Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding

Bodinier, Marie; Peyrat, Marie-Alix; Tournay, Claire; Davodeau, François; Romagné, François; Bonneville, Marc; Lang, François

doi:10.1038/76292

Cited by 142 publications

(150 citation statements)

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“…Positive microcultures from a single donor were pooled and sorted using multimer-coated beads [24,25]. M170 and M117 TIL populations were directly sorted with the same procedure.…”

Section: Resultsmentioning

confidence: 99%

“…HLA-A Ã 0201/ Meloe-1, Melan-A and BMLF1 a3-mutated monomers were generated by our recombinant protein facility (INSERM U892, Nantes, France), as previously described [24]. MELOE-1-specific T-cell lines were then labeled with a panel of 24 anti-Vb mAb (Vb1, À2, À3, À4, À5.1, À5.2, À5.3, À7, À7.2, À8, À9, À11, À12, À13.1, À13.2,À13.6, À14, À16, À17, À18, À20, À21.3, À22 and -23) to analyze their diversity (Immunotech BeckmanCoulter, Marseille, France).…”

Section: Tetramer and Vb Labelingmentioning

confidence: 99%

“…We included in this study, two melanoma TIL populations (M170 and M117), spontaneously enriched for MELOE-1/A2-specific T cells, containing 2.3 and 2.8% antigenspecific T cells, respectively (Fig. 3A, lower panel and 3B).Positive microcultures from a single donor were pooled and sorted using multimer-coated beads [24,25]. M170 and M117 TIL populations were directly sorted with the same procedure.…”

mentioning

confidence: 99%

“…After a 14-day culture period, the specificity of each microculture was tested by double labeling using a specific HLA-A Ã 0201/MELOE-1 36-44 tetramer and an anti-CD8 antibody (BD Biosciences, France). Immunomagnetic sorting of MELOE-1-specific T cells was performed on TIL and specific microcultures derived from PBMC, using M280 streptavidin magnetic beads (Dynal, France) coated with specific recombinant pMHC complexes, as previously described [24,25]. MELOE-1/A2-specific T-cell lines were then cultured, expanded and cloned as described [32,33].…”

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confidence: 99%

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Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

et al. 2010

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We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2 1 melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE-1/A2-specific T-cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE-1-specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1 Â 10 5 CD8 1 cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Va12.1 chain for 17/18 MELOE-1-specific T-cell clones derived from healthy donors or patients, associated with diverse Vb chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE-1 36-44 peptide and against HLA-A2 1 melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE-1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.Key words: Immunotherapy . Melanoma . MELOE-1 . T-cell repertoire . TCR Introduction A key advance in tumor immunology in the past 15 years has been the elucidation of the antigenic basis of tumor cell recognition and destruction, which has opened the way to development of antigen-based immunotherapy in cancer patients. Tumor-associated antigens (TAA) have been grouped into categories according to their expression pattern in neoplastic and normal tissues. TAA can be roughly subdivided into two main classes: proteins expressed specifically by tumor cells, and proteins that are expressed by both tumor cells and by normal cells to a significant level. The first category includes cancergermline antigens, proteins expressed by unconventional gene expression and mutated antigens (see [1] for review). TAA belonging to the second category are the differentiation antigens, mainly expressed in the melanocytic lineage, and the antigens overexpressed by various tumor tissues. Although the immunogenicity of a number of TAA has been documented in clinical Ã These authors contributed equally to this work. We recently showed a correlation between the infusion of T cells reactive against an HLA-A2 epitope derived from another melanoma antigen, MELOE-1, and time to progression of patients treated with autologous tumor infiltrating lymphocytes (TIL) [10]. This antigen is encoded by the meloe gene, overexpressed in human melanoma cell lines, when compared to normal melanocytes, and dramatically underexpressed in other cancer cell lines (colon, renal, breast and lung carcinoma cell lines) and in a variety of normal tissues [10]. This expression profile and the potential involvement of MELOE-1-specific...

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“…Positive microcultures from a single donor were pooled and sorted using multimer-coated beads [24,25]. M170 and M117 TIL populations were directly sorted with the same procedure.…”

Section: Resultsmentioning

confidence: 99%

Section: Tetramer and Vb Labelingmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

et al. 2010

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“…Purified CD8 + T cells were then incubated with the PEconjugated HLA-A*0201-pp65 (495-503) tetramer (Immunotech, Marseille, France) as described elsewhere [35] and anti-CD8-FITC (Becton Dickinson, San Jose, CA) for 45 min at room temperature. One microgram of tetramer-PE was used per 1×10 6 cells in PBS plus 2% FCS and 2 mM EDTA.…”

Section: Purification Of Pbmc By Using the Hla-a*0201-pp65 495-503 Tementioning

confidence: 99%

Characterization of antigen‐specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65

Hamel¹,

Rohrlich²,

Baron³

et al. 2003

Eur J Immunol

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Human cytomegalovirus (HCMV) and adenovirus cause significant morbidity and mortality in immunocompromised hosts undergoing allogeneic stem cell transplantation. We have previously established a procedure for the generation of polyclonal CTL with specificity against adenovirus and HCMV using a recombinant adenovirus encoding the HCMV pp65 protein (RAdpp65). However, specific CTL expanded after in vitro culture steps were subjected to several in vitro restimulations and, depending on the protocol adopted, this could lead to a selection bias, compromising the clinical benefit. To determine which part of the memory repertoire is selected after in vitro restimulation, we have followed the specificity and clonal composition of pp65-peptide-specific CD8 + T cells in HLA-A*201 individuals before and after repeated in vitro restimulation of cells with RAdpp65, combining HLA tetrameric complexes and immunoscope analysis. Tetramer staining showed that, after in vitro restimulation, up to 60% of CD8 + T cells were virus-specific. Immunoscope analysis showed that the predominant TCRBV diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure. Altogether, these results suggest that the use of RAdpp65 to induce CMV-and adenovirus-specific CTL may be appropriate for immunotherapy.

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Identification of Human Antigen‐Specific T Cells Using MHC Class I and Class II Tetramers

2004

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Major histocompatibility complex (MHC) tetramers typically consist of a fluorophore‐streptavidin complex and biotinylated soluble MHC molecules carrying a peptide of interest. Tetramers bind to T cell receptors (TCR) that recognize the MHC molecule/peptide combination with high specificity. Native MHC molecules are expressed as cell‐surface glycoproteins capable of binding a variety of peptides generated from the degradation of self and non‐self proteins for display to T cells. The human MHC gene locus is highly polymorphic, with >800 class I and >500 class II alleles currently identified. This heterogeneity contributes to the uniqueness of each person's immune system. This unit describes procedures for labeling CD8+ T cells with MHC class I tetramers and CD4+ T cells with MHC class II tetramers. The protocols can be used for detecting and enumerating human antigen‐specific T cells. Both CD8+ and CD4+ antigen‐specific T cells are rare events and require that sufficient numbers of cells be evaluated. To minimize nonspecific tetramer binding contributed by irrelevant cell populations, a cumulative gating strategy using positive selection and/or exclusion gating is described.

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Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding

Cited by 142 publications

References 14 publications

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

Characterization of antigen‐specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65

Identification of Human Antigen‐Specific T Cells Using MHC Class I and Class II Tetramers

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