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3 Domain Mutants of Peptide/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells1
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* Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland;
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and
National Centres of Competence in Research Program on Molecular Oncology, Epalinges, Switzerland
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Abstract |
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3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of 3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy. |
Introduction |
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We asked whether fluorescent pMHC class I multimers unable to bind the CD8 coreceptor could identify high avidity T cells among heterogeneous populations. Binding of pMHC to CD8 involves mainly a conserved, negatively charged loop in the 3 domain of the MHC H chain (residues 223–229) (8, 9, 10). Accordingly, double amino acid substitutions such as D/T to K/A in positions 227/228 (thereafter 227,8KA) abrogate binding to CD8 (11, 12). Besides its role in signal transduction (12), CD8 augments the stability of TCR/pMHC interaction by binding cooperatively to the same pMHC molecule, which results in enhanced TCR avidity (11, 13, 14, 15). Previous reports suggest that CD8 dependence for multimer binding may vary according to TCR avidity (13, 16). In particular, when TCR avidity is relatively low, CD8 contributes significantly to pMHC binding. We reasoned that pMHC class I multimers containing 227,8KA-mutant H chain (thereafter peptide/227,8KA-A2 multimers) may fail to stain T cells with low avidity, but still bind to high avidity, tumor-reactive T cells. To address this hypothesis, we chose the repertoire of CD8 T cells reactive to the self-differentiation Ag Melan-A/MART-1 (thereafter Melan-A) (17, 18). We found that 227,8KA-A2 multimers selectively labeled high avidity tumor-reactive T cells and could be used to isolate such T cells even when present at a frequency as low as 1 in 10,000 CD8 T cells.
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Materials and Methods |
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Cells were collected from HLA-A*0201 individuals, as identified by typing with PCR sequence-specific oligonucleotide probe. Melan-A/wt-A2 T cell clones were derived from PBMCs of a healthy individual (HD 009) and a melanoma patient (LAU 203), from a thymus tissue of a child that had undergone corrective cardiac surgery (T 12), from lymphocyte suspensions of vaccine-site sentinel lymph nodes (LNs) (LAU 371 and 445), and a tumor-infiltrated LN (LAU 392) from melanoma patients (19). A clone specific for the influenza matrix flu-MA58–66 peptide was derived from PBMCs of a healthy individual (HD 008). Clones were expanded by stimulation with 1 µg/ml PHA, 105 irradiated allogeneic feeders, and 100 U/ml IL-2. PBMCs from healthy individuals (HD 006, HD 009) and melanoma patients (LAU 155, 203, 233, 269, 337, 465, 567) and tumor-infiltrated LNs from melanoma patient LAU 352 were collected for ex vivo multimer staining.
pMHC multimers
PE-labeled HLA-A2 multimers were synthesized around the Melan-A26–35 A27L (ELAGIGILTV) peptide (20). The HLA-A2 H chain mutants D227K/T228A were produced by PCR mutagenesis using the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA). Mutant proteins were expressed in Escherichia coli, refolded in vitro, and purified as for the wild-type HLA-A2 molecule. ELISA using the w6/32 mAb were performed to ensure correct folding of MHC H chain/peptide/
2-microglobulin complexes.
Flow cytometry immunofluorescence analysis and sorting
CD8 T lymphocytes were negatively selected from PBMCs and tumor-infiltrated LNs using a CD8 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained either with PE-labeled Melan-A/wt-A2 or Melan-A/227,8KA-A2 multimers for 1 h at room temperature, incubated with appropriate mAbs for 20 min at 4°C, washed, and immediately analyzed on a FACSVantage SE, using CellQuest software (BD Biosciences, San Jose, CA). Multimer staining at 4°C showed similar mean fluorescence intensities (MFIs), albeit values were in this case in general lower. Anti-CD3 FITC mAb and goat anti-rat IgG APC were purchased from BD Biosciences; anti-CD45RA-ECD mAb was from Immunotech (Nyon, Switzerland). Anti-CCR7 rat IgG mAb 3D12 was provided by M. Lipp (Max Delbrück Institute, Berlin, Germany). Melan-A/wt-A2 or Melan-A/227,8KA-A2 T cells were cloned by staining with Melan-A/wt-A2 and Melan-A/227,8KA-A2 multimers, respectively, and single cell sorting was performed using a FACSVantage SE.
