Murine Butyrophilin-Like 1 and Btnl6 Form Heteromeric Complexes in Small Intestinal Epithelial Cells and Promote Proliferation of Local T Lymphocytes

Abstract

To date, few molecular conduits mediating the cross-talk between intestinal epithelial cells and intraepithelial lymphocytes (IELs) have been described. We recently showed that butyrophilin-like (Btnl) 1 can attenuate the epithelial response to activated IELs, resulting in reduced production of proinflammatory mediators, such as IL-6 and CXCL1. We here report that like Btnl1, murine Btnl6 expression is primarily confined to the intestinal epithelium. Although Btnl1 can exist in a cell surface-expressed homomeric form, we found that it additionally forms heteromeric complexes with Btnl6, and that the engagement of Btnl1 is a prerequisite for surface expression of Btnl6 on intestinal epithelial cells. In an IEL-epithelial cell coculture system, enforced epithelial cell expression of Btnl1 significantly enhanced the proliferation of IELs in the absence of exogenous activation. The effect on proliferation was dependent on the presence of IL-2 or IL-15 and restricted to IELs upregulating CD25. In the γδ T-cell subset, the Btnl1-Btnl6 complex, but not Btnl1, specifically elevated the proliferation of IELs bearing the Vγ7Vδ4 receptor. Thus, our results show that murine epithelial cell-specific Btnl proteins can form intrafamily heterocomplexes and suggest that the interaction between Btnl proteins and IELs regulates the expansion of IELs in the intestinal mucosa.

Keywords: butyrophilin-like; intestinal epithelial cells; intraepithelial lymphocytes; mucosal immunity; γδ T cells.

Figure 1

Validation of the Btnl6 antibody and Btnl6 protein expression in tissue. (A) A rabbit anti-mouse Btnl6-reactive antibody was developed against a synthetic peptide (residues 79–93). Peptide alignment across Btnl4 and 6 is shown. (B) Lysates from HEK 293 cells transfected with FLAG-tagged Btnl4 cDNA pMX-IRES-GFP, FLAG-tagged Btnl6 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP) were divided and immnoblotted either using anti-FLAG- or anti-Btnl6 antibody. A single band consistent with the theoretical molecular weight of ~64 kDa for FLAG-tagged Btnl4 and ~59 kDa for FLAG-tagged Btnl6 was detected under reducing conditions when immunoblotted with anti-FLAG. Anti-Btnl6 antibody detected Btnl6 transfectants, but not Btnl4 transfectants or HEK 293 cells transfected with empty vector. (C) Tissues from C57BL/6 mice were analyzed for Btnl6 protein expression using a Btnl6-specific polyclonal antibody. A single band consistent with the theoretical molecular weight of ~58 kDa for Btnl6 was detected in large and small intestine, and in small intestinal epithelial cells under reducing conditions. No bands were detected on gels immunoblotted with preimmune serum. The β-actin immunoblot acts as a loading control. IELs, intraepithelial lymphocytes; LPLs, lamina propria lymphocytes. Data are representative of two experiments.

Figure 2

Btnl6 expression. (A) HEK 293 cells were transfected with N-terminal FLAG-tagged Btnl6 cDNA pMX-IRES-GFP. An anti-FLAG antibody detected cell surface expression of Btnl6 but not gfp⁺ HEK 293 cells transfected with empty vector (pMX-IRES-GFP). (B) Murine small intestinal epithelial cells, MODE-K cells, were transfected with N-terminal FLAG-tagged Btnl6 cDNA pMX-IRES-GFP. Cells were stained with an anti-FLAG antibody for cell surface expression of Btnl6, or permeabilized for detection of intracellular Btnl6 localization. Btnl6 could not be detected on the plasma membrane of transfected MODE-K cells. Instead, Btnl6 was retained in the intracellular compartment as evident by anti-FLAG staining. (C) MODE-K cells were cotransfected with untagged Btnl4 cDNA- and N-terminal FLAG-tagged Btnl6 cDNA pMX-IRES-GFP, or untagged Btnl1 cDNA- and N-terminal FLAG-tagged Btnl6 cDNA pMX-IRES-GFP. An anti-FLAG antibody detected cell surface expression of Btnl6 on the Btnl1–Btnl6 cotransfectants, but not on the Btnl4–Btnl6 cotransfectants. Representative plots (A–C) from single experiments out of three independent experiments are shown. (D) MODE-K cells were transfected with N-terminal FLAG-tagged Btnl6 cDNA pMX-IRES-GFP, FLAG-tagged Btnl6 cDNA- and HA-tagged Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP). While no Btnl6 expression was detected on the cell surface of MODE-K cells transfected with Btnl6, Btnl6 was detected on most Btnl1–Btnl6 cotransfectants. Btnl6-, Btnl1-, and double-stained Btnl1 + Btnl6 expressing cells are exemplified by arrows. Anti-FLAG antibody was used for detection of FLAG-Btnl6 (red) and anti-HA antibody was used for detection of HA-Btnl1 (purple). Slides were counterstained with DAPI (blue) to visualize nuclei. The green staining indicates transfected, GFP⁺ cells. Original magnification 20×. Images are representative of three experiments.

