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The Journal of Immunology, 2006, 176: 2330-2336.
Copyright © 2006 by The American Association of Immunologists

Ex Vivo Characterization of Allo-MHC-Restricted T Cells Specific for a Single MHC-Peptide Complex1

Mikaël J. Pittet2,*, Asma Gati{dagger}, Frederic-Anne Le Gal{ddagger}, Gilles Bioley*, Philippe Guillaume§, Magda de Smedt, Jean Plum, Daniel E. Speiser*, Jean-Charles Cerottini§, Pierre-Yves Dietrich{ddagger}, Pedro Romero3,* and Alfred Zippelius3,{dagger}

* Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland; {dagger} Department of Oncology, University Hospital Zurich, Zurich, Switzerland; {ddagger} Division of Oncology, Laboratory of Tumor Immunology, University Hospital, Geneva, Switzerland; § Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and Department of Clinical Chemistry, Microbiology and Immunology, University Hospital, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-126–35 multimers at frequencies that are in the range of 10–4 CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alloreactive T cells mount vigorous responses, reflected by MLR in vitro and the clinical manifestations of graft rejection and graft-vs-host disease after transplantation over MHC barriers (1). In contrast to the autologous peripheral T cell repertoire, which is devoid of high-avidity T cells due to self-tolerance, alloreactive T cells react with allogeneic MHC molecules to which they have not been exposed during thymic selection. The size of the alloreactive T cell repertoire is several orders of magnitude higher than that of T cells specific for a foreign peptide bound to a self-MHC molecule, with 1–10% of peripheral T cells being able to recognize alloantigen (2). To account for these observations and, in addition, to explain the structural basis of alloreactivity, mainly two models representing extremes in the spectrum of TCR/Ag recognition have been put forward. On the one hand, alloreactive T cells may recognize polymorphic residues of allogeneic MHC molecules, allowing T cells with even low affinity to be activated by the unusually high density of antigenic determinants (3). In line with this, previous work has demonstrated the existence of peptide-independent alloreactive CD8 T cells (4, 5, 6). In contrast, a diverse "gemisch" of Ags, now known as naturally processed peptides, presented by non-self-MHC molecules, hence not involved in thymic selection processes, may be recognized by alloreactive T cells (7). In support of the latter mode of recognition, several studies have accumulated compelling evidence that alloreactive T cells recognize MHC-peptide complexes as do nominal Ag-specific T cells. First, the alloreactive T cell repertoire is broad and diverse as demonstrated in studies using synthetic peptide libraries (8, 9). Second, peptide-specific T cells, either alloreactive or autologous, demonstrate similar affinity for MHC-peptide complexes (10, 11, 12). Third, alloreactive T cell clones specific for tumor or viral Ags specifically lyse tumor cell lines (13, 14) or infected cells (15), respectively.

Major hurdles in the isolation and molecular characterization of alloreactive T cells are the generally low frequency of peptide-specific T cells and the difficulty to separate them from cells that recognize foreign MHC. The development of fluorescent MHC class I/peptide-soluble multimeric complexes (multimers) (16) that allow direct visualization and isolation of rare Ag-specific T cells ex vivo has been a major step in tracking monospecific, yet polyclonal T cell populations. Although multimers were initially used to characterize Ag-specific CD8 T cells directed against autologous targets, they have been used recently for the analysis of alloreactive T cells recognizing specific peptides (17, 18).

Applying various MHC class I/peptide multimers synthesized around distinct HLA-A*0201-restricted epitopes, we find that only HLA-A*0201/Melan-A26–35 multimers label a subset of human CD8 T cells (named allo-A2/Melan-A+ cells) in HLA-A*0201-negative individuals. These cells are readily detectable ex vivo within CD8 single-positive thymocyte populations as well as in the peripheral blood of newborns and adults. Similar to their counterparts in HLA-A*0201-positive individuals (19, 20), clones generated from this alloreactive T cell pool exhibit a large heterogeneity in terms of avidity of recognition of the Melan-A peptide, ranging from low to very efficient, and of specific recognition of both Melan-A analog and parental peptides presented by HLA-A*0201. In addition, allo-A2/Melan-A+ cells also exhibit a prominent TCR{alpha} chain restriction. The constraints involved in the shaping of this large alloreactive, yet Ag-specific repertoire are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Tissues and cells

