Diversity in the HLA-I Recognition of HLA-F Monoclonal Antibodies: HLA-F or HLA-Ib Monospecific, HLA-E or HLA-G Bispecific Antibodies with or without HLA-Ia Reactivity.

Previous investigators have used various anti-HLA-F monoclonal antibodies (mAbs) to demonstrate that the tissue distribution of HLA-F is highly restricted. Notably, these mAbs differed in their immunodiagnostic capabilities. Specifically, mAbs Fpep1.1 and FG1 detected HLA-F intracellularly in B cells but not on the cell surface, whereas mAb 3D11 detected HLA-F on the cell surface. The presence of HLA-F on T cells was recognized by mAb FG1 but not by mAb Fpep1.1. mAb 3D11 detected HLA-F on the cell surface of activated B cells and on peripheral blood lymphocytes, but not on the normal cells. Importantly, mAb 3D11 revealed that HLA-F exists as a heavy chain (HC) monomer, rather than as an HC associated with B2m. Although these mAbs are believed to be specific to HLA-F, their monospecificity has not been formally established, which is critical for immunodiagnostic and therapeutic purposes. Previously, we investigated the diversity of HLA class I reactivities of anti-HLA-E mAbs using HLA-I coated multiplex bead assays on a Luminex platform. We reported that more than 80% of the HLA-E mAbs were cross-reactive with other HLA-I molecules, with exceptionally few truly HLA-E-monospecific mAbs. In the present investigation, we generated IgG mAbs against HCs of HLA-F in Balb/C mice and examined the cross-reactivity of anti-HLA-F mAbs with other HLA-I alleles using a multiplex bead assay on the Luminex platform. Beads coated with an array of HLA homo- and heterodimers of different HLA-Ia (HLA-A, HLA-B, and HLA-C) and Ib (HLA-E, HLA-F, and HLA-G) alleles were used to examine the binding of the anti-HLA-F mAbs. Only two mAbs were HLA-F monospecific, and five were HLA-Ib restricted. Several anti-HLA-F mAbs cross-reacted with HLA-E (n = 4), HLA-G (n = 3), HLA-Ia alleles (n = 9), HLA-G and HLA-Ia (n = 2), and HLA-Ib and HLA-Ia (n = 6). This monospecificity and polyreactivity were corroborated by the presence of HLA-F monospecific and HLA-I-shared sequences. This study emphasizes the need to monitor the mono-specificity of HLA-F for reliable immunodiagnostics and passive immunotherapy.

Cell Surface B2m-Free Human Leukocyte Antigen (HLA) Monomers and Dimers: Are They Neo-HLA Class and Proto-HLA?

Cell surface HLA-I molecules (Face-1) consist of a polypeptide heavy chain (HC) with two groove domains (G domain) and one constant domain (C-domain) as well as a light chain, B2-microglobulin (B2m). However, HCs can also independently emerge unfolded on the cell surface without peptides as B2m-free HC monomers (Face-2), B2m-free HC homodimers (Face 3), and B2m-free HC heterodimers (Face-4). The transport of these HLA variants from ER to the cell surface was confirmed by antiviral antibiotics that arrest the release of newly synthesized proteins from the ER. Face-2 occurs at low levels on the normal cell surface of the lung, bronchi, epidermis, esophagus, breast, stomach, ilium, colorectum, gall bladder, urinary bladder, seminal vesicles ovarian epithelia, endometrium, thymus, spleen, and lymphocytes. They are upregulated on immune cells upon activation by proinflammatory cytokines, anti-CD3 antibodies, antibiotics (e.g., ionomycin), phytohemagglutinin, retinoic acid, and phorbol myristate acetate. Their density on the cell surface remains high as long as the cells remain in an activated state. After activation-induced upregulation, the Face-2 molecules undergo homo- and hetero-dimerization (Face-3 and Face-4). Alterations in the redox environment promote dimerization. Heterodimerization can occur among and between the alleles of different haplotypes. The glycosylation of these variants differ from that of Face-1, and they may occur with bound exogenous peptides. Spontaneous arthritis occurs in HLA-B27+ mice lacking B2m (HLA-B27+ B2m-/-) but not in HLA-B27+ B2m+/- mice. The mice with HLA-B27 in Face-2 spontaneous configuration develop symptoms such as changes in nails and joints, hair loss, and swelling in paws, leading to ankyloses. Anti-HC-specific mAbs delay disease development. Some HLA-I polyreactive mAbs (MEM series) used for immunostaining confirm the existence of B2m-free variants in several cancer cells. The upregulation of Face-2 in human cancers occurs concomitantly with the downregulation of intact HLAs (Face-1). The HLA monomeric and dimeric variants interact with inhibitory and activating ligands (e.g., KIR), growth factors, cytokines, and neurotransmitters. Similarities in the amino acid sequences of the HLA-I variants and HLA-II ß-chain suggest that Face-2 could be the progenitor of both HLA classes. These findings may support the recognition of these variants as a neo-HLA class and proto-HLA.

