Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.

Section: Discussionsupporting

confidence: 71%

Visualization of p53264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor

Zhu

Belmont

Price‐Schiavi³

et al. 2006

“…In our previous studies, we showed that the relative peptide binding affinities of both peptides (p11C and p68A) were comparable by the standard iodinated peptide-binding assay (data not shown) as well as by the peptide-binding assay using the TCR tetramer (28,29). Moreover, the immunodominance hierarchy was maintained when the animals were vaccinated with a DNA vaccine construct containing the minimal epitopes of both peptides separated from one another with triple alanine spacers (28,29). This construct likely expresses the same copy number of both epitopes and serves to normalize Ag-processing steps.…”

Section: A Number Of Recent Studies Have Demonstrated the Importance mentioning

confidence: 99%

“…Selected TCR-␣ and TCR-␤ clones were used to screen Ag-specific TCR-␣␤ pairs using the Drosophila expression system as described before (29). In brief, each 5Ј primer specific for sequences of selected clones was designed with addition of a KpnI restriction site, and 3Ј primers specific for TCR-␣ (3Ј BamHTCRA, 5Ј-CCC CAG CCC AGA AAG TGT CTG TGG ATC CGC G-3Ј) and ␤-chain (3ЈBamHTCRB, 5Ј-GAG GCC TGG GGT AGA GCA GAC TGT GGA TCC GCG-3Ј) were designed with a BamHI restriction site at the 3Ј end to amplify the coding sequence upstream of the transmembrane region by PCR.…”

Section: Screening Of Ag-specific Tcr-␣␤ Pairs Using the Drosophila Ementioning

confidence: 99%

Analysis of TCRαβ Combinations used by Simian Immunodeficiency Virus-Specific CD8+ T Cells in Rhesus Monkeys: Implications for CTL Immunodominance

Hasegawa

Moriya

Liu

et al. 2007

Self Cite

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving HIV vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01+ rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-α- and -β-chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of α- and β-chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naive T cell precursor frequency may be predetermined and that this process dictates immunodominance of SIV-specific CD8+ T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.

“…HLA class I-associated Ags have also been detected on cells using Abs (15)(16)(17) or Fab monomers/tetramers (18 -20) specific for given peptide-HLA complexes, allowing semiquantitative determination of surface Ag levels by FACS. A similar approach has used soluble monomers (21,22), dimers (23), or tetramers (24,25) of soluble TCRs to detect class I-restricted Ags, although given the naturally low affinity of TCR for Ag, it remains uncertain how quantitative and Ag sensitive such measurements are.…”

mentioning

confidence: 99%

Quantifying and Imaging NY-ESO-1/LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors

Purbhoo¹,

Sutton²,

Brewer³

et al. 2006

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157–165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10–50 NY-ESO-1/LAGE-1157–165 epitopes per cell.