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. 2018 Dec;19(12):1352-1365.
doi: 10.1038/s41590-018-0253-5. Epub 2018 Nov 12.

The γδTCR Combines Innate Immunity With Adaptive Immunity by Utilizing Spatially Distinct Regions for Agonist Selection and Antigen Responsiveness

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Free PMC article

The γδTCR Combines Innate Immunity With Adaptive Immunity by Utilizing Spatially Distinct Regions for Agonist Selection and Antigen Responsiveness

Daisy Melandri et al. Nat Immunol. .
Free PMC article

Abstract

T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities typify adaptive immunity. Conversely, large compartments of γδTCR+ intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (Vγ chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.

Conflict of interest statement

Competing Interests Statement

O.N. and O.P. are employees of GammdaDelta Therapeutics. O.N., O.P., and A.H.C. are equity holders in GammaDelta Therapeutics.

Figures

Figure 1
Figure 1. Primary Vγ7+ IEL exhibit a semi-invariant TCR usage.
a, TCR deep-sequencing analysis of Vγ7 CDR3 length distribution (number of amino acids) of sorted Vγ7+ cell RNA. Data are expressed as the relative proportion of reads for each length, pooled from three independent sorts from pooled mice IEL (n = 12). Relative amino acid composition is shown for the most common length (13) using WebLogo (black, hydrophobic; green, basic; red, acidic; blue, polar). b, TCR deep-sequencing data from (a) analysed to determine Trdv gene usage by Vγ7+ cells. Data derived from Vγ7+ cells sorted from pooled mice IEL (n = 4). Representative of three independent sorts. c, TCR deep-sequencing data from (a) was further analysed to determine Vδ7, Vδ2-2, and Vδ6D-1/2 CDR3 length distribution and composition for the most common length (16, 16 and 13, respectively), as in (a). d, Flow cytometry analysis of CD25 (left) and CD122 (centre) expression by primary Vγ7+ IEL after co-culture with MODE-K.EV or MODE-K.l1l6 cells overnight. Data expressed as mean±s.d. of the proportion of positive Vγ7+ IEL (CD25) or gMFI of Vγ7+ IEL (CD122) in individual co-cultures (n = 4). Corresponding examples of raw flow cytometry plots are shown (right). Representative of five experiments. e,f, Flow cytometry analysis of CD3 (e) and CD71 (f) expression by Vγ7+ IEL after co-culture with MODE-K.EV or .l1l6 cells. Data expressed as mean±s.d. of gMFI in co-cultures from individual mice (n = 4). Corresponding examples of raw flow cytometry plots are shown (right). Representative of five (CD3) and two (CD71) experiments. *P < 0.05, **P < 0.001.
Figure 2
Figure 2. Expression of murine Vγ7 TCR confers responsiveness to Btnl1+Btnl6.
a, Flow cytometry analysis of γδTCR, and Vγ7 or Vγ5 expression on J76 cells transduced with the indicated TCRs, 72 h post-transduction. Representative of 2 independent transductions. b, Flow cytometry analysis of γδTCR and CD69 expression by J76 cells transduced with the indicated TCRs and co-cultured with control IgG, α-CD3e (OKT3), MODE-K.EV, or MODE-K.l1l6 for 5 h. Representative of three independent experiments. c, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (right) by J76 cells transduced with the indicated TCRs (see Table 1 for details) and co-cultured with MODE-K.l1l6 or α-CD3ε for 5 h. Data expressed as mean±s.d., normalized to MODE-K.EV and control IgG, respectively; pooled from three independent experiments. d, Flow cytometry analysis of Nur77 expression (left) by J76-mo5 cells co-cultured with the indicated antibodies or cell lines for 2 h. Data expressed as mean±s.d. of the proportion of Nur77+ cells; pooled from three experiments. Corresponding examples of raw flow cytometry contour plots are shown (right). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. Expression of human Vγ4 TCR confers responsiveness to BTNL3+BTNL8.
a, RNAscope analysis of TCRδ (TRDC, left), dihydropicolinate reductase (dapB, centre; negative control), or Peptidyl-prolyl Isomerase B (PPIB, bottom; positive control) expression in paraffin-embedded human colon sections. Scale bars, 1 mm. Representative of biopsies from multiple donors (n = 3). b, Flow cytometry analysis of TCR downregulation (top) and CD25 expression (bottom) by human colonic lymphocytes after co-culture with 293T.EV or 293T.L3L8 cells overnight. TCR downregulation data expressed as mean±s.d. of independent co-cultures from multiple donors (n = 11) with 293T.L3L8 cells, normalized to 293T.EV cells. CD25 data shown as the paired proportion of CD25+ cells within Vγ2/3/4+ cells in lymphocytes co-cultured with the indicated cell lines for each donor (n = 11). *P < 0.05, **P < 0.0001. c, Flow cytometry analysis of Vγ2/3/4 and γδTCR (donor 1), or Vγ2/3/4 and CD25 (donors 2 and 3) expression by human colonic lymphocytes after co-culture with the indicated cell lines overnight. Gates used for single-cell sorting are shown. Pre-gated on singlets/live/CD3+Vδ2-/γδTCR+ cells. d, Flow cytometry analysis of γδTCR and CD69 expression by J76-hu17 cells (see Table 2 for details) and co-cultured with the indicated antibodies or cell lines for 5 h. Representative of three independent experiments. e, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (right) by J76 cells transduced with the indicated TCRs (see Table 2) and co-cultured with 293T.L3L8 or α-CD3ε (OKT3) for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV and control IgG respectively. Representative of three independent experiments.
