Human Immune Monitoring Center Services
The HIMC provides consultation regarding optimal project-specific immunologic assessment and innovative approaches to evaluate immune responses in clinical studies.
Our staff of researchers will offer expert advice to assist the investigator in designing and conducting the proper experiment and preparing the appropriate samples for analysis.
We will help define the end points of the immune assay, assay parameters, sample requirements (collection, labeling, storage, handling, and shipping) and processing, reagents and controls, data analysis, data management expectations and estimated costs.
Immune Monitoring Assays:
1. Immune cell profiling with polychromatic flow cytometry
Phenotypic analysis of immune cells are analyzed according to established cell staining panels for effector and regulatory T lymphocyte subsets, B lymphocytes, natural killer (NK) cells, NKT cells, monocytes, neutrophils, dendritic cells (DC), and plasmacytoid dendritic cells. BD LSRFortessa analytical flow cytometer (Becton Dickinson) with high throughput capability (96 well format) to support large clinical study analysis is dedicated to the Immune Monitoring Center
2. Antigen specific T cell responses
- The breadth of T cell repertoire: EpiMax analysis (non HLA-restricted)
Antigen specific T cell activation to specific peptide libraries is determined by analysis of cell proliferation (CFSE dye dilution) and cytokine production using Multiplex Luminex Xmap technology. This technology allows the analysis of TCR repertoire as well as the type of immune responses based on the cytokine/chemokine secretion profiling using multiplex technology
- Frequency and characterization of CD8+ T cells:
Tetramer/pentamer staining for antigen specific CD8+ T cells in combination with intracellular cytokine staining (HLA-restricted). The frequency of antigen reactive T cells is quantified by tetramer staining, whereas the quality of antigen-specific T cells is measured using intracellular cytokine staining using multichromatic flow cytometry.
- CD137 assay (in development)
- Frequency of antigen-specific CD4+ T cells using CD154 assay (in development)
- ELISPOT assay
The quantity of a functional antigen reactive T cells are analyzed by Enzyme Linked ImmunoSPOT assay. PBMCs are stimulated and cultured in a 96 well plate with membrane coated with antibody specific for a protein (i.e., IFN-?). T cell producing antibody targets are measured with as spots.
3. Measure of soluble factors in serum and plasma
Luminex based assay will be used to measure biomarkers, cytokines, chemokines, hormones, nitric oxide and growth factor, cell signalling and phospho-protein detection in pathway specific for immune call activation. A Luminex 200 system capable of multiplexing up to 100 assays simultaneously in a single microtiter plate well.
4. Genomic profiling and Functional Assays of specific Leukocyte subsets (in collaboration with the Genomic Institute) Purification of Leukocyte subsets and RNA isolation
RNA isolation of total PBMC or purified cell subsets for transcriptional studies is provided. Purification of immune cell subsets will be performed either i) using a 5-laser BD™ FACSAria-II cell sorter installed in a Baker Bioprotect® III – Biosafety Cabinet in the Shared Research Facility of Flow Cytometry or, ii) through magnetic cell sorting. Transcriptional profiling will be performed by the genomic core.
Functional Assays for Leukocyte subsets: protocol specifics upon discussion with the PI
5. Technologies for future Development
- Immunofluorecence and Immunohistochemistry analysis of frozen or paraffin embedded tissues
- Mass Cytometry for multiparametric biological analysis (CyTof)
In addition, the following core services are provided for the investigators at MSSM and through collaborative relationships with other performance sites such as Cancer Immunotherapy Trials Network (CITN), Eastern Cooperative Oncology Group (ECOG) and Hoosier Oncology Group (HOG).
- Serum and plasma processing and storage
- PBMC processing and storage
- PBMC cryopreservation
- PAXgene collection and storage
- PAXgene RNA extraction
- Tempus tube collection and storage
- Tempus tube RNA extraction