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Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific γ δ T Cell Compartments

View Article: PubMed Central - PubMed

ABSTRACT

Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7+ γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαβ+ repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7+ cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4+ cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.

No MeSH data available.


Human Intestinal γδ Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted γδ T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR Vγ chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR Vγ2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRVγ chain usage (left) and cell surface TCRγδ expression (right) in gut-derived γδ T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.
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figs7: Human Intestinal γδ Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted γδ T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR Vγ chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR Vγ2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRVγ chain usage (left) and cell surface TCRγδ expression (right) in gut-derived γδ T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.

Mentions: Of six functional human Vγ chain genes (Vγ2, 3, 4, 5, 8, and 9) (Arden et al., 1995), Vγ4 was reported to be the signature chain of intestinal Vδ2− cells (Landau et al., 1995). Indeed, for up to ten donors examined, most intestinal Vδ2− cells reacted with a Vγ2/3/4-specific antibody, but not a Vγ5/3-specific antibody (blue and red bars, Figure 7B; Table S1A), and TCR deep sequencing showed that Vγ4 sequences far outnumbered Vγ2 sequences (Figure S7A). Thus, despite individual variation, most gut γδ T cell compartments included a substantial Vγ4+Vδ2− subset, while some donors also displayed relatively high representation of Vγ8+Vδ1+ cells (Figure 7B; Table S1A).


Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific γ δ T Cell Compartments
Human Intestinal γδ Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted γδ T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR Vγ chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR Vγ2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRVγ chain usage (left) and cell surface TCRγδ expression (right) in gut-derived γδ T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.
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figs7: Human Intestinal γδ Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted γδ T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR Vγ chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR Vγ2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRVγ chain usage (left) and cell surface TCRγδ expression (right) in gut-derived γδ T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.
Mentions: Of six functional human Vγ chain genes (Vγ2, 3, 4, 5, 8, and 9) (Arden et al., 1995), Vγ4 was reported to be the signature chain of intestinal Vδ2− cells (Landau et al., 1995). Indeed, for up to ten donors examined, most intestinal Vδ2− cells reacted with a Vγ2/3/4-specific antibody, but not a Vγ5/3-specific antibody (blue and red bars, Figure 7B; Table S1A), and TCR deep sequencing showed that Vγ4 sequences far outnumbered Vγ2 sequences (Figure S7A). Thus, despite individual variation, most gut γδ T cell compartments included a substantial Vγ4+Vδ2− subset, while some donors also displayed relatively high representation of Vγ8+Vδ1+ cells (Figure 7B; Table S1A).

View Article: PubMed Central - PubMed

ABSTRACT

Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7+ γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαβ+ repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7+ cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4+ cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.

No MeSH data available.