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

2. Diagnosis of Immuodeficit Immunological Diagnosis Molecular Diagnosis Clinical Diagnosis

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

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

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

8. Rpm 30 min

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

11.  TCR Lck CD 4 - 8 CD40L Fyn ZAP 70 CD40 CD28 B27 AG Anti-CD28 APC T CELL PHA, PWM ConA OKT3 IL-2R IL-2  C

12. An example *

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

14. ELISA TEST (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 EEEE

15. ELISA TEST plate

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

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

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

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.

20. TCR gene rearrangements….

21. T-Cell Receptor ß CDR3 Spectratyping Base pair Amminoacidi 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