Peripheral blood from nine healthy donors (HD)4 that were 19 to 82 years of age was collected at the Blood Transfusion Center (Lausanne, Switzerland). Cord blood from 11 newborns and pediatric thymus tissue from 7 children that were 0–12 years of age and underwent corrective cardiac surgery were collected at the University Hospital of Ghent, (Ghent, Belgium) following the guidelines of the Medical Ethical Commission of the Hospital. Lymphocytes were prepared and cryopreserved as previously described (21). Samples were selected based on the absence of HLA-A2 expression.

MHC/peptide multimers and mAbs

Synthesis of PE-labeled HLA-A*0201/peptide multimers was performed as previously described (16, 22). Because of the unstable binding of the Melan-A26–35 natural peptide (EAAGIGILTV) to HLA-A*0201 (23), the analog peptide Melan-A26–35 A27L (ELAGIGILTV) was used. The interchangeability between the HLA-A*0201/Melan-A analog and natural peptide multimers has been previously demonstrated (21, 22). mAbs were obtained from BD Biosciences.

Purification and analysis of cells

CD8 T lymphocytes were positively enriched from PBMCs and cord blood lymphocytes using CD8 microbeads (Miltenyi Biotec), while CD8 single-positive thymocytes were negatively enriched using CD4 microbeads. Cells were stained with PE- labeled multimers for 1 h at room temperature in PBS, 0.2% BSA, 50 µM EDTA, then incubated with appropriate mAbs for 20 min at 4°C. Cells were either immediately analyzed on a FACSCalibur (BD Biosciences) or sorted into defined populations on a FACSVantage SE using CellQuest software.

Cytolytic activity

Single allo-A2/Melan-A+ cells were sorted ex vivo on a FACSVantage SE, then stimulated with 1 µg/ml PHA-L, 150 U/ml IL-2, 10 ng/ml IL-7, and 106/ml allogeneic irradiated PBMCs. Cells were tested for their lytic activity against peptide-pulsed HLA-A2+ TAP-deficient T2 cells or toward the melanoma cells lines Me 290 (HLA-A2+Melan-A+), Na8 (HLA-A2+ Melan-A–), and Me 260 (HLA-A2–Melan-A+) in 4 h 51Cr release assays. The percentage of specific lysis was calculated as previously described (24).

Amplification of TCRVbeta and TCRV{alpha} transcripts and sequencing of the PCR products

Total mRNA was prepared from monoclonal allo-A2/Melan-A+ cell populations using TRIzol (Invitrogen Life Technologies) and converted to cDNA by standard methods using reverse transcriptase and an oligo(dT) primer as previously described (19). cDNAs were amplified in nonsaturating PCR conditions (30 cycles) with a panel of previously validated 5' sense primers specific for 29 Vbeta and 22 V{alpha} subfamilies and one 3' antisense primer specific for the corresponding C gene segment (25). PCR products were cloned with the TOPO TA cloning kit (Invitrogen Life Technologies). One Shot TOP10 chemically competent Escherichia coli (Invitrogen Life Technologies) were transformed and plated for blue/white color selection on medium containing 5-bromo-4-chloro-3-indolyl-D-galactopyranoside. Plasmid DNA was extracted from white colonies using the Qiagen Plasmid Mini kit (Qiagen) and sequenced using a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit with 7-deaza-dGT7 (Amersham Pharmacia Biotech). For each plasmid, the sequencing reaction was performed in both directions using a C primer or the V{alpha}2-specific primer, respectively. The sequence of highly homologous TCR clonotypes (i.e., the ones with only one or two nucleotide differences) was checked by repeating the sequence analysis starting from the total mRNA, to avoid artifacts. The TCR nomenclature used is according to Arden et al. (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
High frequency of circulating, phenotypically naive allo-Melan-A+ CD8 T cells in HLA-A*0201-negative individuals

