1. Measuring the functional activity of T and B lymphocytes Polyclonal activation of T and B cells lectin-induced activation α-IgM, α-CD3 or α-TCR antibody allogeneic T cell activation (examination of the immediate-early activation events) T and B cell response activation markers proliferative response: 3 H-thymidine incorportion CFSE fluorescence decrease cell cycle events Antibody or cytokine production (ELISA, bioassay, CBA) Determinating the number of activated T and B cells after the administration of the antigen ELISPOT, Intracellular cytokine staining MHC tetramers

2. Phases of the humoral immune response (review)

3. Phases of T cell response (review)

4. Lymphocyte function can be investigated by polyclonal T/B-lymphocyte activator materials Immunodeficiencies mainly characterized by different functional immunoassays Lymphocyte activation by specific antigen is hardly detected, because of the low number of the antigen specific cells

5. Polyclonal activation of lymphocytes by LPS, lectins, PMA/ionomycin A B C BCR or TCR-specifc antibodies could activate the lymphocytes also (PMA activates protein kinase C) TLR4 B cell T cell

6. Polyclonal B cell activators In the presence of cytokines yesAnti-Ig yes EBV (transforming effect) yesnoSpA (superantigen, staphylococcus protein A) yesnoPWM (pokeweed mitogen) Human B cells Ig secretionT cell dependencyActivator Polyclonal T cell activators Phytohaemagglutinin (PHA)lectinCanavalia ensiformis Concanavalin A (ConA)lectinPhaseolus vulgaris anti-CD3Monoclonal antibody

7. Pokeweed (PWM) (Phytolacca americana) – formerly used for coloring red wine (toxic: triterpene saponin) Chenopodiales Phytolaccaceae

8. Phytohaemagglutinin (PHA)  Canavalia ensiformis – Jackbean, Sword bean

9. Concanavalin A (ConA)  Phaseolus vulgaris – bean

10. EXAMINATION OF T AND B CELL FUNCTIONS

11. Receptor crosslinking (immediate) phosphorylation steps (seconds-minutes) - Western blot - Bead array I.c Ca 2+ increase - FACS, microscopy Gene activation - RT-PCR Cytokine synthesis Cytokine secretion - i.c cytometry - ELISA, ELISPOT - Bioassay Antigen receptors (TCR, BCR), and different other receptors (eg. cytokine receptors) Cell-cycle/apoptosis - DNA content - IN antigens Cell division- 3 H-thymidine, CFSE, MTT Lymphocyte activation The examination often requires specific Ag-Ab reactions

12. Western blotting Steps: 1.sample preparation (cells, tissues) 2.gel electrophoresis 3.blotting 4.labeling 5.development use: Identification of defined components from protein mixtures by antigen specific antibodies Anode(+) Cathode(-)

13. SDS-PAGE gel  resolved into single protein bands (overlap possible) Presence of a protein is determined by hybridizing the proteins, transferred or applied to a membrane, with the relevant antibody Standard Protein sample SDS-PAGE Membrane Western blot Antibody recognizes epitope in specific protein Western Blot

14. Used to detect specific proteins in a sample Proteins separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS- PAGE), transferred to a membrane Primary (1 st ) antibody (monoclonal or polyclonal) used to detect protein Enzyme linked 2 nd antibody (e.g. horseradish peroxidase-linked) used to detect 1 st antibody

15. Investigation of the presence or absence of Bruton’s tyrosine kinase (BTK) by Western blot X-linked agammaglobulinemia. XLA patients do not generate mature B cells, which manifests as an almost complete lack of antibodies in their bloodstream.

