immune function assays

8/2/2019 Immune Function Assays

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Assays of Immune Function


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Some Definitions

BrdU: bromodeoxyuridine (incorporated into DNAduring cell division)

CBA: cytometric bead array

DC: dendritic cell(s)

ELISA: enzyme-linked immunosorbent assay

ICS: intracellular cytokine staining

LPA: lymphoproliferative assay (using3

H-thymidine incorporation)

MHC: major histocompatibility complex


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Humoral versus Cellular Immunity

Humoral Immunity Antibody production: ELISA, CBA

Cellular Immunity T cell specificity: MHC multimer staining

Cytokines: ELISA, CBA, ICS, ELISPOT

Degranulation: CD107 staining

Cytotoxicity: 51Cr release

Proliferation: BrdU incorporation, LPA


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Early & Late Functions of CellularImmunity

IL-4IL-2

TNFaIFNg

APC-T cell

interactions

Cytokine

expression

Proliferation/

Death

Cyto-

toxicity


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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


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ELISA Assays

Require a matched pair of capture anddetector antibodies for the analyte ofinterest

Wide variety of antibody pairs available formany different analytes

Standards available for assay calibration


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CBA/Luminex Assays

Use multiplexed beads (varying in FL3/FL4intensity) labeled with capture antibodies forspecific analytes

Sample (e.g., serum or cell culture supernatant) isadded together with PE-labeled detector antibody

Software calculates the level of each analytebased on PE fluorescence of each bead population

relative to a standard curve


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Examples of CBA Assays

Day 4Baseline

IL-1b

IL-8

GM-CSF

IL-6


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ELISA versus CBA Assays

ELISA CBA

Types of analytes antibodies,cytokines

Antibodies,cytokines

Number of simultaneousanalytes

One Up to sevenor more

Type of readout Colorimetric Flowcytometry


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Best use of ELISA or CBA

ELISA: defined system where only one ora 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 analytesare of potential interest and the sample islimited

Example: measuring the effect of allergens oncytokines in human tears


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MHC Multimer Assays

Measure binding of T cells to a specificpeptide+MHC combination

Can be used to identify rare populations ofantigen-specific T cells without in vitroactivation


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MHC-peptide Dimers and Tetramers


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Example of Dimer vs. Tetramer Staining

CD8 FITC

HLA-A2:Ig

CMVtetram

er

CD8 FITC

unloaded dimer loaded dimer tetramer


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Advantages of Dimers vs. Tetramers

Dimers:

Investigator can load peptide of interest

Can be used to coat plates for antigen-specific

cell capture/stimulation Tetramers:

More MHC alleles commercially available

Higher affinity binding in some systems

Directly fluorochrome labeled


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ELISPOT Assays

PBMC are plated on a filter-bottom 96-well platecoated 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.


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ELISPOT Assay Principle

Coat plate with anti-cytokine Ab

wash

Add PBMC

24 h

1 h

15 min

Prepare PBMC and count Wash out cells, add detector Ab

Wash, add substrate

Count on dissectingmicroscope

orAnalyze on

automated reader

Add Ag


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ICS Assays

Measure production of cytokines in short-termstimulated whole blood, PBMC, etc.

Can measure multiple cell-surface andintracellular markers in combination, usingmultiparameter flow cytometry

Can detect rare events such as antigen-specificT cells


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Principle of Plate-Based ICS Assays

Antigenic stimulus+ brefeldin A

Incubate 6-24 h

Fix cells Permeabilize Stain

20 ml PBMC/WB sample

Gate on cellsof interest


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Example of ICS Results

pp65 protein peptide mix A2 peptide CMV lysate

CD8

CD4

anti-IFNg FITC

CD69PE


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Correlation of ICS and ELISPOT Assays

CMV Lysate

0 100 200 300 400 5000

500

1000

1500

2000

ELISPOT

r2 = 0.4p < 0.1

CMV pp65 peptide mix

0 100 200 300 4000

300

600

900

1200

r2 = 0.3p < 0.05

ELISPOT

ICS

ICS


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Comparison of Ag-Specific T CellMethods


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CD107 Assays

CD107a and CD107b are proteins found incytotoxic granules of CTL and other cells

Upon degranulation, CD107a and CD107b are

transiently transported to the cell surface

Using labeled antibodies to CD107a and CD107bduring short-term stimulation, the exocytosis ofCD107 is captured on degranulating cells.


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Example of CD107 Assay

Anti-IFNg FITC

CD107a+b

APC

36%


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BrdU Assays

Can measure cell proliferation based onincorporation of fluorescently labeled BrdU

Can be combined with cell-surface andintracellular markers (e.g., cytokines) formultiparameter staining


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Principle of BrdU Assay (with IFNg)


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Example of Ag-Specific BrdU Assay

p55 gagUnstimulated

Anti-BrdU FITC/DNase

HIV-REMUNE


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CFSE Assays

Cells (usually PBMC) are labeled withCFSE dye, then allowed to proliferate invitro (or in vivo in mice)

CFSE is divided equally among daughtercells, so each generation becomes half asintense in CFSE staining


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Example of CFSE Assay

Day 0 Day 4 - IL-4+ cells

No CD81

+ CD81


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Comparison of CFSE and BrdUAssays

unstimulated SEB

CFSE

BrdU


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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

immune function assays

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