INTRODUCTION
In mammals, leukocyte immunoglobulin-like receptors (LILRs) include 11 functional genes, which are classified as activating (LILRA1, 2, 4–6), inhibitory (LILRB1–5), and soluble (LILRA3). They are highly homologous in their extracellular regions but differ in their intracellular regions [
1,
2]. The leukocyte immunoglobulin (Ig)-like receptor subfamily B (LILRB) of humans was first described and cloned in 1997 [
3] and acts as an important group of immunoreceptor tyrosine-based inhibitory motifs [ITIMs: I/V/L/S)XYXX(L/V)] containing receptor in their cytoplasmic tail. It belongs to a group of type I transmembrane glycoproteins with extracellular Ig-like domains that bind ligands and intracellular ITIMs [
4,
5]. The structure of LILRB groups consists of a signal peptide, two or four Ig-like domains, transmembrane domain and a long cytoplasmic tail with ITIMs [
1,
6]. Moreover, the LILRB groups are classified into group 1 (LILRB1 and LILRB2) and group 2 (LILRB3, LILRB4, and LILRB5). They consist of members that interact with the human leukocyte antigen (HLA) class I molecules and non-HLA ligands and are well-characterized and have been increasingly identified in the recent years [
1,
2,
6].
These LILRB groups are expressed on various cell types such as macrophages, T cells, B cells, NK cells, and dendritic cells although the expression patterns are different between them [
1,
2,
6]. ITIMs are activated by various phosphatases such as protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP-1), SHP-2 (PTPN11), or Src homology 2 domain-containing inositol phosphatase (SHIP) [
1]. These phosphatases are capable of dephosphorylating multiple signaling-related molecules such as extracellular-regulated kinase (ERK), Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) or nuclear factor kappa B subunit signaling pathway [
7]. LILRB groups have been shown to control the activity of toll-like receptors (TLR) [
8], antigen-presenting and cytokine production, thus demonstrating that the LILRB groups play an important role in the regulation of innate immune responses [
8]. Moreover, the gene expression and polymorphisms of LILRBs have been reported to be associated with autoimmune and infectious diseases such as those caused by
Salmonella [
8], cytomegalovirus [
9], and
Mycobacterium tuberculosis [
10], as well as rheumatoid arthritis [
11], Alzheimer’s model [
12], psoriatic arthritis [
13], HIV/AIDS [
14], and cancer [
6]. Recently, some members of the Ig superfamily that may be involved in immune responses such as triggering receptor expressed on myeloid cells, cluster of differentiation 300, signal regulatory protein and chicken Ig-like receptors (CHIR)-A, CHIR-B, and CHIR-AB homologs were identified in chicken using bioinformatics approaches [
15]. The studies of the chicken leukocyte immunoglobulin receptor (LIR) indicated that
LIR genes are shown highly polymorphic locus harboring a variable number of
CHIR genes and also play an important role in avian influenza infection in chicken [
15]. However, pertinent information is lacking in chicken; for example, no data are available on the primary structure of LILRB1 and LILRB3, and the functions of those have not yet been investigated.
In this paper, therefore, we have identified novel LILRB1 and LILRB3 genes and described the expression and functional analysis of the variants of LILRB1 and LILRB3 genes in chicken, namely LILRB1R, −B1S, −B3R, and −B3S, which are isolated from two genetically disparate chicken lines in macrophage (HD11) cell lines. Our findings strongly suggest that the LILRB1 and LILRB3 associated with major histocompatibility complex (MHC) class I and non-classical β2-microglobulin (β2m) and controlled the constitutive signaling mediated by SHP-2 that regulates the JAK/STAT signaling pathway. We also described that LILRB1 and LILRB3 were expressed on HD11 cells and they modulated cytokines production.
