DETECTION AND SORTING OF ANTIGEN-SPECIFIC T-CELLS

Volume 41, May 2001 TRANSFUSION 687www.transfusion.org

B A S I C S C I E N C E

Immunologists have long sought methods for trackingspecific T cells in vivo in the course of an immune re-sponse against pathogens. The specificity of T-cell ac-tivation relies on the recognition by the T-cell recep-

tor (TCR) of foreign peptides presented by MHC proteins.Peptides bound to class I MHC molecules stimulate theTCRs of CD8+ cells, whereas class II MHC-peptide com-plexes are recognized by TCRs of CD4+ cells. Until recently,the only available reagents with which to label T-cellclonotypes were idiotypic MoAbs, which are specific for aparticular combination of TCR α and β chains. The produc-tion of such antibodies is sometimes difficult, requires priorknowledge of the TCRs that react to a given epitope, andcannot be used to assess the full extent of a normal hetero-geneous immune response against a particular MHC-pep-tide complex.

Methods for producing soluble MHC proteins loadedwith relevant peptides have been developed,1,2 and re-searchers tried labeling specific T cells with these MHC-peptide complexes. However, because of the low affinity ofthe interaction between the TCR and the MHC-peptidecomplex (approx. 10–5 M, with a dissociation constant of <1min3), initial attempts using soluble monomeric forms ofMHC-peptide were unsuccessful. The problem was solvedby constructing multimers of MHC-peptide to increase theoverall avidity of the reagent. Two strategies were used: theapproach of Dal Porto et al.4 was to make a chimeric MHC-immunoglobulin protein to produce divalent peptide-MHC-immunoglobulin complexes, whereas Altman et al.5

produced tetravalent MHC-peptide complexes by cross-linking biotinylated MHC-peptide monomers withstreptavidin.

GENERATION OF MHC TETRAMERSThe method described by Altman et al.,5 which is favored by mostlaboratories producing class I MHC or class II MHC-peptidemultimers, relies on the use of an Escherichia coli enzyme,called BirA, which specifically recognizes a 15-mer peptideand biotinylates a lysine within this sequence.6 As depictedin Fig. 1, a BirA peptide-tagged MHC chain is produced inE. coli by ligating an oligonucleotide coding for the BirA-specific peptide to the 3´ end of the cDNA coding for theclass I MHC heavy chain and by cloning the construct in theappropriate expression vector. The advantage of using en-zymatic biotinylation is that it will occur only on the spe-cific sequence at the tail of the MHC chain, whereas chemi-cal biotinylation, which is commonly used for antibodies,would couple biotine to any lysine within the sequence andthus would potentially alter TCR recognition sites or resultin a bad positioning of the monomer onto streptavidin.

For class I MHC complexes, the MHC heavy chain andβ2-microglobulin are produced separately as inclusion bod-ies in E. coli and dissolved in 8M urea, and both chains arethen mixed with the peptide of interest in a large volumeof refolding solution for 4 days to allow proper renaturationof the MHC-peptide-β2-microglobulin monomer. This isthe critical step, and the amount of recovered, properly re-folded monomer will vary with the isotype of the heavy chain(HLA-A, B, or C) and with the affinity of the peptide for theMHC molecule. After concentration, monomers are bio-tinylated with recombinant BirA enzyme and then purifiedon an ion exchange column (Mono-Q, Amersham PharmaciaBiotech, Orsay, France). They can be stored at –80°C for subse-quent use or tetramerized by mixing with fluorescent avidinor streptavidin at a molar ratio of 0.8:4. This ratio must beadjusted carefully for each mix, so that tetramers only areobtained and not a mix of monomers, dimers, trimers, andtetramers. Optimal conditions for each preparation aredetermined by checking the end product on a gel filtrationcolumn. The fluorescent tetramer can be stored for severalmonths at 4°C and used to label antigen-specific T cells.

For class II MHC-peptide complexes, the yield of theabove method is so poor that alternative strategies havebeen developed. They rely on the transfection of the two

MHC-peptide multimers:tools of choice for detecting and sorting antigen-specific T-cells

François Lang and Marie Bodinier

ABBREVIATION: TCR = T-cell receptor.

From INSERM U463, Nantes, France.

