Flow Cytometry Based Immune Assessment

Immunophenotyping: We offer standardized multi-parameter cell surface immunophenotyping (and intracellular cytokine assays – see below) for human leukocytes. We have developed and validated a standard set of surface markers for the quantification of the major immune cell populations and their functional subsets, including B, T, NK, DC, and monocyte subsets. Several CLIA-certified panels are available to aid in the diagnosis of primary immunodeficiency (such as common variable immunodeficiency, CVID), follow memory T cell reconstitution upon bone marrow transplantation, and identify senescent CD8+/CD28- T cells in the elderly. Additionally, a key service the core provides is the development of new panels based on the research interests of our clients.

 

We have developed a standard set of surface markers for immunophenotyping of B, T, NK, DC, and monocyte subsets in PBMC (see Table below).

Cell
Population
Phenotype
T Cells CD3+CD4+ or CD3+CD8+
  Naive CCR7+CD45RA+
  Effector Memory CCR7-CD45RA-
  Central Memory CCR7+CD45RA-
  TEMRA CCR7-CD45RA+
  Exhausted KLRG1+/CD57+/CD28-
  Activated HLA-DR+
  Helper  
    Type 1 (Th1) CD4+CXCR3+CCR4-CCR6-
    Type 17 (Th17) CD4+CXCR3+CCR4+CCR6+
    Type 2 (Th2) CD4+CXCR3+CCR4+CCR6-
    Follicular (Tfh) CD4+CXCR5+PD-1+
  Regulatory CD4+CD25++CD127lo
    Treg cells CD4+CD25++CD127lo
    Treg cells (alternative) CD4+CD25+FOXP3+
    Tr1 cells CD4+CD45RA-CD49b+LAG-3+
Antigen Presenting Cells  
  Classical Monocytes CD56-CD14++CD16-
  Non-classical Monocytes CD56-CD14+CD16++
  Intermediate Monocytes CD56-CD14+CD16+
  Myeloid CD1c+ Dendritic Cells CD19-CD1c+
  Myeloid CD141 Dendritic Cells CD19-CD141+
  Plasmacytoid Dendritic Cells CD19-CD123+
  Activated CD86+/HLA-DR+
B cells CD19+
  Immature CD19+CD21-
  Transitional CD19+CD24++CD38++
  Naive CD19+CD27-
  Memory CD19+CD27+
    Unswitched CD19+CD27+IgD+IgM+
    Switched lgM+ CD19+CD27+IgD+IgM+
    Switched CD19+CD27+IgD-IgM-
    Unconventional CD27- Switched CD19+CD27-IgD-
  Plasmablasts CD19+CD27+CD38++IgM-
  Activated CD70+/CD86+
  TACI+/BCMA+
NK Cells CD3-CD56+
  Cytotoxic CD56dimCD16++
  Cytokine-producing CD56++CD16dim

Intracellular cytokine staining (ICS) of antiviral lymphocytes: A consequence of immune cell activation is cytokine production. The frequency of responsive cells and the types of cytokines they produce can be useful biomarkers to monitor immune responses to vaccines or specific antigens, or disease progression. We have developed 10/14-color flow cytometry panels to evaluate cell degranulation (i.e. CD107a) and cytokine production (e.g. IFN-γ, IL-2, IL-10, TNF-α) by CD4+/CD8+ T cells and CD56+ NK cells stimulated with CMV, EBV, BK, or PPD peptides. Other stimuli of your choice can also be used.

 

Direct and indirect allo-recognition assay: In organ transplantation, donor allo-antigen is recognized by the recipient's immune system through direct cross-reactivity of donor HLA molecules with recipient T cell receptors (direct recognition), or through presentation of donor antigen by recipient antigen presenting cells in the context of recipient HLA molecules (indirect recognition). The frequency of T cells capable of responding to donor allo-antigens indicates the breadth of the patient's allo-immunity to the donor organ, allowing for potential prediction of rejection and/or graft loss. By utilizing panels similar to those used in ICS, this assay provides direct measurement of the subject's immune reactivity against allo-antigens presented in the transplanted tissue, and enables risk assessment and mechanistic understanding of graft rejection.