16. Futatani T et al. Blood 1998;91:595-602 Investigation of the presence or absence of Bruton’s tyrosine kinase (BTK) by flow cytometry

17. Fluo-3 AM – excitable by blue light Indo-1 AM – excitable by UV light These indicator dyes bound to apolar groups (e.g. acetoxy-methylester: AM) cross the cell membrane, in the cell, esterases cleve them so the fluorochromes become polar and are trapped in the cell An increase in cytoplasmic Ca 2+ levels can be detected by fluorescent indicator dyes. /Fluo-3 or Indo-1/ Detection of intracellular Ca 2+ concentration

18. Measurement of Ca 2+ signal by flow cytometry Fluorescence proportional with Intracellular Ca 2+ level time basic signal activation of cells e.g. Fluo-3 or Indo-1 You can detect by fluorimeter also

19. Measurement of Ca2+ signal by flow cytometry T cell hybridoma specific for influenza virus hemagglutinin protein-derived peptide - Ca 2+ signal by antigen presentation non-activated T cells T cell activation (APC - T cell) activated T cells

20. Immunohistochemistry Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue Immunofluorescence Fluorescent dye coupled to antibody FITC – fluorescein isothiocyanate (green) PE – phycoerythrin (orange) Immunoenzyme method enzyme-coupled antibody P – peroxidase PA – alkaline phosphatase ( Substrates converted into an insoluble compound)

21. Tissue sample Fixation Section before staining Freezing Sectioning Immunohistochemistry

22. Immunohistochemistry ABC Method Secondary antibody Avidin Cells Slide Primary antibody Biotin Enzim X Tissue sample

23. Classical histochemistry Acute bronchopneumonia (hematoxilin- eosin staining)

24. Immunohistochemistry (CD68 + macrophages and lymphocytes, granuloma)

25. Antinuclear autoantiboies (ANA) from the serum of a SLE patient can be visualized in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence)

26. Immunohistochemistry using fluorescent detection Detection of actin microfilaments

27. A fixed and permeabilized skin fibroblast. Mitochondria were labeled with mouse IgG (anti–OxPhos Complex V) and visualized using goat anti–mouse IgG conjugated with orange-fluorescent Alexa Fluor 555. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (a mushroom toxin). Nucleus was stained with TO-PRO-3 iodide.

28. Peroxisome labeling in fixed and permeabilized pulmonary artery endothelial cell. Peroxisomes were labeled using an antibody directed at peroxisomal membrane protein 70 and detected with Alexa Fluor 488–labeled goat anti–mouse IgG. Mitochondria were stained with MitoTracker Red prior to fixation; Nuclei were stained with blue-fluorescent DAPI.

29. Flow cytometry An immunofluorescent method that mutually complements the fluorescent microscopy Investigation of different cells or particles travelling high velocity in flow Detects fluorescence intensity and scattered light of the labeled cells Can investigate enormous number of cells in short period of time Can investigate enormous number of cells in short period of time

30. Why flow cytometry Most cells in the immune system can be found in free or loosely adherent form. They can be easily suspensed and labeled by fluorescent antigen specific antibodies, and then they can be examined cell by cell. The cells’ light scatter and immunofluorescent properties can be analyzed statistically (eg. percentages of different cell populations) Rare cell populations can be identified and examined (eg. antigen specific lymphocytes) The method provide qualitative and quantitative data – it can detect the presence of different antigens in the cell, and the expression levels of these antigens. Changes in the expression of certain molecules can be followed after different treatment of the specimen. (eg. cell activation, disease progression)

31. Benchtop flow cytometer Sorter - flow cytometer (FACS station)

32. Example Chanel Layout for Laser-based Flow Cytometry PMT 1 PMT 2 PMT 4 The emited fluorescent light can be separeted to components by special mirrors and filters Laser PMT 3 cell suspension in tube photodetectors flow cell forward light scatter detector (PMT=photo-multiplayer tube)

33. Laser Light scatter and fluorescence Forward angle light scatter sensor (FSC, FALS) Side light scatter(SSC) and fluorescence detectors Can be loosely considered as a representation of the particle size represents the granularity of the cells Multocolor staining can be used to identify cell sub-populations * autofluorescence – presence of piridins and flavins.