MATERIALS AND METHODS
Bioinformatic analysis and identification of chicken LILRB1 and B3 genes
Our previous study [
16,
17] reported that more than 15,000 novel transcripts are expressed in the chicken lines 6.3 (R) and 7.2 (S), which associated with Marek’s disease (MD) resistance and shared the same MHC haplotype. These sequences were then used to search all the current chicken genome assemblies through the
Gallus_gallus-5.0 reference Annotation Release 103 nucleotide BLAST program (
https://blast.ncbi.nlm.nih.gov/Blast.cgi). The chromosomal locations were clarified using the BLAST-Like Alignment Tool (BLAT) (
http://asia.ensembl.org/Gallus_gallus/Info/Index) (
Supplementary Table S1). The genomic DNA identified as encoding potential chicken
LILRB1 and
LILRB3 genes was then analyzed using GeneScan [
18] to predict the coding DNA sequence (CDS) and protein domains. To confirm the sequences of two genetically disparate chicken lines, primers were designed using the Lasergene software (DNASTAR Inc. Madison, WI, USA) and synthesized by Genotech Co. Ltd. (Daejeon, Korea) (
Supplementary Table S2). The polymerase chain reaction (PCR) products from 10 individual samples of chicken line 6.3 and line 7.2 were purified using the QIAQuick gel Extraction Kit (QIAgen, Hilden, Germany), and sub-cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA), followed by transformation into
Escherichia coli (
E. coli) TOP10 (Invitrogen, USA) high-efficiency competent cells. Through blue-white screening, the positive clones were selected and sequenced by Genotech (Korea). Protein identification was conducted using the Expert Protein Analysis System (ExPASy;
http://www.expasy.org/tools/) and the multiple sequence alignment was analyzed using the Lasergene software (DNASTAR Inc., USA). To draw a phylogenetic tree of the amino acid sequences of LILRB1 and LILRB3 groups, the neighbor-joining method with a bootstrap value of 1,000 in the MEGA6 program (
https://www.megasoftware.net/) was used. Signal peptides were predicted by SignalP 4.1 Server v.4.1 (
http://www.cbs.dtu.dk/services/SignalP/) and glycosylation motifs were predicted using NetOGlyc 4.0 Server (
www.cbs.dtu.dk/services/NetOGlyc/). The Ig domains, transmembrane domain, and the cytoplasmic regions were predicted by InterPro v.56.0 (
https://www.ebi.ac.uk/interpro/). The ITIMs were mapped to each peptide sequence using the established V/L/S/NxYxxL/V ITIM motif [
4,
5]. Full-length CDS of
LILRB1 and
LILRB3 genes were cloned in pCR2.1 (Invitrogen, USA) and excised from pCR2.1 using
NotI/
Xbal (Bioneer Corp, Daejeon, Korea), and ligated into the eukaryotic expression vector, pcDNA3-eGFP (Addgene, Cambridge, MA, USA), and then, followed by the transformation of
E. coli BL21 (Invitrogen, USA). The positive clones were sequenced, and the structure and the ligand-binding site of the protein were determined by molecular replacement using the program RaptorX web server (
http://raptorx.uchicago.edu/). To identify the function, the ligands of
LILRB1 and
LILRB3 genes were mapped and searched using the RCSB protein data bank (
http://www.rcsb.org).
Transfection into the macrophage (HD11) cell line
The chicken macrophage (HD11) cells [
19] were transiently transfected with a pcDNA3-eGFP vector containing either the LILRB1R-, LILRB1S-, LILRB3R-, or LILRB3S-encoding sequence and also mock control with empty pcDNA3-eGFP vector using Lipofectamine 3000 transfection reagent (Invitrogen, USA), as per the manufacturer’s recommended protocols. A total of 1.0×106 cells per well in six-well plate (Thermo Scientific, Waltham, MA, USA) were transfected with 4.0 μg of plasmid using the Lipofectamine 3000 reagent and the transfected cells were harvested after 48 h for further analysis.
