Immuno-Technology Core Hours: 9:00 AM - 5:00 PM Locations: Forchheimer Building, Room 403 Phone: Teresa P. DiLorenzo, PhD 718.430.2014 Email: firstname.lastname@example.org Leadership Teresa P. DiLorenzo, PhD Co-Core Director Location: Forchheimer Building, Room 403 Dirk Homann, MD Co-Core Director Location: Icahn School of Medicine at Mount Sinai Overview The new Immuno-Technology Core (ITC) has been conceived as an education, coordination and service core that offers access to advanced cellular, imaging and immunomodulation technology platforms operated across both Einstein and Mount Sinai. Specifically, the ITC supports experimental design, execution and analysis for investigations that seek to employ a tailored approach to mass cytometry; multiplexed tissue imaging, automated tissue staining and whole slide image acquisition; biophotonic in vivo imaging; and immunomodulatory T cell targeting through synTAC technology. Objectives To meet the complex demands for tailored assay development, performance and interpretation, the ITC will harness the combined technology infrastructure at Einstein and Mount Sinai for the following principal objectives: To raise awareness about select cellular, imaging, and immunomodulation technology platforms; to provide consultation and advice about the potential and limitations of respective technologies; to assist investigators with experimental planning, design and considerations about expected financial investments; and to facilitate interactions with managers, application scientists and data analysis specialists working with each technology platform. To provide support services required for the effective and efficient implementation of tailored experimental protocols including specific reagent selection, testing and validation; protocol adjustment and optimization; and integration with established assay workflows. To facilitate access and provide practical support for the usage of advanced technology platforms: Cellular (Mount Sinai): High Dimensional Mass Cytometry / Cytometry by Time Of Flight (CyTOF) Tissue imaging (Mount Sinai): MICSSS platform (multiplexed immunohistochemical consecutive staining on a single slide) Automated tissue staining (Leica BOND RX Automated IHC Research Stainer) Whole slide image acquisition (Hamamatsu NanoZoomer S60 Digital Slide Scanner) In vivo imaging (Einstein and Mount Sinai): IVIS Spectrum In Vivo Imaging System Immunomodulation (Einstein): synTac platform (artificial immunological synapse for T cell activation; unique technology developed at Einstein) ITC Technology Platforms High Dimensional Mass Cytometry / CyTOF Mass cytometry combines the high-throughput single cell analysis capabilities of flow cytometry with the specificity and resolution of mass spectrometry for a broad and detailed characterization of heterogeneous cell samples according to ~50 discrete parameters at the single-cell level. Sample processing, staining, acquisition and basic QA/QC are performed and administered through the Mount Sinai Human Immune Monitoring Center (HIMC). Due to the fundamentally modular nature of the assay technology, specific experimental strategies need to be precisely tailored according to the nature of biological samples, preferred composition of antibody staining samples, sample processing protocols, data analysis requirements, and potential need for training. Thus, unless noted otherwise, overall cost estimates can be developed only in detailed discussions with interested investigators and typically align with the similarly tailored fee schedules implemented by HIMC. The ITC provides individualized consultation about specific experimental design, execution and analysis that is free of charge. To initiate a consultation, please email ITC Co-Director Dr. Homann (email@example.com). Specific topics covered in the consultation include: Biological samples (single cell preparations): Tissue origin (PBMC, pancreas, other) Tissue availability and quantity Processing requirements Quantity of samples Composition of antibody staining panels: >500 metal-conjugated antibodies available through the HIMC. Multiple novel pancreatic cell-specific metal-conjugated antibodies available through the ITC. Commercial metal-conjugated antibodies. Custom metal conjugations of investigator-provided antibodies (ITC service provided at $450/100 µg antibody) Sample processing protocols: Cell culture (e.g., immune cell stimulation) Bar coding Cell surface staining Intracellular staining Sample acquisition Data analysis: Basic data processing and QA/QC Data storage and sharing Advanced data analysis strategies and platforms Investigator training Overall expense considerations and cost estimates Tissue Imaging The ITC provides individualized access to three tissue staining platforms covering a multiplexed tissue staining approach, automated tissue staining, and whole slide image acquisition. MICSSS technology platform: Multiplex Immunohistochemical Consecutive Staining on a Single Slide, originally developed at Mount Sinai, is a chromogen-based IHC staining assay independent of proprietary reagents or equipment. It is performed with formalin-fixed, paraffin-embedded (FFPE) tissue sections using up to 10 iterative cycles of