34. B NK Th Tc Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (eg. monitoring AIDS progression) Labeling: FITC labeled anti-CD4 antibody(α-CD4-FITC) PE labeled anti-CD8 antibody (α-CD8-PE) Lymphocytes in the periferial blood Fluorescent microscopy Immunophenotyping

35. focused laser beam Th high velocity flow stream (in cuvette or stream in air) CD4 FITC CD8 PE detector detecting CD4-FITC labeled (T H ) cell signal processing unit screen a dot representing a CD4+ CD8- cell increasing light intensity microscopy:

36. Tc detector detecting the PE labeled cell (CD8-PE) CD8 PE CD4 FITC signal processing unit increasing light intensity

37. B detector detecting the unlabeled cell (eg.B cell) by autofluorescence CD8 PE CD4 FITC Signal processing unit increasing light intensity microscopy: dim (autofluorescent) cell

38. quadrant statistics CD8 PE CD4 FITC 38% 0% 44% 18%

39. Graphical representations 1. dot-plot contour- plot density- plot

40. Graphical representations 2. Histogramm homogenous cell population is normally distributed (Gaussian) Numeral intensity values: ~ 7 ~ 1300

41. Different cell types - characteristic light scattering forward light scattering (FSC) („size”) side light scattering (SSC) (e.g. granulated) granulocytes monocytes lymphocytes

42. Examination of peripheral blood by haematology automats Measured parameters: peroxydase staining (the presence of myeloperoxydase, x – axis) light scatter (high on large granular cells, y – axis) Only the major cell types can be identified 1 Noise 2 Nucleated Red Blood Cells 3 Platelet Clumps 4 Lymphocytes and Basophils 5 Large Unstained Cells 6 Monocytes 7 Neutrophils 8 Eosinophils

43. Characterisation of immune cells using cell surface markers Cell types, differentiation stages can be identified using a combination of cell surface markers. Used in diagnostics: - ratio of different cell types - altered expression of cell surface markers Examples: - Inflammatory processes – increased neutrophil numbers - HIV progression – decrease of CD4 + T cell count CD4 + : CD8 + = 1.6 Normal CD4 + T cell count = 600 – 1400/  l AIDS = CD4 + T cell count <200/  l - increase of CD5 + B cells – typical for some B cell Leukemias

44. WAS: Wiscott-Aldrich Syndrome XLA: X-linked Agammaglobulinemia Inhibited B cell development: Lack of CD19+ B cells A typical symptom: Lacking or decreased CD43 expression Diagnosis of immunodeficiency by flow cytometry

45. Intracellular cytokine detection by immunofluorescence cytokine specific antibody with fluorescent labelling - the cell membrane should be permeabilized (detergent) - the cells should be fixed previously avoiding the decomposition of the cells (e.g. aldehyde fixation) cytokines - optionally the cells could be labelled by some cell type specific antibody in the beginning (e.g. CD4)

46. One can determine which cell type produced the cytokines!

47. ELISA ELISA plate E nzyme L inked I mmune S orbent A ssay well

48. enzyme linked immune sorbent Antibody conjugated with enzyme Antigen/antibody adsorbed to solid surface

49. Enzyme activity in ELISA is directly proportional to the amount of antigen present Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods

50. Basic setups in ELISA, immunohistochemistry, flow cytometry Direct method Indirect method Antigen Primary antibodies Label Secondary antibodies

51. For antigens present at low concentration in complex biological samples Removal of unbound material Blocking free plastic surface with inert protein Removal of unbound protein Addition of biotinylated antibody specific to a different epitope on target protein Removal of unbound material Addition of avidin- conjugated enzyme Addition of substrate Coating with Ag- specific „capture” antibody Addition of antigen- containing solution Removal of excess enzyme Steps of combined sandwich ELISA

52. May be you have already met such kind diagnostic tools, or you are going to meet them during your career Eg. detection of human chorionic gonadotropin in serum or urine (pregnancy test) The principles of these tools are similar as the ELISA assay you have met before.

