Methods to measure functional of the immunocompetent cells

blast transformation (LPS and ConA activation), polyclonal B and T lymphocyte activation,

ELISPOT

9. seminar/practice

Measuring the functional activity of T and B lymphocytes

Polyclonal activation of T and B cells via non-antigen-specific stimulation

lectin-induced activationα-IgM, α-CD3 or α-TCR antibodyallogeneic T cell activation(examination of the immediate-early activation events)

Characterization of responses by activated T and B cells activation markersproliferative response: 3H-thymidine incorporation

CFSE fluorescence decreasecell cycle events

Antibody or cytokine production (ELISA, CBA)

Determination of the number of activated T and B cells after the administration of the antigen

ELISPOT, Intracellular cytokine stainingPentamer (or tetramer) technicsPentamer (or tetramer) technics

Topics:

Phases of the humoral immune response(review)

Phases of T cell response(review)

(review) BCR signaling

TCR signaling(review)

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

Polyclonal activation of lymphocites by LPS, lectins, PMA/ionomycin

BCR- or TCR-specific antibodies may also activate the lymphocytes

(PMA activates protein kinase C)

TLR4

B cell (mouse) T cell T cell

Polyclonal B cell activators

In the presence of cytokinesyesAnti-Ig

yesyesEBV (transforming effect)

yesnoSpA (superantigen, staphylococcus protein A)

yesnoPWM (pokeweed mitogen)

Human B cells

Ig secretionT cell dependencyActivator

Polyclonal T cell activatorsPhytohaemagglutinin (PHA) lectin Canavalia ensiformis

Concanavalin A (ConA) lectin Phaseolus vulgaris

anti-CD3 Monoclonal antibody

Mouse B cells

In the presence of cytokinesnoAnti-Ig

yesnoPPD (purified protein derivate, mycobacterium)

yesyesPWM

yesnoLPS

Pokeweed (PWM)(Phytolacca americana) – formerly used for colouring red wine(toxic: triterpene saponin)ChenopodialesPhytolaccaceae

Phytohaemagglutinin (PHA) Canavalia ensiformis – Jack-bean, Sword bean

Concanavalin A (ConA) Phaseolus vulgaris – bean

Receptor crosslinking(immediate)

phosphorilation steps(seconds-minutes)

- Western blot- Bead array

ic Ca2+ increase - FACS, microscopy

Gene activation - RT-PCR

Cytokine synthesis

Cytokine secretion

- IC cytometry

- ELISA, ELISPOT

Antigen receptors (TCR, BCR), and different other receptors (e.g. cytokine receptors)

Cell-cycle/apoptosis - DNA content- IN antigens

Cell division - 3H-thymidine, CFSE, MTTLymphocyte activationThe examination often requires specific

Ag-Ab reactions

Western blotWestern blot

It can detect the presence or even phosphorIt can detect the presence or even phosphoryylation lation state of specific proteinsstate of specific proteins

The cells’ activation stage can be „frozen” at different times, so The cells’ activation stage can be „frozen” at different times, so the the events events of the activation can be monitored in parallel of the activation can be monitored in parallel samples.samples.

at least 105-106 cells required

Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by by Western blotWestern blot

X-linked agammaglobulinemia. XLA patients do not generate mature B cells, which manifests as an almost complete lack of antibodies in their bloodstream.

Futatani T et al. Blood 1998;91:595-602

Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by flow cytometryby flow cytometry

An inrease in cytoplasmic Ca2+ levels can be detected by

fluorescent indicator dyes./Fluo-3 or Indo-1/

Detection of intracellular (cytoplasmic) Ca2+ concentration

for example – ic Ca2+ signal in a single cell

antigen presentation by B cell to T cell(click)

method: RT-PCR, QRT-PCR

Investigation of gene activationInvestigation 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)

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)

The result:

You can determine which cell type has produced the cytokines!

