Assays of Immune Function

Some Definitions

BrdU: bromodeoxyuridine (incorporated into DNA during cell division) CBA: cytometric bead array DC: dendritic cell(s) ELISA: enzyme-linked immunosorbent assay ICS: intracellular cytokine staining LPA: lymphoproliferative assay (using 3Hthymidine incorporation) MHC: major histocompatibility complex

Humoral versus Cellular Immunity

Humoral Immunity

Antibody production: ELISA, CBA

Cellular Immunity

T cell specificity: Cytokines: Degranulation: Cytotoxicity: Proliferation:

MHC multimer staining ELISA, CBA, ICS, ELISPOT CD107 staining 51Cr release BrdU incorporation, LPA

Early & Late Functions of Cellular Immunity

IL-2 IL-4

IFNg APC-T cell interactions

TNFa Cytotoxicity Proliferation/ Death

Cytokine expression

Categories of Cellular Assays

Bulk Assays

Radioactive:

51Cr

release LPA (3H-thymidine incorporation)

Non-Radioactive:

ELISA CBA Single-cell Assays

For Specificity:

MHC-peptide tetramer staining MHC-Ig dimer staining

For Function:

ELISPOT ICS CD107 staining BrdU incorporation CFSE assay

ELISA Assays

Require a matched pair of capture and detector antibodies for the analyte of interest Wide variety of antibody pairs available for many different analytes Standards available for assay calibration

CBA/Luminex Assays

Use multiplexed beads (varying in FL3/FL4 intensity) labeled with capture antibodies for specific analytes Sample (e.g., serum or cell culture supernatant) is added together with PE-labeled detector antibody Software calculates the level of each analyte based on PE fluorescence of each bead population relative to a standard curve

CBA Assay Principle

Y Y Y

IL-2

IL-4

IL-5

DETECTOR ANTIBODY

IL-10

READ ON FLOW CYTOMETER

Y Y

WASH

Y Y Y

Y Y Y Y

Y Y

TNF

Y Y Y

Y Y Y Y Y Y

Y Y Y

IFN

BEADS

LYSATE, SERUM OR SUPERNATANT

Y Y

Y
Y Y Y Y Y Y Y Y

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Y Y Y Y

Y Y

Examples of CBA Assays

Baseline
IL-1b IL-8 GM-CSF IL-6

Day 4

ELISA versus CBA Assays

ELISA Types of analytes Number of simultaneous analytes Type of readout antibodies, cytokines One Colorimetric

CBA Antibodies, cytokines Up to seven or more Flow cytometry

Best use of ELISA or CBA

ELISA: defined system where only one or a few analytes are to be measured

Example: testing the effect of various conditions on IL-12 production from purified DC

CBA: systems in which multiple analytes are of potential interest and the sample is limited

Example: measuring the effect of allergens on cytokines in human tears

MHC Multimer Assays

Measure binding of T cells to a specific peptide+MHC combination Can be used to identify rare populations of antigen-specific T cells without in vitro activation

MHC-peptide Dimers and Tetramers

Example of Dimer vs. Tetramer Staining

unloaded dimer

loaded dimer CMV tetramer

tetramer

HLA-A2:Ig

CD8 FITC

CD8 FITC

Advantages of Dimers vs. Tetramers

Dimers:

Investigator can load peptide of interest Can be used to coat plates for antigen-specific cell capture/stimulation More MHC alleles commercially available Higher affinity binding in some systems Directly fluorochrome labeled

Tetramers:

ELISPOT Assays

PBMC are plated on a filter-bottom 96-well plate coated with anti-cytokine antibody. The plate is cultured 24-48 hours to allow cytokine secretion and capture on the plate. Cells are washed off and detector antibody is added, followed by enzyme substrate. Cytokine-secreting cells are identified as spots of secreted cytokine.

ELISPOT Assay Principle

Prepare PBMC and count wash Add PBMC Wash out cells, add detector Ab 24 h

Coat plate with anti-cytokine Ab

Add Ag 15 min

1h

Count on dissecting microscope or Analyze on automated reader Wash, add substrate

ICS Assays

Measure production of cytokines in short-term stimulated whole blood, PBMC, etc. Can measure multiple cell-surface and intracellular markers in combination, using multiparameter flow cytometry Can detect rare events such as antigen-specific T cells

Principle of Plate-Based ICS Assays

Incubate 6-24 h 20 ml PBMC/ WB sample Antigenic stimulus + brefeldin A Fix cells Permeabilize Stain

Gate on cells of interest

Example of ICS Results

pp65 protein peptide mix A2 peptide CMV lysate

CD4 CD69 PE

CD8

anti-IFNg FITC

Correlation of ICS and ELISPOT Assays

CMV Lysate
2000 1500
r 2 = 0.4 p < 0.1

CMV pp65 peptide mix

1200 900
r 2 = 0.3 p < 0.05

ICS

ICS

1000
500 0

600 300 0

100 200 300 400 500

100

200

300

400

ELISPOT

ELISPOT

Comparison of Ag-Specific T Cell Methods

CD107 Assays

CD107a and CD107b are proteins found in cytotoxic granules of CTL and other cells Upon degranulation, CD107a and CD107b are transiently transported to the cell surface Using labeled antibodies to CD107a and CD107b during short-term stimulation, the exocytosis of CD107 is captured on degranulating cells.

Example of CD107 Assay

CD107a+b APC

36%

Anti-IFNg FITC

BrdU Assays

Can measure cell proliferation based on incorporation of fluorescently labeled BrdU Can be combined with cell-surface and intracellular markers (e.g., cytokines) for multiparameter staining

Principle of BrdU Assay (with IFNg)

Example of Ag-Specific BrdU Assay

Unstimulated

HIV-REMUNE

p55 gag

Anti-BrdU FITC/DNase

CFSE Assays

Cells (usually PBMC) are labeled with CFSE dye, then allowed to proliferate in vitro (or in vivo in mice) CFSE is divided equally among daughter cells, so each generation becomes half as intense in CFSE staining

Example of CFSE Assay

Day 0 Day 4 - IL-4+ cells

No CD81

+ CD81

Comparison of CFSE and BrdU Assays

unstimulated

SEB

BrdU

CFSE

Categories of Cellular Assays

Bulk Assays

Radioactive:

51Cr

release LPA (3H-thymidine incorporation)

Non-Radioactive:

ELISA CBA Single-cell Assays

For Specificity:

MHC-peptide tetramer staining MHC-Ig dimer staining ELISPOT ICS CD107 staining BrdU incorporation CFSE assay

For Function: