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Antibodies for β2-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
Antibodies for β2-Microglobulin and the Heavy Chains of HLA-E, HLA-F, and HLA-G Reflect the...
Ravindranath, Mepur H.;Ravindranath, Narendranath M.;Amato-Menker, Carly J.;El Hilali, Fatiha;Selvan, Senthamil R.;Filippone, Edward J.;Morales-Buenrostro, Luis Eduardo
2023-03-31 00:00:00
antibodies Article Antibodies for 2-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 1 , 2 , 3 4 5 Mepur H. Ravindranath * , Narendranath M. Ravindranath , Carly J. Amato-Menker , Fatiha El Hilali , 6 7 8 Senthamil R. Selvan , Edward J. Filippone and Luis Eduardo Morales-Buenrostro Department of Hematology and Oncology, Children’s Hospital, Los Angeles, CA 90027, USA Emeritus Research Scientist, Terasaki Foundation Laboratory, Santa Monica, CA 90064, USA Norris Dental Science Center, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA 90089, USA Department of Microbiology, Immunology, and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV 26506, USA Medico-Surgical, Biomedicine and Infectiology Research Laboratory, The Faculty of Medicine and Pharmacy of Laayoune & Agadir, Ibnou Zohr University, Agadir 80000, Morocco Division of Immunology and Hematology Devices, OHT 7: Office of In Vitro Diagnostics, Office of Product Evaluation and Quality, Center for Devices and Radiological Health, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA Division of Nephrology, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19145, USA Department of Nephrology and Mineral Metabolism, Institute of Nacional Medical Sciences and Nutrition Salvador Zubirán, Vasco de Quiroga 15, Sección XVI, Mexico City 14000, Mexico * Correspondence: thiruranganath04@gmail.com Citation: Ravindranath, M.H.; Ravindranath, N.M.; Amato-Menker, Abstract: Rheumatoid arthritis (RA) is a progressive, inflammatory, autoimmune, symmetrical C.J.; El Hilali, F.; Selvan, S.R.; polyarticular arthritis. It is characterized by synovial infiltration and activation of several types of Filippone, E.J.; Morales-Buenrostro, immune cells, culminating in their apoptosis and antibody generation against “altered” autoantigens. L.E. Antibodies for 2-Microglobulin 2-microglobulin ( 2m)-associated heavy chains (HCs) of HLA antigens, also known as closed and the Heavy Chains of HLA-E, conformers (Face-1), undergo “alteration” during activation of immune cells, resulting in 2m-free HLA-F, and HLA-G Reflect the structural variants, including monomeric open conformers (Face-2) that are capable of dimerizing as HLA-Variants on Activated Immune either homodimers (Face-3) or as heterodimers (Face-4). 2m-free HCs uncover the cryptic epitopes Cells and Phases of Disease Progression in Rheumatoid Arthritis that can elicit antibodies (Abs). We report here the levels of IgM and IgG Abs against both 2m Patients under Treatment. Antibodies and HCs of HLA-E, HLA-F, and HLA-G in 74 RA patients receiving immunosuppressive drugs. 2023, 12, 26. https://doi.org/ Anti- 2m IgM was present in 20 of 74 patients, whereas anti- 2m IgG was found in only 8 patients. 10.3390/antib12020026 Abs against 2m would be expected if Abs were generated against 2m-associated HLA HCs. The majority of patients were devoid of either anti- 2m IgM or IgG but had Abs against HCs of different Academic Editor: Marino Paroli HLA-Ib molecules. The paucity of anti- 2m Abs in this cohort of patients suggests that Abs were Received: 24 December 2022 developed against 2m-free HLA HCs, such as Face-2, Face-3, and Face-4. While 63 of 68 patients had Revised: 18 February 2023 IgG Abs against anti-HLA-F HCs, 36 and 50 patients showed IgG Ab reactivity against HLA-E and Accepted: 27 March 2023 anti-HLA-G HCs, respectively. Evidently, anti-HLA-F HC Abs are the most predominant anti-HLA-Ib Published: 31 March 2023 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. Copyright: © 2023 by the authors. These results underscore the need for further studies to unravel the nature of HLA-F variants on Licensee MDPI, Basel, Switzerland. activated immune cells and synoviocytes of RA patients. This article is an open access article distributed under the terms and Keywords: antibodies; IgM; IgG; 2-microglobulin; HLA-Ib; HLA-E; HLA-F; HLA-G; heavy chains; conditions of the Creative Commons Face-1; Face-2; Face-3; Face-4; homodimers; heterodimers; immunosuppressive drugs Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Antibodies 2023, 12, 26. https://doi.org/10.3390/antib12020026 https://www.mdpi.com/journal/antibodies Antibodies 2023, 12, 26 2 of 22 1. Introduction Chronic inflammatory, autoimmune, life-long debilitating diseases may predomi- nantly involve activation of (i) the adaptive immune system (rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis), (ii) the innate immune system (Crohn’s disease, ulcerative colitis), and (iii) a combination of both adaptive and innate immune systems (ankylosing spondylitis, psoriasis). The pathogenesis of these diseases is multifactorial due to complex interactions among genetic, environmental, and therapeutic factors. Therefore, it is difficult to identify a single or specific biomarker that could be used to define disease progression or identify an effective target for therapy. Developing personalized therapy for these diseases depends on distinguishing the shared and unshared events during their immunological and inflammatory progression. This investigation fo- cuses on antibody responses to HLA structural variants in RA patients receiving a variety of immunomodulatory therapies. For a better understanding of the diversity of the antibody responses, it is necessary to identify different stages of disease progression. RA in genetically susceptible individ- uals commences with asymptomatic synovial inflammation, followed by infiltration and activation of immune cells, proliferation, and antibody production against “altered” au- toantigens, and culminates in hyperplasia of the joints with bone and cartilage degradation. Immunological events can be generally distinguished into three phases during disease progression [1–4]. Phase-I commences with infiltration of immune cells into the synovium, which is further accelerated during Phase-II [5,6]. Phase-II involves further infiltration; hyperactivation; and proliferation of T and B lymphocytes, neutrophils, macrophages, macrophage-like synoviocytes (MLSs), and fibroblast-like synoviocytes (FLSs) in the syn- ovium. Furthermore, in this phase, pro-inflammatory cytokines are produced in the synovium, cartilage, and bone [7–9]. During this phase, abnormal B cell recognition leads to the production of autoantibodies. Some of those are generated against rheumatoid factor, anti-citrullinated protein, mutant citrullinated vimentin, and several other “altered” auto-proteins [6,10]. Phase-III demarcates deterioration of RA due to the induction of cell death, primarily by synovial apoptosis mediated by activated T cells and NK cells through the interaction of cell surface molecules of the TNF family, namely, the Fas antigen (CD95) and