The sensitivity could reach that of the Western blot.(e.g. with chilled CCD camera mounted microscope – but you need only

one cell for detection)

ELISPOTEnzyme Linked Immuno-Spot

the principles are similar to ELISAcapable to determine the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector moleculesthe sensitivity allows the determination 1 activated cell among 300 000 other, so it can reveal activated effector cells not only after policlonal-, but after antigen specific activationthe first steps should be done in aseptic conditions

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 chromogenic substrate (AEC 3-amino-9-ethylcarbazol)

(activation, incubation)

- washing

A spot showing the place of the

cytokine producing cell

upper view of a well on an ELISPOT plate with the generated spots

The process

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

Stimuli(e.g. antigen)

resting lymphocyte(G0)

changes in the RNA- and protein synthesis, in the cell membrane and in the transports

DNA-synthesis

cell division

effector cell memory cell

Cell-cycle

- transcription (RT-PCR)

- protein synthesis (Immunoassay)

change in the number of the cells (MTT, CFSE)

The size of the cycling cells are increased –

called blast transformation

DNA quantification(fluorescent DNS intercalating agents, 3H-thymidine)

Possibility of the examination

GG22

MMGG00

GG11

ss

0 200 400 600 800 1000

GG00GG11

ss GG22 MM

DNA analysisDNA analysis

DNA content

cell number

2N2N 4N4N

The cell cycle can be examined by fluorescent dye

that intercalates stoechiometrically into the double stranded DNA (e.g. propidium iodide, PI)

Distribution of a normal cycling cell-population by DNA content (flow cytometry)

Methods for determinating the B/T cell proliferation

3H-labeled thymidine incorporation – measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number

thymidine-analog bromodeoxyuridin (BrdU) can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS)

Carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescent stain can be used to tracking the cell divisions:

Tracking the cell divisionsTracking the cell divisions„Cell tracer” dye enter the cell, and trapped there. The apolar CFSE can bind covalently to the cellular proteins. Later the stain can only be diluted by the cell divisions: distributed equally between the two daughter cells – the fluorescence intensity decreases to the half also.

cell divisions:7 6 5 4 3 2 1 0

T cell antigen specificityT cell antigen specificityIdentifying the antigen specific T cells

immunization

The efficiency of an immunization can be evaluated by the increase of the

antigen specific cell number

antigen antigen specific specific

T cellT cell

If you can identify the specificity of the T cell receptors then you can If you can identify the specificity of the T cell receptors then you can monitor the increase of the antigen specific T cells’ numbermonitor the increase of the antigen specific T cells’ number

T cell clones with the same T cell receptor

..but the MHC binds the TCR with low affinity

The interaction The interaction between one MHC between one MHC

molecule and one TCR molecule and one TCR is not strong enough is not strong enough

for labellingfor labelling

Labelled MHC-peptide complex can be used to identify the matching (specific) T cell receptor

MHC

T cell receptors

T cellT cell

Pentamer (or tetramer) technicsPentamer (or tetramer) technics

The pentamerThe pentamer

self assembling coiled-coil-domain

MHCmolecule

peptide

fluorescent label

One part of the pentamer

The multimerized MHC-peptide complex can have enough avidity

peptide specific

T cell

MHC pentamer

Binding of the MHC pentamer to the T-cell

The MHC-peptide oligomer can bind the specific T-cell receptors

with high avidity

T cell receptors

Click here to watch the animation

The number of the antigen-specific T cells can be evaluated by MHC

multimers. So the efficiency of an immunization or a therapy can be

estimated.

CMV specific T cells in healthy HLA-A2 donor

EBV BZLF-1 (RAKFKQLL/ HLA-B*0801) specific T cells

(90-95% of the human population are carrier)

Influenza epitope (GILGFVFTL/HLA-A0201) specific T cells in a

healthy donor

Tetramer (pentamer) tests

The number of microbe specific T cells can be increased in the body because

of the persistent (e.g. herpesviruses) or repeated infections

allele sequence Tumour (associated) epitopeA*0201 GVLVGVALI Carcinogenic Embryonic Antigen (CEA) 694-702

