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

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.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

HLA-DQ-Specific Recombinant Human Monoclonal Antibodies Allow for In-Depth Analysis of HLA-DQ Epitopes.

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation.

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8+ T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8+ T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions.

Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies.

The Ig-genes of the heavy chains in anti-D-specific hybridomas and Fab/scFv-fragments selected from phage-display libraries are restricted to a group of closely related genes (IGHV3s genes). We analyzed the Ig-gene repertoire in anti-D-specific B cells of two hyperimmunized donors using a completely different method. Single B cells were cultured for 10 days in an EL4.B5 culture system. mRNA from anti-D-producing B cells was reverse transcribed into cDNA. Heavy- and light-chain gene rearrangements were amplified by PCR reactions, sequenced and cloned into a pNUT-vector system, thereby allowing the production of complete IgG and IgM. Eleven anti-D-specific B-cell clones were isolated and analyzed. Eight of these clones (including IgM-producing clones) had IGHV3s genes. We demonstrated that functional anti-D-specific IgM (4 clones) and IgG (2 clones) was produced. Using a new method, we analyzed the IGHV gene repertoire of anti-D-specific B cells of hyperimmunized donors and showed that it is indeed restricted. Moreover, we found a high frequency (1:100 and 1:500) of anti-D-specific B cells in the peripheral B cells of hyperimmunized donors. We suggest that this approach could be applied for the selection of human mAbs from immunized donors and for the analysis of Ig-gene repertoires at the single-B cell level.

The single antigen expressing lines (SALs) concept: an excellent tool for screening for HLA-specific antibodies.

Definition of the antibody specificity in the serum of patients waiting for a renal transplant or in need for platelet transfusion is a crucial step for finding adequate donors. Confounding factors are the complexity of the serum antibodies and the expression of several, up to six, different human leukocyte antigens (HLA) on peripheral blood lymphocytes used as target cells in the antibody screening. Single antigen-expressing (SAL) cell lines were generated by transfecting human major histocompatibility complex (MHC) class I sequences into K562, an erythroleukemia-derived cell line lacking MHC class I and II expression. Thirty-seven different SALs have been generated so far. In this study, we present the validation of 16 of those SALs by flow cytometry against a panel of 84 human HLA-specific monoclonal antibodies (30 HLA-A [8 IgG/22 IgM], 45 HLA-B [18 IgG/27 IgM], 6 HLA-A, B [3 IgG/3 IgM], and 3 HLA-C [all IgM]) developed in our laboratory. The SALs proved to be suitable tools to determine acceptable mismatches for highly sensitized patients. This concept of transfecting target sequences in immortalized cell lines opens up new avenues in the definition of serum and cellular reactivity for sensitized patients awaiting a suitable organ or blood component.

Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers.

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.