Reagents
Mouse monoclonal anti-GFPuv/eGFP antibody was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-chicken MHC class I-PE and mouse anti-chicken β2m-PE antibodies were purchased from Southern Biotech (Birmingham, AL, USA). Rabbit anti-chicken p-STAT1 (Ser727), anti-chicken p-STAT3 (Ser727), and anti-chicken p-JAK2 (Tyr1007/Tyr1008) were purchased from Santa Cruz Biotech (Dallas, TX, USA). Rabbit anti-chicken suppressor of cytokine signaling 1 (SOCS1), anti-chicken STAT1, anti-chicken STAT3 antibodies, horseradish peroxidase (HRP)-linked anti-rabbit secondary antibodies, and protein G–sepharose beads were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-chicken p-SHP2 (Tyr542), rabbit anti-chicken JAK2, and rabbit anti-chicken TYK2 antibodies were purchased from Biorbyt (San Francisco, CA, USA). Rabbit anti-chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-His (C-Term)-HRP and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibodies were purchased from Invitrogen (USA). Mouse monoclonal anti-chicken interferon (IFN)-γ antibody, mouse monoclonal anti-chicken interleukin (IL)-17A antibody, mouse monoclonal anti-chicken IL-12p40 antibody and recombinant versions of these proteins (kindly provided by Dr. Hyun S. Lillehoj, USDA), EZ-Link Sulfo-NHS-LC-Biotin, Goat anti-mouse IgG secondary antibody linked HRP conjugate, and HRP-conjugated Streptavidin were purchased from Thermo Scientific (USA).
Flow cytometry analysis
The cells were incubated in a flow cytometer buffers (10% fetal calf serum), 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2 mM ethylenediaminetetraacetic acid [EDTA] in phosphate buffered saline [PBS]) and primary antibodies were added to 1.0×106 cells and then, kept for 30 min on ice. To evaluate the LILRB1- and LILRB3-eGFP binding with MHC class I and β2m, anti-MHC class I-PE and anti-β2m-PE antibodies were incubated with the cells as described above, followed by a single wash with the flow cytometer buffers. For assessing LILRB1- and LILRB3-eGFP constitutive signaling mediated by SHP-2, anti-SHP-2 antibody was added and incubated for 30 min on ice, followed by a single wash with the flow cytometer buffers before adding Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen, USA). The control groups were incubated with goat anti-chicken IgG-PE or rabbit IgG only (data not show). Flow cytometry analysis was performed with BD FACS Aria II cell analyzer (BD Biosciences, San Jose, CA, USA). Data were acquired with BD FACS Diva Version 6.1.3 and analyzed using FlowJo 7.6.1. Forward and side-scatter gating removed contaminants such as cell debris.
Immunoprecipitation and western blotting
Cells were washed twice with ice-cold PBS, harvested in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 100 mM sodium fluoride, 0.1% [w/v] sodium dodecyl sulfate [SDS], 0.5% [w/v] sodium deoxycholate, 1% Triton X-100, 10 mM sodium pyrophosphate, and 10 mM sodium orthovanadate) containing complete EDTA-free protease inhibitor cocktail (Thermo Scientific, USA), then gently lysed for 30 min at 4°C, and centrifuged at 13,000 g for 15 min at 4°C. The concentration of protein was evaluated using the Coomassie protein assay kit (Thermo Scientific, USA) in microplates as per the manufacturer’s instructions. The protein (100 μg) was incubated with the anti-eGFP mAb or anti-GAPDH antibody at 4°C overnight and then added to 50 μL of protein G–Sepharose (Invitrogen, USA) for 2 h. The immunoprecipitates were then washed three times in RIPA buffer and solubilized in 2×SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Samples were electrophoresed on Tris-glycine SDS-PAGE gels, and the proteins were transferred on to polyvinylidene fluoride membranes (GE Healthcare, Rydalmere, Australia). Membranes were blocked with 5% non-fat milk (Thermo Scientific, USA) or 3% bovine serum albumin (BSA) (Sigma-Aldrich, USA) in PBS pH 7.4 containing 0.05% Tween 20 (PBST) for 2 h at room temperature (RT, 25°C), washed with PBST, and incubated with anti-MHC class I, anti-β2m or anti-SHP-2 antibody or signaling antibodies overnight at 4°C and the relevant secondary antibody in 2% non-fat milk or 0.5% BSA in PBST for 2 h at RT. Finally, the membranes were developed using Western Lightning ECL Plus (Thermo Scientific, USA) and Hyperfilm (GE Healthcare, Australia).
