Amino acid sequences of MDV vIL-8 and all known chicken CXC chemokines were aligned using the ClustalW (Vector NTI 9.1, Invitrogen) and MEGA 6 software (Tamura et al., 2013). Phylogenetic analyses of vIL-8, chicken and human CXC chemokines were performed based on their amino acid sequences applying neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods using the MEGA 6 software (Tamura et al., 2013). Full length amino acid sequences of MDV vIL-8 (AAN60433.1), the known chicken CXC chemokines, cCXCL1 (XP_420608.1), cCXCL8L1 (NP_990349.1), cCXCL8L2 (NP_990829.1), cCXCL12 (NP_989841.1), CXCL13L1 (XP_004941081.1), CXCL13L2 (CCC15119.1), CXCL13L3 (CCC15120.1), cCXCL14 (NP_990043.1) and human CXC chemokine orthologues, hCXCL1 (AAH11976.1), hCXCL8 (NP_000575.1), hCXCL12 (AAH39893.1), hCXCL13 (AAH12589.1), hCXCL14 (XP_527018.2) were obtained from Genbank.

Cells for flow cytometric analysis were obtained from M11 (B^x2x/ B^x2x) white leghorn chicken. Birds were housed under conventional conditions in groups of up to 10 birds. All animal experiments were approved by the appropriate governmental agencies (Regierung von Oberbayern, Az.: 55.2-1-54-2532.6-12-09). HEK293 and HEK293-T cells were grown in DMEM high glucose supplemented with 10% FBS and 1% penicillin/streptomycin (Biochrom, Germany), DT40 cells were grown in IMDM (Biochrom, Germany) with 10% FBS, 1% chicken serum (ThermoFisher Scientific, USA) and 1 mM ß-Mercaptoethanol at 37°C. Primary leukocytes were obtained by dissociation of the organs using a stainless steel sieve and density centrifugation on Biocoll (1,077 g/ml, Biochrom, Germany).

To generate recombinant proteins for binding and chemotaxis assays, expression plasmids were generated. Full-length coding sequences of MDV-vIL-8, CXCL13L1, L2, or L3 were cloned into a modified pCR3.1 expression vector containing a C-terminal huFc-tag. cDNA from RB-1B infected cells or spleen cells of a Rhode Island Red (RIR) chicken served as a template for cloning the viral and cellular genes, respectively. HEK293 cells were transfected with the expression plasmids constructs using polyethyleneimine (PEI) as described previously (Boussif et al., 1995) or X-tremeGENE® 9 DNA transfection reagent (Sigma-Aldrich) according to the manufacturer's protocol. To generate protein for chemotaxis assays, cells were cultured in serum free HEK293 A medium (Bio&SELL, Germany). Supernatants containing the secreted huFc tagged chemokines or huFc control protein were harvested after 24 h and subjected to further analysis.

Recombinant chemokines and huFc control were quantified by ELISA. Briefly, 96-well ELISA plates (MaxiSorp, Nunc, Wiesbaden, Germany) were coated overnight with 1 μg/ml of donkey anti-huIgG (Jackson ImmunoResearch Europe Ltd, UK) in carbonate buffer (pH 9.6) and blocked with 4% skim milk. Plates were then incubated with serial dilutions of supernatants containing huFC-tagged chemokines, followed by incubation with HRP coupled rabbit anti-huIgG (SouthernBiotech, USA) 1:4,000 in PBS-T and tetramethylbenzidine (TMB). To confirm the correct size of the recombinant chemokines and huFc control protein we performed SDS-PAGE and Western blotting using a goat anti-human Fc HRP-conjugated secondary Ab (Invitrogen).

To generate cell lines expressing CXCR5 with an N-terminal extracellular Flag-tag, we amplified the full-length receptor from cDNA of bursal cells and cloned it into the p3XFLAG-myc-CMV™-25 expression vector (Sigma, USA). The CXCR5 expression plasmid was transfected into HEK293 cells using X-tremeGENE® 9 DNA transfection reagent (Sigma-Aldrich) according to the manufacturer's protocol and stable clones were selected with 250 μg/ml of neomycin (Biochrom, Germany). Receptor expression was confirmed by flow cytometry using an Alexa647 conjugated mouse anti-Flag antibody (AbD Serotec, Germany).