the Fas ligand (Fas-L). Apoptosis occurs in more than 50% of synoviocytes and T cells from synovial tissue and synovial fluid of RA patients [11]. While hyperactivation of immune cells promotes the proliferation and expression of cell surface “altered autoantigens”, culminating in their shedding, the occurrence of apoptosis-mediated cellular degeneracy also results in the release of cytoplasmic and cell-surface intact and “altered autoantigens” into the synovial fluid and then into circulation. The “altered autoproteins” expose the antigenic, cryptic domains of amino acids (epitopes) of the intact native proteins. Such exposure elicits the production of Abs. As usual, the first formed Abs against newly released “altered autoantigens” are IgM, followed by IgG Abs. The present investigation focuses on one such group of “altered auto-antigens”, namely, cell-surface HLA class-I. HLA class-I has diversified isomers, namely, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. A pair of the same or different alleles of each of the six isomers is expressed on the cell surfaces of naïve and activated T and B lym- phocytes, neutrophils, and macrophages. HLA-I molecules are expressed as heterodimers consisting of 2-microglobulin ( 2m) non-covalently associated with the HLA heavy chain (HC). The intact 2m-associated HLA is referred to as a closed conformer [12] or Face-1 [13]. However, 2m-free HCs (Face-2) are also expressed on the cell surface during activation under different pathological conditions, such as inflammation, autoimmune diseases, and malignancy, as illustrated in Figure 1. Such Face-2 molecules may homodimerize (2-HCs of the same allele) to form Face-3 or heterodimerize (HCs of different alleles of same or different isomers) to form Face-4. Antibodies 2023, 12, x FOR PEER REVIEW 3 of 23 activation under different pathological conditions, such as inflammation, autoimmune diseases, and malignancy, as illustrated in Figure 1. Such Face-2 molecules may homodi- Antibodies 2023, 12, 26 3 of 22 merize (2- HCs of the same allele) to form Face-3 or heterodimerize (HCs of different al- leles of same or different isomers) to form Face-4. Figure 1. All immune cells, particularly lymphocytes, express cell-surface HLA class-I molecules as heterodimers consisting of HLA HC polypeptide and 2m, together with a peptide in the groove. They are considered trimers. They are also known as closed conformers (Face-1). Upon activation, these cells express monomeric variants of HLA class-I molecules and are referred to as open con- Figure 1. All immune cells, particularly lymphocytes, express cell-surface HLA class-I molecules as heterodimers cons formers (Face-2). isting of HLA The Face-2 HC polypeptide an versions of HLA-C, d β2m, together with HLA-F, and HLA-G a peptide are also in observed the groov naturally e. on They are consi normald cells ered trimers. [14–17]. Face-2 They are also k is also observed nown as clo in the monocytes sed conformers ( of patients Face-1). Upon with spondylo-arthr activation, opathies these cells express monomeric variants of HLA class-I molecules and are referred to as open con- and RA [18–20]. The monomeric version (Face-2) may dimerize with its own allele (homodimers or formers (Face-2). The Face-2 versions of HLA-C, HLA-F, and HLA-G are also observed naturally on Face-3) or with other alleles of the same or different isomers (heterodimers or Face-4) [21–24].The normal cells [14–17]. Face-2 is also observed in the monocytes of patients with spondylo-arthropa- figure shows different -domains of the HC and different alleles in different colors. thies and RA [18–20]. The monomeric version (Face-2) may dimerize with its own allele (homodi- mers or Face-3) or with other alleles of the same or different isomers (heterodimers or Face-4) [21– We hypothesize that when Face-1 molecules are shed into synovial and body fluids, 24].The figure shows different α-domains of the HC and different alleles in different colors. both IgM and IgG can be formed against 2m, whereas when only 2m-free HCs (Face-2) are shed or recognized, the propensity for the appearance of anti- 2m can be virtually We hypothesize that when Face-1 molecules are shed into synovial and body fluids, absent. Possibly, the IgM and IgG profiles of serum anti-HLA-HC Abs with or without both IgM and IgG can be formed against β2m, whereas when only β2m-free HCs (Face-2) anti- 2m Abs may reflect the different phases of disease progression and may serve as are shed or recognized, the propensity for the appearance of anti-β2m can be virtually biomarkers for RA-progression-based therapies. absent. Possibly, the IgM and IgG profiles of serum anti-HLA-HC Abs with or without anti-β2m Abs may reflect the different phases of disease progression and may serve as 2. Material and Methods biomarkers for RA-progression-based therapies. 2.1. Information on the Patient Cohort The sera of 74 patients (57 females, 17 males) and sera of normal controls (26 males 2. Material and Methods and 26 females) obtained from clinical facilities in Mexico were provided by Professor Dr. 2.1. Information on the Patient Cohort Luis Eduardo Morales-Buenrostro, Departamento de Nefrología y Metabolismo Mineral, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga The sera of 74 patients (57 females, 17 males) and sera of normal controls (26 males 15, Sección XVI, Tlalpan, DF. 14000. MEXICO to Dr. Mikki Ozawa at TFL. The sera were and 26 females) obtained from clinical facilities in Mexico were provided by Professor Dr. collected after obtaining written informed consent approved by the Institutional Review Luis Eduardo Morales-Buenrostro, Departamento de Nefrología y Metabolismo Mineral, Boards in Mexico by Dr. Morales-Buenrostro, and further approved for research by the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga ethics committee at Terasaki Foundation Laboratory, by the late Professor Paul Terasaki. All 15, Sección XVI, Tlalpan, DF. 14000. MEXICO to Dr. Mikki Ozawa at TFL. The sera were patients were seropositive for rheumatoid factor. All clinical details, including demographic collected after obtaining written informed consent approved by the Institutional Review characteristics of the patients studied and different combinational treatment regimens with Boards in Mexico by Dr. Morales-Buenrostro, and further approved for research by the the drugs received prior to the time of blood drawing and the information available and ethics committee at Terasaki Foundation Laboratory, by the late Professor Paul Terasaki. permissible by ethics committee are presented as a Supplemental Table S1 and discussed in All patients were seropositive for rheumatoid factor. All clinical details, including demo- appropriate context. All sera were kept at 20 C before shipment and analyses of the sera. Antibodies 2023, 12, 26 4 of 22 2.2. Immunoregulatory Drugs Received by the Patient Cohort The specific immunomodulatory therapies received by the cohort of patients are presented in Table 1. Serum IgM and IgG Abs were studied a month or more after adminis- tration of the drugs. Since the drugs are capable of suppressing antibody production, their mechanisms of action are summarized hereunder: Methotrexate [25,26]: It inhibits dihydrofolate reductase, blocks the folic acid-dependent synthesis of purines and pyrimidines, inhibits T cell proliferation, diminishes macrophage recruitment and function, and promotes the release of adenosine, an anti-inflammatory mediator. Folic Acid [26]: It reduces methotrexate’s adverse effects. Chloroquine [27]: It inhibits MHC-II expression, antigen presentation, immune ac- tivation, and the production of pro-inflammatory cytokines. It interferes with Toll-like receptor 7 (TLR7) and TLR9 signaling pathways, and interferes with cyclic GMP-AMP synthase activity. Leflunomide (ARAVA) [28]: It suppresses cell proliferation in activated lymphocytes; inhibits (i) dihydro-orotate dehydrogenase activity and protein tyrosine kinase activity in actively dividing cells, (ii) nuclear factor B (NFB) activation and NFB-dependent reporter gene expression, (iii) oxygen free-radical generation, and (iv) immunoglobulin IgG and IgM production; and (v) lowers IL-1 and IL-2 levels. Prednisone [29]: It inactivates NFB, decreasing proinflammatory cytokine produc- tion; inhibits cyclooxygenase-2, adhesion molecules, and other inflammatory mediators, and importantly, suppresses IgM and IgG production. Azulfidine (Sulfasalazine) [30]: It inhibits the release of IL-2 produced by T cells, and IL-1, IL-8, IL-12, and TFN- produced by monocytes and macrophages; induces apoptosis of macrophages; inhibits the production of serum IgM and IgG; and suppresses infiltration of fibroblasts, neutrophils, and plasma cells. Azathioprine [31]: It decreases leukocyte proliferation; promotes apoptosis of acti- vated T cells and macrophages; reduces the expression of leukocyte adhesion molecules; and impairs NFkB signaling. Omeprazole [32]: Omeprazole inhibits both basal and drug-stimulated gastric acid secretion in a dose-dependent fashion. ACEI (Angiotensin Convertase Enzyme Inhibitor) [33]: It prevents the risk of hyper- tension and cerebrovascular disease, which are increased in RA patients. 2.3. Antigen Source Only HLA-Ib proteins were investigated in this study. Recombinant HLA-E, HLA-F, HLA-G, and 2m folded HCs (10 mg/mL in 2-[N-morpholino] ethanesulfonic acid [MES] buffer) were obtained from the Immune Monitoring Laboratory, Fred Hutchinson Cancer Research Center (University of Washington, Seattle, WA). Recombinant HCs of HLA-Ib R107 alleles (HLA-E , HLA-F1, and HLA-G1) were folded and made available for coating microbeads. Figure 2 shows the amino acid sequences of the HCs of HLA-E , HLA-F, HLA-G, and 2m used for coating the beads. All HLA-Ib alleles have only the extracellular domain without the leader peptide containing 21 amino acids, and no transmembrane or intracellular domains. 2.4. Immunoassay with Single Antigen Beads To detect IgM and IgG reactivity to HCs of HLA-Ib isomers and 2m in the sera of RA patients and in normal males and females of the same ethnicity, a multiplex Luminex - based immunoassay (One Lambda, Canoga Park, CA, USA) was used, as described ear- lier [34–37]. The recombinant HLA-E, HLA-F, and HLA-G HCs (10 mg/mL in MES buffer) and recombinant 2m (same concentration) were individually attached by a process of sim- ple chemical coupling to differently fluorochromed 5.6 m polystyrene microspheres inter- nally dyed with infrared fluorophores. The sera were diluted 1:10 with phosphate-buffered ® ® saline (PBS, pH 7.2). Using dual-laser flow cytometry (Luminex xMAP multiplex tech- nology), single antigen (AG) assays were carried out for data acquisition and analysis of Antibodies 2023, 12, 26 5 of 22 anti-HLA-Ib Abs (39–40). For HLA-E, HLA-F, HLA-G, and 2m, positive (coated with IgG) and negative (coated with human or bovine albumin) controls were added separately. IgG and IgM screenings were performed using secondary anti-human IgG (One Lambda, cat. no. LS-AB2) and secondary anti-human IgM (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, cat. no. 709-116-073, USA), respectively. The secondary Ab was used at a dilution of 1/100. Data generated with Luminex Multiplex Flow Cytometry (LABScan 100, Thermo Fisher Scientific, Waltham, MA, USA) were analyzed using computer software, the protocol being the same as that reported earlier [33–39]. The mean and SD of the mean fluorescence intensity (MFI) for each allele were recorded. Table 1. Specific immunomodulatory drug combinations administered to patients in different groups included methotrexate, folic acid, chloroquine, leflunomide, prednisone, azulfidine (Sulfasalazine), azathioprine, omeprazole and IECA (angiotensin convertase inhibitor). (+) indicates usage of the drug in the patients. Number of Patients with Serum IgM & IgG Chemotherapy Regimens for RA Antibodies Reacting to 2M, HLA-E/-F & G in Each Group Group 1 Group2 Group 3 Group 4 Group 5 Group 6 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 14/16 23/24 15/15 6/6 5/5 2/3 1 [+] - - - - - - - - - 2 2 [+] [+] - - - - - - - - 2 2 3 [+] [+] [+] - - - - - - - 3 6 3 1 4 [+] [+] [+] [+] - - - - - - 2 5 [+] [+] [+] [+] [+] - - - - - 1 6 [+] [+] [+] - - - [+] - - - 1 7 [+] [+] [+] - [+] - - - - - 1 1 8 [+] [+] - [+] - - - - - - 1 2 9 [+] [+] - [+] [+] - - - - - 1 10 [+] [+] - [+] [+] [+] - - - - 1 11 [+] [+] - [+] - - [+] - - - 1 12 [+] [+] - [+] [+] [+] [+] [+] - - 1 13 [+] [+] - - [+] - - - - - 1 14 [+] [+] - - [+] - [+] - [+] - 1 15 [+] [+] - - - - [+] [+] - - 1 16 [+] [+] - - - - [+] - - - 1 17 [+] [+] - - - - [+] - [+] - 1 18 [+] [+] - - - - - - [+] - 1 19 [+] [+] - - - [+] - - - - 1 1 1 20 [+] - [+] - - - - - - - 3 2 1 1 21 [+] - [+] - - - [+] - - - 1 22 [+] - [+] [+] - - - - - - 1 23 [+] - [+] - - [+] - - - - 1 24 [+] - - - [+] - - - - - 1 25 [+] - - - - [+] - - - - 2 26 [+] - - - - - [+] - - - 2 1 27 [+] - - - - - - - [+] - 1 28 - - [+] - - - - - - - 2 29 - - [+] - - - - - [+] - 1 30 - - [+] [+] [+] - [+] - [+] - 1 31 - - - [+] - [+] - - - - 1 1 32 - - - [+] - [+] - - [+] [+] 1 RA, Rheumatoid Arthritis; ACEI, Angiotensin Convertase Enzyme Inhibitor. Methotrexate Folic Acid Chloroquine ARAVA Azulfidine ACEI Prednisone Azathioprine Omeprazole Thyroid enzyme Antibodies 2023, 12, 26 6 of 22 Table 2. Profiles of serum Abs in Group 1 with 16 RA patients, one of whom had both RA and SLE. These patients did not have Abs against 2m, but had IgM against HCs of HLA-E (n = 13), HLA-F (n = 7), and HLA-G (n = 4) and IgG against HCs (HLA-E, n = 8; HLA-F, n = 14; and HLA-G, n = 12). The presence of Abs against HCs but not against 2m suggests that the immunogen was 2m-free HCs. Such a possibility can occur if the immune cells are hyperactivated and express 2m-free HCs, as Face-2, or even Face-3 and Face-4, as illustrated in Figure 1. When these 2m-free HCs are shed or released, the intact expression of 2m-associated HCs may remain unaffected on the cell surface. The shed 2m-free HCs may have elicited IgG. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Treatment at Sampling Patient ID Complications IgM IgM IgM IgM IgG IgG IgG IgG 1 Alb-RA136F39 Met/Chlrqn/Predns 0 953 0 0 0 0 0 0 2 Alb-RA018M30 Met/Folic/Azulf 0 3721 1359 1747 0 0 0 1012 3 0 771 0 0 0 0 585 0 Alb-RA121F60 Met/Chlrqn/ 4 Alb-RA112F39 Hypertns Met/Folic/Azat/Predns 0 1172 553 0 0 0 585 0 Met/Folic/ 5 Alb-RA 106F47 0 2286 1011 0 0 0 518 777 ACEI 6 Alb-RA113F77 Met/Chlrqn/ 0 669 0 0 0 0 558 1227 7 0 923 0 0 0 0 888 862 Alb-RA082F51 8 Alb-RA045F32 0 651 0 0 0 867 1218 0 9 Alb-RA 125F43 Met/Folic 0 500 0 0 0 736 2099 1027 Met/Chlrqn/ 10 Alb-RA 034F48 0 1695 0 622 0 987 774 1407 Folic/Azulf/ Met/Chlrqn/ 11 Alb-RA129F28 0 2207 615 0 0 748 2330 1392 Folic Folic/Met/ 12 Alb-RA127M47 0 2780 0 0 0 607 653 2957 Chlrqn/ 13 Alb-RA 095F64 Hypertns/diabet Met/Folic 0 0 0 1498 0 0 753 779 14 Alb-RA098F64 Hypertns Met/Chlrqn/Folic/ 0 0 666 0 0 602 627 1317 15 Alb-RA012F57 Met/Chlrqn/ 0 0 1258 0 0 811 1357 1306 SLE/Hypertns 16 Alb-RA 021F65 Azulf 0 1806 769 1035 0 1175 1242 1380 diabetics Table 3. Profile of serum Abs in Group 2 with 24 RA patients, one of whom had both RA and SLE. Since these patients did not have IgM or Abs against 2m and only IgG against HCs of HLA-E (n = 13), HLA-F (n = 23), and HLA-G (n = 16), this group may represent a more advanced stage disease than that represented by Group 1. Shedding of HLA-F seems to have been more prevalent. The presence of Abs against HCs but not against 2m suggests that the immunogen should be 2m-free HCs, a possibility that can occur if the immune cells are hyperactivated and express 2m-free HCs, as Face-2 or even Face-3 and Face-4. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Patient ID Treatment at Sampling Complications IgM IgM IgM IgM IgG IgG IgG IgG 1 Alb-RA032F54 Met/Chlrqn 0 0 0 0 0 0 520 0 2 Alb-RA039M31 0 0 0 0 0 0 682 0 3 Alb-RA031M71 Met 0 0 0 0 0 0 817 0 4 0 0 0 0 0 0 1952 0 Alb-RA107F65 Met/chlrqn 5 Alb-RA 096F41 Met/Chlrqn/Folic 0 0 0 0 0 0 647 0 6 Alb-RA126M26 Met/Chlrqn/Folic/ 0 0 0 0 0 0 0 1416 Met/Folic/ 7 Alb-RA102F61 Hyprtns/Thrmbss 0 0 0 0 0 0 668 547 ARAVA/Azulf/ 8 Alb-RA094M66 Dyslipid Met/Folic/ 0 0 0 0 0 0 660 4138 9 Alb-RA037F51 Met/Chlrqn/Folic/ARAVA 0 0 0 0 0 0 1163 1763 SysVasc/ 10 Alb-RA049M66 Hyprtns/ Met/Folic 0 0 0 0 0 0 1294 946 dslIpid/Ren Dis 11 Alb-RA002F35 Met/Folic/Arav 0 0 0 0 0 0 1587 872 12 Alb-RA115M40 Met/Omprz 0 0 0 0 0 512 645 0 Met/Chlrqn/ 13 Alb-RA076M60 Hyprtns/dyslipid 0 0 0 0 0 514 773 0 ACEI 14 Alb-RA111F39 Hyprtns Met/ACEI 0 0 0 0 0 758 849 0 15 Alb-RA058M32 Met/Folic/Azulf Predns/Omprz 0 0 0 0 0 540 559 508 16 Alb-RA020F26 Met 0 0 0 0 0 591 680 552 17 Alb-RA133F37 Met/Chlrqn/Folic/ARAVA 0 0 0 0 0 743 603 549 Hyprtns 18 Alb-RA038F55 Met/Chlrqn/Folic 0 0 0 0 0 524 957 809 Hypothyr Met/Folic/ 19 Alb-RA103F72 Hyprtns 0 0 0 0 0 512 661 1206 ACEI 20 Alb-RA118F30 Met/Chlrqn/Folic 0 0 0 0 0 577 1148 510 21 Alb-RA099M76 Hyprtns Met/AECI 0 0 0 0 0 561 1165 866 22 Alb-RA132F58 Hyprtns Met/Chlrqn/Folic 0 0 0 0 0 651 1829 1528 23 Alb-RA135F55 Met/ Chlrqn/ Folic 0 0 0 0 0 1563 1084 1477 Met/Folic/Azulf/ARAVA/Azat/ 24 Alb-RA109F50 SLE/Hyprtns 0 0 0 0 0 1133 719 934 Predns/ ACEI Antibodies 2023, 12, 26 7 of 22 Table 4. Profile of serum Abs in Group 3 with 14 patients. These patients had IgM Abs against 2m. They had also developed IgM against HCs of HLA-E (n = 14), HLA-F (n = 7), and HLA-G (n = 8) and IgG against HCs (HLA-E, n = 5; HLA-F, n = 13; and HLA-G, n = 10). The presence of IgM against 2m indicates that the immunogen was an intact or 2m-associated HC of HLA-I (closed conformer or Face-1), as illustrated in Figure 1. The intact HLA were possibly released due to cell death. Shedding of HLA-F seems to have been more prevalent than other HLA-Ib molecules. HLA-F HLA-E HLA-E HLA-F HLA-G HLA-G 2M 2M Other Treatment at Sampling Patient ID Heavy Chains Heavy Chains Complications IgM IgM IgM IgM IgG IgG IgG IgG 1 Alb-RA 110F63 Met/Chlrqn/Folic 695 581 0 0 0 0 0 0 Met/Folic/ 2 Alb-RA 114F61 2817 1910 0 0 0 0 510 0 IECA Met/Folic/ 3 Alb-RA 052F57 3293 636 0 0 0 0 980 0 Predns/Omprz 4 Alb-RA 092M56 Met/Predns 1134 618 0 0 0 0 1469 0 5 Alb-RA 051F44 Met/Chlrqn/Folic 1296 740 0 0 0 734 685 1106 Azulf/ 6 Alb-RA 015F35 Hypothyrd ARAVA/ 850 2001 500 0 0 1020 766 1251 Chlrqn/Predns/ Met/Chlrqn/ 7 Alb-RA 105F24 Folic/ 593 4042 1209 2522 0 0 1022 829 8 Alb-RA 033F26 Met/Folic/Ompraz 603 632 1512 763 0 559 1239 1842 9 Alb-RA 014F73 Hypertens Met/IECA 940 841 0 837 0 0 1207 1247 10 Alb-RA 100F21 Met/Chlrqn/ARAVA/Ompr 1008 1824 495 673 0 0 624 4518 11 Alb-RA 019F25 Chlrqn 1034 624 531 1016 0 0 1234 1093 12 Alb-RA 060F44 Met/Azulf 1317 1430 1541 3545 0 2076 1834 9985 13 Alb-RA 047F50 Chlrqn 1718 1117 973 1441 0 1104 1122 973 Met/Folic/Azulf 15 Alb-RA 138F55 1728 786 0 2784 0 0 940 861 /Predns/ Table 5. Profile of serum Abs in Group 4 with 6 patients. These patients have IgM but not IgG Abs against 2m (n = 6). These patients did not have IgM Abs against any of the HLA-Ib antigens. They had IgG Abs against HCs of HLA-E (n = 2), HLA-F (n = 6), and HLA-G (n = 5). The presence of IgM against 2m indicates that the immunogen could have been intact or 2m-associated HCs (closed conformers or Face-1) of HLA-Ia (HLA-A, HLA-B, and HLA-C) but not HLA-Ib. The intact HLA-Ia molecules were possibly released due to cell death. However, shedding of 2m-free HLA-F (Face-2, Face-3 and Face-4) seems to have been more prevalent than other 2m-free HLA-Ib molecules. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Treatment at Sampling Patient ID Complications IgM IgM IgM IgM IgG IgG IgG IgG Met/Chlrqn/ 1 Alb-RA 085F47 Dyslipid/diabet 596 0 0 0 0 540 1626 1580 ARAVA/Statins ARAVA/Ompr/ Thyrd enzys/ 2 Alb-RA 048F41 Hpothyr/Hyprtns 638 0 0 0 0 0 1267 1464 IECA/B-block Met/Folic/ 3 Alb-RA 063M60 859 0 0 0 0 769 730 591 ARAVA Met/Chlrqn/ 4 Alb-RA 124F29 953 0 0 0 0 0 1289 683 Folic/Azulf/ Met/Folic/ 5 Alb-RA 005M75 979 0 0 0 0 0 1602 1771 ARAVA Met/Chlrqn/ 6 Alb-RA 043F24 1516 0 0 0 0 0 1142 0 Folic/ Antibodies 2023, 12, 26 8 of 22 Table 6. Profile of serum Abs in Group 5 with 5 patients. These patients had no IgM but only IgG against 2m. Both IgM and IgG Abs against HCs of HLA-E/HLA-F/HLA-G were present. Anti-HLA-E and anti-HLA-F IgM were present in 4 patients and anti-HLA-G in 2 patients. Possibly, the intact HLA were released due to cell death. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Treatment at Sampling Patient ID Complications IgM IgM IgM IgM IgG IgG IgG IgG Met/ 1 Alb-RA 027F37 ARAVA/ 0 0 1816 0 578 1438 1356 2580 Predns Met/Folic/ 2 Alb-RA084M62 0 763 637 0 825 822 1568 2829 predns Met/Folic/azulf 3 Alb-RA 137F33 0 1381 0 0 500 1086 1183 987 Chlrqn Met/Chlrqn/ 4 Alb-RA134F45 0 1607 840 654 861 2700 2763 3726 Folic/ 5 Alb-RA 088F34 Diabetes Met/Predns 0 2237 1360 2799 754 1218 1525 3166 Table 7. Profile of serum Abs in Group 6 with 3 patients. None of the patients had IgM Abs against 2m or HCs, as observed in Group 2. However, in contrast to Group 2, the patients in this group had IgG Abs against 2m and HCs. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Treatment at Sampling Patient ID Complications IgM IgM IgM IgM IgG IgG IgG IgG 1 Alb-RA090F41 Met/Chlrqn/ 0 0 0 0 752 1474 1577 1104 2 Alb-RA030M58 0 0 0 0 782 1812 3138 1531 3 Alb-RA097F47 Thrombosis Met/Chlrqn/ 0 0 0 0 573 778 0 0 Trimmed mean fluorescence values for the SAB reactions were obtained from the output (.csv was converted to .xls) file generated by the flow analyzer and were adjusted for blank and background signals using the formula below. To express the values of anti-HLA Antibodies 2023, 12, x FOR PEER REVIEW 9 of 23 Abs at specified dilution, the sample specific fluorescent value (trimmed MFI) for each bead was taken into consideration. Four different kinds were obtained: (1) trimmed MFI with serum Abs; (2) MFI for HLA-coated beads added with PE-conjugated 2nd antibody only; 2.3. Antigen Source (3) trimmed MFI of LABSCreen negative control (LSNC) sera with minimal or no HLA Abs; and (4) MFIs for the negative control beads (with PE-conjugated 2nd antibody only) used Only HLA-Ib proteins were investigated in this study. Recombinant HLA-E, HLA-F, for LSNC. Normalized MFI is calculated as follows: {MFI (1)—MFI (2)}—{(MFI of LSNC HLA-G, and β2m folded HCs (10 mg/mL in 2-[N-morpholino] ethanesulfonic acid [MES] (3)—MFI (4)}. The control beads included those coated with human IgG (positive control) buffer) were obtained from the Immune Monitoring Laboratory, Fred Hutchinson Cancer and serum albumin (HSA/BSA) (negative control). For each analysis, at least 100 beads Research Center (University of Washington, Seattle, WA). Recombinant HCs of HLA-Ib R107 were counted. Mean and standard deviation of the MFI for each allele were recorded. alleles (HLA-E , HLA-F1, and HLA-G1) were folded and made available for coating mi- All the data were stored and archived at TFL. Origin Graphics Softwar R e (OriginLab, crobeads. Figure 2 shows the amino acid sequences of the HCs of HLA-E , HLA-F, HLA- Northampton, MA, USA.) was used to plot the data. Basic statistical analyses were carried G, and β2m used for coating the beads. All HLA-Ib alleles have only the extracellular do- out with Excel software. Only an MFI above 500 was considered positive at a 1/10 dilution main without the leader peptide containing 21 amino acids, and no transmembrane or of sera. MFI values <500 were recorded as “0” value in the tables. intracellular domains. β −Microglobulin https://www.uniprot.org/uniprotkb/P61769/entry Last updated 1986-07-21 v1 10 20 30 40 45 50 MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKS NFLNC YVSGF /H 60 70 80 90 100 PSDIEVDLL KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNH VTLSQP KIVKWDRDM Figure 2. Cont. HLA-E Source: https://www.uniprot.org/uniprotkb/P13747/entry (see also Wikipedia) LP MVDGTLLLLLSEALALTQTWA 30 40 50 60 70 α1GSHSLKYFH TSVSRPGRGE PRFISVGYVD DTQFVRFDND AASPRMVPRA 80 90 100 110 PWMEQEGSEY WDRETRSARD TAQIFRVNLR TLRGYYNQSE AGSH 120 122 130 140 150 α2TLQWMH GC ELGPDGRF LRGYEQFAYD GKDYLTLNED LRSWTAVD 160 170 180 185 190 200 TA AQISEQKSND ASEAEHQRAY LEDTC VEWLH KYLEKGKETL 210 220 224 230 240 α3LHLEPPKTH THHPISDHEA TLRC WALGFY PAEITLTWQQ DGEGHTQ 250 260 270 280 290 DTE LVETRPAGDG TFQKWAAVVV PSGEEQRYTC HVQHEGLPEP VTLR 300 310 320 330 340 WKPASQ PTIPIVGIIA GLVLLGSVVS GAVVAAVIWR KKSSGGKGGS YSKA 350 358 EWSDSA QGSESHSL HLA-F Source: https://www.uniprot.org/uniprotkb/P30511/entry (see also Wikipedia) LP MAPRSLLLLLSGALALTDTWA 30 40 50 60 70 α1GSHSLRYFS TAVSRPGRGE PRYIAVEYVD DTQFLRFDSD AAIPRMEPRE P 80 90 100 11O WVEQEGPQY WEWTTGYAKA NAQTDRVALR NLLRRYNQSE AGSH 120 122 130 140 150 α2TLQGMN GC DMGPDGRL LRGYHQHAYD GKDYISLNED LRSWTAA 160 170 180 185 190 200 DTV AQITQRFYEA EEYAEEFRTY LEGEC LELLR RYLENGKETL 210 220 224 230 240 α3QRADPPKAHV AHHPISDHEA TLRC WALGFY PAEITLTWQR DGEEQT 250 260 270 280 290 QDTE LVETRPAGDG TFQKWAAVVV PPGEEQRYTC HVQHEGLPQP LILR 300 310 320 330 340 WEQSPQ PTIPIVGIVA GLVVLGAVVT GAVVAAVMWR KKSSDRNRGS Y SQAAV HLA-G Source: https://www.uniprot.org/uniprotkb/P17693/entry (see also Wikipedia) LP MVVMAPRTLFLLLSGALTLTETWA 30 40 50 60 7 α1GSHSMRYFS AAVSRPGRGE PRFIAMGYVD DTQFVRFDSD SACPRMEPRA 0 80 90 100 110 PWVEQEGPEY WEEETRNTKA HAQTDRMNLQ TLRGYYNQSE ASSH 120 122 130 140 150 α2TLQWMI GC DLGSDGRL LRGYEQYAYD GKDYLALNED LRSWTAADT 160 170 180 185 190 200α2 A AQISKRKCEA ANVAEQRRAY LEGTC VEWLH RYLENGKEML α3 210 220 224 230 240 QRADPPKTHV THHPVFDYEA TLRC WALGFY PAEIILTWQR DGEDQT 250 260 270 280 290 QDVE LVETRPAGDG TFQKWAAVVV PSGEEQRYTC HVQHEGLPEP LML 300 310 310 320 α3 RWKQSSL PTIPIMGIVA GLVVLAAVVT GAAVAAVLWR KKSSD Antibodies 2023, 12, x FOR PEER REVIEW 9 of 23 2.3. Antigen Source Only HLA-Ib proteins were investigated in this study. Recombinant HLA-E, HLA-F, HLA-G, and β2m folded HCs (10 mg/mL in 2-[N-morpholino] ethanesulfonic acid [MES] buffer) were obtained from the Immune Monitoring Laboratory, Fred Hutchinson Cancer Research Center (University of Washington, Seattle, WA). Recombinant HCs of HLA-Ib R107 alleles (HLA-E , HLA-F1, and HLA-G1) were folded and made available for coating mi- crobeads. Figure 2 shows the amino acid sequences of the HCs of HLA-E , HLA-F, HLA- G, and β2m used for coating the beads. All HLA-Ib alleles have only the extracellular do- main without the leader peptide containing 21 amino acids, and no transmembrane or intracellular domains. β −Microglobulin https://www.uniprot.org/uniprotkb/P61769/entry Last updated 1986-07-21 v1 10 20 30 40 45 50 MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKS NFLNC YVSGF /H Antibodies 2023, 12, 26 9 of 22 60 70 80 90 100 PSDIEVDLL KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNH VTLSQP KIVKWDRDM HLA-E Source: https://www.uniprot.org/uniprotkb/P13747/entry (see also Wikipedia) LP MVDGTLLLLLSEALALTQTWA 30 40 50 60 70 α1GSHSLKYFH TSVSRPGRGE PRFISVGYVD DTQFVRFDND AASPRMVPRA 80 90 100 110 PWMEQEGSEY WDRETRSARD TAQIFRVNLR TLRGYYNQSE AGSH 120 122 130 140 150 α2TLQWMH GC ELGPDGRF LRGYEQFAYD GKDYLTLNED LRSWTAVD 160 170 180 185 190 200 TA AQISEQKSND ASEAEHQRAY LEDTC VEWLH KYLEKGKETL 210 220 224 230 240 α3LHLEPPKTH THHPISDHEA TLRC WALGFY PAEITLTWQQ DGEGHTQ 250 260 270 280 290 DTE LVETRPAGDG TFQKWAAVVV PSGEEQRYTC HVQHEGLPEP VTLR 300 310 320 330 340 WKPASQ PTIPIVGIIA GLVLLGSVVS GAVVAAVIWR KKSSGGKGGS YSKA 350 358 EWSDSA QGSESHSL HLA-F Source: https://www.uniprot.org/uniprotkb/P30511/entry (see also Wikipedia) LP MAPRSLLLLLSGALALTDTWA 30 40 50 60 70 α1GSHSLRYFS TAVSRPGRGE PRYIAVEYVD DTQFLRFDSD AAIPRMEPRE P 80 90 100 11O WVEQEGPQY WEWTTGYAKA NAQTDRVALR NLLRRYNQSE AGSH 120 122 130 140 150 α2TLQGMN GC DMGPDGRL LRGYHQHAYD GKDYISLNED LRSWTAA 160 170 180 185 190 200 DTV AQITQRFYEA EEYAEEFRTY LEGEC LELLR RYLENGKETL 210 220 224 230 240 α3QRADPPKAHV AHHPISDHEA TLRC WALGFY PAEITLTWQR DGEEQT 250 260 270 280 290 QDTE LVETRPAGDG TFQKWAAVVV PPGEEQRYTC HVQHEGLPQP LILR 300 310 320 330 340 WEQSPQ PTIPIVGIVA GLVVLGAVVT GAVVAAVMWR KKSSDRNRGS Y SQAAV HLA-G Source: https://www.uniprot.org/uniprotkb/P17693/entry (see also Wikipedia) LP MVVMAPRTLFLLLSGALTLTETWA 30 40 50 60 7 α1GSHSMRYFS AAVSRPGRGE PRFIAMGYVD DTQFVRFDSD SACPRMEPRA 0 80 90 100 110 PWVEQEGPEY WEEETRNTKA HAQTDRMNLQ TLRGYYNQSE ASSH 120 122 130 140 150 α2TLQWMI GC DLGSDGRL LRGYEQYAYD GKDYLALNED LRSWTAADT 160 170 180 185 190 200α2 A AQISKRKCEA ANVAEQRRAY LEGTC VEWLH RYLENGKEML α3 210 220 224 230 240 QRADPPKTHV THHPVFDYEA TLRC WALGFY PAEIILTWQR DGEDQT 250 260 270 280 290 QDVE LVETRPAGDG TFQKWAAVVV PSGEEQRYTC HVQHEGLPEP LML 300 310 310 320 α3 RWKQSSL PTIPIMGIVA GLVVLAAVVT GAAVAAVLWR KKSSD Figure 2. Amino acid sequences of 2m, and 1, 2, and 3 domains of the HCs of HLA-Ib isomers HLA-E, HLA-F, and HLA-G. Each sequence shown above from the 1 domain is the leader peptide (LP) of an HLA isomer. Letters in bold and italics refer to monospecific sequences for HLA-E (in black) that are specific for HLA-E and B*8201 (in green), shared with all HLA-Ia and Ib isomers (in blue), or shared with all HLA isomers except HLA-A and HLA-F (in red). Letters in red in HLA-F and HLA-G are the amino acids that differ from HLA-E. Red letters in yellow in HLA-G denote similarity to HLA-F. Note that HLA-E, HLA-F, and HLA-G have cysteine in the same position, as shown in the bold letter C in dark red with the position indicated in the superscript. The cysteine noted at positions 122, 185, 224, and 280 may facilitate homo- and heterodimerization of 2m-free HCs (Face-2), as shown in Figure 1. 3. Results Abs were observed in the sera of 68 of 74 patients while receiving immunomodulatory drugs. These immunomodulatory drugs included methotrexate (63/74 patients) supple- mented with folic acid (44/74), and chloroquine with methotrexate (14) or alone (3). In addition, several of the patients had also received combination therapy with leflunomide (ARAVA) and/or prednisone and/or azulfidine (sulfasalazine) and/or omeprazole and/or ACEI, as presented in Table 1. IgM and IgG Abs reacting to 2m and HCs of HLA-E, HLA-F, and HLA-G coated on microbeads were assessed for MFIs. These MFIs are considered to be Antibodies 2023, 12, x FOR PEER REVIEW 11 of 23 F, and HLA-G coated on microbeads were assessed for MFIs. These MFIs are considered to be semi-quantitative, as the MFI may vary with the lot of bead sets used, and each Lab- screen bead set is admixed with both β2m-associated and β2m-free HCs [40–42]. 3.1. Categorization of Sera Based on the Distributions of Anti-β2m and HCs Abs Based on MFIs obtained for IgM and IgG Abs, the patient sera were divided into Antibodies 2023, 12, 26 10 of 22 categories based on the presence or absence of anti-β2m or anti-HC Abs, and the class of antibody (IgM or IgG). The major serum groups are shown in Figure 3, as follows: Group 1. Sera (n = 16) with no anti-β2m IgM or IgG but with HLA-Ib HC IgM and semi-quantitative, as the MFI may vary with the lot of bead sets used, and each Labscreen IgG; bead set is admixed with both 2m-associated and 2m-free HCs [40–42]. Group 2. Sera (n = 24) with no anti-β2m IgM or IgG but only with HLA-Ib HC IgG; Group 3. Sera (n = 14) with anti-β2m IgM but not IgG; 3.1. Categorization of Sera Based on the Distributions of Anti-b2m and HCs Abs Group 4. Sera (n = 6) with anti-β2m IgM but not IgG and with HLA-Ib HC IgG; Based on MFIs obtained for IgM and IgG Abs, the patient sera were divided into Group 5. Sera (n = 5) with only anti-β2m IgG together with HLA-Ib HC IgM and IgG; categories based on the presence or absence of anti- 2m or anti-HC Abs, and the class of Group 6. Sera (n = 3) with only anti-β2m IgG and HLA-Ib HC IgG; antibody (IgM or IgG). The major serum groups are shown in Figure 3, as follows: Group 7. Sera (n = 6) with neither anti-β2m nor HLA-Ib HC Abs. Figure 3. Profile of anti-HLA-Ib Abs in the sera of 74 RA patients. Sera can be broadly classified as Figure 3. Profile of anti-HLA-Ib Abs in the sera of 74 RA patients. Sera can be broadly classified those having anti-β2m Abs (n = 28, 20 with IgM, 8 with IgG) and those without anti-β2m Abs (n = as those having anti- 2m Abs (n = 28, 20 with IgM, 8 with IgG) and those without anti- 2m Abs 47). The table in the figure illustrates the detailed profile of IgM and IgG Abs against HCs of HLA- (n = 47). The table in the figure illustrates the detailed profile of IgM and IgG Abs against HCs of E, HLA-F, and HLA-G. HLA-E, HLA-F, and HLA-G. 3.2. Group 1: Sera with No Anti-β2m IgM or IgG but with Anti-HLA-Ib HC IgM and IgG Group 1. Sera (n = 16) with no anti- 2m IgM or IgG but with HLA-Ib HC IgM and IgG; Group 2. Sera (n = 24) with no anti- 2m IgM or IgG but only with HLA-Ib HC IgG; The sera of 16 of 74 patients had neither anti-β2m IgM nor IgG but had both IgM and Group 3. Sera (n = 14) with anti- 2m IgM but not IgG; IgG against HCs of HLA-E, HLA-F, and HLA-G (Table 2). IgM Abs formed against HLA- Group 4. Sera (n = 6) with anti- 2m IgM but not IgG and with HLA-Ib HC IgG; E were present in more patients (n = 13) compared to those formed against HLA-F (n = 7) Group 5. Sera (n = 5) with only anti- 2m IgG together with HLA-Ib HC IgM and IgG; and HLA-G (n = 4). In contrast, IgG against HCs of HLA-F (n = 14) and HLA-G (N = 12) Group 6. Sera (n = 3) with only anti- 2m IgG and HLA-Ib HC IgG; were present in more patients compared to those against HC HLA-E (n = 8). The presence Group 7. Sera (n = 6) with neither anti- 2m nor HLA-Ib HC Abs. of Abs against only HCs of HLA-Ib loci and not against β2m suggests that the immunogen may have been β2m-free HLA variants (Face-2, Face-3, and Face-4), instead of an intact 3.2. Group 1: Sera with No Anti-b2m IgM or IgG but with Anti-HLA-Ib HC IgM and IgG HLA (Face-1). The sera of 16 of 74 patients had neither anti- 2m IgM nor IgG but had both IgM and IgG against HCs of HLA-E, HLA-F, and HLA-G (Table 2). IgM Abs formed against HLA-E were present in more patients (n = 13) compared to those formed against HLA-F (n = 7) and HLA-G (n = 4). In contrast, IgG against HCs of HLA-F (n = 14) and HLA-G (N = 12) were present in more patients compared to those against HC HLA-E (n = 8). The presence of Abs against only HCs of HLA-Ib loci and not against 2m suggests that the immunogen may have been 2m-free HLA variants (Face-2, Face-3, and Face-4), instead of an intact HLA (Face-1). 3.3. Group 2: Sera with No Anti-b2m IgM or IgG but Only with Anti-HLA-Ib HC IgG Sera of 24 of 74 patients had neither anti- 2m IgM nor IgG, but had only IgG against HCs of HLA-E (n = 13), HLA-F (n = 23), and/or HLA-G (n = 16) (Table 3). Once again, Antibodies 2023, 12, 26 11 of 22 the prevalence of IgG against HCs of HLA-Ib loci without any IgM or IgG against 2m strongly suggests that the immunogen may have been 2m-free HCs (Face-2) of the HLA, rather than intact HLA molecules (Face-1). 3.4. Group 3: Sera with Anti-b2m IgM Only but with Anti-HLA-Ib HC IgM and IgG The sera of 14 patients had only anti- 2m IgM but not anti- 2m IgG. However, both IgM and IgG Abs against the HCs of HLA-E (IgM n = 14, IgG n = 5), HLA-F (IgM n = 7, IgG n = 13), and HLA-G (IgM 8, IgG n = 10) were detectable (Table 4). The presence of high MFIs of IgM against 2m and HLA-E suggests the release or shedding of intact HLA-E molecules (Face-1) from selected immune cells, possibly due to cell death. The prevalences of IgM Abs against 2m and HCs indicate not only the commencement of cell death but also Phase-III of immunological progression (Phase IIIa). The high prevalence of anti-HLA-F HC IgG in 14 of 15 patients suggests that one or more of the activated immune cell types may express 2m-free HLA-F variants (Face-2, Face-3, and Face-4). 