BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation

Abstract

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.

FIGURE 1

Cloning and sequence analysis of BTNL2. A, Full-length BTNL2 cDNA was cloned from a spleen cDNA library. Sequence analysis revealed four polymorphic differences from the existing sequence in NCBI database, which are shown in bold. The predicted mouse BTNL2 amino acid sequence was aligned with human BTNL2 sequence. The bar symbol (|) marks the intron/exon border, and leader and transmembrane domains are underlined. An asterisk (*) denotes identity, a colon (:) denotes conservation of strong groups, and a period (.) denotes conservation of weak groups between mouse and human sequences. B, Phylogenetic analysis of the full-length BTNL2 protein among B7/butyrophilin superfamily members. C, Genomic organization of the human and mouse BTNL2 gene were adapted from Stammers et al. (35) and Valentonyte et al. (30) with our sequence confirmation.

FIGURE 2

Expression of BTNL2 mRNA. Real-time PCR analysis was conducted using primers specific for the transmembrane and intracellular regions of BTNL2. A, Tissues harvested from a B6 mouse were analyzed for BTNL2 expression, and the expression level in thymus was set at 1. B, CD4⁺, CD8⁺ T cells and B cells purified from spleen and lymph nodes by AutoMACS sorting (90–95% purity), bone marrow-derived dendritic cells, and peritoneal macrophages were used in the analysis. The expression level by CD4⁺T cells was set at 1. The data are representative of three experiments.

FIGURE 3

Expression of a BTNL2 receptor in lymphocytes. BTNL2-Ig fusion protein was biotinylated to stain B cells before and after LPS treatment, and CD4⁺ and CD8⁺ T cells before and after activation with Con A, which was revealed by streptavidin-PE. Histogram analysis was performed on gated B cells (A), wild-type, CD28- or ICOS-deficient CD4⁺ or CD8⁺ T cells (B). The lighter line represents human IgG1 isotype control staining; the darker line, BTNL2-Ig staining. Con A-activated splenocytes were preincubated with 20-fold excess of B7.1-Ig before stained with biotinylated BTNL2-Ig (C). 293 cells were transfected with a PD-1 expression vector, and subsequently stained with biotinylated PD-L1-Ig or BTNL2-Ig (D). The data are a representative of at least three experiments.

FIGURE 4

BTNL2 inhibits T cell proliferation. Purified CD4⁺ T cells were treated for 3 days, and [³H]thymidine uptake was examined. A, T cells activated with 1 μg/ml plate-bound anti-CD3 and anti-CD28 in the presence of indicated doses of BTNL2-Ig or control human IgG (hIg). B, T cells activated with indicated doses of plate-bound anti-CD3 and 5 μg/ml BTNL2-Ig or hIg for 24 h before measuring activation induced cell death. C, T cells were activated with anti-CD3 and anti-CD28 as above in the presence of 10 μg/ml plate-bound or soluble BTNL2-Ig or control hIg. Proliferation of T cells treated with hIg was set at 100%. D, T cell proliferation after activation with indicated doses of anti-CD3 with or without 1 μg/ml CD28 and in the presence of 10 μg/ml hIg, BTNL2-Ig, B7S1-Ig, or B7H3-Ig. The data are representative of more than three experiments, with error bars indicating the SD of triplicate samples in each experiments.

FIGURE 5

IL-2 regulation by BTNL2. A, CD4⁺ T cells were activated with different doses of plate-bound anti-CD3 in the presence of 10 μg/ml BTNL2-Ig or hIg. IL-2 production was measured at 24 h after treatment by ELISA. B, CD4⁺ T cells were activated with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 in the presence of different doses of BTNL2-Ig, and IL-2 production was examined by ELISA. C, CD4⁺ T cells were treated with 1 μg/ml anti-CD3 in the presence of 10 μg/ml hIg, BTNL2-Ig, B7S1-Ig, or B7H3-Ig with or without IL-2 (100 U/ml) and/or 1 μg/ml anti-CD28 for 3 days, and [³H]thymidine uptake was measured. The data are a representative of more than three experiments, with error bars indicating the SD of triplicate samples in each experiments.

FIGURE 6

BTNL2 inhibits TCR signaling pathways. DO11.10 hybridoma was transfected with luciferase reporter constructs for each individual signaling pathway, and stimulated with 1 μg/ml plate-bound anti-CD3 in the presence of 10 μg/ml BTNL2-Ig or hIg for 4 h before luciferase activity measurement. The data are representative of at least three experiments, with error bars indicating the SD of triplicate samples in each experiment.