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.

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).

Effects of Taurine Supplementation on Neuronal Excitability and Glucose Homeostasis.

In this study we examined the role of chronic taurine supplementation on plasma glucose homeostasis and brain excitability through activation of the insulin receptor. FVB/NJ male mice were supplemented with taurine in drinking water (0.05% w/v) for 4 weeks and subjected to a glucose tolerance test (7.5 mg/kg BW) after 12 h fasting. We found that taurine-fed mice were slightly hypoglycemic prior to glucose injection and showed significantly reduced plasma glucose at 30 and 60 min post-glucose injection when compared to control mice. Previously, we reported that taurine supplementation induces biochemical changes that target the GABAergic system. Those studies show that taurine-fed mice are hyperexcitable, have reduced GABAA receptors expression and increased GAD and somatostatin expression in the brain. In this study, we found that taurine-fed mice had a significant increase in insulin receptor (IR) immuno-reactivity in the pancreas and all brain regions examined. At the mRNA level, we found that the IR showed differential regional expression. Surprisingly, we found that neurons express the gene for insulin and that taurine had a significant role in regulating insulin gene expression. We propose that increased insulin production and secretion in taurine-fed mice cause an increase activation of the central IR and may be partially responsible for the increased neuronal excitability observed in taurine supplemented mice. Furthermore, the high levels of neuronal insulin expression and its regulation by taurine implicates taurine in the regulation of metabolic homeostasis.