for mRNA expression levels of 96 different genes that have been preselected based on their involvement in immune related functions. Real-time PCR curves are analyzed for each single cell sorted into a well to generate a gene expression profile. Results are displayed as heat maps based on the level of gene expression and statistical clustering algorithms are used to define the association of individual cells into distinct functional subsets. This technology is currently being used in an IAL research project to characterize cell populations related to T follicular helper cells that are a major viral reservoir in HIV infected patients.
Mass cytometry is an emerging technology that is being newly implemented within the IAL in 2013. This technique employs some of the same principles as flow cytometry analysis. However, rather than fluorochrome labeled antibodies to characterize cells, antibodies are conjugated with elemental isotopes such as the lanthanide series of rare earth metals. Cells stained with antibodies are then introduced into an inductively coupled plasma mass spectrometer (ICP-MS) for elemental analysis. Significant advantages of the mass cytometry technique are the large number of elements that can be used to label different antibodies in a given panel and the absence of overlap in mass between the elemental isotopes used. This allows for >37 parameters to be followed simultaneously without the need for compensations between different antibody mass labels. By comparison, careful compensation for the overlap in fluorescence signals from different fluorochrome labeled antibodies is necessary for standard flow cytometry and limits the number of parameters that can be followed. The highly multi-parametric capability of mass cytometry facilitates the exploration of complex pathways in individual cell populations. In addition to these extensive analytical capabilities, the mass cytometry community is a highly collaborative group of scientists that have developed sophisticated software tools for data analysis. These tools assist in the discovery of cellular relationships, the characterization of responses to therapy and the elucidation of developmental pathways. In addition to deep profiling of the cell systems of interest, samples can be multiplexed in order to substantially increase the cell analysis throughput.
Several seminal studies have been published in recent years using mass cytometry that highlight the capabilities of this emerging technology. One such study performed by Bendall, Simonds et al. was able to map the differential immune and drug responses across the human hematopoietic system. Human bone marrow cells were incubated under one of 22 different conditions and analyzed for 18 functional markers relating to signaling pathways and 13 core surface markers relevant to the immune system, all in a small number of samples. This study provides a comprehensive functional profile of the entire human hematopoietic system and demonstrated that drug effects can be both treatment and cell-type specific. These experiments show that it is possible to predict the efficacy of pharmacological intervention in a disease model where the inflammatory mechanism is known.
The investigative power of the mass cytometry technique was further demonstrated in experiments performed by members of the Neel and Nolan labs. This study analyzed primary tumors samples from 40 patients with serous ovarian cancer. After high throughput FACS analysis to investigate the heterogeneous tumor cell populations for 356 different cell surface markers, subpopulation screening was performed by mass cytometry. Part of this study focused on identifying the mechanism by which ovarian tumors developed drug resistance. In ovarian cancer, 70-90% of patients who initial respond to standard of care treatment develop a drug resistance tumor leading to death of the patient. Two competing models have been put forward to explain the high rate of tumors that emerge with drug resistance. These are the stochastic model where tumor cells can self-renew indefinitely to acquire drug resistant mutations and the cancer stem cell model where a subpopulation of cells is responsible for maintaining tumor growth. Their mass cytometry studies support the cancer stem cell hypothesis were this cell subpopulation has different sensitivities to chemotherapeutic drugs compared to the bulk tumor population. Mass cytometry allows for the deep profiling of these populations to characterize their attributes, functions and response to varying drug treatments. As a result, these studies can identify a therapy which specifically targets the cancer stem cell subpopulation and thus may decrease the rate of morbidity and mortality from these gynecological malignancies.
Analysis of these multidimensional data sets would not be feasible without the development of analysis algorithm such as SPADE (Spanning tree Progression of Density normalized Events). SPADE has been used to extract cellular hierarchies and provide a two dimensional map of all the immune cell lineages based on surface expression markers. Software packages such as this are available and supported through Cytobank which is a critical component to make full use of the multi-parameter data sets generated using mass cytometry.
The clinical immune-monitoring platform at the IAL, consisting of flow cytometry, Fluidigm and mass cytometry analysis, will provides both broad and in depth analysis of patient’s samples. These technologies have different strengths that can combine to provide critical profiling support for the diagnostic laboratories, vaccine trials, and transplantation center activities in addition to fundamental and translational laboratory research. Flow cytometry attributes include the phenotypic and functional analysis of live cells with up to 18 parameter analysis and diagnostic capabilities that are widely applicable to different diseases. The Fluidigm system provides a transcriptional profile of up to 96 genes at the single cell levels that can be used to study a variety of diseases and the transcriptional response of different cell populations to treatment therapy. The mass cytometer is a new innovative technology that will be applied to immune-monitoring and excels at multi-parameter analysis of immunological markers and signaling pathways. The multiplexing capabilities allow for profiling the immunological response to different drug therapies and treatment prognosis for a variety of diseases. Collectively, these analysis methods within the immune-monitoring platform will provide extensive profiling capabilities for immunologic, autoimmune, hematologic diseases and cancer.
The Division of Immunology and Allergy (Departments DL and DM) and the Department of Oncology plan to work together to develop a common immune-monitoring platform to support the advancement of patient care towards improved therapy and quality of life.