Tetramer analysis

by

Sadegh feizollahzadeh

1. Monitoring vaccine studies2. Monitoring effect of Immunotherapy3. Monitoring T-cell responses in infectious disease, cancer, autoimmunity

and other malignancies4. Detection of antigen-specific T-cell responses in immunological research

Phases of the humoral immune response(review)

Phases of T cell response(review)

Antigen presentin

g cell

Peptide: 9 – 10 a.a.

MHC class I

Antigen Recognition by Antigen-Specific CD8 T Cells

CD8

CD8 T cell

TCR

The affinity of TCR for its ligand is low (10-5 M)The CD8 coreceptor binds to the same ligandand provides a 10-fold increase (10-6 M)

..but the MHC binds the TCR with low affinity

The interaction between one The interaction between one MHC molecule and one TCR is MHC molecule and one TCR is

not strong enough for labellingnot strong enough for labelling

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

MHC

T cell receptors

T cellT cell

peptide specific

T cell

MHC pentamer

Binding of the MHC multimermer to the T-cell

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

T cell receptors

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.

• Peptid/MHC (pMHC) tetramers were invented and first reported by Altman and Davis in 1996.

• This novel reagents have revolutionized the study of T cell responses by allowing fore accurate and rapid:

1. enumeration of antigen-specific T cells, 2. The phenotypic characterization of such cells and 3. Their viable isolation for further analysis.

BIOLOGY OF TCR-pMHC INTERACTIONS ANDIMPLICATIONS IN TETRAMERS

• The affinity of the TCR-pMHC interaction in solution is much lower than that of an antibody–antigen interaction.

• The typical dissociation constant (Kd) of an antibody–antigen interaction is around 1 nM, a typical TCR-pMHC interaction may be in the range of 10 M.

• Previous efforts to use soluble pMHC complexes as a staining reagent to identify antigen-specific T cells were unsuccessful.

• Potentially three pMHC complexes can interact with three TCRs simultaneously on a T cell surface, such that the avidity of this interaction becomes very strong, making staining possible.

COMPARISONS TO OTHER ASSAYS

• limiting dilution assays (LDA), cytokine flow cytometry (CFC) and ELISPOT analyses.

• Typically, tetramer analyses have provided frequency estimates that exceed those detected using these alternate assay systems.

• when combined with a functional readout, this yields important additional information

• Such a combination was used successfully to demonstrate for the first time that a tumor-specific T cell population was anergic in vivo

PRODUCTION OF TETRAMERS

• The MHC class I molecule is engineered with a C-terminal substrate peptide for BirA-mediated biotinylation, biotin signal peptide (bsp).

• A BirA substrate peptide (usually the 15-mer, LHHILDAQKMVWNHR) is fused to the extracellular portion of the MHC molecule.

• This MHC I-bsp recombinant protein (as well as β2m) is generally produced using E. coli in inclusion bodies (BL21DE3 systems).

• Briefly, recombinant MHC I-bsp, 2m and the peptide of interest are mixed together in a folding reaction which generally goes over 48–72 h.

• Resulting in a 1–5 per cent final yield.• Properly folded pMHC complexes are extensively purified

from unfolded materials via size exclusion columns (FPLC) and further cleaned up with anion exchange.

• During this process, the biotinylation reaction is carried out via the addition of biotin-ligase (BirA enzyme), biotin and ATP.

• Streptavidin directly conjugated to a fluorophore (generally phycoerythrin or APC) is used to facilitate their use in flow cytometry or sorting.

• Under optimal conditions, the lower level of detection for tetramer-based assays is approximately 1/8000–1/10 000 (i.e. 0.01–0.0125 per cent).

• periodic retesting of tetramers by retitration analyses on a regular (every 6–8 weeks) basis depending on the peptide used.

BASIC METHODOLOGY AND IMPORTANTFACTORS

• Quality and numbers of cells• Staining volume, temperature and time• Tetramer concentration• Anti-CD8 and other antibodies• Data interpretation

• The same numbers of cells should be added per stain in all tetramer assays.

• one stains 1–2 million PBMCs per condition and collects as many events as possible (105–106) for each analysis (0.01 per cent of CD8+ T cells).

• As an example, if one collects 5× 105 events and CD8+T cells represent 10 per cent of PBMC, then a 0.01 per cent antigen-specific population would represent five events.

• frozen/thawed PBMC samples may be used without compromising the quality of tetramer staining data.

• To maximize cell viability and quality, thawed cells may be ‘rested’ in complete media (containing human serum) for several hours to overnight and then enriched by discontinuous gradient centrifugation (Ficoll/Percoll) prior to staining.

• As an example, CD62L (an important marker for the distinction between naive versus memory T cells) contains glycosylations which may be cleaved during the freeze/thaw process.

Staining volume, temperature and time

• To conserve reagents, it is advisable to stain in a small volume, generally 30–50μ l. This is adequate for 1–2 million PBMCs.

• The temperature at which the staining occurs may significantly impact the degree of specific tetramer staining.

• The main reason to perform tetramer staining on ice rather than room temperature is to favor detection of low avidity tetramer–T cell interactions.