Antibodies for ß2-Microglobulin and the Heavy Chains of HLA-E, HLA-F, and HLA-G Reflect the HLA-Variants on Activated Immune Cells and Phases of Disease Progression in Rheumatoid Arthritis Patients under Treatment.

Rheumatoid arthritis (RA) is a progressive, inflammatory, autoimmune, symmetrical polyarticular arthritis. It is characterized by synovial infiltration and activation of several types of immune cells, culminating in their apoptosis and antibody generation against "altered" autoantigens. ß2-microglobulin (ß2m)-associated heavy chains (HCs) of HLA antigens, also known as closed conformers (Face-1), undergo "alteration" during activation of immune cells, resulting in ß2m-free structural variants, including monomeric open conformers (Face-2) that are capable of dimerizing as either homodimers (Face-3) or as heterodimers (Face-4). ß2m-free HCs uncover the cryptic epitopes that can elicit antibodies (Abs). We report here the levels of IgM and IgG Abs against both ß2m and HCs of HLA-E, HLA-F, and HLA-G in 74 RA patients receiving immunosuppressive drugs. Anti-ß2m IgM was present in 20 of 74 patients, whereas anti-ß2m IgG was found in only 8 patients. Abs against ß2m would be expected if Abs were generated against ß2m-associated HLA HCs. The majority of patients were devoid of either anti-ß2m IgM or IgG but had Abs against HCs of different HLA-Ib molecules. The paucity of anti-ß2m Abs in this cohort of patients suggests that Abs were developed against ß2m-free HLA HCs, such as Face-2, Face-3, and Face-4. While 63 of 68 patients had IgG Abs against anti-HLA-F HCs, 36 and 50 patients showed IgG Ab reactivity against HLA-E and anti-HLA-G HCs, respectively. Evidently, anti-HLA-F HC Abs are the most predominant anti-HLA-Ib HC IgG Abs in RA patients. The incidence and intensity of Abs against HLA-E, HLA-F, and HLA-G in the normal control group were much higher than those observed in RA patients. Evidently, the lower level of Abs in RA patients points to the impact of the immunosuppressive drugs on these patients. These results underscore the need for further studies to unravel the nature of HLA-F variants on activated immune cells and synoviocytes of RA patients.

Role of HLA-I Structural Variants and the Polyreactive Antibodies They Generate in Immune Homeostasis.

Cell-surface HLA-I molecules consisting of ß2-microglobulin (ß2m) associated heavy chains (HCs), referred to as Face-1, primarily present peptides to CD8+ T-cells. HCs consist of three α-domains, with selected amino acid sequences shared by all alleles of all six isoforms. The cell-surface HLA undergoes changes upon activation by pathological conditions with the expression of ß2m-free HCs (Face-2) resulting in exposure of ß2m-masked sequences shared by almost all alleles and the generation of HLA-polyreactive antibodies (Abs) against them. Face-2 may homodimerize or heterodimerize with the same (Face-3) or different alleles (Face-4) preventing exposure of shared epitopes. Non-allo immunized males naturally carry HLA-polyreactive Abs. The therapeutic intravenous immunoglobulin (IVIg) purified from plasma of thousands of donors contains HLA-polyreactive Abs, admixed with non-HLA Abs. Purified HLA-polyreactive monoclonal Abs (TFL-006/007) generated in mice after immunizing with Face-2 are documented to be immunoregulatory by suppressing or activating different human lymphocytes, much better than IVIg. Our objectives are (a) to elucidate the complexity of the HLA-I structural variants, and their Abs that bind to both shared and uncommon epitopes on different variants, and (b) to examine the roles of those Abs against HLA-variants in maintaining immune homeostasis. These may enable the development of personalized therapeutic strategies for various pathological conditions.