3.5. Group 4: Sera with Anti-b2m IgM Only without Anti-HLA-Ib HC IgM but with IgG The sera of six patients had anti- 2m IgM only and not anti- 2m IgG. No IgM Abs against the HCs of HLA-Ib were noted. IgG Abs against HLA-E (IgG n = 2), HLA-F (IgG n = 6), and HLA-G (IgG n = 5) were detectable in patients (Table 5). The presence of IgM against 2m without any IgM against HCs of HLA-Ib suggests that the Abs against 2m may have been due to the release or shedding of 2m from the cell surface as intact HLA-Ia molecules (Face-1), possibly due to cell death in Phase III. The presence of IgG Abs against HCs of HLA-Ib without anti- 2m IgG suggests that the immunogen may have been from 2m-free HCs (Face-2) of the HLA-Ib. The absence of IgM Abs against HCs in the presence of IgG Abs against HCs supports the contention that the patients may have represented a phase subsequent to Phase-IIIa (Phase-IIIb). 3.6. Group 5: Sera with Anti-b2m IgG but Not IgM Together with HLA-Ib HC IgM and IgG Sera of five patients had only anti- 2m IgG but not IgM, and had both IgM and IgG against the HCs of HLA-E (IgM n = 4, IgG n = 5), HLA-F (IgM n = 4, IgG n = 5), and HLA-G (IgM n = 2, IgG n = 5) (Table 6). The presence of anti- 2m IgG and the high MFIs of IgG and IgM against HLA-E, HLA-F, and HLA-G suggest that they could have been a consequence of the release or shedding of intact HLA-Ib molecules (Face-1) from several types of immune cells, possibly due to their cell death. The MFIs of IgG Abs against HLA-E, HLA-F, and HLA-G were higher than for other groups of patients. Since the patients had no IgM but only IgG against 2m, and they had both IgM and IgG against HCs of HLA-Ib, they may represent a phase subsequent to Phase-IIIb (Phase-IIIc). 3.7. Group 6: Sera with Anti-b2m IgG but Not IgM and with HLA-Ib HC IgG but Not IgM Sera of three patients had anti- 2m IgG but no IgM and had IgG but no IgM Abs against the HCs of HLA-E, HLA-F, and HLA-G (Table 7). It appears that this group reflects a more advanced disease stage than the one represented by Groups 4 and 5, as IgM was no longer detectable. These patients may represent a phase subsequent to Phase-IIIc (Phase-IIId). 3.8. Group 7: Sera with Neither Anti-b2m Nor HLA-Ib HC Abs In this group of sera (n = 6), neither anti- 2m nor anti-HLA-Ib HC IgM or IgG Abs were observed (Table 8). These patients received almost the same therapy as those in other groups. Could it be possible that these patients reflect those receiving therapies for longer durations? Or could the immunosuppressive therapies have been more efficacious in this group of patients? Antibodies 2023, 12, 26 12 of 22 Table 8. Profile of serum Abs in Group 7 with 6 patients. Most unusually, both IgM and IgG Abs against 2m and HLA-Ib HCs were totally absent, possibly due to the durations or types of therapies the patients received. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G 2M 2M Other Heavy Chains Heavy Chains Treatment at Sampling Patient ID Complications IgM IgM IgM IgM IgG IgG IgG IgG 1 Alb-RA091F30 Met 0 0 0 0 0 0 0 0 2 Alb-RA024F54 Hpothyr Met/Thyoid Enz 0 0 0 0 0 0 0 0 3 Alb-RA 065F59 Hpertns/Diabet Met/Chlrqn/Folic 0 0 0 0 0 0 0 0 Met/Chlrqn/ 4 Alb-RA104F22 0 0 0 0 0 0 0 0 Predns Met/Chlrqn/ 5 Alb-RA130F51 0 0 0 0 0 0 0 0 Folic/Omprz Met/Chlrqn/ 6 Alb-RA074F37 0 0 0 0 0 0 0 0 Folic/Azul 3.9. High Levels of Anti-HLA-Ib IgM and IgG Abs in Normal Males and Females of the Same Ethnicity as the RA Patients For the purpose of comparison of the profiles of anti-HLA-Ib Abs with the normal controls of the same ethnicity of the patients, we examined serum IgM and IgG Abs of normal males and females using Luminex multiplex flow cytometry. The profiles of Abs in normal individuals against HLA-E, HLA-F, and HLA-G are presented in Table 9. It is important to note that the incidence and strength (MFI) of anti-HLA-E, anti-HLA-F, and HLA-G IgM and IgG Abs were consistently high in almost all patients, in contrast to the patient cohort. When comparing the Ab-profile normal cohort with patients, it can be noted that none of the patients in Group1 (Table 2), Group2 (Table 3), Group4 (Table 5), or Group6 (Table 7) had both IgM and IgG against all three HLA-Ib molecules. Both IgM and IgG against all three HLA-Ib molecules were observed in 3 of the 15 patients in Group3 (Table 4, Alb-RA 033F26, Alb-RA 060F44, and Alb-RA 47F50) and 2 of the 5 patients in Group5 (Table 6, Alb-RA 134F454 and Alb-RA 088F34). Table 9. Profiles of IgM and IgG antibodies formed against HCs of HLA-E, HLA-F, and HLA-G in normal male and female Mexicans, representing the control cohort. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G Males Females IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG AT-252 6524 0 2334 1441 3103 2585 AT-63 7369 704 5729 1396 7493 1995 AT-126 3172 884 2139 1506 6177 4761 AT-212 6827 653 3236 947 4835 1701 AT-48 6157 1106 1743 2086 4906 4228 AT-56 4972 728 781 2086 2209 2397 AT-364 3953 730 1146 1511 1989 2331 AT-343 4739 915 1722 1452 3086 1974 AT-400 3948 632 1305 1002 2616 2464 AT-372 4280 1075 1778 2199 5917 2895 AT-449 2923 886 749 2069 1969 3717 AT-1 3510 936 1166 1873 3738 2156 AT-154 2817 803 1621 1728 1988 2995 AT-323 3255 897 1534 1315 3715 2423 AT-222 2230 1154 923 1387 1120 3765 AT-253 3130 717 1841 1430 4206 3511 AT-229 2144 1798 665 2002 1260 3578 AT-362 2377 0 1492 1105 3773 1915 AT-359 1804 1143 872 1982 2170 3216 AT-38 2666 1313 1761 1823 2486 3427 AT-393 1764 1115 919 1455 2213 3138 AT-18 2448 1071 1994 1466 3690 2526 AT-304 1623 0 980 1428 1403 2932 AT-374 2212 789 685 1555 1483 3112 AT-54 1490 1849 1469 2186 1762 3413 AT-109 2128 1510 1004 2612 1941 3882 AT-277 1360 1134 769 2263 1556 2487 AT-330 2091 928 574 1927 2192 2923 AT-82 1279 1028 875 1649 1259 3051 AT-290 2000 2203 1060 2333 2521 3849 AT-88 1227 834 0 1693 987 4700 AT-150 1887 533 1347 1127 3287 3554 Antibodies 2023, 12, 26 13 of 22 Table 9. Cont. HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G Males Females IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG AT-239 1216 652 784 1402 2019 3236 AT-300 1866 1018 961 1800 2815 4152 AT-338 1118 1141 0 1788 1009 3430 AT-191 1663 1728 1476 3156 2788 2995 AT-438 1049 877 0 2402 1005 2818 AT-78 1621 685 823 1248 1580 1637 AT-392 741 1314 1219 1963 1379 3477 AT-89 1483 670 734 1404 2284 3811 AT-386 960 1121 0 1945 1038 3220 AT-200 1476 1042 577 1875 1362 2933 AT-133 822 1738 979 3049 624 4756 AT-181 1402 623 1052 1350 1938 1775 AT-242 735 1206 554 1600 1680 2626 AT-70 1302 790 0 1832 1225 2514 AT-145 667 1258 0 1626 1263 2405 AT-112 1137 0 995 935 1780 1750 AT-219 0 639 0 2249 778 2321 AT-140 802 1007 516 2083 1555 3042 AT-354 0 1437 0 2778 641 3670 AT-412 802 994 0 1794 1823 2495 Mean 1989 1018 848 1853 1843 3281 Mean 2671 905 1340 1697 2912 2744 Median 1425 1111 874 1758 1479 3218 Median 2110 906 1056 1675 2504 2711 SD 1650 446 673 460 1258.4 729.99 SD 1712 454 1123 520 1433 767 4. Discussion 4.1. Anti-HLA-F IgG without Anti-b2m-IgG Is Most Prevalent in RA Patients The highest prevalence (63/68) of anti-HLA-F IgG and the lowest prevalence (8/69) of