Cytolytic activity
T cell clones were tested for their lytic activity in 4-h 51Cr release assays (20). The HLA-A,B-negative B cell line C1R expressing wild-type or mutant HLA-A2 molecules (provided by A. Sewell, Nuffield Department of Clinical Medicine, Oxford, U.K.), the melanoma cell lines Me 290 (Melan-A+/A2+), and Na 8 (Melan-A-/A2+) were used as target cells.
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Results |
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Initially, we selected two Melan-A-specific T cell clones (clones 1 and 5) that were derived from the same individual (T 12). Although both clones were efficiently stained with Melan-A/wt-A2 multimers (Fig. 1a), clone 1 specifically lysed Melan-A-expressing tumor cells, whereas clone 5 did not (Fig. 1b). With the latter, lysis was only observed when saturating doses of the Melan-A peptide were exogenously added. Functional avidity of Ag recognition defined as concentration of peptide required for 50% maximal cytolytic activity was assessed in a 51Cr release assay using C1R cells expressing wild-type HLA-A2 as target cells in the presence of serial dilutions of the Melan-A peptide (Fig. 1c). Clone 1 efficiently recognized the Melan-A peptide (IC50, 10-11 M), whereas the functional avidity of clone 5 was significantly lower (IC50, 10-7 M). We tested whether the two clones could be distinguished based on the staining efficiency obtained with serial dilutions of multimers. However, the MFI of staining with Melan-A/wt-A2 multimers was similar and equally dose dependent for both clones (Fig. 1e). To abrogate the participation of CD8 in multimer binding, we then produced multimers containing 3 domain mutants of the HLA-A2 H chain. Clone 1 was positively stained with Melan-A/227,8KA-A2 multimers, whereas the MFI of clone 5 was similar to that observed with an irrelevant influenza matrix flu-MA58–66 T cell clone (Fig. 1, d and e).
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To further document these findings, we derived a series of Melan-A-specific T cell clones from healthy individuals and melanoma patients by Melan-A/wt-A2 multimer-assisted single cell sorting. We obtained 35 clones that were specifically stained with 300 ng/ml of Melan-A/wt-A2 multimers; this concentration did not result in staining above background of T cell clones with irrelevant specificity (Fig. 1, d and e). All clones expressed comparable levels of TCR and CD8, as indicated by staining with specific mAbs (data not shown).
The Melan-A-specific T cell clones were further analyzed for tumor reactivity, as assessed by lysis of Me 290 (Melan-A+/A2+) or Na 8 (Melan-A-/A2+) melanoma tumor cells in 4-h 51Cr release assays (Fig. 2a). A total of 16 of 35 clones efficiently killed Melan-A-expressing tumor cells (• and gray circles), whereas the 19 remaining clones did not (
). Functional avidity of Ag recognition was assessed on wild-type HLA-A2-transfected C1R cells as targets in the presence of serial dilutions of Melan-A peptide (Fig. 2b). The functional avidity was extremely variable among the clones, with IC50 values varying between 10-13 and >10-6 M. As expected, tumor-reactive clones exhibited lower IC50 (median, 10-11 M) than nontumor-reactive clones (median, 10-8 M). All clones showed comparable intensity of staining with Melan-A/wt-A2 multimers. By contrast, when clones were incubated with Melan-A/227,8KA-A2 multimers (Fig. 2c), none of the 19 nontumor-reactive clones were stained above background (group I, ). These clones exhibited low functional avidity, but nevertheless could be stained with Melan-A/wt-A2 multimers. Among the tumor-reactive T cell clones, 9 of 16 were positively stained with Melan-A/227,8KA-A2 multimers (group II, •), whereas the 7 remaining clones were not (group III, gray circles). Accordingly, lack of multimer labeling in the latter group suggests that a significant fraction of T cells with high functional avidity was dependent on CD8/pMHC interaction. We used a blocking anti-CD8 mAb to confirm CD8-dependent binding of Melan-A/wt-A2 multimers to clones from group III. In contrast to clones from group II, multimer staining of clones from group III decreased when increasing doses of anti-CD8 mAb were added (data not shown).