It was shown previously that in vitro stimulation of PBMC from HLA-A*0201-negative individuals with HLA-A*0201-restricted peptides derived from foreign (i.e., Flu-MP58–66) (17) and self (i.e., Melan-A/MART-126–35 A27L; Ref.18)-Ags leads to the proliferation of alloreactive, yet Ag-specific, CD8 T cells that can be identified with the respective HLA-A*0201/peptide multimers. To determine the frequency of such cells in fresh peripheral blood of HLA-A*0201-negative individuals, i.e., to elucidate the possibility to readily track those cells without prior amplification, we used A2/peptide multimers synthesized around a total of 10 immunodominant epitopes derived from viral and tumor Ags. CD8 T cells specific for a variety of HLA-A*0201-restricted self- and non-self peptides, including gp100208–218, tyrosinase368–376, MAGE-4130–139; MAGE-10254–262; NY-ESO-1157–165, CMV pp65495–503, EBV280–288, FLU-MA58–66, and HIV-POL476–484, were undetectable ex vivo (percent HLA-A*0201/peptide multimer+ cells in CD8 T cells was 0.002 ± 0.005 (mean ± SD) whereas detection limit was <0.01%, data not shown). In sharp contrast, frequencies above detection limit were often found when multimers synthesized around the Melan-A26–35 peptide were used (Fig. 1). Circulating allo-A2/Melan-A+ cells were detectable in CD8 T cells from eight of nine HLA-A*0201-negative individuals (mean ± SD, 0.017 ± 0.010). These cells exhibited a CCR7+CD45RAhigh surface phenotype (mean ± SD, 95 ± 4%, data not shown), compatible with a naive functional status. Indeed, allo-A2/Melan-A+ CD8 cells were also readily detectable in 9 of 11 cord blood samples of HLA-A*0201-negative newborns (mean ± SD, 0.010 ± 0.006; Fig. 1).


Figure 1
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  		FIGURE 1. Ex vivo detection of allo-A2/Melan-A+ cells in HLA-A*0201-negative HD. CD4-depleted thymocytes (a) and purified CD8 T cells from newborn cord blood (b) and adults (c) were stained with HLA-A*0201/Melan-A or HLA-A*0201/Flu-MA58–66 multimers, anti-CD8, and anti-CD3. The percentage of allo-A2/Melan-A+ cells is given in gated CD3+CD8+ lymphocytes.

 
High frequency of allo-Melan-A+ cells in CD8 single-positive thymocytes of HLA-A*0201-negative individuals

To investigate whether allo-A2/Melan-A+ cells are detectable in the thymus, we performed multimer staining of lymphocytes obtained from human thymi of HLA-A*0201-negative individuals. Frequencies above detection limit were found among CD8 single-positive thymocytes in seven of seven individuals (mean ± SD, 0.014 ± 0.008; Fig. 1). CD8 T cells specific for peptides other than Melan-A (see above) were not detected in cord blood lymphocytes nor in CD8 single-positive thymocytes of HLA-A*0201-negative individuals (data not shown).

The functional repertoire of ex vivo-sorted allo-A2/Melan-A+ CD8 T cell clones

To dissect the functional heterogeneity of the allo-A2/Melan-A+ repertoire, we derived 41 clones from HLA-A*0201-negative individuals (Table I) by HLA-A*0201/Melan-A multimer-assisted flow cytometry single-cell sorting ex vivo. Following expansion, all 41 clones were specifically stained with HLA-A*0201/Melan-A multimers, either synthesized around the analog Melan-A26–35 A27L peptide or the parental Melan-A26–35 peptide (mean fluorescence ± SD, 2200 ± 1160 and 1140 ± 1140, respectively), but were not stained with HLA-A*0201/peptide multimers synthesized around an irrelevant peptide (influenza matrix flu MA58–66, mean fluorescence: 4 ± 1). HLA-A*0201/Melan-A multimers did not label clones with unrelated specificity (mean fluorescence ± SD, 5 ± 2). Furthermore, the mean fluorescence intensity of A2/Melan-A+ clones obtained from HLA-A*0201-negative or -positive individuals was similar (representative clone 1.5 derived from an HLA-A2+ individual in Fig. 2 and Ref.27). Fig. 2 shows a representative staining of clones derived from one individual (CB886). Altogether, these results illustrate the specificity of the labeling with HLA-A*0201/Melan-A multimeric constructs.