Enzyme-linked immunosorbent assay
We coated a 96-well plate (Nunc MaxiSorp, Nunc, Wiesbaden, Germany) with dilutions (1:500) of monoclonal anti-IFN-γ, anti-IL-12p40, or anti-IL-17A antibodies for 7 d at 4°C as previously described [
20]. Next, the plates were blocked with 5% non-fat milk for 2 h at 25°C, the plates were incubated overnight at 4°C with the cell supernatant, or different dilutions of the recombinant IFN-γ, IL-17A, and IL-12p40. Following incubation with a biotinylated monoclonal IFN-γ, IL-17A, or IL-12p40 antibody, HRP-conjugated Streptavidin was added. The plates were washed three times with PBST at each step. Then, 3,3′,5,5′-tetramethylbenzidine (Thermo Scientific, USA) was used as the chemiluminescent substrate and the luminescence was measured using a Hybrid Microplate Reader (Epoch, BioTek Instruments, Inc, Winooski, VT, USA).
Quantitative real-time polymerase chain reaction
After 48 h of transfection with a pcDNA3-eGFP vector containing either the LILRB1R, −B1S, −B3R, or −B3S encoding sequence and a mock control, RNA was extracted using the TRIzol kit (Invitrogen, USA) as per manufacturer’s instructions. Next, 2 μg total RNA was reverse transcribed to cDNA by using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, USA), according to manufacturer’s protocols. Quantitative PCR (qPCR) was performed using Light Cycler 96 Real-time PCR System (Roche Diagnostics, Indianapolis, IN, USA) in a 20-μL reaction mixture containing 1 μL cDNA, 10 μL 2× FastStart Universal SYBR Green Master Mix (Roche Diagnostics, USA), and 10 pmol each of forward and reverse primers of genes (
Supplementary Table S2). The primers were designed using the Lasergene software (DNASTAR Inc., USA) and were synthesized by Genotech Co. Ltd. (Korea). Thermal conditions for performing qPCR are as follows: initial incubation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 50°C to 55°C for 30 s, and extension at 72°C for 30 s; and termination by final incubation at dissociation temperatures 95°C (10 s), 65°C (60 s), 97°C (1 s), and 37°C (30 s). Genes expression were quantified after normalization with the chicken
GAPDH gene and were calculated using the 2
−ΔΔCt method.
Bioactivity assays
To determine the cytotoxicity of LILRB1R, −B1S, −B3R, and −B3S linked eGFP vector and empty vector transfected into HD11 cell line after 48 h transfection, cell proliferation and NO production assay was performed in 96-well plates according to well-established protocols [
20]. To measure the nitrite content, 100 μL of the culture medium was incubated with 100 μL of Griess reagent (Sigma-Aldrich, USA) at RT for 10 min. Then, the absorbance was measured at 540 nm using a microplate reader as described [
20]. The nitrite content was calculated based on a standard curve constructed with NaNO
2. Cell proliferation was determined with the cell counting Kit-8 (CCK-8) assay according to the manufacturer’s protocol as described [
20] (Dojindo Molecular Technologies, Inc., Mashikimachi, Kumamoto, Japan). Lipofectamine 3000 transfection reagent and culture medium were used as controls.
Statistical analysis
Data are represented as the mean±standard error of the mean of three independent experiments for each group (n = 3) and were analyzed with the SAS 9.4 statistical program (SAS Institute Inc., 100 SAS Campus Drive Cary, NC, USA). Comparison between the experimental groups was carried out using Duncan’s multiple comparison method. Differences were considered significant when p<0.05.