Ramifications of the HLA-I Allelic Reactivity of Anti-HLA-E*01:01 and Anti-HLA-E*01:03 Heavy Chain Monoclonal Antibodies in Comparison with Anti-HLA-I IgG Reactivity in Non-Alloimmunized Males, Melanoma-Vaccine Recipients, and End-Stage Renal Disease Patients.

Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E HC-affinity columns, suggesting that the HLA-I reactivity is due to antibodies formed against HLA-E. Hence, we examined whether anti-HLA-E antibodies can react to HLA-I alleles. Monoclonal IgG antibodies (mAbs) against HCs of two HLA-E alleles were generated in Balb/C mice. The antibodies were analyzed using multiplex bead assays on a Luminex platform for HLA-I reactivity. Beads coated with an array of HLA heterodimers admixed with HCs (LABScreen) were used to examine the binding of IgG to different HLA-Ia (31-HLA-A, 50-HLA-B, and 16-HLA-C) and Ib (2-HLA-E, one each of HLA-F and HLA-G) alleles. A striking diversity in the HLA-Ia and/or HLA-Ib reactivity of mAbs was observed. The number of the mAbs reactive to (1) only HLA-E (n = 25); (2) all HLA-Ib isomers (n = 8); (3) HLA-E and HLA-B (n = 5); (4) HLA-E, HLA-B, and HLA-C (n = 30); (5) HLA-E, HLA-A*1101, HLA-B, and HLA-C (n = 83); (6) HLA-E, HLA-A, HLA-B, and HLA-C (n = 54); and (7) HLA-Ib and HLA-Ia (n = 8), in addition to four other minor groups. Monospecificity and polyreactivity were corroborated by HLA-E monospecific and HLA-I shared sequences. The diverse HLA-I reactivity of the mAbs are compared with the pattern of HLA-I reactivity of serum-IgG in non-alloimmunized males, cancer patients, and ESKD patients. The findings unravel the diagnostic potential of the HLA-E monospecific-mAbs and immunomodulatory potentials of IVIg highly mimicking HLA-I polyreactive-mAbs.

Four Faces of Cell-Surface HLA Class-I: Their Antigenic and Immunogenic Divergence Generating Novel Targets for Vaccines.

Leukocyte cell-surface HLA-I molecules, involved in antigen presentation of peptides to CD8+ T-cells, consist of a heavy chain (HC) non-covalently linked to ß2-microglobulin (ß2m) (Face-1). The HC amino acid composition varies across all six isoforms of HLA-I, while that of ß2m remains the same. Each HLA-allele differs in one or more amino acid sequences on the HC α1 and α2 helices, while several sequences among the three helices are conserved. HCs without ß2m (Face-2) are also observed on human cells activated by malignancy, viral transformation, and cytokine or chemokine-mediated inflammation. In the absence of ß2m, the monomeric Face-2 exposes immunogenic cryptic sequences on these cells as confirmed by HLA-I monoclonal antibodies (LA45, L31, TFL-006, and TFL-007). Furthermore, such exposure enables dimerization between two Face-2 molecules by SH-linkage, salt linkage, H-bonding, and van der Waal forces. In HLA-B27, the linkage between two heavy chains with cysteines at position of 67 of the amino acid residues was documented. Similarly, several alleles of HLA-A, B, C, E, F and G express cysteine at 67, 101, and 164, and additionally, HLA-G expresses cysteine at position 42. Thus, the monomeric HC (Face-2) can dimerize with another HC of its own allele, as homodimers (Face-3), or with a different HC-allele, as heterodimers (Face-4). The presence of Face-4 is well documented in HLA-F. The post-translational HLA-variants devoid of ß2m may expose several cryptic linear and non-linear conformationally altered sequences to generate novel epitopes. The objective of this review, while unequivocally confirming the post-translational variants of HLA-I, is to highlight the scientific and clinical importance of the four faces of HLA and to prompt further research to elucidate their functions and their interaction with non-HLA molecules during inflammation, infection, malignancy and transplantation. Indeed, these HLA faces may constitute novel targets for passive and active specific immunotherapy and vaccines.