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  		Table I. HLA typing of HLA-A*0201-negative individuals (HD001, HD002, HD 010, and CB886)

 

Figure 2
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  		FIGURE 2. Multimer staining of 11 T cell clones from a HLA-A*0201-negative cord blood (CB886) using ex vivo HLA-A*0201/Melan-A multimer-assisted flow cytometry single-cell sort. T cell clones were stained with multimers synthesized around the A27L Melan-A26–35 A27L analog, the Melan-A26–35 natural decapeptide, the Melan-A27–35 natural nonapeptide, and the irrelevant flu-MA58–66 peptide. Each circle represents one T cell clone, a T cell clone (clone 1.5) derived from a HLA-A*0201 melanoma patient was used as positive control.

 
Avidity of Ag recognition of all clones was assessed by titrating Melan-A parental and analog peptides in functional CTL assays on T2 (HLA-A2+TAP–/–) target cells (Fig. 3). Three groups of clones were clearly distinguishable according to their functional activity: 1) clones that recognized both Melan-A26–35 A27L analog and Melan-A parental peptides with high avidity (Fig. 3a); 2) clones that recognized Melan-A26–35 A27L analog peptide but only very poorly or not Melan-A parental peptide (Fig. 3b); and 3) clones that failed to recognize both Melan-A analog and parental peptides, at least within the range of peptide concentration used (10–4 M or lower, Fig. 3c). There was no correlation between the ability of A2/Melan-A multimers to bind the specific clones (as defined by mean fluorescence intensity of multimer+ cells) and the functional avidity of the latter (as defined by peptide concentration required to obtain 50% maximal lysis in peptide titration experiments; data not shown). The distribution of cells in the different groups was 27% in group 1 (11 clones), 39% in group 2 (16 clones), and 24% in group 3 (10 clones), respectively. These observations are similar to those reported for the Melan-A-specific CD8 T cell repertoire in HLA-A2+ individuals that is composed of three subsets that are indistinguishable based on their capacity to be stained with A2/Melan-A multimer+ CD8 T cells, but that reveal distinct functional activities (28). As observed previously for at least some alloreactive T cell clones (4, 12), the remaining 7% of clones (3 clones) exhibited a peptide-independent recognition based on a high lytic activity against peptide-processing defective T2 cells (data not shown). This activity could not be enhanced by addition of exogenous Melan-A26–35 A27L analog peptide and Melan-A parental peptides. NK-sensitive K562 cells were not lysed by these T cell clones, suggesting that these cells recognize either HLA-A*0201 in a peptide-independent fashion or TAP-independent peptides ubiquitously expressed in human cells.


Figure 3
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  		FIGURE 3. Functional avidity of ex vivo-sorted allo-A2/Melan-A+ T cell clones from HLA-A*0201-negative individuals. Clonal populations were tested for peptide recognition in chromium release assays using T2 cells as targets at a lymphocyte:target ratio of 10:1 in the presence of serial dilutions of the indicated peptides. Three groups of clones were distinguishable: a, clones recognizing the Melan-A26–35 A27L analog and the Melan-A parental peptides; b, clones recognizing only the Melan-A26–35 A27L analog peptide; and c, clones without reactivity against the Melan-A26–35 A27L analog and the Melan-A parental peptides. Representative examples are shown.

