Alanine Mutagenesis in the Complementarity Determining Region 3 of the MTB and HIV-1 Peptide-Bispecific T Cell Receptor Beta Chain Affects Ligand Recognition

Abstract

Mycobacterium tuberculosis/human immunodeficiency virus (MTB/HIV) coinfection presents a special challenge to the prevention and treatment of tuberculosis and HIV/AIDS. Adoptive transfer of high-affinity T cell receptor (TCR) gene-modified T cells against MTB and HIV antigens is a promising approach to treating MTB/HIV coinfected patients whose cellular immunity is obviously disordered. We have previously successfully identified that a bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A*0201+ healthy individual using the complementarity determining region 3 (CDR3) spectratype analysis recognizes both MTB Ag85B199–207 and HIV-1 Env120–128 peptide. However, it has not been known how residues on CDR3 loops, which have been shown to play a leading role in antigen binding and specificity contribute to the bispecific TCR contact with the peptide–major histocompatibility complex (MHC) complexes. In this study, we provided an extensive investigation of residues in the predicted CDR3 of the bispecific TCR beta (β) chain using alanine scanning mutagenesis. Our data showed that three of the five substituted residues (G115A, T116A, A117G) in CDR3β of the bispecific TCR caused a significantly diminished T cell response to antigen, whereas the remaining two substituted residues (D114A, S118A) resulted in completely eliminated response, thus identifying the two residues that were particularly critical for the recognition of peptide–MHC in the bispecific TCR. These findings will provide an imperative foundation for generating an improved high-affinity bispecific TCR for use in T cell adoptive immunotherapy for MTB/HIV coinfected individuals.

Functional analysis of the antigen binding sites on the MTB/HIV-1 peptide bispecific T-cell receptor complementarity determining region 3α

“…The furin cleavage site could remove the P2A peptide remaining on the TCR β chain, and the SGSG linker has been demonstrated to efficiently synthesize functional TCRs compared to the 2A sequence only [ 17 ]. To promote preferential pairing and improve cell surface expression of the exogenous TCR, nine critical amino acids (AAs) in the TCR α and β C regions were replaced by murine counterparts as described previously [ 15 ]. The amino acid residues 112–116 (ESPYS) of the TCR CDR3α were targeted for alanine substitution, which designated E112A, S113A, P114A, Y115A and S116A, respectively (Fig.…”

“…The research had been carried out in accordance with the World Medical Association Declaration of Helsinki and was approved by the ethics committee of the Southern Medical University. Monocyte-derived dendritic cells (DCs) and CD8 + T cells were generated as previously described [ 15 ]. The 293T, T2 and J.RT3-T3.5 (kindly provided by Dr Wei He, Peking Union Medical College, Beijing, China) cells were routinely cultured.…”

“…The wild-type (WT) bispecific TCR has generated as described in previous study [ 15 ]. Briefly, for generating the variant TCR α-chains, the 5’-end β15-fu-SGSG-P2A-α17 fragment was amplified from the WT plasmid pHAGE-β15-fu-SGSG-P2A-α17-IRES-IZsGreen using the forward primer P1 and the reverse primer P3, P5, P7, P9, or P11, and the 3’-end β15-fu-SGSG-P2A-α17 fragment was generated using the forward primer P4, P6, P8, P10, or P12 and the reverse primer P2.…”

“…Intracellular cytokine staining was performed to assess the level of IFN-γ produced by T cells and was performed as previously described [ 15 ].…”

“…Previously, we have successfully identified a MTB Ag85B 199–207 and HIV-1 Env 120–128 peptide-bispecific TCR screened out from peripheral blood mononuclear cells (PBMCs) of a HLA-A∗0201 + healthy individual and have further studied that how residues on the predicted CDR3 of the β chain contribute to the bispecific TCR contact with the peptide-MHC [ 14 , 15 ]. However, it is not clear which residues in the predicted CDR3α of the bispecific TCR play a crucial role in ligand recognition.…”