Identification of Human T Cell Receptor γδ-recognized Epitopes/Proteins via CDR3δ Peptide-based Immunobiochemical Strategy

Abstract

Human T lymphocytes, bearing T cell receptor (TCR) ␥␦, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCR␥␦ have been defined so far. To investigate antigenic epitopes/proteins recognized by ␥␦T cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR ␦ chain (CDR3␦) peptide-mediated epitope/protein-binding assays. CDR3␦ peptides synthesized using the CDR3 region in TCR V␦2 chain were validated for their binding specificity to target cells or tissues. These CDR3␦ peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionizationtandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCR␥␦, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to ␥␦T cells but also functionally activate ␥␦T cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCR␥␦. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of V␦2 ␥␦T cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCR␥␦. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by ␥␦T cells. T lymphocytes are classified structurally into two types according to different T cell receptors (TCR)3 ␣␤ or ␥␦. TCR␣␤ specifically recognize antigenic peptides (epitopes) presented by MHC I/II molecules. Although many peptide ligands have been identified for TCR␣␤ (1), only a few ligands have been identified for TCR␥␦ so far (2, 3).Human ␥␦T cells account for ϳ5% of CD3 ϩ T cells in the peripheral blood, but constitute a major T cell subset in other anatomic locations, such as the intestine. In the peripheral blood of healthy individuals, TCR V␦2 chain pairing with one particular V␥9 chain is expressed on 50 -90% of the ␥␦T cells, whereas intestinal intraepithelial ␥␦T cells frequently express the V␦1 gene, which can associate with different V␥ elements (4). The antigen-binding site of TCR␥␦ is formed primarily from three complementarity-determining regions (CDRs) contributed by each V␥ and V␦ domain. CDR1 and CDR2 fragments are encoded by germ line V genes, whereas the CDR3 is formed by somatic rearrangement of V(D) and J fragments. Sequence diversity in antigen receptors is not evenly distributed among all six CDRs but is highly concentrated in one or two CDR3. It had been proposed that the principal antigen specificity of an immunoglobulin or TCR is derived from its most diverse CDR3 fragments (5).In addition to TCR diversity, structures of the TCR-CD3 complex, and tissue distribution, the pattern of antigenic recognition is another key difference between ␥␦T cel...

Antigen Specificity of γδ T Cells Depends Primarily on the Flanking Sequences of CDR3δ

“…With this novel strategy, we have successfully identified seven tumor-related epitopes, two hepatitis B virus (HBV) infection-related antigenic epitopes, and two self proteins including heat shock protein (HSP) 60 and human mutS homolog 2 (hMSH2) that are recognized by human ␥␦ TCR (7). These results further support that the primary sequence of CDR3␦ in ␥␦ TCR determine the specificity of antigen binding.…”

“…Nine peptides as putative epitopes were identified, among which seven were tumor-related and two were HBV infection-related. BLAST searches revealed that most matched proteins were conserved proteins of prokaryotes (7). Results suggest the possibility that ␥␦ TCR recognizes some conserved molecules.…”

“…We mutated V, N-D-N, or J fragments of a V␦2 TCR CDR3 sequence (OT3) in peptide and engineered ␥␦ TCR. We found that the conserved flanking regions of CDR3␦ play a critical role in antigenic binding to OEC cells/tissues or hMSH2 protein, a new ligand for ␥␦TCR we found recently (7). Furthermore, we have identified the cysteine residue in V fragment and the leucine residue in J fragment as critical residues in the binding activity of ␥␦ TCR.…”

Defining the nature of human γδ T cells: a biographical sketch of the highly empathetic

“…76 More recently, another putative Vc9Vd2 T cell tumor antigen, human MutS homologue 2 (MSH2), was identified using a peptide-based affinity screening system for the CDR3 of the d chain. 77 MSH2 is normally located in the nucleus where it functions as a DNA mismatch repair gene, but it is often mutated in a number of different types of epithelial cancers and can be ectopically expressed. 78 Transformation of normal human B cells by Epstein-Barr virus or subjecting renal carcinoma cell lines to oxidative stress also led to an increased surface expression of MSH2 and rendered them susceptible to Vc9Vd2 T cell-mediated cell lysis that could be blocked by the use of anti-MSH2 antibodies or downregulation of its gene expression.…”

CDR3δ -grafted γ9δ2T cells mediate effective antitumor reactivity

“…We found that synthesized OEC-derived CDR3d-peptides could mimic the TCRcd and bind specifically to tumor cell lines and tissues 25 A CDR3d-grafted antibody also showed specific binding to several tumor cells 19 We used synthesized OEC-derived CDR3d peptides as probes to search for putative TCR-binding epitopes by screening a 12-mer random peptide phage-displayed library and identified several putative TCR ligands within tumor cell extracts by affinity chromatography and liquid chromatography/electrospray ionization tandem mass spectrometry analysis. Nine peptides and two proteins that bound the cdTCR been identified including human mutS homolog 2, a novel cdTCR ligand 15,31 Another research group also found that the CDR3d region was responsible for the T22 protein binding specificity of the Vd2 T cells clone G8 32 We had previously constructed full-length PBMCs-derived c9 and d2 chains using overlapping PCR. The fulllength c9 and d2 chains with tumor antigen-specific CDR3 regions were stably transfected into J.RT3-T3.5 by electroporation and expressed functional c9d2TCR 26 In this study, we induced c9 and d2 (OT3) expression in PBLs using a retroviral vector and generated efficient tumor reactive T cells.…”