 
A total of nine allo-A2/Melan-A+ CTL clones derived from a single individual (HD 009) that exhibited different functional avidities toward the analog and the parental Melan-A peptides based on experiments using peptide-pulsed T2 cells as targets were further selected and analyzed for their functional activity toward HLA-A2+Melan-A+ tumor cells (Me 290). Similarly to Melan-A-specific CTL derived from HLA-A2+ individuals (28), we found that only clones with high avidity toward the Melan-A analog and parental peptides (two of two clones from group 1 as defined above) were able to lyse Me 290 (Fig. 4a). Furthermore, three of five clones with high avidity toward the Melan-A analog peptide only (group 2) could selectively lyse Me 290 tumors pulsed with the analog peptide (Fig. 4b). Finally, two of two clones with no avidity toward the Melan-A analog and parental peptides did not lyse Me 290 (group 3) pulsed or not with the analog peptide (Fig. 4c). Hence, the capacity of these allo-A2/Melan-A+ CTL clones to kill HLA-A2+Melan-A+ tumor cells was related to their intrinsic functional avidity toward the Melan-A peptides. However, we also found that functional recognition by allo-A2/Melan-A+ CTL was not restricted to HLA-A2+Melan-A+ tumors. For instance, six of the nine clones described above (two of two clones from group 1, four of five clones from group 2, and zero of two clones from group 3) were also able to lyse HLA-A2+Melan-A– tumors (NA8-MEL), while two of nine clones (one of two clones from group 1, one of five clones from group 2, and zero of two clones from group 3) lysed HLA-A2–Melan-A+ tumors (Me 260; data now shown). These findings suggest that some clones can recognize antigenic determinants other than HLA-A2/Melan-A complexes, e.g., distinct HLA/peptide complexes, with sufficient avidity to mediate lysis, and that alloreactive CTL from groups 1 and 2 exhibit extended cross-reactive patterns. In contrast, the use of T2 (HLA-A2+TAP–/–) cells pulsed with exogenous peptide as targets as described above in this study offered us the possibility to precisely characterize the functional avidity of the allorestricted Melan-A-specific repertoire upon recognition of HLA-A2/Melan-A complexes only.


Figure 4
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  		FIGURE 4. Cytotoxic activity of allo-A2/Melan-A+ CTL clones toward HLA-A2+Melan-A+ tumor cells. Three groups of clones were functionally tested in chromium release assays using Me 290 tumor cells as targets at the indicated CTL:tumor cell ratios: a, clones recognizing both the Melan-A26–35 A27L analog and parental peptides; b, clones recognizing only the Melan-A26–35 A27L analog peptide; and c, clones without reactivity against the Melan-A26–35 A27L analog and parental peptides. Representative examples are shown.

 
Prominent TCR-{alpha} chain conservation in allo-A2/Melan-A+ CD8 T cell clones

To analyze the TCR repertoire displayed by allo-A2/Melan-A+ clones, RNA was extracted from 31 clones and cDNA was subjected to RT-PCR using a panel of V{alpha} and Vbeta oligonucleotides covering virtually 100% of the TCR repertoire. In line with studies reporting the heterogeneity of the Melan-A-specific TCR repertoire in HLA-A*0201-positive healthy individuals and melanoma patients (19, 20, 29, 30), allo-A2/Melan-A+ clones from three HLA-A*0201-negative individuals (HD 001, HD 002, HD 010; Table I) were found to rearrange many distinct Vbeta gene segments (16 different Vbeta gene segments used; Table II). A slightly increased proportion of clones (5 of 28, 18%) used in this case a single Vbeta (Vbeta14.1). There were no evident constraints on CDR3beta size or sequence. Likewise, Jbeta segment usage was diverse and no significant similarity was found when comparing sequences of defined Vbeta-Jbeta rearrangements.


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  		Table II. TCR-{alpha} and -beta chain amino acid sequences of ex vivo-sorted allo-A2/Melan-A+ T cell clones from HLA-A*0201-negative individuals (HD 001, HD 002, HD 010 and CB886)a

 
Next we asked whether the selective use of V{alpha}2.1 in the Melan-A-specific TCR repertoire observed in HLA-A*0201-positive healthy individuals and melanoma patients was also found in HLA-A*0201-negative individuals. As assessed by RT-PCR analysis, 93% of clones (38 of 41) expressed the V{alpha}2.1 gene segment (Fig. 5 and Table II). The latter was rearranged with a large number (15 segments) of different J{alpha} gene segments, albeit some of them were slightly overrepresented (e.g., J{alpha}31, J{alpha}43). CDR3{alpha} size ranged from four to eight amino acids, and CDR3{alpha} sequences were rather diverse. However, sequence similarities were found within defined V{alpha}-J{alpha} gene segments (e.g., V{alpha}2.1-J{alpha}31 and V{alpha}2.1-J{alpha}43-using clones). Moreover, as reported for high-avidity T cells selected by chronic exposure to Ag (31, 32), some of the identified CDR3{alpha} sequences were either identical (public sequences) or highly homologous among clones derived from different donors (Table II), while no public or highly homologous sequences were found in the CDR3beta region. Notably, these clones that exhibited peptide-independent recognition as indicated above also expressed the V{alpha}2.1 gene segment (Table II).