Address reprint requests to: François Lang, DVM, PhD,

INSERM U463, Institut de Biologie, 9 quai Moncousu, 44093

Nantes Cedex 01, France; e-mail: flang@nantes.inserm.fr.

Received for publication October 13, 2000, and accepted

November 15, 2000.

TRANSFUSION 2001;41:687-690.

LANG AND BODINIER

688 TRANSFUSION Volume 41, May 2001 www.transfusion.org

chains of the class II molecule in insect cells that have theability to produce properly folded, soluble class II MHCcomplexes in the absence of antigen processing. Crawfordet a1.2 described an approach in which the peptide epitopeis linked to the amino terminus of the class II MHC β chainthrough a short, flexible linker, and the biotinylation tag isadded at the carboxy terminus of the β chain. They reportthe production of several class II MHC-peptide tetramersthat could stain T-cell hybridomas in the mouse.2 However,this approach is cumbersome, as a new cDNA constructmust be engineered for each peptide. More recently, Novaket a1.7 described the production in insect cells of emptyclass II molecules that can subsequently be loaded with thepeptide of interest. To obtain better association of the α andβ chains, they added leucine zipper sequences to eachchain. They report the production of DR0401 and DQ0602tetramers that could stain T-cells that are specific for influ-enza A or HSV2 epitopes, respectively.7,8

DETECTION OF ANTIGEN-SPECIFICT CELLS WITH TETRAMERS

The staining of specific T cells within a polyclonal lympho-cyte population with MHC-peptide tetramers is one of themajor applications of these new tools. It allows a simple and

accurate monitoring of the timing and magnitude of the invivo T-cell response against viral, bacterial, or tumoralepitopes, and it thus provides critical information to immu-nologists and clinicians for evaluating the shaping of theTCR during primary and recall immunization, evaluatingthe efficiency of the immune response against a givenepitope, and assessing the intensity of a specific responsein a given patient.

Of course, the prerequisite to tetramer analysis is theknowledge of immunodominant epitopes and their MHCrestriction, and great efforts are being made by many labo-ratories to define viral or tumoral epitopes that are re-stricted by the most frequent HLA alleles. The best-studiedMHC context to date is HLA-A0201, and a number of viraland tumoral epitopes have been defined in this context andused to make tetramers. In Fig. 2 (top panels) are showntypical stainings of polyclonal T-cell populations with HLA-A0201 (mutated, see below) tetramers loaded with a pep-tide from the CMV protein pp65 or from the melanomaantigen Melan-A/MART I. More recently, tetramers withother HLA alleles, such as HLA-B8 and HLA-B35, have beensuccessfully developed,9,10 and it is likely that, in the nearfuture, the list of available tetramers will include the mostfrequent HLA-A and -B alleles.

The first surprising observation that came out of analy-ses with class I MHC-peptide tetramers had to do with thefrequencies of specific CD8+ cells in peripheral blood dur-ing acute viral infection. In both the mouse11 and hu-mans,10,12 it was found that specific T cells accounted for apercentage of blood CD8+ cells that is at least 10 times theprevious estimates obtained from limiting-dilution analy-sis. This discrepancy is likely due to the fact that, in limit-ing-dilution analysis, clones have to survive and proliferateto be detected, when, in fact, most of these expanded T cellsare short-lived and do not proliferate after in vitrorestimulation. The specificity and functionality of thesetetramer-positive cells were further documented by testingthem for cytolysis and INFγ production against peptide-loaded target cells after FACS.10,12

Detection of specific CD4+ cells in human blood bydirect staining with class II MHC-peptide tetramers has notyet been reported. In both reports describing staining withhuman class II tetramers,7,8 specific T cells had to be am-plified in vitro by restimulation with the relevant peptidebefore they could be detected and sorted by FACS. It re-mains to be determined whether this truly reflects a lowerfrequency of circulating CD4+ memory cells than of CD8+memory cells or whether class II MHC-peptide tetramershave a lower avidity for the TCR and thus a higher detec-tion threshold than class I MHC-peptide tetramers.

In this respect, it should be noted that class I and classII MHC tetramers differ in their ability to engage the co-receptor. In physiologic conditions, CD4+ and CD8+ co-receptors participate in TCR recognition of the MHC-pep-

Fig. 1. Schematic description of the production of class I MHC-

peptide tetramers in E. coli.

