Epitope Mapping of Anti-mouse ACKR4 Monoclonal Antibodies Developed by N-Terminal Peptide Immunization

Introduction

Immune cell priming, effector responses, and memory formation are governed by chemokines and the spatially restricted expression of G protein–coupled receptors (GPCRs). ¹ Chemokine receptors constitute a major class of seven-transmembrane receptors and are categorized as canonical GPCRs and atypical chemokine receptors (ACKR1–ACKR4).^2,3 Ligand binding to canonical chemokine receptors activates heterotrimeric G proteins.^1,4 In contrast, ACKRs, despite structural homology to GPCRs, do not couple to G protein signaling. Instead, they mediate β-arrestin–dependent internalization and degradation of chemokines, thereby functioning as scavenger receptors that regulate chemokine bioavailability.^2,5 Cryogenic-electron microscopy analyses of agonist-bound ACKR3 revealed a distinct chemokine-binding mechanism and provided a structural basis for β-arrestin-biased receptor.^6,7

ACKR4 is expressed in T lymphocytes ⁸ and in stromal compartments^9,10 and binds CCL19, CCL20, CCL21, CCL22, and CCL25. ² Through ligand sequestration, ACKR4 modulates CCR7-, CCR6-, CCR4-, and CCR9-dependent migratory responses. ² This activity establishes chemokine gradients that direct dendritic cell trafficking from peripheral tissues to draining lymph nodes.^9,11,12 ACKR4 is also expressed in a flow-dependent manner in afferent lymphatic collectors, where it removes CCL21 from the luminal surface and limits aberrant T-cell entry into inflamed dermal collectors. Consistent with this function, ACKR4 deficiency impairs T-cell migration to draining lymph nodes. ¹³

For functional analysis of ACKR4-expressing cells in mouse preclinical models, monoclonal antibodies (mAbs) against mouse ACKR4 (mACKR4) are required. Using immunization with the N-terminal mACKR4 peptide, we previously generated anti-mACKR4 mAbs, including A₄Mab-1, A₄Mab-2, and A₄Mab-3. ¹⁴ This study determined their specific binding epitopes using an alanine scanning strategy.

Materials and Methods

Results

Determination of A₄Mab-1, A₄Mab-2, and A₄Mab-3 epitopes by flow cytometry using alanine scanning

Alanine scanning was performed on the N-terminal region (aa 2–19) of mACKR4. Eighteen mutants with alanine (or glycine) substitutions in mACKR4 with an N-terminal MAP tag were created (Fig. 1). The MAP tag is essential for confirming the cell surface expression of each mutant. These mutant proteins were transiently expressed in CHO-K1 cells. Reactivity with A₄Mab-1, A₄Mab-2, and A₄Mab-3 was assessed by flow cytometry. As shown in Figure 2A, A₄Mab-1 did not react with the Y12A mutant. A₄Mab-2 did not react with three mutants (Y12A, E15A, and N16A) (Fig. 2B). Moreover, A₄Mab-3 did not react with three mutants (E14A, N16A, and E17A) (Fig. 2C). The cell surface expression of each mutant was confirmed with PMab-1, an anti-MAP tag mAb (Fig. 2D). The normalized reactivity (geometric mean of A₄Mabs divided by the geometric mean of PMab-1) was calculated in Supplementary Fig. S1.

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FIG. 1.

The illustration of alanine- (or glycine)-substituted mutants of mACKR4. The N-terminal amino acids of mACKR4 were substituted with alanine (or glycine). PMab-1 recognizes the N-terminal MAP tag and confirms the cell surface expression of each mutant.

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FIG. 2.

Determination of A₄Mab-1, A₄Mab-2, and A₄Mab-3 epitopes by flow cytometry using alanine scanning. CHO-K1 cells transiently expressing mACKR4 mutants and wild-type (WT) were treated with A₄Mab-1 (1 µg/mL, A), A₄Mab-2 (1 µg/mL, B), A₄Mab-3 (1 µg/mL, C), PMab-1 (1 µg/mL, D), or blocking buffer for 30 minutes at 4°C. The cells were then incubated with Alexa Fluor 488-conjugated anti-rat IgG. Red lines indicate cells treated with the primary mAbs, while black lines show cells treated with the blocking buffer. The normalized reactivity (geometric mean of A₄Mabs/geometric mean of PMab-1) was calculated in Supplementary Fig. S1. Experiments were conducted three times, and representative histograms were shown.

Determination of the A₄Mab-1 and A₄Mab-2 epitopes using Western blotting

We previously reported that A₄Mab-1 and A₄Mab-2 are suitable for Western blotting, but A₄Mab-3 did not work in Western blotting. ¹⁴ Therefore, mutant lysates were prepared and analyzed by Western blotting using A₄Mab-1 and A₄Mab-2. As shown in Figure 3A, A₄Mab-1 detected about 45 kDa major bands, which completely disappeared in the lysates of five mutants (Y11A, Y12A, E14A, E15A, and E17A). Furthermore, A₄Mab-2 also detected 45 kDa major bands, which completely disappeared in the lysates of five mutants (Y11A, Y12A, Y13A, E15A, and N16A) (Fig. 3B). PMab-1 was used to confirm the expressions (Fig. 3C). The normalized band intensity (A₄Mabs/PMab-1) was calculated in Supplementary Fig. S2.