Figure 5
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  		FIGURE 5. Selective V{alpha}2.1 TCR usage of ex vivo-sorted allo-A2/Melan-A+ T cell clones from HLA-A*0201-negative individuals. Amplified TCR V{alpha}2.1 PCR products from 10 clones were resolved on a 1% agarose gel and revealed by ethidium bromide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We document here the presence of a large T cell repertoire labeled with allo-HLA-A*0201/Melan-A/MART-126–35 multimeric complexes in HLA-A*0201-negative individuals. Strikingly, this T cell pool shares various properties of the previously described Melan-A-specific CD8 T cell repertoire in HLA-A*0201-positive healthy individuals (for review, see Ref.33): 1) those allo-A2/Melan-A+ cells are detectable ex vivo in the thymus and maintained at high frequency in the peripheral circulation, albeit its frequency is one order of magnitude lower; 2) they exhibit a naive surface phenotype; 3) they are endowed with a large functional heterogeneity in terms of functional avidity ranging from low to very efficient recognition of both Melan-A analog and parental peptides presented by HLA-A*0201; and 4) they preferentially use the V{alpha}2.1 TCR gene segment. The existence of a large repertoire of alloreactive, yet Ag-specific CD8 T cells is peculiar to Melan-A, as allorestricted CD8 T cells specific for other self- and non-self peptides were not directly identifiable ex vivo in the same individuals.

Because of the low frequency of alloreactive T cells directed against specific peptides, previous studies have reported the use of several methods to expand them in vitro before analysis. These methods include stimulation with TAP-deficient cell lines loaded with either peptide libraries or peptides, e.g., specific for tumor Ags (8, 9, 12, 15, 17, 18, 34) and, very recently, stimulation with allogeneic monomeric HLA class I-peptide complexes presented on autologous B cells (35). In particular, those studies that used HLA/peptide multimers to successfully isolate alloreactive T cells after stimulation examined the molecular basis of Ag recognition. It could be demonstrated that the generated T cell clones are indeed capable of specifically recognizing the related peptide, but, unexpectedly, may exhibit full cross-recognition of antigenically unrelated peptides (17, 18).

In this study, we elucidate the functional and structural diversity of clonotypes composing the repertoire of an alloreactive, Ag-specific T cell repertoire ex vivo. We show that the vast majority of this repertoire (>90%) is highly specific for the Melan-A analog and parental peptides. Although the relative frequency of peptide-specific vs -nonspecific alloreactive T cells may vary depending on the relationship between the foreign and the autologous T cell’s MHC (36), the results presented here are compatible with previous studies demonstrating a dominance of peptide-dependent alloantigen recognition in both mice and humans (37, 38, 39, 40). Unexpectedly, the Melan-A alloreactive repertoire described here is functionally broad and diverse in that individual clones exhibit a large range of functional avidities, from low to very efficient recognition of both Melan-A analog and parental peptides, and shares these features with its counterpart repertoire in HLA-A*0201-positive individuals (28). In contrast to previous studies that report the possibility to generate T cells from the alloreactive T cell repertoire with particularly high avidity for the allogeneic MHC (9, 12), we demonstrate here by analysis at the clonal level that the avidity for the allo-MHC-peptide complexes is never higher than that of autologous Melan-A-specific T cells.

Interestingly, only a minority (<10%) of allo-A2/Melan-A+ cells appeared to recognize determinants that do not include/involve Melan-A, i.e., clones capable of lysing TAP–/– T2 cells either unpulsed or pulsed with peptides other than Melan-A. These clones may exhibit a peptide-independent activity and recognize polymorphic differences on the MHC molecules themselves (4). Strikingly, HLA-A*0201 multimers incorporating peptides other than Melan-A (e.g., Flu-MA58–66) failed to stain those clones (data not shown), contradicting a peptide-independent interaction with HLA-A2. It is possible that target recognition is rather dependent on TAP-independent peptides presented by HLA-A*0201. Alternatively, those clones may exhibit a low affinity for foreign MHC and require a high density of MHC-peptide complexes (3) that may not be attained by the HLA-A*0201 multimers. In consequence, it is formally possible that multimer staining underestimates the frequency of clones belonging to this category.

