How to evaluate the immune response and

identify immunedeficit…

Dr M.L. Romiti

Diagnosis of Immuodeficit

•Immunological Diagnosis

•Molecular Diagnosis

•Clinical Diagnosis

Immunologic test: first level

•Cell number

•Cell typeCD3

CD4 CD3CD8 CD1

4 CD19

CD20 CD5

6 CD16

T BT Mo Nk

Flow cytometry

Dimension

Structure and complessitySpecific 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

An Exemple on Peripheral Blood Cells

Forward Scatter (linear)

Sid

e S

catte

r (l

ine

ar)

Granulocytes

Monocytes

Lymphocytes

• Proliferation

• Cytokine production

• Effector functions

Immunologic test: second level

Rpm 30 min

PBMC are co-cultured with mitogens /antigens. Quantification by -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

cpm

SI = cpm stimulated PBMC

cpm unstimulated PBMC

1.PROLIFERATION TEST

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d e c o mp re s s o re T IF F (L Z W)s o n o n e c e s s a ri p e r v is u a liz z a re q u e s t'imma g in e .

Lck

CD 4 - 8

CD40L

Fyn

ZAP 70

CD40 QuickTime™ e undecompressore TIFF (LZW)sono necessari per visualizzare quest'immagine.CD28

B27

AG

Anti-CD28

APC

T CELL

PHA, PWM ConA

OKT3

IL-2R

IL-2C

An exampleNormal ranges

0 20000 40000 60000

K

PHA

OKT3

PWM

Candida

Tetanus

*

•ELISA

•ELISPOT

•INTRACELLULAR STAINING (al FACS)

2.Cytokine measurement

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

ELISPOTIt 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

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

3.Effector function Cytotoxicity: 51Cr release

Killing

51Cr

51Cr

51Cr51Cr

51Cr

Un infecte

d

W

WAg

51Cr

virus infecte

d

51Cr

51Cr

51Cr

51Cr

CD8CTL W

W51Cr is retained by intact cells 51Cr is realised from lysed cell

51Cr

51Cr51Cr

51Cr

Cytotoxic cells co-cultured with

potentially infected target cells

Target cells marked with 51Cr

The radioactivity is measured in the supernatant of cell cultured

cpm

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

Base pairAmminoacidi

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.

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