How to evaluate the immune response and identify immunedeficit…

1. How to evaluate the immune response and identify immunedeficit… Dr M.L. Romiti

2. Clinical Diagnosis Diagnosis of Immuodeficit • Immunological Diagnosis • Molecular Diagnosis

3. Immunologic test: first level • Cell number • Cell type CD8 CD3 CD14 CD20 CD3 CD4 CD19 CD56 CD16 Mo B Nk T T

4. Flow cytometry Dimension Structure and complessity Specific surface molecules

5. Immune phenotype by Flow Cytometry The cells are aligned and crossed by a laser beam. The deviation of the beam is converted into electrical signals by photomultipliers. Two main signals are analysed: • Scattering : • - Side scatter (refraction, reflection): cell structure and complexity • - Forward scatter (refraction): dimensions • Fluorescence: phenomenon in which, when molecules called fluorophores are hit by a certain wavelenght emit a longer wavelenght in the visible spectrum

6. Side Scatter (linear) Forward Scatter (linear) An Exemple on Peripheral Blood Cells Granulocytes Monocytes Lymphocytes

7. Immunologic test: second level • Proliferation • Cytokine production • Effector functions

8. Rpm 30 min

10. 1.PROLIFERATION TEST PBMC are co-cultured with mitogens /antigens. Quantification by b-scintillator of Timidine 3H incorporated into cells DNA after 3/7 days is measured in (cpm) and Stimulation Index (SI) . 3 / 7 days Tim 3H SI = cpm stimulated PBMC cpm unstimulated PBMC cpm

11. APC PHA, PWM ConA Anti-CD28 MHC B27 OKT3 CD40 IL-2 CD 4 - 8 CD40L AG CD28 a b TCR IL-2R T CELL Fyn Lck ZAP 70

12. An example *

13. 2.Cytokine measurement • ELISA • ELISPOT • INTRACELLULAR STAINING (al FACS)

14. E E E E ELISATEST (Enzyme Linked Immunosorbent Assay) PBMC co-cultured in vitro with a suitable stimulus, secrete cytokines. Each cytokine can be capture by a specific antibody linked to an enzyme that reacts with a specific substrate and generates a colored product detectable as assorbance

15. ELISA TEST plate

16. CELL CYTOKINE SPOT ELISPOT It is based on, and was developed from a modified version of the ELISA TEST allowing the quantitation of the secreted Cytokines (IFNg, IL-2, IL-10, IL-4…) by individual cells(SPOT)

17. - Ag (cytokine) cells + Ag (cytokine) fluorescence INTRACELLULAR STAINING Stimulated Lymphocytes treated with Brefeldin or Monensin produce but do not secrete cytokines The cells are then permeabilized with detergents and cytokines are detected using fluorescent mAbs FACS analysis

18. CD8 CTL W W 51Cr 51Cr 51Cr Un infected virus infected 51Cr 51Cr 51Cr 51Cr Ag 51Cr 51Cr 51Cr 51Cr 51Cr W 51Cr 51Cr W cpm The radioactivity is measured in the supernatant of cell cultured - + infection 3.Effector function Cytotoxicity: 51Cr release Target cells marked with 51Cr Cytotoxic cells co-cultured with potentially infectedtarget cells Killing 51Cr is retained by intact cells 51Cr is realised from lysed cell

19. TCR SPECTRATYPING ANALYSIS • Distinct T cells have different TCell Receptor (TCR) to recognize a huge amount of antigens. • T repertoire represents the specificity of T cells to different antigens. CDR3

20. TCR gene rearrangements….

21. Base pair Amminoacidi T-Cell Receptor ß CDR3 Spectratyping Spectratyping is a molecular analysis using RNA amplification allows to quantitatively evaluate the clonal diversity of T cells and their potential of antigen recognition. Each spectrum obteined represents a gaussian distribution of frequencies.

22. Four main patterns of peaks distribution A.B.SKEWED (SK): less than 4 peaks or a strongly altered distribution with wide deletions or single expantions with more than 50% of the total area under the curve C. POLYCLONAL ALTERED (pa): five or more peaks having am altered distribution D. POLYCLONAL (p): 5 or more peaks with a Gaussian-like distribution

23. Thank you for your attention