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Ex vivo identification, isolation, and characterization of Melan-A/227,8KA-A2 multimer+ cells in healthy individuals and melanoma patients
To evaluate the potential of Melan-A/227,8KA-A2 multimers for adoptive T cell therapy, we investigated whether tumor-reactive T cells could be directly identified and isolated from PBLs. To directly compare binding of Melan-A/227,8KA-A2 multimers with naive and Ag-experienced Melan-A-specific T cells, we used PBLs from healthy individuals (n = 2) as well as from melanoma patients with detectable Ag-experienced Melan-A-specific T cells (n = 8). We used both wild-type and mutated multimers in association with anti-CCR7 and anti-CD45RA mAbs (Fig. 3, a–c). The frequency of Melan-A/227,8KA-A2 multimer+ cells in CD8 T cells was reduced as compared with that of Melan-A/wt-A2 multimer+ cells (mean ± SD, 0.3 ± 0.5% and 1.0 ± 1.3%, respectively), i.e., 
30% of Melan-A-specific T cells stained positively with Melan-A/227,8KA-A2 multimers. These cells were detectable ex vivo in all 10 individuals analyzed. Detailed analysis revealed that 20% of naive Melan-A-specific T cells stained positively with Melan-A/227,8KA-A2 multimers, both in healthy individuals and melanoma patients (Fig. 3c). Conversely, 40% of Ag-experienced Melan-A-specific T cells stained positively with Melan-A/227,8KA-A2 multimers (Fig. 3c). It is likely that these cells exhibit a high avidity, CD8-independent TCR/pMHC interaction, similar to the clones from group II identified in Fig. 2. The fraction of Ag-experienced Melan-A-specific T cells that were not stained with mutated multimers may resemble clones from group III in Fig. 2, i.e., exhibit a high avidity, CD8-dependent TCR/pMHC interaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a model, we investigated the T cell repertoire specific for the Melan-A26–35 tumor-antigenic peptide (17). We previously found that a large pool of Melan-A-specific CD8 T cells (10-3 CD8 T cells) is generated and maintained as a naive CCR7+CD45RAhigh population in healthy individuals (25). It is composed of T cells that exhibit a broad range of functional avidities of Ag recognition and significant differences in tumor reactivity (26). A fraction of these naive T cells may become activated and shift toward an Ag-experienced CCR7-CD45RAlow phenotype in 30% of melanoma patients (20, 27). During such an immune response, only T cells with high TCR avidity become part of the Ag-experienced T cell repertoire (A. Zippelius et al., manuscript in preparation). As wild-type multimers do not provide information on the functional avidity of T cell clones (this study and 24, 26), such reagents are ineffective to reliably isolate high avidity Melan-A-specific tumor-reactive CD8 T cells. We explored a novel approach by means of soluble pMHC multimers incorporating mutations in the 3 domain (D227K/T228A) that abrogate binding with the CD8 coreceptor. These reagents have been used recently to label low CD8-expressing Ag-specific T cells (28). As compared with wild-type multimers, 3 domain mutant multimers did not stain CD8 T cells with low functional avidity for Melan-A peptide. These findings corroborate previous observations that CD8 exerted a critical role in pMHC binding to low avidity TCR (13, 29). By contrast, a significant fraction of T cells with high functional avidity did not require CD8 for binding of pMHC multimers. Based on these novel insights into CD8 coreceptor participation of high avidity TCR/pMHC interactions, 3 domain mutant multimers allowed to selectively identify tumor-reactive T cells from freshly obtained lymphocytes of melanoma patients as well as healthy blood donors. We document a high sensitivity and specificity of such multimers, as the frequency (0.01%) of Melan-A/227,8KA-A2 multimer+ cells in CD8 T cells represented the lower limit of detection under the conditions used. The finding that a small fraction of naive tumor-reactive Melan-A-specific T cells could be stained with Melan-A/227,8KA-A2 multimers is compatible with the recent observation that high avidity T cells only compose a minority of the naive Melan-A-specific T cell pool. As the majority of melanoma patients do not exhibit Ag-experienced Melan-A-specific T cells in the peripheral blood (20, 27), 3 domain mutant multimers may open the avenue for generating tumor-reactive T cells from the vast majority of melanoma patients.
