Cloning

The cDNA sequences for the expression of γδ TCRs outside the CDR3 were obtained from the international ImMunoGeneTics information system (http://www.imgt.org) and the HLA-DR cDNA sequences were obtained from the Immuno Polymorphism Database. The CDR3 sequences for the TCR02, TCR04, and TCR05 were described in Ravens et al. (2017). The cDNA sequences were synthesized by BIOCAT and cloned into the retroviral Vectors pBulletIRESneo and pBulletIRESpuro (both vectors kindly provided by Jürgen Kuball’s lab; University Medical Center Utrecht, Utrecht, Netherlands) via the restriction sites for NcoI and BamHI. For the soluble TCR expression, the extracellular domains of the respective γδ TCRs were amplified by PCR by using the NEBNext 2× PCR Master Mix from New England Biolabs and the following primers: Vγ3-forward: 5′-GAA​GAT​CTT​CCA​GCA​ATC​TGG​AGG​G-3′; Vγ3-reverse: 5′-GCT​CTA​GAC​TGC​AGC​AGC​AGG​GTA​TC-3′; Vδ1-forward: 5′-GAA​GAT​CTG​CCC​AGA​AGG​TGA​CCC​AGG-3′; Vδ1-reverse: 5′-GCT​CTA​GAC​TTC​TCT​GTG​TGC​ACG​ATG​GC-3′.

The PCR product was cloned into the vector pT1205 (Krey et al., 2010) via the restriction sites XbaI and BglII. For the in silico design of CDR-exchange mutants, the CDR1 of Vδ1 was replaced by the CDR1 of Vδ3, the CDR2 of Vδ1 by the CDR2 of Vδ2, and the HV4 of Vδ1 by the HV4 of Vδ2. The γ-chain hybrid Vγ8_04* was designed by replacing the CDR3 of a Vγ8-chain with the CDR3 of the Vγ3-chain of the TCRs 02, 04, and 05. The CDR-exchange mutants as well as the hybrid Vγ8_04* were then synthesized by BIOCAT and cloned into the pBullet vectors, respectively.