Immune Monitoring and Flow Cytometry

Director

Jacqueline Y. Channon Smith, PhD 

Research Assistant Professor of Microbiology & Immunology

Dartmouth Medical School

Staff

John DeLong, BS

Xiaosong Wang, MD, PhD, Research and Development

Contact Information

Rubin Building - DHMC, Room 710

E-Mail: Jacqueline.Y.Smith@Dartmouth.edu

Website: http://dms.dartmouth.edu/iml/

Lab Phone: 603-653-9913
CCOPs
Shared Resource Service Requests: https://freedom7.dartmouth.edu/login

Mission

The Immune Monitoring Laboratory provides

  • State-of-the-art immunoassays to allow NCCC clinicians to monitor the immune responses of patients enrolled in clinical trials;
  • Expert guidance in the choice and interpretation of immunoassays most relevant to a particular clinical trial;
  • Quality control by rigorous analysis of a reference population to define baseline responses as well as validation of the reproducibility of the result; and
  • A resource for all NCCC Investigators and Dartmouth community to use various dedicated instruments not normally available in individual laboratories, e.g. ELISPOT reader, Robosep, autoMACS, Luminex.

Overview of Services Provided

Shared Instruments

  • Guava
  • Robosep

Immunoassays

  • ELISPOT assays
  • CFSE-labeled cell proliferation assays
  • Cytokine assays including Luminex multiplex assays
  • Leukocyte subset analysis
    • intracellular staining
    • pentamer staining
  • T regulatory cell assays:
    • inhibition assay
    • FoxP3+ phenotype
    • Treg conversion assay
  • Whole blood cytokine assay
  • COPD assay

Blood Isolation Services

  • PBMC isolation and cryopreservation
  • Serum isolation and cryopreservation
  • Plasma isolation and cryopreservation
  • Cell subset enrichment (using magnetic beads)

Forms

To book time on Shared Instruments or to request an assay or other service, please login to https://gemini.dartmouth.edu/login.

E-mail Tom.Caldwell@Dartmouth.Edu to set up a user name and password.

Luminex Assay Request Form

Services Provided by the Immune Monitoring Laboratory

Sample Preparation, Enrichment, and Storage

  • Separation, aliquoting and labeling of cells, sera and plasma for immunological assays or storage.
  • Enrichment of specific cell populations for functional analysis, e.g. CD8+ cells for ELISPOT assays. Cell subtypes can be isolated from any source, e.g. blood, spleen, lymph node, solid tumor, ascites. Cells are labeled with antibody conjugated to magnetic nanobeads and separated in a magnetic field. Purity and yield are determined by flow cytometry. The isolated cells are not activated by this procedure and can be used in functional assays.
  • Long-term storage of peripheral blood mononuclear cells and plasma/serum in liquid nitrogen freezers for longitudinal studies.

Assays

  • The Enzyme-linked immunospot (ELISPOT) assay determines the precursor frequency of antigen-specific T cells that secrete a given cytokine, e.g. IFNgamma.
  • The Dye Dilution Proliferation Assay determines the precursor frequency of CFSE-labeled proliferating cells. For example, antigen-specific T cells, antigen-specific B cells, or tumor cells. Since this is a flow cytometry-based assay, up to six different measurements can be determined simultaneously. For example, proliferation of cell subsets can be analyzed, so CD4+ and CD8+ proliferating T cells can be measured. Furthermore, intracellular cytokines can also be measured in these same cells, or cells can be stained with specific pentamers.
  • Measurement of cytokines and chemokines in serum, plasma, or in supernatants from peripheral blood mononuclear cells (PBMC's) stimulated ex vivo with specific antigen. Single cytokines can be measured by ELISA, or multiple cytokines can be analyzed in a single well using the Luminex array reader. Cytokine production can also be determined using flow cytometry by intracellular staining of PBMC's stimulated ex vivo with specific antigen.

LUMINEX vs ELISA

  • No endogenous standard exists for cytokines. Some cytokine suppliers state how their sample concentration compares relative to NIBSC standards and other suppliers do not. However, the NIBSC standards do not qualify as true reference standards because many are recombinant proteins that may differ from natively expressed proteins found in a biological sample. (Click here to see article in BioRadiations on this topic.) As a result standards from different suppliers are not equal. Unless the same standard is used for Luminex and ELISA, a direct comparison of concentration in pg/ml is not possible. For this reason, the actual protein value of each sample you obtain from the standard curve may have no significance other than its relative value to other samples within the same assay run on the same day. This ratio should be similar for Luminex and ELISA, but will also depend on the sensitivity of the antibody pairs used for each assay method.
  • Cells can be phenotyped using 6-color antibody cocktails (e.g. CD4+CD25hiFoxP3+ regulatory T cells, activated CD4+ T cells, B cell subsets, activated neutrophils or monocytes).
  • Cell signaling, in particular measuring phosphorylation levels of kinases and other cell signaling molecules, can be carried out using the Luminex array reader.
  • The functional regulatory T cell assay measures inhibition of CFSE-labeled T effector cell proliferation following anti-CD3 stimulation and uses fresh or cryopreserved cells.

Equipment

  • Zeiss ELISPOT reader
  • Miltenyi autoMACS
  • Luminex array reader
  • Guava CPA
  • Robosep

Facility

The Immune Monitoring Laboratory is located on the seventh floor of the Rubin Building, Norris Cotton Cancer Center, Lebanon NH. It is open 8 am to 5 pm Monday through Friday and is also available by e-mail and telephone. The Core is managed by the Norris Cotton Cancer Center as a shared resource on behalf of the university and is available to all investigators within the greater Dartmouth research community. This Shared Resource is supported by funds from the Norris Cotton Cancer Center Core Grant, the Dartmouth Immunology COBRE Program Project, and funds from Dartmouth. It is overseen by the Experimental and Translational Models Group, an oversight and advisory committee of the Cancer Center.

Charges

Most services of this shared resource are on a fee-for-service basis. Charges for services will be made directly to investigator accounts.  

Publications

Fahey et al. (2005) Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract. Human Reproduction 20:1439-1446.

Zhang et al. (2005) Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood:106:1544-1551.

Zhang et al. (2006) Generation of Antitumor Responses by Genetic Modification of Primary Human T Cells with a Chimeric NKG2D Receptor. Cancer Res. 66:5927-5933.

Barber et al. (2007) Chimeric NKG2D Receptor-Bearing T Cells as Immunotherapy for Ovarian Cancer. Cancer Res. 67:5003-5008.

Bak et al. (2007) Calreticulin requires an ancillary adjuvant for the induction of efficient cytotoxic T cell responses. Mol. Immunol. 45:1414-1423.

Huarte, et al. (2008) Pilar is a novel modulator of human T cell expansion. Blood (in review).

Huarte et al. (2008) Depletion of dendritic cells delays ovarian cancer progression by boosting anti-tumor immunity. Cancer Cell (in review).

Presentations

Channon et al. (2008) Conversion of naïve effector CD4+ cells to regulatory T cells ex vivo: a new immunotherapy for treatment of relapsing multiple sclerosis? American Association of Neuroloy 60th Annual Meeting, April 2008.

Abstracts

Wolf et al. (2008) Serum cytokine profile in metastatic renal cell carcinoma (RCC) patients undergoing immunotherapy. American Society of Clinical Oncology (ASCO) 2008 Annual Meeting.