Abstract
SummaryVγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1’s P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.
Highlights
Radiation Hybrids Identify BTN2A1 as Essential for P-Ag Sensing We showed previously that a T cell hybridoma expressing the Vg9Vd2 MOP T cell receptor (TCR) produced interleukin (IL)-2 in co-culture with BTN3A1-transduced Chinese hamster ovary (CHO) cells incubated with the anti-BTN3A1 monoclonal antibody 20.1 but exhibited a complete lack of response to hydroxy-3-methyl-but2-enyl pyrophosphate (HMBPP) or Zol (Riano et al, 2014; Starick et al, 2017)
To identify Factor X, we used an unbiased genome-based approach involving generation of radiation hybrids between CHO-Chr 6 cells and BTN3A1-transduced hypoxanthineaminopterin-thymidine (HAT)-sensitive rodent fusion partners and subsequent analysis of their capacity to stimulate P-Ag sensing by Vg9Vd2 T cells (Figure 1A)
We postulated that comparison of the human gene products transcribed in stimulatory radiation hybrids would allow mapping of the gene(s), which alongside BTN3A1 are mandatory for PAg-mediated stimulation
Summary
Generation of radiation hybrids CHO Chr 6 (106 or 107 cells) were irradiated at Faxitron CP160 (program 160 kV, 6.3 mA, 300 Gy: 60 min, 100 Gy: 20 min). The cell pellet was gently tapped and 1 mL PEG1500 was added slowly over a minute with gentle mixing in a prewarmed water-bath. After addition of PEG, cells were resuspended in 50 mL warm serum free RPMI and incubated for 30 min, followed by centrifugation at 461 g for 5 min and careful resuspension in RPMI supplemented with 10% FCS at 104 cells/mL. 100 ml of 2X HAT was added and cells were selected for two weeks. The selected clones were supplemented with HT medium and further seeded at limiting dilutions to obtain single cell clones which were tested for P-Ag mediated activation of our Vg9Vd2 TCR (MOP) transductants.
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