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FIG. 3.

Determination of A₄Mab-1 and A₄Mab-2 epitopes by Western blotting. Cell lysates from CHO-K1 cells transiently expressed mACKR4 mutants and wild-type (WT) were electrophoresed on 5%–20% polyacrylamide gels and transferred onto PVDF membranes. Membranes were then treated using A₄Mab-1 (1 µg/mL, A), A₄Mab-2 (1 µg/mL, B), and PMab-1 (1 µg/mL, C). Then, the membranes were treated with horseradish peroxidase-conjugated anti-rat IgG. Protein bands were detected with Pierce™ ECL Plus or ImmunoStar LD. The normalized intensity (A₄Mabs/PMab-1) was calculated in Supplementary Fig. S2. Experiments were conducted three times, and representative images are shown.

Figure 4 summarizes the results and illustrates the epitope of A₄Mab-1, A₄Mab-2, and A₄Mab-3 in the N-terminal region of mACKR4.

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FIG. 4.

The schematic illustration of A₄Mab-1, A₄Mab-2, and A₄Mab-3 epitopes. (A) Tyr12 of mACKR4 is required for recognition by A₄Mab-1 in flow cytometry (yellow), and Tyr11, Tyr12, Glu14, Glu15, and Glu17 are required for recognition by A₄Mab-1 in Western blotting (underlined). (B) Tyr12, Glu15, and Asn16 of mACKR4 are required for recognition by A₄Mab-2 in flow cytometry (yellow), and Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are required for recognition by A₄Mab-2 in Western blotting (underlined). (C) Glu14, Asn16, and Glu17 of mACKR4 are required for the recognition by A₄Mab-3 in flow cytometry (yellow).

Discussion

This study conducted the epitope mapping of anti-mACKR4 mAbs (A₄Mab-1, A₄Mab-2, and A₄Mab-3) by alanine scanning. Results revealed that Tyr12 is required for recognition by A₄Mab-1 in flow cytometry (Fig. 2A), while Tyr11, Tyr12, Glu14, Glu15, and Glu17 are required in Western blotting (Fig. 3A). Since Tyr12 is a commonly identified residue required for A₄Mab-1 recognition, Tyr12 is thought to be a center of epitope. In A₄Mab-2, Tyr12, Glu15, and Asn16 are required in flow cytometry (Fig. 2B), while Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are required in Western blotting (Fig. 3B). Since Tyr12, Glu15, and Asn16 are commonly identified residues required for A₄Mab-2 recognition, these are thought to be the center of epitope. Additionally, Glu14, Asn16, and Glu17 are required for recognition by A₄Mab-3 in flow cytometry (Fig. 2C). The recognition mode by A₄Mab-1 and A₄Mab-2 may be different between the native structure in flow cytometry (Fig. 2) and the denatured structure in Western blotting (Fig. 3). Chemokine receptors and ACKRs possess a conserved disulfide bond between the N-terminus and the third extracellular loop. ¹⁷ Disruption of the disulfide bond may change the accessibility of A₄Mabs in Western blotting. These results would contribute to the understanding of mAb–epitope interaction.

ACKR4 has been established as a critical regulator of dendritic cell trafficking through modulation of CCR7-dependent pathways via regulation of the abundance of CCR7 ligands, CCL19 and CCL21. ¹⁸ The N-terminal region of CCR7 contains sulfated tyrosine residues that are essential for high-affinity binding to these ligands.^19,20 Although ACKR4 also binds to these ligands, the requirement for tyrosine sulfation in the ligand binding has not been investigated. A₄Mabs were established by immunization with the N-terminal 1–19 peptide without any modification, including sulfation. ¹⁴ The epitope of A₄Mab-1 and A₄Mab-2 includes a Tyr triplet (Fig. 4), suggesting that A₄Mab-1 and A₄Mab-2 recognize nonsulfated tyrosine residues.

Unlike canonical GPCRs, ACKR4 does not elicit classical G protein–mediated signaling; instead, it functions as a scavenging receptor that facilitates chemokine clearance.^21,22 Mechanistically, ACKR4 is coupled to the endocytic machinery via β-arrestin. Upon ligand engagement, ACKRs undergo internalization into endosomal compartments, followed by lysosomal degradation of the bound chemokine. ⁵ Although the N-terminal region of ACKR4 has not been definitively characterized as a ligand-binding interface, it remains of interest to determine the relationship between A₄Mab epitopes and the neutralizing or receptor internalization effects. These activities would be expected to attenuate mACKR4 function, potentially leading to an accumulation of its cognate chemokines.

Authors’ Contributions

M.H., M.Y., and T.N. performed the experiments. T.T., M.K.K., and Y.K. designed the experiments. H.S. and Y.K. wrote the article. All authors have read and agreed to the published version of the article.