53. hCG Rapid One-Step Immunochromatographic Assay strip nitrocellulose membrane (signal detection pad) glass fiber membrane with visually labeled detection antibodies front view side view hCG capture antibody lane control antibody lane (detection antibody capture) absorbtion pad (cellulose) sample application pad urine

54. hCG capture antibody line control antibody line detection antibodies hCG control line (C) test line (T) hCG + hCG negative detection antibody capture antibodies

55. hCG line ( bound hCG ) control line detection antibody capture antibody control line test line hCG positive hCG negative Competitive system

56. ELISA plates - results

57. ELISPOT Enzyme Linked Immuno-Spot The principles are similar to ELISA Capable to determine the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector molecules The sensitivity allows the determination of 1 activated cell among 300,000 others, so it can reveal activated effector cells not only after polyclonal but after antigen specific activation The first steps should be done in aseptic conditions

58. ELISPOT - coating with antigen specific capture antibodies - blocking - administration of the cells - administration of biotin conjugated antigen specific secondary antibody - avidin-enzyme conjugate - administration of the unsoluble chromogenic substrate (AEC 3-amino-9-ethylcarbazol ) (activation, incubation) - washing A spot showing the place of the cytokine producer cell Upper view of a well on an ELISPOT plate with the generated spots The process

59. It can be evaluated by microscopy (slow, manual process) or you can use “ELISPOT plate reader” (fast + standardizable spot number and size determination)

60. Very sensitive cytokine determination can be achieved by cytokine sensitive or cytokine dependent cell lines. The presence or absence of cytokines determines the fate of the indicator cells that could be cell proliferation or cell death. Bioassay Wehi 164 cell line CTLL2 cell line IL-2 is presentno IL-2 TNF is presentno TNF

61. Bioassays could be equivocal, because of the cytokine cross reactivity of indicator cells: eg. the IL-2 dependent CTLL2 cells can proliferate in the presence of high IL4 concentration also. Living cells can be visualized by colorimetric assay (eg. MTT), or their proliferation can be measured by other methods. cytokine concentration IL-2 TNF CTLL2 Wehi 164 MTT assay living cells convert the stain to purple-blue

62. Cytokine array (The process is similar to the procedures of Western-blot after the blotting step) ( - ) IFN  ( + ) IL-2IL-4IL-5 … IL-12… multiple antigen specific antibodies bound to membrane unknown cytokine containing solution „Luminescent antibody” mixture Multiple cytokines can be detected rapidly in the same procedure Disadvantage – defined volume sample needed to cover the surface of the membrane

63. cytokine production of moDC activated by CD40L and CD40L+SLAM combination Réthi és mtsi. 2006

64. method: RT-PCR, QRT-PCR Investigation of gene activation Activation of T cells can be monitored by the detection of the transcribed mRNA of the activated genes. e.g. activation of cytokine genes cells  RNA isolation RNA  (reverse transcriptase)  cDNA cDNA  (PCR)  determination of the length and quantity RT-PCR: agarose gel (densitometry) QRT-PCR: fluorescent method (TaqMan probe (FRET) or dsNA intercalating fluorochrome  SYBR green)

66. PCR

67. Characteristics of the separation: purity recovery, yield, lost viability of the cells Physical isolation of the cells of interest from a heterogeneous population Differences in the physical, biological or immunological properties of the cells are utilized to separate the cells (differences in cell surface receptor expression is often available – there is a possibility to further investigate the separated living cells ) physical – density, size cell biological – adherence, phagocytosis, sensitivity to the medium immunological – antigen differences (surface!) Cell separation

68. Base strategies: positive separation – labeling and separation of the cells of interest eg. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody. The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high. negative separation – get rid of the labeled unwanted cells (depletion) The cells become affected only by the separation environment This is the preferred strategy in the functional examinations. Separation

69. The different density parts of the anticoagulated blood has been separating to three parts in undisturbed tube: bottom: sedimented red blood cells top: cell free plasma the intermediate layer is called „buffy coat” contains the leukocytes, platelets The process can be accelerated by centrifugation

70. (Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg) (from Google pictures)

71. Ficoll-Paque: density based cell separation peripheral blood pipetting cells on ficoll centrifugation separated cells plasma ficoll Red blood cells mononuclear cells (PBMC) neutrophil granulocytes pipettig the „ring” containing the mononuclear cells to a new tube to get rid of ficoll

72. Magnetic immunoseparation (MACS) paramagnetic bead antigen specific antibody

73. MACS

74. Magnetic cell separation (MACS) MAGNET column depleting or selecting unlabeled cells (negative separation) separation of labeled cells (positive separation)

75. Magnetic column