CXCR5 expressing HEK293 cells were used for repeated intraperitoneal immunizations of a BALB/c mouse. Murine spleen cells were fused to SP2/0-Ag14 hybridoma cells. Specificity of resulting hybridomas was examined by flow cytometry using undiluted hybridoma supernatant and goat anti-mouse-IgG-FITC (Sigma-Aldrich, USA, 1:200) on primary spleen cells as well as HEK293-CXCR5 cells and untransfected HEK293. Clone 6A9 was selected, its isotype was determined as IgG1 and monoclonality was ensured by subcloning using the single-cell limited dilution method.

Receptor binding of recombinant chemokines was examined by flow cytometry on the chicken B cell line DT40 cells, HEK293-CXCR5 cells and primary chicken leukocytes from different lymphoid organs. Cells were incubated on ice with chemokine containing supernatants for 30 min. Chemokine binding was detected using a mouse anti-huFC-Alexa647 secondary antibody. Subpopulations of primary leukocytes were assessed by staining with RPE conjugated anti-Bu1 (clone AV20, B cells), anti-CD4 (CT4), anti-CD8 (CT8) and anti-Kul01 (myeloid cells) (Southern Biotechnology, USA).

Migration assays were performed at room temperature with endotoxin-free single-use material. DT40 cells were washed twice in warm RPMI (Biochrom, Germany) and seeded at a concentration of 10⁵ cells/ml in chemotaxis medium (RPMI containing 0.5% bovine serum albumin). Twenty-four-well-Transwell® Permeable Supports (Corning, New York, USA) with a pore size of 5 μm were coated with bovine fibronectin (10 μg/ml) (Life Technologies) dissolved in endotoxin-free distilled H₂O (Sigma-Aldrich) and incubated for 1 h at 37°C and 5% CO₂. Plates were air-dried at 37°C for 2 h. Different dilutions of serum free supernatant from chemokine-transfected HEK293-T cells were added to the bottom of the transwell inserts. Chemotaxis medium served as a control. 100 μl of DT40 cell suspension was added to the transwell inserts. After a migration time of 90 min, cells were taken from the lower chamber and transferred directly into FACS Trucount® tubes (Becton Dickinson) and the number of cells was determined by flow cytometry. The chemokinesis index was calculated by dividing the number of migrated cells in stimulated wells by the chemokinesis control.

Flow cytometric analyses were performed with a BD FACSCanto II (Becton Dickinson, Heidelberg, Germany) using DIVA and FlowJo (Tree Star Inc., Oregon, USA) software. Plots were gated for viable cells upon doublet discrimination.

Statistical analyses were performed using GraphPad Prism. Binding assays data of were analyzed using paired Student's t-test and migration assays by One-way ANOVA. Results were considered significant when p < 0.05.

To identify potential cellular orthologs of the viral chemokine, we analyzed the amino acid sequences of all chicken CXC chemokines present in the recently published chicken genome (Figure 1A). Surprisingly, vIL-8 had the highest homology to the CXCL13 variants and not the two chicken IL8 chemokines CXCL8L1 (K60) and CXCL8L2 (9E3/CEF4) it was initially named after. CXCL13L1 shared the highest sequence identity of 53.8% with vIL-8, suggesting that this variant is the true orthologue of the viral chemokine. CXCL13L2 (33.0%) and CXCL13L3 (36.1%) also shared a higher sequence identity with vIL-8, compared to the IL-8 variants CXCL8L1 (28.8%) and CXCL8L2 (31.1%). Importantly, vIL-8 and all three CXCL13 variants lack the conserved ELR motif present in granulocyte attracting, inflammatory chemokines such as IL-8. Furthermore, vIL-8, CXCL13L1, and CXCL1L2 are the only genes with three exons, while all other chicken CXC chemokines have four exons (Wang et al., 2005). In addition, we performed phylogenetic analyses of vIL-8 and cellular CXC chemokines (Figure 1B), in which vIL-8 clustered with the human and the chicken CXCL13 variants and not the inflammatory IL-8 chemokines, again indicating that vIL-8 is a CXCL13 orthologue.

To confirm the in silico analysis, we compared the biological properties of vIL-8 and the chicken CXCL13 variants. To obtain functional chemokines, we cloned huFC-tagged vIL-8 and CXCL13 variants into expression plasmids and produced recombinant proteins in HEK293 cells. Recombinant protein in culture supernatants was quantified by ELISA (Figure 2A). Western blot analysis confirmed the correct size of the monomeric form of CXCL13L1, L2, and L3 of approximately 38 kDa (Figure 2B). For CXCL13L1, an additional lower molecular weight band was observed that most likely represents a degradation product of the chemokine. As observed previously, the size of huFC and vIL-8 was larger than predicted (Engel et al., 2012), which is likely due to glycosylation of the proteins.