DISCUSSION
In this study, we identified and characterized novel
LILRB1 and
B3 genes from two genetically disparate chicken lines using a bioinformatics approach and
in vitro analysis. Our initial analysis yielded 4 group transcripts as potential
LILRB1 and
LILRB3 genes from chicken lines 6.3 and 7.2, which were then analyzed using phylogeny alongside other structurally similar Ig-superfamily receptors. Comparison of amino acids identities and similarities between LILRB1 and LILRB3 in line 6.3 to those proteins in line 7.2 ranked between 97%–99%, and also between LILRB1 and LILRB3 to the corresponding mammalian proteins ranked between 13%–19% and 13%–69%, respectively. Moreover, phylogenetic analysis displaying amino acid similarity placed the LILRB1R, −B1S, −B3R, and −B3S together with members of the
LILRB1 and
LILRB3 genes of mammals suggesting that the 4 receptors are closely associated with
LILRB1 and
LILRB3 genes from other species. Together with the high degree of sequence similarity, this indicates that the LILRB1R, −B3R, and −B1S, −B3S amino acids isolated from the disease-resistant line 6.3 and susceptible line 7.2, respectively, represent homologs of the novel receptor in the chicken LILRB family. Moreover, the human LILRB1 and LILRB3 receptors contain 4 canonical and permissive ITIMs with NxYxxV, VxYxxV, VxYxxL and SxYxxL being present on every receptor [
5], whereas the chicken LILRB1R, −B1S, −B3R, and −B3S receptors only contain one canonical ITIMs (LxYxxL) on every receptor. This possibly provides a greater diversity of signaling abilities by human LILRB1 and LILRB3 than that of chicken
LILRB1 and
LILRB3 genes. Our findings indicate that the
LILRB1 and
LILRB3 genes can be subdivided into two groups: transmembrane molecules with two ITIM motifs (LxYxxL and SxVxxV) in the resistant line 6.3 and two ITIM motifs (LxYxxL and LxYxxV) in the susceptible line 7.2. On the other hand, LILRB1R and −B1S have two Ig domains whereas LILRB3R and −B3S contain only one Ig domain (
Figure 1C). The results may be implicated in the diversity of signaling abilities for
LILRB1 and
LILRB3 genes in resistant line 6.3 and susceptible line 7.2. In addition, at least one of these motifs contains the sequence V/YxxL/V, which has been shown to be the consensus sequence for binding by the SH2 domains of the SHP-1 or SHP-2 phosphatase. Another report had shown that tyrosine-phosphorylated LILRB1 associates and coimmunoprecipitates SHP-1 and SHP-2 [
23]. Evidence from other immunoreceptors suggests that the phosphorylation of such motifs generates binding sites for other signaling molecules, including the SHIP [
23]. To demonstrate this point, we analyzed the
LILRB1 and
LILRB3 genes-transfected cell from two chicken lines associated with SHP-2 by immunoprecipitation, FACS analysis, and qRT-PCR. The results indicate that the
LILRB1 and
LILRB3 genes-transfected cell in the two chicken lines associated with SHP-2 was significantly higher in LILRB3-containing cell line than in LILRB1-containing cell line. In addition, they were higher in the chicken line 6.3 than in line 7.2. Recently, some researchers suggested that SHP-2, a cytoplasmic SH2 domain-containing protein tyrosine phosphatase, is involved in the signaling pathways of a variety of growth factors and cytokines such as JAK-STAT and MAPK signaling pathway, and plays an important role in transducing the signal from the cell surface to the nucleus, and is a critical intracellular regulator of cell proliferation and differentiation [
4]. Taken together,
LILRB1 and
LILRB3 genes in the two chicken lines could differentially regulate the signaling pathways and cytokines production.
In humans, the binding of LILRB1, but not LILRB3, to HLA-A, -B, and -C, and other non-classical class I molecules such as HLA-E, HLA-G, and HLA-F, is currently being evaluated in several cell types [
2]. There are at least two predicted LILRB1 and LILRB3 ligands for each of the
LILRB1 and
LILRB3 genes, and most of the ligand candidates of these genes bind to MHC class I and β2m antigens. Moreover, these genes are believed to have other ligands or binding residues that differ in the two genetically disparate chicken lines.
By immunoprecipitation, FACS analysis, and qRT-PCR, it was confirmed that LILRB1 and LILRB3 genes in the two chicken lines strongly bind to MHC class I and β2m. The LILRB1 and LILRB3 proteins binding to MHC class I was significantly higher than those binding to β2m. In addition, the LILRB1 and LILRB3 genes-transfected cell binding to MHC class I and β2m in line 6.3 were markedly higher than those in line 7.2. Moreover, LILRB1 and LILRB3 genes significantly upregulated MHC class I-related genes such as TAP-1 and TAP-2, which are essential for antigen presentation by the MHC class I pathway, by more than 100-fold. The finding that MHC class I is recognized by inhibitory receptor genes LILRB1 and LILRB3 in two genetically disparate chicken lines expressed on macrophage cells expands the concept of self-recognition in immune response. Taken together, our results show that LILRB1 and LILRB3 genes are necessary and sufficient for the induction of MHC class I expression. Future analyses of the in vivo function of LILRB1 and LILRB3 genes are required to reveal if these two molecules play redundant or more exclusive roles in MHC class I-dependent immune responses.