• At room temperature (23˚C) or 37˚C, tetramer staining generally saturates at 15–30 min, while on ice (or at 4˚C) saturation does not occur until at least 2 h.

Tetramer concentration

• Staining with suboptimal tetramer concentrations may lead to an underestimate of antigen-specific T cell frequencies, while staining with excessive tetramer concentrations may lead to high backgrounds and overestimate of antigen-specific T cell frequencies.

• For most tetramers, they are generally used at a 1–20 g/ml final concentration.

Anti-CD8 and other antibodies

• The MHC I molecule naturally has a low affinity for CD8.• Depending on the CD8 determinant recognized by the

antibody, anti-CD8 counterstaining may block, have little impact on, or even augment the intensity of pMHC tetramer staining of T cells.

• Tetramers from Beckman Coulter Immunomics are made with non-CD8 binding mutant MHC I molecules.

• Antibodies to markers not present on the cells of interest – for CD8+T cells, useful ‘dump’ antibodies may include anti-CD4, 14, or 19.

Data interpretation

• Specific tetramer staining of T cells may be confirmed via competition with unlabeled pMHC monomers or anti-CD3 antibodies.

• Tetramer-positive events are generally ‘normalized’ as a percentage of total CD8+ T cells.

• Data may be reported as the absolute number of tetramer+, CD8+cells per amount (i.eμ/ml) of blood.

ADVANCED USES OF TETRAMERS

• Isolation by sorting and cloning• Phenotypic analysis by multi-color flow cytometry• Intracellular staining• Analysis of ‘structural avidity’• Functional analysis• In situ hybridization

Isolation by sorting and cloning

• Care should be taken to keep the cells cold and in serumrich media to maximize their function before and after sorting.

Phenotypic analysis by multi-color flow cytometry

• Even with three- or four- color FACS, the expression of several markers by an antigen-specific T cell population of interest may be determined by performing a few different stains.

• With the increasing availability of six- and even 10-color FACS (Table 22.2), large numbers of markers could be determined from only a few stains.

• Not all antibodies work well in combination, so considerable effort must be made to optimize phenotypic panels which work well together.

Intracellular staining

• Tetramers can be combined successfully with intracellular staining to analyze expression of cytokines (e.g. IFN-γ, IL- 2, IL-4, TNF-α) or cytolytic granules (perforin, granzyme A, granzyme B) by antigen-specific T cells.

• Start with large numbers of cells.• Tetramer staining could be performed prior to cell activation.

Analysis of ‘structural avidity’

• ‘Functional avidity’ or ‘recognition efficiency’ is emerging as a key factor in the effectiveness of an antigen-specific T cell response.

• The rate of dissociation of bound pMHC tetramers from antigen-specific T cells upon addition of a competing antibody (such as anti-TCR) may be used as a relative measure of the difference in TCR affinities between T cells.

Functional analysis

• Staining for cytokine production, cytolytic granules and direct ex vivo testing for cytolytic activity after sorting.

• CD107 mobilization could be combined with peptide/MHC tetramer staining to directly correlate antigen-specificity and cytolytic ability on a single-cell level.

In situ hybridization

• This is a technically challenging procedure which requires extensive optimization of staining conditions.

• Most successful in situ tetramer staining has been achieved using fresh or frozen tissue sections, although some success has also been achieved with ‘lightly fixed’ tissue samples.

USE IN CLINICAL TRIALS AND FUTUREPROSPECTS

• Certain hurdles prevent the widespread use of tetramers in the clinical setting.

• In cancer vaccination, peptide-specific T cell responses as detected by tetramers have largely not correlated with clinical outcome.

• Peptide-specificity does not necessarily guarantee tumor-reactivity of a T cell response – recognition efficiency of the T cells is a key factor.

• One cannot simply rely on enumeration of antigen-specific T cells to predict clinical outcome in clinical trials.

• It is critical to be able to study the biology of such cells in terms of recognition efficiency, in vivo functional status and homing patterns.

1. Monitoring vaccine studies2. Monitoring effect of Immunotherapy3. Determination of immune status (e.g. in connection with transplantation

and other immunodeficiency)4. Monitoring T-cell responses in infectious disease, cancer, autoimmunity

and other malignancies5. Detection of antigen-specific T-cell responses in immunological research

1.65%

2.34%

0.29%

5.3% 0.02%

0.01%0.54%

0.44%

PATIENTS CONTROLS

CD4

Tetramer

AD5

AD6

AD10

AD22AD25

AD18

AD9

AD14

A

0.03%

N

J

A

0.02%19.13%

9.9%

AD controls0.001

0.01

0.1

1

10

100 P<0.05

Perc

enta

ge C

D3+

and

Tetr

amer

+

Individuals with atopic dermatitis have higher frequencies of circulating Der p 1-specific CD4+ T cells than non-atopics

(short term culture)

Multimer-based enumeration of antigen–specific T cells

(Peripheral blood, HLA-A2+healthy donor)

<0.01% 0.1% 0.1% 1%

HIV Melan-A Influenza virus Cytomegalovirus

A2

/Me

lan

-A

A2

/Flu

–M

A

A2

/CM

V

A2

/HIV

pp65 495-503Melan-A 26-35 Flu-MA 58-66Pol 476-484

CD8 CD8 CD8 CD8

<0.01%

0.1%

0.1%

1%

Staining Whole Blood ex vivo. Example of direct whole blood staining with EBV Class II iTAg MHC Tetramer. Figure 2a-Irrelevant Tetramer. Figure 2b-Positive Tetramer.

