1. Assays of Immune Function

2. 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 3 H- thymidine incorporation) MHC: major histocompatibility complex

3. 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: 51 Cr release Proliferation:BrdU incorporation, LPA

4. Early & Late Functions of Cellular Immunity IL-4IL-2 TNF  IFN  APC-T cell interactions Cytokine expression Proliferation/ Death Cyto- toxicity

5. Categories of Cellular Assays Bulk Assays Radioactive: 51 Cr release LPA ( 3 H-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

6. 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

7. 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

8. CBA Assay Principle

9. Examples of CBA Assays Bead Intensity Detector Ab Intensity Day 4 Baseline IL-1  IL-8 GM-CSF IL-6 Effect of Rhinovirus inoculation on cytokines in nasal lavage: Spiking of single cytokines to show assay specificity:

10. ELISA versus CBA Assays ELISACBA Types of analytesantibodies, cytokines Antibodies, cytokines Number of simultaneous analytes OneUp to seven or more Type of readoutColorimetricFlow cytometry

11. 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

12. 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

13. MHC-peptide Dimers and Tetramers

14. Example of Dimer vs. Tetramer Staining CD8 FITC HLA-A2:Ig CMV tetramer CD8 FITC unloaded dimerloaded dimer tetramer

15. 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

16. 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.

17. 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 dissecting microscope or Analyze on automated reader Add Ag

18. 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

19. 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 cells of interest

20. Example of ICS Results pp65 proteinpeptide mixA2 peptideCMV lysate CD8 CD4 anti-IFN  FITC CD69 PE

21. Correlation of ICS and ELISPOT Assays CMV Lysate 0100200300400500 0 1000 1500 2000 ELISPOT r 2 = 0.4 p < 0.1 CMV pp65 peptide mix 0100200300400 0 300 600 900 1200 r 2 = 0.3 p < 0.05 ELISPOT ICS

22. Comparison of Ag-Specific T Cell Methods

23. 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.

24. Example of CD107 Assay Anti-IFN  FITC CD107a+b APC 36%

25. 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

26. Principle of BrdU Assay (with IFN  )

27. Example of Ag-Specific BrdU Assay p55 gag Unstimulated CD71PE Anti-BrdU FITC/DNase HIV-REMUNE

28. 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

29. Example of CFSE Assay Day 0Day 4 - IL-4 + cells No CD81 + CD81

30. Comparison of CFSE and BrdU Assays unstimulated SEB CFSE BrdU

31. Categories of Cellular Assays Bulk Assays Radioactive: 51 Cr release LPA ( 3 H-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