A deeper understanding of the intracellular signal transduction pathways initiated by
LILRB1 and
LILRB3 genes is necessary to know how these genes can activate the expression of immune-related genes. Concurrent with a previous report showing that JAK/STAT pathway plays a key role in cytokine-induced biological responses, we demonstrated the effects of
LILRB1 and
LILRB3 genes on the JAK/STAT signaling pathway in the activated HD11 cell line [
24]. Moreover, LILRB groups have been shown to regulate TLR activity [
8], antigen-presenting phenotype and cytokine production [
8]. Thus, the LILRB groups play an important role in the regulation of innate immune responses. The expression levels of phosphorylated STAT1 (p-Ser
727), STAT3 (p-Ser
727), and JAK2 (p-Tyr
1007/Tyr
1008) molecules and un-phosphorylated (STAT1/3, JAK2, TYK2, and SOCS1) molecules in response to
LILRB1 and
LILRB3 gene-transfected HD11 cell line compared to mock control by western blotting were increased, and also significantly upregulated in STAT1/3, JAK2, TYK2, and SOCS1 mRNA by qRT-PCR. Our results indicate that LILRB1 and LILRB3 activate and regulate the JAK/STAT signaling pathway and also control the immune system.
This study provides the first evidence indicating the possible role of JAK/STAT pathway in the signaling mechanism of chicken
LILRB1 and
LILRB3 genes in the chicken HD11 cell line. To investigate the function of
LILRB1 and
LILRB3 genes for growth factor activation, 3 chemokine (
CCL-4,
CXCL-13, and
CXCL-14) and 12 cytokine (
IL-1β,
IL-4,
IL-6,
IL-10,
IL-12p40, I
L-17A,
IL-17F,
IFN-α,
IFN-β,
IFN-γ,
LITAF, and
TGFβ4) genes in the immune cell were measured by qRT-PCR. The expression of these cytokine genes was increased in LILRB1 and LILRB3-transfected cells of line 6.3 than those of line 7.2. As previously reported, chemokines appear to promote host resistance by mobilizing leukocytes and activating immune functions that kill, expel, or sequester pathogens [
25] and IFN-α, IFN-β, IFN-γ, and IL-17 families play important roles in conferring resistance against pathogens. They also increase the expression of class I MHC molecules on cells making them more resistant to pathogens and are highly produced from Th1 and Th17 cells in response to pathogens [
26]. IFN and IL-17 induce
IL-1β,
IL-6,
LITAF [
27] and
TGFβ4 mRNA production [
26]. Moreover, Th2 cytokine (IL-10) and Treg cytokine (TGFβ4) mRNA have been reported to upregulate the cell surface expression of the inhibitory receptors on monocytes and dendritic cells and these mRNA also associated with the LILRB group in response to pathogens such as HIV [
28]. Our result indicates that chicken
LILRB1 and
LILRB3 genes activate the JAK/STAT signaling pathway, and upregulate the expression of chemokines such as Th1, Th2, and Th17 cytokines. Expression levels of chemokines and cytokines were significantly higher in LILRB1- and LILRB3-transfected cells of the resistant line 6.3 than those of the susceptible line 7.2. Therefore, we demonstrated that
LILRB1 and
LILRB3 genes can induce several chemokines, such as Th1, Th2, and Th17 cytokine mRNA, which may be essential in the pathogenesis of diseases, highlighting the importance of this novel pathway in chicken disease.
In summary, this is the first report of the cloning, structural analysis, and function of the novel chicken LILRB1 and LILRB3 genes isolated from two genetically disparate chicken lines. Like other LILRB family members, LILRB1 and LILRB3 genes possess ITIM motifs in the cytoplasmic domain that bind to cytosolic tyrosine kinases such as SHP-2. We also showed that the LILRB1 and LILRB3 genes bind to MHC class I and β2m as well as other genes involved in class I antigen presentation, processing and regulation of immune responses. Moreover, LILRB1 and LILRB3 genes induce and regulate the JAK-STAT signaling pathway and upregulate Th1, Th2, and Th17 cytokine genes in the chicken cell line.