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. 2020 Dec;20(12):3341-3353.
doi: 10.1111/ajt.15950. Epub 2020 May 15.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR

Cynthia S M Kramer  1 , Marry E I Franke-van Dijk  1 , Kim H Bakker  1 , Merve Uyar-Mercankaya  1 , Gonca E Karahan  1 , Dave L Roelen  1 , Frans H J Claas  1 , Sebastiaan Heidt  1
Affiliations

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR

Cynthia S M Kramer et al. Am J Transplant. 2020 Dec.

Abstract

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor-specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA-DR-specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA-DR antigen-reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*07:01 antigen-reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA-DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Keywords: alloantibody; basic (laboratory) research / science; histocompatibility; immunogenetics; major histocompatibility complex (MHC); recipient selection; sensitization.

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Conflict of interest statement

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

FIGURE 1
FIGURE 1
HLA‐DR‐specific memory B cell clones isolated from peripheral blood. A, Representative example of 3 independent experiments depicting the flow cytometry gating strategy to live single cell sort CD3‐CD27+IgD‐HLA‐DR tetramer double positive B cells from PBMC. B, IgG antibody production by the clones was determined by ELISA. C, IgG positive clones were screened with HLA class II Lifecodes Lifescreen Deluxe kit to detect HLA antibody. The kit contains 5 groups of HLA class II beads and each data point represents a single bead group. D, HLA‐specific B cell clones were tested with SAB assays to confirm tetramer specificity used for cell sorting. Each dot presents 1 clone and only MFI of bead with tetramer specificity is depicted. E, Percentage of sorted HLA‐specific B cells from total memory B cells. F, Percentage of HLA antibody‐producing B cell clones from sorted B cells. On the x‐axis are the specificity of the tetramers used depicted. FSC, forward scatter; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; SAB, single antigen beads; SSC, side scatter [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 2
FIGURE 2
Recombinant human HLA‐DR monoclonal antibodies have the same reactivity as the B cell clones. HLA‐DR specificities in serum, supernatants of B cell clones (italic) and generated IgG1 mAbs. A, Serum of individual used for HLA‐DRB1*01:01 sort and the generated LB_DR1_B mAb and with its respective B cell clone. B, Serum of individual used for HLA‐DRB1*04:01/04:05 sort and the generated LB_DR4_A mAb and its respective B cell clone. C, Serum of individual used for HLA‐DRB1*07:01 sort and the generated LB_DR7 mAbs with their respective B cell clones. Only DRB1/3/4/5 beads are shown as all other loci were negative for the B cell clones and mAbs. Purified recombinant monoclonal antibody concentration tested was 62.5 nM. BCM, background corrected mean fluorescence intensity; mAbs, monoclonal antibodies
FIGURE 3
FIGURE 3
Reactivity analysis of LB_DR1_B monoclonal antibody. A, Comparison of the amino acid positions of interests of the reactive HLA‐DR alleles of LB_DR1_B mAb and a selection of the nonreactive HLA‐DR alleles. B, Locations of amino acid 70Q (yellow), 71R (magenta), 73A (green), and 74A (orange) are indicated on crystal structure of DRB1*01:01 (PDB: 3PDO). C, LB_DR1_B mAb interacts with HLA‐DR alleles containing the functional epitope 70Q 73A. D, Schematic representation of the footprint of LB_DR1_B mAb that is highly reactive for HLA‐DR alleles containing the functional epitope 70Q 73A (cyan) and additional amino acids 71R and 74A. In crystal structures the β chain is colored dark blue, α chain light blue, and peptide is gray. Purified recombinant monoclonal antibody concentration tested was 62.5nM. BCM, background corrected mean fluorescence intensity, negative values are presented as zero. mAbs, monoclonal antibodies; PDB, Protein Data Bank; s, self HLA alleles of antibody‐producer
FIGURE 4
FIGURE 4
Reactivity analysis of LB_DR4_A monoclonal antibody. A, Comparison of the amino acid positions of interests of the reactive HLA‐DR alleles of LB_DR4_A mAb and a selection of the nonreactive HLA‐DR alleles. B, Locations of amino acid 31F (red), 32F (yellow), 33H (magenta), 37Y (orange), 38V (lilac), and (C) 13H (green) are indicated on crystal structure of DRB1*04:01 (PDB: 4MD4). D, Schematic representation of the footprint of LB_DR4_A mAb interacting with the functional epitope 31F 32Y 37Y (cyan) (E) LB_DR4_A mAb weakly binds to HLA alleles containing 31F 32Y 37S. F, Schematic representation of the footprint of LB_DR4_A mAb interacting with the highly reactive HLA‐DR4 alleles. In crystal structures the β chain is colored dark blue, α chain light blue, and peptide is gray. Purified recombinant monoclonal antibody concentration tested was 62.5nM. BCM, background corrected mean fluorescence intensity, negative values are presented as zero. mAbs, monoclonal antibodies; PDB, Protein Data Bank; s, self HLA alleles of antibody‐producer
FIGURE 5
FIGURE 5
Reactivity analyses of LB_DR7_A, and LB_DR7_B and LB_DR7_D monoclonal antibodies. A, Comparison of the amino acids of the reactive HLA‐DR alleles of LB_DR7_A mAb with nonreactive HLA‐DR alleles. B, Positions 30 (green), and 11 (magenta) and (C) 25 (yellow), and 14 (orange) are indicated on the crystal structure of DRB1*01:01 (PDB: 3PDO). D, A schematic representation of the footprint of LB_DR7_A mAb with 14K 25Q as functional epitope (cyan). E, Comparison of the amino acids of the reactive HLA‐DR alleles of LB_DR7_B and LB_DR7_D with the nonreactive HLA‐DR alleles, of which only a selection is shown. Only BCM of LB_DR7_B are depicted. F, Location of position 78 (yellow) and (G) of 96 (orange), 98 (magenta), and 120 (green) on the DRB1*01:01 crystal structure. H, A schematic representation of LB_DR7_B and LB_DR7_D footprint with 78V or (I) with 96H 98E 120S as the functional epitope (cyan) of the mAb. In crystal structures the β chain is colored dark blue, α chain light blue, and peptide is gray. Purified recombinant monoclonal antibody concentration tested was 62.5nM. BCM, background corrected mean fluorescence intensity, negative values are presented as zero. mAbs, monoclonal antibodies; PDB, Protein Data Bank; s, self HLA‐DR alleles of antibody‐producer
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