We previously demonstrated that vIL-8 binds to and induces chemotaxis of B cells (Engel et al., 2012). Therefore, we assessed the binding of the recombinant chemokines to the chicken B cell line DT40. vIL-8 and all three CXCL13 variants efficiently bound to chicken B cells (Figures 3A,B), while no binding was observed for the control protein. Interestingly, vIL-8 showed a clearly higher MFI than the CXCL13 variants.

To determine if vIL-8 and the CXCL13 variants induce B cell migration, we performed chemotaxis assays using DT40 cells. vIL-8 and the CXCL13 variants efficiently induced chemotaxis (Figure 3C), suggesting that these chemokines have similar biological functions.

To confirm that vIL-8 and the CXCL13 variants also target the same cell types in vivo, we performed binding assays with primary PBMCs. vIL-8, CXCL13L1 and CXCL13L2 bound to Bu1 positive B cells (Figure 4A), indicating that these chemokines also target the same cells in vivo. As on DT40 cells, strongest binding was observed for vIL-8. Surprisingly, CXCL13L3 did not bind primary B cells. In addition to B cells, vIL-8 was also previously shown to bind a certain subset of CD4⁺ T cells (Engel et al., 2012). Therefore, we assessed binding of vIL-8 and the CXCL13 variants to CD4⁺ T cells in primary PBMCs. vIL-8, CXCL13L1 and CXCL13L2 bound to a subpopulation of CD4⁺ T cells. As observed for B cells, vIL-8 bound these cells more efficiently than the cellular chemokines, while only minimal binding was observed for CXCL13L3 (Figure 4B). Taken together, our data demonstrate that vIL-8 has binding and chemotaxis properties comparable to CXCL13L1 and CXCL13L2, further supporting our in silico data that the viral chemokine is a CXCL13 ortholog.

Next, we set to determine the receptor of vIL-8 and its cellular orthologs. In humans and mice, CXCL13 has only a single receptor, CXCR5. Hence we examined whether the chicken CXCL13 orthologs bind to the recently discovered chicken CXCR5 (DeVries et al., 2006). Therefore, we generated HEK293 cells that express the receptor with an N-terminal Flag-tag and used these cells to produce a monoclonal antibody against chicken CXCR5, which stains HEK293-CXCR5 as well as an anti-Flag positive control (Figure 5A). To assess binding of vIL-8 and CXCL13 variants, CXCR5 expressing cells were mixed with parental cells at a 1:2 ratio as an internal control for binding specificity. As the anti-CXCR5 antibody, vIL-8 efficiently bound the CXCR5 expressing cells but not the control cells (Figure 5B), demonstrating that CXCR5 is the receptor of the viral chemokine. Similarly, all three CXCL13 variants interacted with CXCR5, highlighting that this receptor-ligand pair is conserved from mammals to chickens.

Next, we assessed if the CXCR5 is expressed on the natural target cells of MDV in vivo. We isolated primary cells from blood and different lymphoid tissues and detected CXCR5 expression using our CXCR5 antibody. CXCR5 was efficiently expressed on B cells and a subset of CD4⁺ cells as shown for vIL-8 (Figure 5C). In addition, we assessed CXCR5 expression in cells isolated from blood (Figure S1A), spleen (Figure S1B), bursa of Fabricius (Figure S1C) and thymus (Figure S1D). CXCR5 was found on all mature B cells in the spleen and all immature B cells in the Bursa of Fabricius. Very few CXCR5 positive T cells were observed in the blood, while about 10% of splenic CD4 and CD8 positive cells do express CXCR5. Among thymic T cells, CXCR5 expression is limited to small subpopulations of CD4 and CD8 single positive cells. Intriguingly, all KUL01⁺ monocytes in the blood and most of the KUL01⁺ macrophages in the spleen do also express CXCR5, suggesting the vIL-8 could also recruit these cells for MDV infection. Taken together, our data demonstrate that CXCR5 is the receptor of vIL-8 and CXCL13 and is expressed by B cells, CD4+ T cell subsets and monocytes /macrophages.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.