A*0201 LLGRNSFEV p53 261-269

A*0201 LLLLTVLTV MUC-1 12-20

A*0201 RLLQETELV HER-2/neu 689-697

A*0201 RMFPNAPYL Wilm's Tumour (WT1) 126-134

A*0201 SLLMWITQV NY-ESO-1 157-165

A*0201 STAPPVHNV MUC-1 950-958

A*0201 VISNDVCAQV Prostate Specific Antigen-1 (PSA-1) 154-163

A*0201 VLQELNVTV Leukocyte Proteinase-3 (Wegener's autoantigen) 169-177

A*0201 VLYRYGSFSV gp100 (pmel17) 476-485

A*0201 YLEPGPVTA gp100 (pmel17) 280-288

A*0201 YLSGANLNL Carcinogenic Embryonic Antigen (CEA) 571-579

A*0201 KVLEYVIKV MAGEA1 278-286

A*0201 KVAELVHFL MAGEA3 112-120

A*0201 KTWGQYWQV gp100 (pmel17) 154-162

A*0201 HLSTAFARV G250 (renal cell carcinoma) 217-225

A*0201 ILAKFLHWL Telomerase 540-548

A*0201 ILHNGAYSL HER-2/neu 435-443

A*0201 IMDQVPFSV gp100 (pmel17) 209-217

A*0201 KIFGSLAFL HER-2/neu 348-356

A*0201 LMLGEFLKL Survivin 96-104

A*0201 ALQPGTALL Prostate Stem Cell Antigen (PSCA) 14-22

A*0201 CMTWNQMNL Wilm's Tumour (WT1) 235-243

A*0201 ELAGIGILTV MelanA / MART 26-35

A*0201 FLTPKKLQCV Prostate Specific Antigen-1 (PSA-1) 141-150

A*0201 GLYDGMEHL MAGEA-10 254-262

A*0301 KQSSKALQR bcr-abl 210 kD fusion protein 21-29

A*0301 ATGFKQSSK bcr-abl 210 kD fusion protein 259-269

A*0301 ALLAVGATK gp100 (pmel17) 17-25

A*2402 VYGFVRACL Telomerase reverse transcriptase (hTRT) 461-469

A*2402 TYLPTNASL HER-2/neu 63-71

A*2402 TYACFVSNL Carcinogenic Embryonic Antigen (CEA) 652-660

A*2402 TFPDLESEF MAGEA3 97-105

A*2402 EYLQLVFGI MAGEA2 156-164

A*2402 CMTWNQMNL Wilm's Tumour (WT1) 235-243

A*2402 AFLPWHRLF Tyrosinase 188-196

B*0801 GFKQSSKAL bcr-abl 210 kD fusion protein 19-27

allele sequence EBV epitopeA*0201 CLGGLLTMV EBV LMP-2 426-434

A*0201 GLCTLVAML EBV BMLF-1 259-267

A*1101 IVTDFSVIK EBV EBNA-4 416-424

A*2402 TYGPVFMCL EBV LMP-2 419-427

B*0702 RPPIFIRRL EBV EBNA-3A 247-255

B*0801 FLRGRAYGL EBV EBNA-3A 193-201

B*0801 RAKFKQLL EBV BZLF-1 190-197

B*3501 HPVGEADYFEY EBV EBNA-1 407-417

allele sequence Influenza A epitopeA*0101 CTELKLSDY Influenza A (PR8) NP 44-52

A*0201 GILGFVFTL Influenza A MP 58-66

A*0301 ILRGSVAHK Influenza A (PR8) NP 265-274

allele sequence HIV epitopeA*0201 ILKEPVHGV HIV-1 RT 476-484

A*0201 KLTPLCVTL HIV-1 env gp120 90-98

A*0201 SLYNTVATL HIV-1 gag p17 76-84

A*0201 TLNAWVKVV HIV-1 gag p24 19-27

A*0301 QVPLRPMTYK HIV-1 nef 73-82

A*0301 RLRPGGKKK HIV-1 gag p17 19-27

A*2402 RYLKDQQLL HIV-1 gag gp41 67-75

B*0702 IPRRIRQGL HIV-1 env gp120 848-856

B*0702 TPGPGVRYPL HIV-1 nef 128-137

B*0801 FLKEKGGL HIV-1 nef 90-97

B*0801 GEIYKRWII HIV-1 gag p24 261-269

B*2705 KRWIILGLNK HIV-1 gag p24 265-274

H-2Kd AMQMLKETI HIV-1 gag p24 199-207

MHC-peptid pentamers for detecting MHC-peptid pentamers for detecting antigen specific T cellsantigen specific T cells

Case study

Helen Burns was the second child born to her parents. She thrived until 6 months of age when she developed pneumonia in both lungs, accompanied by a severe cough and fever. Blood and sputum cultures for bacteria were negative but a tracheal aspirate revealed the presence of abundant Pneumocystis carinii. She was treated successfully with the anti-Pneumocystis drug pentamidine and seemed to recover fully.

As her pneumonia was caused by the opportunistic pathogen Pneumocystis carinii, Helen was suspected to have severe combined immunodeficiency.

What kind of laboratory test should be performed to make or rule out the diagnosis

of severe combined immunodeficiency?

A blood sample was taken and her peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA) to test for T cells function by 3H-thymidine incorporation into DNA.