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

Pro5™ MHC Pentamers contain 5 MHC-peptide complexes that are multimerised by a self-assembling coiled-coil-domain. All 5 MHC-peptide complexes are held facing in the same direction, similar to a bouquet of flowers. Therefore, with Pro5 MHC Pentamer technology, all 5 MHC-peptide complexes are available for binding to T cell receptors (TCRs), resulting in an interaction with very high avidity.

Each Pro5 MHC Pentamer also contains up to 5 fluorescent molecules yielding an improved fluorescence intensity of the complex.

For optimum flexibility Pro5® Pentamers may be ordered labeled with R-PE, APC or biotin Any combination of R-PE-labeled, APC-labeled and biotin-labeled Pentamers may be used

Pro5® Pentamers are available in 50, 150 and 500 test quantities. One test of Pentamer is sufficient to stain approximately 1-2 x 106 PBMCs

• Pro5 MHC Pentamers feature:- Higher avidity for detecting low affinity T cells- Reduced background staining / improved specificity- Brighter overall fluorescence- Increased flexibility for long-term storage and labelingPro5® Pentamers can be used to detect and separate antigen-specific CD8+ T cell populations as rare as 0.02% of lymphocytes.

allelesequenceTumour (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

allelesequenceEBV 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

allelesequenceInfluenza 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

allelesequenceHIV epitopeA*0201ILKEPVHGVHIV-1 RT 476-484

A*0201KLTPLCVTLHIV-1 env gp120 90-98

A*0201SLYNTVATLHIV-1 gag p17 76-84

A*0201TLNAWVKVVHIV-1 gag p24 19-27

A*0301QVPLRPMTYKHIV-1 nef 73-82

A*0301RLRPGGKKKHIV-1 gag p17 19-27

A*2402RYLKDQQLLHIV-1 gag gp41 67-75

B*0702IPRRIRQGLHIV-1 env gp120 848-856

B*0702TPGPGVRYPLHIV-1 nef 128-137

B*0801FLKEKGGLHIV-1 nef 90-97

B*0801GEIYKRWIIHIV-1 gag p24 261-269

B*2705KRWIILGLNKHIV-1 gag p24 265-274

H-2KdAMQMLKETIHIV-1 gag p24 199-207

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

Immudex Dextramer™ Technology

• The MHC Dextramer™ reagents are a new generation of fluorescent MHC multimers that are particularly efficient in the detection of T-cells carrying T-cell receptors with very low affinity for MHC.

• The Dextramer™ reagents carry more MHC-molecules and more fluorochromes than conventional MHC multimers

• MHC Dextramer™ reagents thus provide the following benefits:Brighter stainingHigher resolution

Lower background Higher stability

Minimal lot-to-lot variation

• The MHC Dextramer™ consists of a dextran polymer backbone carrying an optimized number of MHC and fluorochrome molecules.

• MHC Dextramer™ reagents carry more MHC molecules and more fluorochromes than conventional MHC multimers. This increases their avidity for the specific T-cell and enhances their staining intensity, thereby increasing resolution and the signal-to-noise ratio. An optimized formulation of the MHC Dextramer™ products ensures minimal background staining.

• The dextran polymer backbone stabilizes the conformation of the attached proteins, the MHC-peptide complexes and fluorochromes. Thus, MHC Dextramer™ reagents are highly stable reagents.

• MHC Dextramers are suitable for basic and clinical research in a number of applications including drug and vaccine development.

• The MHC Dextramer™ reagents can be used to detect antigen-specific T-cells in fluid cell samples (e.g. blood, cultured cell lines, CSF, lymph, synovial fluid) using flow cytometry, or antigen-specific T-cells may be detected in situ in tissue sections.

PRO5™ PENTAMERSTETRAMERS

AVIDITY & CONFIGURATION

5 MHC-peptide complexes per Pro5™ Pentamer in the same orientation results in enhanced avidity interaction with TCR.

MHC-peptide complex held in tetrahedral configuration allowing only 3 MHC-peptide complexes to bind TCR.

BRIGHTNESSUnique labelling technology allows up to 5 fluorescent labels to attach to each Pro5™ Pentamer yielding unprecedented brightness of signal.

By nature, MHC Tetramers will have one fluorescent label located at the centre of the complex.

STORAGE & LABELLING FLEXIBILITY

Unlabelled Pro5™ Pentamers can be stored long-term at -80°C. Thawed pentamers can be labelled in a flexible manner with your fluorochrome of choice, maximizing the range of possible applications.

Tetramers are made by multimerising biotinylated MHC monomers with fluorescent labelled streptavidin. As a consequence tetramers are always sold labelled and for this reason they must be stored at 4°C, which reduces their shelf-life to about 6-12 months on average.