In contrast to the selection of an autologous T cell repertoire, which is shaped by the interaction of the TCR with self-peptide-MHC complexes on thymic APCs, alloreactive T cells react to allogenic MHC molecules not used during their selection. In this respect, the mechanisms involving thymic selection of the large HLA-A*0201/Melan-A-specific repertoire in the absence of HLA-A*0201 (i.e., in HLA-A*0201-negative individuals) are unknown. However, a valuable clue is the observation that >90% of allo-A2/Melan-A+ CD8 single-positive thymocytes and circulating CD8 cells in HLA-A2-negative individuals express the V{alpha}2.1 gene segment, while displaying a large variety of different Vbeta chains. It is tempting to speculate that the V{alpha}2.1 gene segment is prone to interact with HLA-A*0201/Melan-A multimers, though additional critical interactions with other structural TCR elements are required for successful Melan-A multimer binding. Indeed, ~10% of CD8 cells express V{alpha}2.1 (41) although only 1/1000 of them (0.01% of CD8 cells) are allo-A2/Melan-A+. The other structural TCR elements necessary for providing specificity to Melan-A are not immediately apparent since the other TCR elements used by Melan-A multimer+ T cells are highly diverse. Altogether, we suggest that the prevalence of the allo-A2/Melan-A+ repertoire is the consequence of the random combination of the V{alpha}2.1 sequence with a set of various but distinct Vbeta sequences that provide specificity to Melan-A. Interestingly, in HLA-A*0201-positive individuals, i.e., in the presence of self-MHC on thymic APCs, the frequency of these cells is one order of magnitude higher than in HLA-A*0201-negative individuals. Although it is not possible to directly measure the contribution of negative selection in eliminating some HLA-A2/Melan-A-reactive thymocytes, this difference provides a minimal estimate of the impact of positive selection in humans.

In conclusion, using HLA-peptide multimer complexes, we identified an ex vivo-detectable alloreactive T cell repertoire specific for the differentiation Ag Melan-A. In-depth characterization of this T cell repertoire shows that it exhibits mainly a peptide-specific recognition pattern, but a broad diversity regarding functional avidity. Considering the dominance of the TCR{alpha} chain in Ag recognition, we document a structural constraint with preferential usage of V{alpha}2.1. Altogether, these data substantiate the importance of peptides presented by allo-MHC and provide new insights into the molecular basis for alloreactivity.


   Acknowledgments
 
We are grateful to Dr. I. Luescher (Ludwig Institute for Cancer Research, Epalinges, Switzerland) for synthesis of multimers and Dr. A. Knuth and Dr. D. Jäger (Department of Oncology, University Hospital Zurich, Zurich, Switzerland) for critical reading of this manuscript. We thank Céline Baroffio, Andrée Porret, Danielle Minaidis, Christine Geldhof, and Martine van Overloop for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the National Center for Competence in Research-Molecular Oncology, a Swiss National Science Foundation special program, and a grant from the Cancer Research Institute/Ludwig Institute for Cancer Research Cancer Vaccine Collaborative. A.Z. and A.G. were supported in part by the Emmy-Noether Program (Zi685-2/3) of the Deutsche Forschungsgemeinschaft. Back

2 Current address: Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129. Back

3 Address correspondence and reprint requests to Dr. Alfred Zippelius, Department of Oncology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. E-mail address: alfred.zippelius{at}usz.ch or Dr. Pedro Romero, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Niveau 5, Aile Est, Avenue Pierre Decker 4, 1005 Lausanne, Switzerland. E-mail address: pedro.romero{at}isrec.unil.ch Back

4 Abbreviation used in this paper: HD, healthy donor. Back

Received for publication June 28, 2005. Accepted for publication December 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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