A normal T-cell proliferative response was obtained, with her T cells incorporating 114,050 counts/min of 3H-thymidine (normal control 75,000 counts/min).

Helen had received routine immunizations with orally administrated polio vaccine and DPT (diphtheria, pertussis, and tetanus) vaccine at 2 months old. However, in further tests, her T cells failed to respond to tetanus toxin in vitro, although they responded normally in the 3H-thymidine incorporation assay when stimulated with allogeneic B cells (6730 counts/min incorporated compared with 783 counts/min for unstimulated cells).

When it was found that Helen's T cells could not respond to a specific antigenic stimulus, her serum immunoglobulins were measured and found to be very low.

IgG levels: 96 mg/dl (normal: 600-1400 g/dl)IgA levels: 6 mg/dl (normal: 60-380 mg/dl)IgM levels: 30 mg/dl (normal: 40-345 mg/dl)

Helen's white blood cell count was elevated at 20,000 cells/μl (normal range 4000-7000/μl).

Of these, 82% were neutrophils, 10% lymphocytes, 6% monocytes,

and 2% eosinophils.

The calculated number of 2000 lymphocytes/μl is low for her age (normal >3000 μl-1).

Of her lymphocytes, 7% were B cells (CD20+) (normal 10-12%) 57% reacted with antibody to the T cell marker CD3.

At 388 cells/μl her number of CD8+ T cells was within the normal range, but the number of CD4+ T cells (288/μl) was much lower than the normal (her CD4+ T-cell count would be expected to be twice her CD8+ T-cell count).

The presence of substantial numbers of T cells, and thus a normal response to PHA, ruled out a diagnosis of sever combined immunodeficiency.

Helen's paediatrician referred her to the Children's Hospital for consideration for a bone marrow transplant, despite the lack of diagnosis. When an attempt was made to HLA type Helen, her parents and her healthy 4-year-old brother, a DR type count not be obtained from Helen's white blood cells. A long-term culture of her B cells was made by transforming them with Epstein-Barr virus and the transformed B cells were then examined for expression of MHC class I and class II molecules with fluorescent-tagged antibodies. It was found that her B cells did not express HLA-DQ or HLA-DR molecules and a diagnosis of MHC class II deficiency was established.

Detection of MHC class IImolecules by fluorescent antibody. Helen’s transformed B-cell line was examined by using a fluorescent antibody to HLA-DQ and –DR. Helen (left panels) expressed approximately 1℅ of the amount of MHC class II molecules compared with atransformed B-cell line from a normal control (right panels).

Cells from a patient withclass II histocompatibility deficiency

Immunofluorescence of normalEBV-transformed cells

Transformed B cells Transformed B cells

Transformed B cells Transformed B cells

As her brother did not have the same HLA type as Helen, it was decided to use her mother as a bone marrow donor. The maternal bone marrow was depleted of T cells to diminish the chance of graft-versus-host disease developing and was administered to Helen by transfusion. The graft was successful and immune function was restored.

Discussion and questions

1. Why did Helen lack CD4 T cells in her blood?

The maturation of CD4 T cells in the thymus depends on the interaction of thymocytes with MHC class II molecules on thymic epithelial cells. When the MHC class II genes are deleted genetically in mice, the mice also exhibit a deficiency of CD4 T lymphocytes.

2. Why did Helen have a low level of immunoglobulins in her blood?

The polyclonal expansion of B lymphocytes and their maturation to immunoglobulin-secreting plasma cells requires helper cytokines from CD4 T cells, such as IL-4. Helen’s hypogammaglobulinemia is thus a consequence of her deficiency of CD4 T lymphocytes.

3. In SCID , lymphocytes fail to respond to mitogenic stimuli. Although Helen was first thought to have SCID, this diagnosis was eliminated by her normal response to PHA and an allogenic stimulus. How do you explain these findings?

Helens’ T cells, although decreased in number, are normal and are not affected by the defect. They are capable of normal responses to nonspecific mitogens and to an allogenic stimulus in which the antigen is presented by the MHC molecules on the surface of the (nondefective) allogeneic cells and thus does not require to be processed and presented by the defective cells. However, the failure of her lymphocytes to respond to tetanus toxin in vitro resulted from the fact that, in this situation, there were no cells that could present antigen on MHC class II molecules to the CD 4 T cells.

4. If a skin graft were to be placed on Helen’s forearm do you think she would reject the graft?

Yes. Helen’s T cells would be capable of recognizing the foreign MHC molecules on the grafted skin cells and would reject the graft.