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. 2018 Jan 30;115(5):1039-1044.
doi: 10.1073/pnas.1701237115. Epub 2018 Jan 16.

Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology

Pierre Vantourout  1   2 , Adam Laing  1   2 , Martin J Woodward  1   2 , Iva Zlatareva  1   2 , Luis Apolonia  3 , Andrew W Jones  4 , Ambrosius P Snijders  4 , Michael H Malim  3 , Adrian C Hayday  5   2
Affiliations

Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology

Pierre Vantourout et al. Proc Natl Acad Sci U S A. .

Abstract

The long-held view that gamma delta (γδ) T cells in mice and humans are fundamentally dissimilar, as are γδ cells in blood and peripheral tissues, has been challenged by emerging evidence of the cells' regulation by butyrophilin (BTN) and butyrophilin-like (BTNL) molecules. Thus, murine Btnl1 and the related gene, Skint1, mediate T cell receptor (TCR)-dependent selection of murine intraepithelial γδ T cell repertoires in gut and skin, respectively; BTNL3 and BTNL8 are TCR-dependent regulators of human gut γδ cells; and BTN3A1 is essential for TCR-dependent activation of human peripheral blood Vγ9Vδ2+ T cells. However, some observations concerning BTN/Btnl molecules continue to question the extent of mechanistic conservation. In particular, murine and human gut γδ cell regulation depends on pairings of Btnl1 and Btnl6 and BTNL3 and BTNL8, respectively, whereas blood γδ cells are reported to be regulated by BTN3A1 independent of other BTNs. Addressing this paradox, we show that BTN3A2 regulates the subcellular localization of BTN3A1, including functionally important associations with the endoplasmic reticulum (ER), and is specifically required for optimal BTN3A1-mediated activation of Vγ9Vδ2+ T cells. Evidence that BTNL3/BTNL8 and Btnl1/Btnl6 likewise associate with the ER reinforces the prospect of broadly conserved mechanisms underpinning the selection and activation of γδ cells in mice and humans, and in blood and extralymphoid sites.

Keywords: butyrophilins; endoplasmic reticulum; evolutionary conservation; gamma delta T cells; zoledronate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Requirements for BTN3 proteins for Vγ9Vδ2+ T cell responses to PAgs. (A) Schematic domain organization of BTN3A proteins (blue, IgV; gray, IgC; yellow, TM; red/pink B30.2 domain). (B) RT-PCR for the BTN genes listed on the left and for GAPDH, using RNA of denoted cell lines (Upper). (C) Cell lines listed were pulsed for 12 h with Zol (0.04–50 µM, 5× dilution steps) and cocultured for 5 h with a polyclonal Vγ9Vδ2+ T cell line in the presence of a CD107a antibody: results are means of duplicate stimulations, representative of two independent experiments. (D) CRA123 cells were transfected with pCSIGPW, either EV or encoding the indicated BTN3 proteins, single or in combinations, before treatment with Zol and assayed for their capacity to promote Vγ9Vδ2+ T cell degranulation as in C.
Fig. 2.
Fig. 2.
Heteromeric interactions regulate BTN3 surface expression. (A) CRA123 cells were lysed 48 h posttransfection with the indicated constructs (Upper); one fraction was used to measure total protein expression (INPUT), and the remainder was immunoprecipitated with protein G beads coated with an anti-FLAG antibody (IP). Samples were loaded onto 12% SDS/PAGE and analyzed by Western blot (WB) with the indicated antibodies. Representative of three experiments. (B) IP-MS volcano plot of protein intensity ratios comparing anti-FLAG pull-downs performed on lysates from CRA123 cells transduced with FLAG-BTN3A1+HA-BTN3A2 versus EV control (Top) or FLAG-BTNL3+HA-BTNL8 versus EV (Bottom). X-axis: log2 LFQ protein intensity ratios (Welch Difference); y axis: log10 sum of intensities. (C) CRA123 cells were transfected with the indicated constructs and BTN3 expression assessed by flow cytometry (with anti-pan-BTN3A) 12, 24, or 48 h later. Representative of five independent experiments. (D and E) CRA123 cells were cotransfected with FLAG-tagged constructs (indicated on the right-hand side) and WT constructs (indicated in each quadrant). Anti-FLAG-reactive proteins expression was assessed by flow cytometry 24 h posttransfection. Representative of five independent experiments.
Fig. 3.
Fig. 3.
BTN3A1 surface expression versus functional competence. (A) Schematic representation of the BTN3A1 constructs used to transfect CRA123 cells before treatment with 5 µM Zol. (B) Expression was monitored by flow cytometry on a fraction of the samples, whereas the remainder (C) was coincubated with Vγ9Vδ2+ cells in the presence of an anti-CD107a antibody to assay function. Data are means of three stimulations ± SD. Representative of three independent experiments. P values are relative to 3A1.
Fig. 4.
Fig. 4.
Motifs regulating BTN3 surface expression and function. (A) Wild-type and BTN3A1/3A2 chimaeras used to transfect CRA123 cells, pulsed with 5 µM Zol. (B) Expression was monitored by flow cytometry on a fraction of the samples, whereas the residual (C) was coincubated with Vγ9Vδ2+ cells and assayed as described for Fig. 3. (D) CRA23 cells were transfected with the indicated WT or deletion mutants of BTN3A2; expression was monitored by flow cytometry on a fraction of the samples, whereas the residual (E) were treated as in C. Data are means of three stimulations ± SD. Representative of three independent experiments. (F) Partial amino acid sequences of BTN3A1 (241–360) and BTN3A2 (241–334). TM domains, yellow; start of BTN3A1 B30.2 domain, red; putative ER association motifs, purple.
Fig. 5.
Fig. 5.
BTN3A1 and BTN3A2 associate with the ER and regulate each other’s trafficking. (A) CRA123 cells were transfected with the indicated constructs (Left) and permeabilized and stained with anti-FLAG-APC/anti-PDI-PE (ER marker) or anti-FLAG-PE/anti-Gm130-APC (Golgi marker) 48 h posttransfection. Data were acquired on ImageStreamX and processed in IDEAS, and histograms were generated in R. Representative of three independent experiments. BDS, colocalization score; BF, bright field; O/L, overlay. (B) CRA123 cells were cotransfected with the indicated BTN3 constructs and mCherry-tagged Sec61β. Cells were permeabilized 48 h posttransfection, stained with anti-FLAG, and analyzed by confocal microscopy. (Scale bars, 20 µm.)
Fig. 6.
Fig. 6.
ER association of BTN3 is critical for its function. (A) CRA123 cells were transfected with the indicated WT or ER-retention motif mutants of BTN3A1 (Upper) and BTN3A2 (Bottom), and cell surface expression assessed by flow cytometry. Representative of three independent transfections. (B) CRA123 cells were cotransfected with the indicated constructs before treatment with 5 µM Zol. Cell surface BTN3A expression was monitored and (C) the functional potential of the transfectants measured as described for Fig. 4. Data are the mean of three stimulations ± SD. Representative of three independent experiments. P values are relative to the combination of WT BTN3A1+BTN3A2. Purple arrow in C denotes functional deficiency of cells whose BTN3A expression is shown in purple-boxed plot in B. (D) CRA123 cells were transfected with pCSIGPW, either as an EV or encoding indicated BTN3 WT or ER motif mutants before a 12-h treatment with Zol (0.04–50 µM, 5× dilution steps) and assayed for their capacity to promote Vγ9Vδ2+ T cell degranulation. Data points are the mean of duplicate stimulations and representative of two independent experiments.
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