The Impact of Inflammation on the Immune Responses to Transplantation: Tolerance or Rejection?

Transplantation (Tx) remains the optimal therapy for end-stage disease (ESD) of various solid organs. Although alloimmune events remain the leading cause of long-term allograft loss, many patients develop innate and adaptive immune responses leading to graft tolerance. The focus of this review is to provide an overview of selected aspects of the effects of inflammation on this delicate balance following solid organ transplantation. Initially, we discuss the inflammatory mediators detectable in an ESD patient. Then, the specific inflammatory mediators found post-Tx are elucidated. We examine the reciprocal relationship between donor-derived passenger leukocytes (PLs) and those of the recipient, with additional emphasis on extracellular vesicles, specifically exosomes, and we examine their role in determining the balance between tolerance and rejection. The concept of recipient antigen-presenting cell "cross-dressing" by donor exosomes is detailed. Immunological consequences of the changes undergone by cell surface antigens, including HLA molecules in donor and host immune cells activated by proinflammatory cytokines, are examined. Inflammation-mediated donor endothelial cell (EC) activation is discussed along with the effect of donor-recipient EC chimerism. Finally, as an example of a specific inflammatory mediator, a detailed analysis is provided on the dynamic role of Interleukin-6 (IL-6) and its receptor post-Tx, especially given the potential for therapeutic interdiction of this axis with monoclonal antibodies. We aim to provide a holistic as well as a reductionist perspective of the inflammation-impacted immune events that precede and follow Tx. The objective is to differentiate tolerogenic inflammation from that enhancing rejection, for potential therapeutic modifications. (Words 247).

Antibodies to cryptic epitopes on HLA class I and class II heavy chains bound to single antigen beads: Clinically relevant?

Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta - 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post - transplant desensitization procedures.

Therapeutic Potential of HLA-I Polyreactive mAbs Mimicking the HLA-I Polyreactivity and Immunoregulatory Functions of IVIg.

HLA class-I (HLA-I) polyreactive monoclonal antibodies (mAbs) reacting to all HLA-I alleles were developed by immunizing mice with HLA-E monomeric, α-heavy chain (αHC) open conformers (OCs). Two mAbs (TFL-006 and TFL-007) were bound to the αHC's coated on a solid matrix. The binding was inhibited by the peptide 117AYDGKDY123, present in all alleles of the six HLA-I isoforms but masked by ß2-microglobulin (ß2-m) in intact HLA-I trimers (closed conformers, CCs). IVIg preparations administered to lower anti-HLA Abs in pre-and post-transplant patients have also shown HLA-I polyreactivity. We hypothesized that the mAbs that mimic IVIg HLA-I polyreactivity might also possess the immunomodulatory capabilities of IVIg. We tested the relative binding affinities of the mAbs and IVIg for both OCs and CCs and compared their effects on (a) the phytohemagglutinin (PHA)-activation T-cells; (b) the production of anti-HLA-II antibody (Ab) by B-memory cells and anti-HLA-I Ab by immortalized B-cells; and (c) the upregulation of CD4+, CD25+, and Fox P3+ T-regs. The mAbs bound only to OC, whereas IVIg bound to both CC and OC. The mAbs suppressed blastogenesis and proliferation of PHA-activated T-cells and anti-HLA Ab production by B-cells and expanded T-regs better than IVIg. We conclude that a humanized version of the TFL-mAbs could be an ideal, therapeutic IVIg-mimetic.