To our knowledge, we show for the first time that mutated pMHC class I multimers allow rapid identification and isolation of T cell clones with sufficient functional avidity for in vivo efficacy. The finding that tumor-reactive Melan-A-specific T cells can be stained with the mutated multimers may permit expeditious generation of T cell populations for adoptive transfer therapy in HLA-A2 melanoma patients.
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Acknowledgments |
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Footnotes |
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2 M.J.P. and V.R.-G. equally contributed to this work.
3 Current address: Center for Molecular Imaging Research, Harvard Medical School, Charlestown, MA 02129.
4 Address correspondence and reprint requests to Dr. Alfred Zippelius at the current address: 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
5 Abbreviations used in this paper: pMHC, peptide/MHC; LN, lymph node; MFI, mean fluorescence intensity.
Received for publication April 7, 2003. Accepted for publication June 13, 2003.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T-cell receptors and coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands. Trends Immunol. 23:408.[Medline]
3 domain of HLA-A2. Nature 345:41.[Medline]
() and HLA-A2. Nature 387:630.[Medline]
3 domain mutants of MHC class I/peptide complex. Immunity 14:591.[Medline]
Lys substitution in the acidic loop of major histocompatibility complex class I 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions. J. Exp. Med. 184:1671.This article has been cited by other articles:
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|
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|
|
|
|
 |
|
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|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
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|
|
|
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M. Cebecauer, P. Guillaume, P. Hozak, S. Mark, H. Everett, P. Schneider, and I. F. Luescher Soluble MHC-Peptide Complexes Induce Rapid Death of CD8+ CTL J. Immunol., June 1, 2005; 174(11): 6809 - 6819. [Abstract] [Full Text] [PDF]
|
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 S. Oh, L. P. Perera, D. S. Burke, T. A. Waldmann, and J. A. Berzofsky IL-15/IL-15R{alpha}-mediated avidity maturation of memory CD8+ T cells PNAS, October 19, 2004; 101(42): 15154 - 15159. [Abstract] [Full Text] [PDF]
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25%, marked with a bar on this axis, was considered significant. The y-axis shows the MFI obtained by staining with 300 ng/ml. The bar on this axis represents background MFI obtained by staining all Melan-A-specific T cell clones with irrelevant influenza matrix/A2-wt multimers, defined as mean MFI + 3 x SD = 43. Each circle represents one T cell clone, shown as open (group I), filled (group II), and gray circles (group III). b and d, Correlation between functional avidity and efficiency of multimer staining using Melan-A/wt-A2 multimers (b) or Melan-A/227,8KA-A2 multimers (d). The x-axis shows the concentration [M] of Melan-A26–35 A27L peptide required to obtain 50% maximal lysis in peptide titration experiments (IC50) using C1R cells expressing either wild-type (b) or mutant (d) HLA-A2 molecules as target cells. The y-axis shows the MFI obtained by multimer staining. As described above, each circle represents one T cell clone.