DETECTION AND SORTING OF ANTIGEN-SPECIFIC T-CELLS

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tide complex and stabilize TCR binding by interacting withconstant regions of the MHC. It is therefore important toevaluate the ability of MHC tetramers to engage the co-re-ceptor on T-cells, especially when tetramers are used tostudy TCR transduction events. To address this point,Crawford et al.2 stained CD4+ and CD4– cell hybridomassharing the same TCR and demonstrated that the bindingof their class II tetramers was independent of co-receptorengagement. As a consequence, they observed a direct cor-relation between the intensity of tetramer staining and TCRaffinities.2 This correlation was not evaluated for the humanclass II tetramers constructed by Novak et al.,7 but onewould anticipate that the introduction of leucine zippersequences alters interactions with the co-receptor CD4even further.

In contrast, we13 and others14 clearly demonstrated thatHLA class I tetramers could interact with the co-receptorCD8. Therefore, the intensity of tetramer binding reflectsthe affinity of the TCR-CD8 complex for the MHC-peptiderather than the sole affinity of the TCR. In addition, we haveshown that HLA-A0201 tetramers at concentrations above10 µg per mL gave significant background staining on irrel-evant CD8+ cell clones because of interactions with CD8alone. We therefore introduced a mutation in the α3 domainof HLA-A0201 to decrease binding to CD8 and obtained

mutated tetramers with no backgroundstaining that retained their TCR specific-ity.14 We now routinely use a single stain-ing procedure with these mutated HLA-A0201 tetramers to detect specific, rareT cells, as a double-staining procedurewith a CD8 antibody is no longer re-quired to accurately estimate the per-centage of positive cells (Fig. 2).

SORTING SPECIFIC T CELLSWITH MHC MULTIMERS

It is obviously of great interest to immu-nologists to be able to sort specific T cellsfrom a polyclonal population by usingMHC tetramers. Sorted T cells can thenbe amplified nonspecifically in vitro andanalyzed structurally (phenotype, TCRusage) and functionally (cytokine pro-duction, cytolysis). In most reports, FACSafter staining with MHC tetramers waschosen to isolate specific T cells. Themain advantage of this method is that itis possible to visualize the intensity oftetramer staining and thus to make fineadjustments of the sort window. In thismanner, Yee et al.15 demonstrated thatthey could isolate subpopulations withdifferent avidities for the MHC-peptide

complex from in vitro-generated specific cell lines on thebasis of intensity of tetramer staining. However, FACS canbecome rather inconvenient when large-scale isolation ofspecific, rare (<1%) T cells is required. We therefore devel-oped an immunomagnetic sorting procedure using biotin-ylated MHC-peptide monomers bound to streptavidin-coated beads. We demonstrated that the use of mutatedMHC-peptide monomers with low CD8 binding greatly en-hanced the efficiency of this immunomagnetic sorting.14

Figure 2 shows two examples of the purity of the popula-tions obtained after one round of immunomagnetic sort-ing and then polyclonal amplification. Moreover, we docu-mented that this procedure allowed the recovery of most(approx. 90%) of the specific cells present in the initialsample. Thus, we assert that immunomagnetic sorting withmutated MHC-peptide multimers should be easier to adaptto clinical settings and more convenient than FACS for pro-ducing clinical-grade T cells for immunotherapy.

CONCLUSIONMHC-peptide multimers represent a major technicalbreakthrough in the analysis of T-cell responses, as theyallow the labeling and/or sorting of T-cells according totheir antigen specificity. It can be anticipated that, as more

Fig. 2. Single stainings of polyclonal T-cell populations with 20 µg per mL of mutated

HLA-A0201 loaded with peptides from the CMV protein pp65 and the EBV protein

BMLF-l or melanoma antigens Melan-A/MART I or NA 17-A (top panels). These popu-

lations were sorted with the appropriate multimer (A2-pp65 or A2-Melan-A) and ex-

panded in vitro by unspecific polyclonal stimulation, and purity was assessed by tet-

ramer staining (bottom panels).

LANG AND BODINIER

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MHC-peptide combinations become available commer-cially, they will become routinely used as antibodies for themonitoring of specific T-cell responses in patients. In ad-dition, production of monomers in Good ManufacturingPractices conditions would allow the rapid isolation by im-munomagnetic sorting of clinical-grade T cells for immu-notherapy.

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