Enhancing Natural Killer and CD8+ T Cell-Mediated Anticancer Cytotoxicity and Proliferation of CD8+ T Cells with HLA-E Monospecific Monoclonal Antibodies.

Cytotoxic NK/CD8+ T cells interact with MHC-I ligands on tumor cells through either activating or inhibiting receptors. One of the inhibitory receptors is CD94/NKG2A. The NK/CD8+ T cell cytotoxic capability is lost when tumor-associated human leukocyte antigen, HLA-E, binds the CD94/NKG2A receptor, resulting in tumor progression and reduced survival. Failure of cancer patients to respond to natural killer (NK) cell therapies could be due to HLA-E overexpression in tumor tissues. Preventing the inhibitory receptor-ligand interaction by either receptor- or ligand-specific monoclonal antibodies (mAbs) is an innovative passive immunotherapeutic strategy for cancer. Since receptors and ligands can be monomeric or homo- or heterodimeric proteins, the efficacy of mAbs may rely on their ability to distinguish monospecific (private) functional epitopes from nonfunctional common (public) epitopes. We developed monospecific anti-HLA-E mAbs (e.g., TFL-033) that recognize only HLA-E-specific epitopes, but not epitopes shared with other HLA class-I loci as occurs with currently available polyreactive anti-HLA-E mAbs. Interestingly the amino acid sequences in the α1 and α2 helices of HLA-E, critical for the recognition of the mAb TFL-033, are strikingly the same sequences recognized by the CD94/NKG2A inhibitory receptors on NK/CD8+ cells. Such monospecific mAbs can block the CD94/NKG2A interaction with HLA-E to restore NK cell and CD8+ anticancer cell cytotoxicity. Furthermore, the HLA-E monospecific mAbs significantly promoted the proliferation of the CD4-/CD8+ T cells. These monospecific mAbs are also invaluable for the specific demonstration of HLA-E on tumor biopsies, potentially indicating those tumors most likely to respond to such therapy. Thus, they can be used to enhance passive immunotherapy once phased preclinical studies and clinical trials are completed. On principle, we postulate that NK cell passive immunotherapy should capitalize on both of these features of monospecific HLA-E mAbs, that is, the specific determination HLA-E expression on a particular tumor and the enhancement of NK cell/CD8+ cytotoxicity if HLA-E positive.

Significance of the intraindividual variability of HLA IgG antibodies in renal disease patients observed with different beadsets monitored with two different secondary antibodies on a Luminex platform.

The accurate measurement of anti-HLA alloantibodies in transplant candidates is required for determining the degree of sensitization and for the listing of unacceptable antigens for organ allocation. Both the configuration of the HLA molecules coated on the beads and the nature of detection antibodies may impede assessment of the presence and strength of anti-HLA IgG- with the Luminex single-antigen-bead assay. Sera antibodies of the end-stage renal disease patients were compared using LIFECODES (LC) and LABScreen (LS) beadsets monitored with polyclonal-Fab (IgHPolyFab) and monoclonal-IgG (FcMonoIgG) second antibodies. Positive results at mean fluorescence intensity (MFI) > 500 (at serum dilution 1/10) were used to calculate panel reactive antibody (cPRA) levels. LS-beadsets are coated with monomeric variants in addition to intact HLA antigens with or without peptides, while LC-beadsets are devoid of monomeric variants and with lesser levels of peptide-free heterodimers. Consequently, IgG antibodies against both classes of HLA were reactive to more antigens with LS than with LC-beadsets. For both classes, MFIs were also frequently higher with LS than with LC. For HLA-I, MFIs were higher with IgHPolyFab than with FcMonoIgG with the exception of sera with MFIs > 5000 where they were comparable. For HLA-II, the reverse occurred, with significantly higher levels with FcMonoIgG regardless of the beadsets. The intraindividual variability observed between beadsets with two detection antibodies elucidates that antigens found as acceptable with one beadset may end up unacceptable with the other beadsets, with the possibility of denying potentially compatible transplants to candidates.