 

Lymphocyte proliferation assay (LPA): This test is useful for assessing T-cell function, particularly in the context of impairment of cellular immunity, immunosuppressive therapies, primary or secondary immunodeficiency, or post-transplant (bone marrow or hematopoietic stem cell) recovery. Antigens used in recall assays measure the ability of T cells bearing specific T-cell receptors (TCR) to respond to such antigens when processed and presented by antigen-presenting cells. The antigens used for assessment of the cellular immune response are selected to represent antigens seen by a majority of the population, either through natural exposure or as a result of vaccination. Almost everyone’s lymphocytes can be stimulated to proliferate nonspecifically by stimulating them in vitro with the mitogens. However, these substances provide strong stimuli that are not antigen specific, and usually do not discriminate as well as antigens in reflecting different levels of immunodeficiency. The proliferative capacity of total CD3+, CD4+, and CD8+ T cell subsets and of CD19+ B cells is evaluated by stimulating total peripheral blood mononuclear cells (PBMCs) with Phytohemagglutinin, Concanavalin A, Poke Weed, C. albicans and Tetanus Toxoid. PBMCs are pre-labeled with a fluorescent dye (CFSE) that is diluted upon cell proliferation. Other stimuli of your choice can also be used (for instance, viral antigens).

 

Natural killer cell cytotoxicity (NKCC) assay: This test allows for the quantitative determination of NK cell cytotoxic activity. This is useful for the assessment of patients with recurrent, severe herpes viral infections, primary or secondary hemophagocytic lymphohistiocytosis (HLH), and suspected or known monogenic defects affecting the NK cell compartment; and for the evaluation of immune reconstitution post-hematopoietic stem cell transplantation and post-immunomodulatory therapy. The cytotoxic activity of NK cells can be evaluated by co-incubating patient PBMCs with a target tumor cell line known to be sensitive to NK cell-mediated killing. The target cell line (K562) is pre-labeled with a fluorescent dye (CFSE) to allow discrimination from the effector cells (NK cells) contained within patients’ PBMCs. After the co-incubation period, killed target cells are identified by Sytox Red, which is specifically retained by dead cells.

 

Granulocyte/monocyte oxidative burst assay: This assay determines the oxidative burst of granulocytes and monocytes in heparinized whole blood upon stimulation with PHA or opsonized E.coli by flow cytometry. Overall, this assay is useful for assessing granulocyte immune competency to aid in assessing a patient's relative risk for infections. Impaired oxidative burst is found in patients with AIDS, elderly people, patients with severe infections, patients undergoing therapies with N-acetylcysteine or in recipients of bone marrow and blood transfusions. Additionally, oxidative burst is impaired or even absent in patients with chronic granulomatous disease (CGD) and as such can be used to identify female CGD carriers.

 

Custom immunophenotyping and functional assays: In addition to the above assays and validated cocktails, the IAC can also aid in the development of panels based on the particular needs and interests of the client, as well as on the fluorochromes compatible with the house cytometers.

 

Assisted flow cytometry acquisition: The IAC also provides an acquisition-only service, in which time may be reserved for the running of pre-prepared and stained with fluorochromes compatible with the house cytometers. For consultation services for the development of custom panels and assays, please contact us for inquiries. Our cytometer configurations can be found here:

 

Cytometer-Compatible Fluorochromes for BD LSRFortessa
APC-Cy7, APC-H7
APC, Alexa Fluoro647
Alexa Fluor700
PerCP, PerCP-Cy5.5, 7AAD
FITC, Alexa Fluor488, GFP
PE-Cy5, Propidium Iodide
PE
PE-Cy7
PE-Texas Red, PE-CF594
AmCyan, Aqua Live/Dead, BD Horizon V500, Brilliant Violet 510
Qdot 655, Brilliant Violet 650
Brilliant Violet 785, Qdot 800
Pacific Blue, BD Horizon V450, Brilliant Violet 421
Brilliant Violet 710, Brilliant Violet 711
Brilliant Violet 570 or Qdot 605, Brilliant Violet 605*

*Brilliant Violet 570 and Brilliant Violet 605 can be used alternatively, not in the same panel.