staining, revelation, scanning and bleaching of labile chromogenic substrate. The method can be implemented with standard staining protocols (thus benefitting from previously determined specificity and sensitivity parameters); works regardless of antibody isotype or species; is impervious to staining order for the majority of antigens evaluated; is not limited by antibody crossreactivity, photobleaching or autofluorescence; relies on brightfield images that present more detail about the microanatomical organization of the tissue section; and permits prolonged slide storage for future use as new markers become available. The fee schedule for MICSSS stains performed by ITC personnel are dependent on the number of slides, the availability or need for adjustment of MICSSS staining protocols, the number of iterative staining cycles, and image acquisition modes and aligns with current HIMC fee schedules. To initiate a free consultation, please email ITC Co-Director Dr. Homann (firstname.lastname@example.org); specific topics covered in the consultation include: Survey of MICSSS-adapted staining protocols (including reagents and antibody targets) Development/adaptation of MICSSS-adapted staining protocols Development/adaptation of MICSSS staining protocols compatible with the autostainer (see item II.B.) Investigator training Overall expense considerations and cost estimates Automated tissue staining: the ITC coordinates access and usage of a fully automated Leica BOND RX Automated IHC Research Stainer that can stain up to 30 slides per run while testing multiple protocols simultaneously (including both IHC and IF staining). The instrument offers speed, flexibility and consistency to process numerous slides in timely manner with high-quality results, and permits a streamlined approach to optimize new and adjusted staining protocols. To initiate a consultation, please email ITC Co-Director Dr. Homann (email@example.com). All consultation and training is free of charge; pricing for autostainer usage is contingent on the nature of staining protocols, number of slides, and usage modality (ITC personnel-assisted or unassisted), and complies with current HIMC fee schedules. Specific ITC-provided services include: Tissue staining protocol and reagent review Development/adaptation of standard staining protocols compatible with the autostainer MICSSS stain development and/or execution by ITC personnel Investigator training Overall expense considerations and cost estimates Whole slide image acquisition: the ITC provides access to a Hamamatsu NanoZoomer S60 Digital Slide Scanner that uses NDP.view2 viewer software (downloadable free of charge at https://www.hamamatsu.com/us/en/product/type/U12388-01/index.html). The instrument is a next-generation, high-throughput digital scanner that delivers rapid and high-quality (20x and 40x) image acquisition of whole slides in both brightfield and/or four parameter fluorescence modes. Pricing is tailored according to magnification and type/number of image acquisition modes and corresponds to the current HIMC fee schedule. Additional services facilitated by the ITC include: In vivo imaging The ITC will coordinate access and usage of two Perkin Elmer IVIS Spectrum biophotonic imaging systems, one located in Einstein’s In Vivo Imaging System Facility and the other in Mount Sinai’s Translational and Molecular Imaging Institute. The IVIS Spectrum enables longitudinal three-dimensional intravital imaging of small animals or tissues expressing or containing bioluminescent or fluorescent probes across the blue to near-infrared wavelength region. The instrument is equipped with a high-sensitivity charge-coupled device camera which is ideal for imaging single or multiple probes or reporters. Please schedule a free consultation with ITC Co-Director Dr. DiLorenzo (firstname.lastname@example.org) regarding instrument usage and animal housing for longitudinal imaging studies. synTac technology platform ES-DRC member and Einstein investigator Dr. Steven Almo recently developed innovative reagents to manipulate disease-relevant T cells under a variety of conditions, including type 1 diabetes. Termed synTacs (for “artificial immunological synapse for T cell activation”), these inventive reagents are soluble, precision biologics in which non-exchangeable single-chain peptide-class I MHC (pMHC) is covalently linked to a modulatory domain (e.g., PD-L1, a ligand for the T cell-inhibitory checkpoint receptor PD-1) in a manner that recapitulates the proximity, orientation, and overall organization experienced at the immunological synapse. This strategy not only allows for the explicit targeting of disease-relevant T cells, but also greatly reduces the devastating side effects associated with the global immune modulation directed by all biologics currently in the clinic. Given the emergent, early-stage, and cutting-edge nature of the synTac technology, ES-DRC investigators utilizing these reagents will initially do so in collaboration with ITC Co-Director Dr. DiLorenzo and/or Dr. Almo as appropriate, in order to maximize the likelihood of success of the research. Please schedule a free consultation with ITC Co-Director Dr. DiLorenzo (email@example.com) regarding the synTac technology.