Monitoring native HLA-I trimer specific antibodies in Luminex multiplex single antigen bead assay: Evaluation of beadsets from different manufacturers.

Luminex single antigen bead (SAB) assay utilizes beadsets coated with a set of cloned and purified HLA molecules, for monitoring serum anti-HLA antibodies. Particularly, the level of serum IgG against native HLA-I trimers (heavy chain (HC) and ß2-microglobulin (ß2m) with a peptide), expressed in allograft tissues is correlated with graft failure. In addition to native trimeric HLAI, the beadsets may carry HC only or the dimeric variants, peptide-free HC with ß2m and ß2m-free HC with or without peptides. Currently, three different HLA-I coated beadsets have been produced commercially. The HLA antigen density on one beadset was reported to be approximately 50% of that present on another beadset as evidenced by the binding of an anti-HLA-I mAb W6/32. To date, no efforts have been made to compare the relative distribution of HLA-I variants in these three beadsets. In this study, using monoclonal antibodies (W6/32, HC-10 and TFL-006) that can distinguish the structural variants based on their epitope specificities, the nature of the variants in the three beadsets were comparatively evaluated. One beadset (Beadset A, see Materials and methods for Brand and Manufacturer's names) (W6/32+/HC-10+/TFL-006+) carried at least three variants, while beadset B (W6/32+/HC-10+/TFL-006-) carried two (peptide-associated and peptide-free ß2m-HC) and the beadset C (W6/32+/HC-10-/TFL-006-) carried exclusively the HLA-I trimer suggesting its usefulness for specific monitoring native HLA-I trimer antibodies. Because of the salient differences in the variants coated on the different beadsets, it would be warranted to investigate, if these differences are clinically relevant for monitoring serum anti-HLA antibodies in sensitized patients waiting for donor organs and in allograft recipients (274).

HLA Class Ia and Ib Polyreactive Anti-HLA-E IgG2a Monoclonal Antibodies (TFL-006 and TFL-007) Suppress Anti-HLA IgG Production by CD19+ B Cells and Proliferation of CD4+ T Cells While Upregulating Tregs.

The anti-HLA-E IgG2a mAbs, TFL-006 and TFL-007, reacted with all HLA-I antigens, similar to the therapeutic preparations of IVIg. Indeed, IVIg lost its HLA reactivity, when its HLA-E reactivity was adsorbed out. US-FDA approved IVIg to reduce antibodies in autoimmune diseases. But the mechanism underlying IVIg-mediated antibody reduction could not be ascertained due to the presence of other polyclonal antibodies. In spite of it, the cost prohibitive high or low IVIg is administered to patients waiting for donor organ and for allograft recipients for lowering antiallograft antibodies. A mAb that could mimic IVIg in lowering Abs, with defined mechanism of action, would be highly beneficial for patients. Demonstrably, the anti-HLA-E mAbs mimicked several functions of IVIg relevant to suppressing the antiallograft Abs. The mAbs suppressed activated T cells and anti-HLA antibody production by activated B cells, which were dose-wise superior to IVIg. The anti-HLA-E mAb expanded CD4+, CD25+, and Foxp3+ Tregs, which are known to suppress T and B cells involved in antibody production. These defined functions of the anti-HLA-E IgG2a mAbs at a level superior to IVIg encourage developing their humanized version to lower antibodies in allograft recipients, to promote graft survival, and to control autoimmune diseases.

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab')2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients' sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.

Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies: Problems and Solutions.

BACKGROUND: Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection. METHODS: The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10). RESULTS: The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated ß2-microglobulin-associated HLA HC (pepA-ß2aHC), peptide-free-ß2aHC (pepF-ß2aHC), and ß2-free HC (ß2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-ß2aHC, low density of pepF-ß2aHC, and are lacking ß2fHC. The FCXM analyses confirmed the prevalence of pepA-ß2aHC, but not pepF-ß2aHC or ß2fHC on resting T cells. CONCLUSIONS: The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the ß2fHC or pepF-ß2aHC normalized donor-specific antibody level would reveal the true anti-pepA-ß2aHC reactivity associated with positive FCXM.