Figure 3

Btnl6 forms plasma membrane-tethered heteromeric complexes with Btnl1 in small intestinal epithelial cells. MODE-K cells were transfected with FLAG-tagged Btnl6 cDNA pMX-IRES-GFP, FLAG-tagged Btnl6 cDNA- and HA-tagged Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP). Cell surface proteins were biotinylated prior to cell lysis, and coimmunoprecipitated using FLAG or HA tags. Biotinylated material, immunoprecipitated with anti-FLAG or anti-HA, was either immunoblotted using anti-FLAG or anti-HA (A), or detected with streptavidin-HRP (B,C). GFP immunoblot acts as a loading control. The lanes were reorganized (dashed lines) from the same gel without any image manipulation. Data are representative of two experiments.

Figure 4

Btnl1 and Btnl6 form heteromeric interactions in primary small intestinal epithelial cells. Lysate from isolated primary intestinal epithelial cells were immunoprecipitated with anti-Btnl1 or preimmune rabbit serum-coated beads. Bound protein was released from the beads using alkaline conditions and separated by SDS-PAGE under non-reducing conditions. (A) Separated eluates after immunoblot with anti-Btnl1 antibody followed by HRP-conjugated anti-rabbit antibody. The unspecific band in the preimmune serum lane indicates that some bead-coupled antibodies were released in the elution step. Squares (1–4) indicate excised bands on a corresponding Coomassie stained gel subjected to in-gel digestion by trypsin followed by mass spectrometry analysis. (B) The table shows identified butyrophilin-like proteins in corresponding excised bands in the two independent experiments performed. Neither Btnl1 nor Btnl6 was detected in samples immunoprecipitated with preimmune serum (band 3 and 4). Data are representative of two independent experiments. AC, accession number; SP, signal peptide. A complete list of identified proteins in the excised gel bands is given in Table S1 in Supplementary Material.

Figure 5

Btnl proteins regulate IEL proliferation. (A) MODE-K cells transfected with Btnl6 and Btnl1 cDNA pMX-IRES-GFP, Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP) were cocultured with CFSE-labeled IELs in the presence of anti-CD3 activation. IELs were left to proliferate, and the cell division was monitored after 96 h. Graphs show the mean ± SD, and data are pooled from five independent experiments, each performed in duplicates. (B,C) MODE-K cells transfected with Btnl6 and Btnl1 cDNA pMX-IRES-GFP, Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP) were cocultured with CFSE-labeled IELs in the absence of anti-CD3 activation, with or without IL-2 or IL-15. IELs were left to proliferate, and cell division was monitored after 72 and 96 h (B) or 96 h (C). Proliferation was assessed on total IELs (B) or IELs gated on αβ TCR or γδ TCR IEL subsets (C). Representative histograms from single experiments out of seven independent experiments for IL-2, each performed in duplicates, and out of two independent experiments for IL-15, each performed in triplicates, are shown. Graphs show the mean ± SD. Proliferative response was assessed by flow cytometry after staining with anti-CD45 to exclude GFP⁺ MODE-K cells, and after gating on LIVE/DEAD Fixable Red negative cells to exclude non-viable cells. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 as determined by unpaired two-tailed t-test.

Figure 6

Btnl proteins induce CD25 expression. (A,B) MODE-K cells transfected with Btnl6 and Btnl1 cDNA pMX-IRES-GFP, Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP) were cocultured with IELs without anti-CD3 activation in the presence of IL-2 (A) or IL-15 (B). CD25 expression was assessed on gated αβ TCR and γδ TCR IELs, after gating on LIVE/DEAD Fixable Red negative cells to exclude non-viable cells subsets and on CD45⁺ to exclude GFP⁺ MODE-K cells, after 96 h. Graphs show the mean ± SD, and data are pooled from seven independent experiments, each in duplicates, for IL-2, and two independent experiments, each performed in triplicates, for IL-15. (C) Culture of total IELs or splenocytes, without anti-CD3 in the presence of IL-2, with HEK 293 cells or MODE-K cells transfected with empty vector (pMX-IRES-GFP). Splenocytes and IELs gated on αβ TCR or γδ TCR IEL subsets, after gating on LIVE/DEAD Fixable Red negative cells to exclude non-viable cells subsets and on CD45⁺ to exclude GFP⁺ MODE-K cells, were analyzed for CD25 expression after 96 h. Plots shown are representative of seven independent experiments for IELs and MODE-K cells, and of two independent experiments for IELs and HEK 293 or splenocytes and MODE-K cells. (D) Culture of total IELs, without anti-CD3 in the presence of IL-15, with MODE-K cells transfected with Btnl-pMX-IRES-GFP or empty vector control (pMX-IRES-GFP). IFN-γ was assessed by cytometric bead array. Bars show the mean ± SD, and data are pooled from two independent coculture experiments, each performed in triplicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 as determined by unpaired two-tailed t-test.

Figure 7

The Btnl1–Btnl6 complex specifically induces the proliferation of IELs bearing the Vγ7Vδ4 receptor. Murine MODE-K cells transfected with Btnl6 and Btnl1 cDNA pMX-IRES-GFP, Btnl1 cDNA pMX-IRES-GFP, or empty vector (pMX-IRES-GFP) were cocultured with CFSE-labeled IELs in the absence of anti-CD3 activation in the presence of IL-2. The proliferating population among the γδ TCR IELs in the various coculture conditions was analyzed for the expression of Vγ7, Vγ1, and Vδ4 chains. Bars show the mean ± SD, and data are pooled from four independent experiments in duplicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 as determined by unpaired two-tailed t-test.