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How to evaluate the immune response and identify immunedeficit… Dr M.L. Romiti Diagnosis of Immuodeficit •Clinical Diagnosis •Immunological Diagnosis •Molecular Diagnosis Immunologic test: first level •Cell number •Cell type CD4 CD3 T CD8 CD3 T CD14 Mo CD19 CD20 B CD56 CD16 Nk Flow cytometry Dimension Structure and complessity Specific surface molecules 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 Side Scatter (linear) An Exemple on Peripheral Blood Cells Granulocytes Monocytes Lymphocytes Forward Scatter (linear) Immunologic test: second level • Proliferation • Cytokine production • Effector functions Rpm 30 min 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) . SI = 3/7 days Tim 3H cpm stimulated PBMC cpm unstimulated PBMC cpm APC OKT3 PHA, PWM ConA MHC CD40 B27 Anti-CD28 IL-2 CD 4 - 8 CD40L AG CD28 a b TCR Q uick Tim e™ e un decom pr essor e TI FF ( LZW) sono necessar i per visu alizzar e qu est 'im m a gine. sd Qoeuno co ic kn mTi epcm reeess s™ sao ri re e puTe nIF r vis F (L u aZlizza W ) r e q u e s ts 'im d Qoeuno m co icakn m gTi ep ic ne m reee.ss s™ sao ri re e puTe n IF r vis F (L u aZlizza W) IL-2R sd Qoe uic no c o m ne pi c m s re d Q e o suic s no d Q ™ cse o oa o uic no cm ne ri e re Tp k ire p m un ne cm T T re ee p IF ie rcm s re ™ vi e F sres a o s ™ (L sri e re ua o Z p un ri e re liT W e IF p un rW T )evi FIF r)re s(L vi Fua q sZ (L ue ua liq W zza Z li)W t'st'i re im ) re q ue aqga ue sine t' im sint'.eim m. amg sdQ oeu no ck ic oTkm n T e p im cere e se ™ sk a o ri e pu T e n IF vi F sa (L ua Zzza lizza re us ezza m m g T CELL sd Qoeu sno d Q co ic oeuno kco n m ic Ti ep kn m cm rTi eee pe css m sr™ ee sea ss s o™ ri sre eaopu rire e Te nIF pu rTe n vis FIF r vis (L F u aZ (L ulizza W aZlizza )Wr)e rqeuqe u se t'im s Lck Fyn ZAP 70 An example Normal ranges Tetanus Candida PWM OKT3 PHA K 0 20000 * 40000 60000 2.Cytokine measurement •ELISA •ELISPOT •INTRACELLULAR STAINING (al FACS) 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 E E E E ELISA TEST plate 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) CELL CYTOKINE SPOT 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 3.Effector function Cytotoxicity: 51Cr release Cytotoxic cells co-cultured with potentially target cells infected 51Cr Target cells marked with 51Cr CD8 CTL Killing 51Cr 51Cr Un 51Cr infected 51Cr 51Cr 51Cr 51Cr 51Cr 51Cr is retained by intact cells virus 51Cr infected 51Cr 51Cr 51Cr 51Cr 51Cr is realised from lysed cell cpm The radioactivity is measured in the supernatant of cell cultured Ag - + infection 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. TCR gene rearrangements…. 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. Base pair Amminoacidi 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 Thank you for your attention