Figure 4
Figure 4. Human Vγ4HV4 is a critical determinant of the response to BTNL3+8.
a, Alignment of human TCR Vγ chain amino acid sequences (divergence from Vγ4 in red). Variable regions are highlighted (green, CDR1; yellow, CDR2; pink, HV4; blue, CDR3). Horizontal arrow delineates Framework Region 3 (FR3). b,c,d, Flow cytometry analysis of TCR downregulation (x-axis) and CD69 upregulation (y-axis) by J76 cells transduced with hu17 or the indicated variants (see Supplementary Fig. 4a,b,c,d) and co-cultured with 293T.L3L8 for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV. Representative of two (b), three (c) and four (d) independent experiments.
Figure 5
Figure 5. Cross-species conservation of the critical role of HV4γ in the response to Btnl/BTNL.
a, Alignment of mouse Vγ7 and Vγ6 sequences (top), and of mo5 variants (bottom, differences from wild-type Vγ7 sequence in red). Due to space constraints the most C-terminal region sequence (YYCASWA, identical between all constructs), is not depicted. CDR1/2/3 regions are highlighted in green, yellow and cyan, respectively. b,c, Flow cytometry analysis of TCR downregulation (x-axis) and CD69 upregulation (y-axis) by J76 cells transduced with mo5 or the indicated variants (see Supplementary Fig. 5a,b,c,d) and co-cultured with MODE-K.l1l6 for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to MODE-K.EV. Representative of four independent experiments. d, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (centre) by J76 cells transduced with the indicated TCRs and co-cultured with 293T.l1l6 or 293T.L3L8 for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV. Corresponding raw flow cytometry plots are shown (right). Representative of four independent experiments. *P < 0.01, **P < 0.001.
Figure 6
Figure 6. A proposed model for BTNL3 engagement by Vγ4+ TCRs.
a, Flow cytometry analysis of TCR downregulation (top) and CD69 upregulation (bottom) by J76-hu17 co-cultured with the indicated stimulants. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV. Representative of three independent experiments. b, Heterodimeric model of BTNL3 (green) / BTNL8 (teal), derived with 3D-JIGSAW from a BTN3A1 homodimer (PDB 4F80). Candidate motifs (see Supplementary Fig. 6c,d) are highlighted in orange, yellow, blue and red. c, Flow cytometry analysis of TCR downregulation (top) and CD69 upregulation (bottom) by J76-hu17 cells co-cultured with the indicated 293T transfectants for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to EV. Representative of three independent experiments. d, SwarmDock best-fit of TCR Vγ4 V-domain (light grey, PDB 4MNG) docking to BTNL3 IgV-domain (green). Motifs validated by functional assays (see Fig. 4c; Fig. 6c) are highlighted (TCR Vγ4: pink [HV4γ]; BTNL3: orange [NQFHA], blue [WF], red [DEEAT]) with side-chains displayed. e, Flow cytometry analysis of the indicated soluble TCRs (sTCR; pre-incubated with α-His antibody) binding to 293T.EV or 293T.L3L8 cells after incubation at 4°C for 1 h. Representative of three independent experiments. f, Flow cytometry analysis of the indicated sTCR+α-His stainings. Data expressed as gMFI mean±s.d. of individual stainings (n = 3), normalized to α-His alone. g, Flow cytometry analysis of TCR downregulation (top) and CD69 upregulation (bottom) by J76-mo5 co-cultured with the indicated 293T transfectants for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to empty vector transfectants. Representative of three independent experiments.
Figure 7
Figure 7. Human Vγ4+ and mouse Vγ7+ TCRs exhibit dual-reactivity.
a, SwarmDock model showing the crystal structure of a Vγ4Vδ1 TCR binding CD1d-sulfatide (PDB 4MNG) docking to the complete BTNL3/BTNL8 heterodimer model (Fig. 6b). The docking solution is derived from Fig. 6d. b, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (right) by JRT3 cells transduced with LES or hu12 TCRs and co-cultured with the indicated cell lines or α-CD3ε (OKT3) for 5 h. Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV or control IgG respectively. Representative of two independent experiments. c, Flow cytometry analysis of CD3ε and γδTCR expression (left), and staining with Streptavidin alone (center) or pre-incubated with CD1c-PC (right) on J76 cells transduced with hu17 or hu20 TCRs. Representative of three experiments. d, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (right) by J76 cells transduced with hu17 or hu20 TCRs and co-cultured with 293T.L3L8 cells or α-CD3ε Data expressed as mean±s.d. of individual co-cultures (n = 3), normalized to 293T.EV or control IgG, respectively. Representative of three independent experiments. e, Flow cytometry analysis of TCR downregulation (left) and CD69 upregulation (right) by J76 cells transduced with the mo8 (T22-specific) or mo5 (control) Vγ7+ TCRs and co-cultured with the indicated cell lines for 5 h. Data expressed as mean±s.d. (n = 3), normalized to MODE-K.EV or 293T.EV. Representative of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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