anti- 2m IgG suggest that the anti-HLA-F IgG may not be formed against intact HLA-F molecules or Face-1 HLA-F, but rather appears to be formed against 2m-free HLA-F (Face-2, Face-3, or Face-4 of HLA-F). Based on our findings [41,42] on the categories of monoclonal Abs (mAbs) generated after immunizing HLA-E HCs in mice, it was clarified that the Abs formed could be HLA-E-monospecific (e.g., mAbs TFL-033, TFL-034, and TFL-145) or polyreactive to many or all HLAs, as illustrated in Table 10. Highly polyreactive mAbs (e.g., TFL-006 and TFL-007) raised against Face-2 of HLA-E react to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. These Abs recognized the most shared amino acid sequences (AYDGKDY and LNEDLRSWTA) of other HLA-I molecules, as shown in blue letters in Figure 2. Table 10. Diversity of the monoclonal Abs generated after immunizing mice with recombinant HC of G107 R107 HLA-E (HLA-E or HLA-E ) revealed monospecific (Category 1) and different categories of polyreactive mAbs, which included HLA-Ib-specific and HLA-Ia polyreactive mAbs. [ ]; Negative or not reactive; [+] Positive or reactive. HLA-Ia HLA-Ib Categories of mAbs HLA-A HLA-B HLA-C HLA-E HLA-F HLA-G Category 1 [ ] [ ] [ ] [+] [ ] [ ] Category 2 [ ] [ ] [ ] [+] [+] [ ] Category 3 [ ] [ ] [ ] [+] [ ] [+] Category 4 [ ] [ ] [ ] [+] [+] [+] Category 5 [ ] [+] [ ] [+] [ ] [ ] Category 6 [ ] [+] [+] [+] [ ] [ ] Category 7 [+] [+] [+] [+] [ ] [ ] Category 8 [+] [+] [+] [+] [+] [ ] Category 9 [+] [+] [+] [+] [ ] [+] Category 10 [+] [+] [+] [+] [+] [+] However, the higher prevalence of anti-HLA-F IgG compared to those of HLA-E and HLA-G suggests that the Abs may have been formed specifically against HLA-F, such as 164 173 the 2m domain sequence TQRFYEAEEY , shown in red in Figure 2. In support of this contention, we can observe the following patterns. (1) Anti-HLA-F IgG was observed in 64 of 69 patients. Only anti-HLA-F IgG was observed in 12 of 69 patients, indicating that these IgG Abs were specific for HLA-F. Thus, only Antibodies 2023, 12, 26 14 of 22 anti-HLA-F IgG was observed in 2 of 16 Group 1 patients (Table 2), 5 of 24 patients in Group 2 (Table 3), 4 of 15 Group 3 patients (Table 4), and 1 of 6 Group 4 patients (Table 5). (2) The MFI of anti-HLA-F IgG was higher than the MFI of anti-HLA-E or anti-HLA-G in 7 of 16 patients in Group 1 (Table 2), 17 of 24 patients in Group 2 (Table 3), 8 of 15 patients in Group 3 (Table 4), 3 of 6 patients in Group 4 (Table 5), 1 of 5 patients in Group 5 (Table 6), and 2 of 3 patients in Group 6 (Table 7). These findings suggest that the IgG Abs may formed against sequences of HLA-F shared with other HLA HCs. Formation of HLA-F IgG Abs in the absence of anti- 2m IgG strongly favors the view that these Abs are generated against 2m-free HLA-F variants (Face-2, Face-3, and/or Face-4) rather than from the 2m-associated HLA-F (Face-1). A primary alteration in activated cells involves the formation of a persistent HLA variant without 2m, referred to as open conformer [12] or Face-2 [13]. An earlier study performed [43] on a murine HLA (H-2D ) found that 2m is not required for cell surface expression of HLA, shattering the previously held dogma [44] that the HCs of HLA-I can be conformationally stable on the cell surface only as heterodimers with 2m. Using the 2m-free, HLA-HC-specific mAb LA45, Schnabl et al. [45] first reported the presence of the Face-2 variant on the cell surfaces of both in vitro and in vivo activated human T lymphocytes. Several monoclonal Abs were developed against Face-2, which include LA45, L31, TFL-006, and TFL-007. Madrigal et al. [46] observed the expression of the LA45 epitope on lectin-activated T cells. After phytohemagglutinin (PHA) activation of T cells isolated from 12 healthy individuals, the cell surface became distinctly positive with LA45. Demaria et al. [47] observed the formation of the Face-2 variants on human peripheral blood T cells stimulated with phorbol myristate acetate (PMA), anti-CD3 Ab, or PHA. Indeed, all of the HLA-I isomers are expressed as Face-2 variants on activated cells. Using mAb L31, HLA-C was first observed to express Face-2 naturally on a subpopulation of “normal cells”; however, the density of Face-2 was higher on 20 different kinds of EBV- transformed B lymphoid cell lines, expressing CW1 through CW8 [48] and on activated T cells of transgenic mice [49]. Similarly, using LA45 and other mAbs, the presence of HLA-F Face-2 variants was documented on activated T lymphocytes [50–52]. The presence of HLA-G Face-2 was observed on trophoblast cells in first-trimester human placental tissues with a HLA-G Face-2-specific mAb MEM-G/01 [53]. Face-2 variants were also observed on several human cancers (neuroblastoma cell lines IMR-32 and LAN-1) [14] and on colon [54], breast, ovarian, renal, and bladder carcinoma and human melanoma cell lines [15,16]). Studies on Face-2 of HLA-G revealed that the exposed amino acids may once again get masked due to homodimerization of Face-2 molecules to become Face-3 or due to heterodimerization of Face-2 of different alleles to become Face-4 [17,55,56]. Similar findings are observed when studying HLA-Ia loci. Face-2 of HLA-B27 in thymic epithelial cells and a subpopulation of peripheral blood lymphocytes of B27- transgenic mice contributed to the development of arthritis [57,58]. Indeed, Bix and Raulet [59] established that functionally conformed, free class-I HCs (Face-2) existed on the surfaces of 2m-negative cells. Face-2 of HLA-B27 exposes cysteine at position 67 in the extracellular 1-domain, which is otherwise masked by 2m. Most importantly, the increased expression of Face-2 was observed not only with HLA- B27, but also with other HLA-Ia loci (HLA-B and HLA-C) on monocytes of patients with spondylo-arthropathies (SA) and RA [18–20,60–62]. Ding et al. [19] observed that the Face-2 expression was more “strongly associated with synovial fluid (SF) cells than peripheral blood (PB) cells and closely associated with SA disease activity” (page 8). However, they found no correlation between free HCs (FHC) and HLA-B27 expression in SA patients, as has been reported earlier by others [60–62]. This is possibly due to the presence of free HCs of other HLA-I isomers, including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Therefore, they [19] prudently pointed out “the increased relative FHC expression that (they) observed on the surface of SA monocytes may not have all been ascribed to HLA-B27 alone” (page 9). Antibodies 2023, 12, 26 15 of 22 Allen et al. [21] documented the formation of Cys -dependent HC homodimers 101 164 of Face-3 in 2m-free HLA-B27. Subsequently, exposed Cys and Cys in 2m-free HCs were also shown to participate in B27 homodimer (Face-3) formation [22]. San- tos et al. [23] reported the induction of HLA-I Face-3 after activation of dendritic cells. HLA-B27 Face-2 and Face-3 were observed to form strong ligands for leukocyte Ig-Like receptors (LILRB2) [63] and KIR3DL2 [24]. Such binding of KIR and LILR family receptors with Face-3 and Face-4 of HLA can down-regulate T cell receptor-mediated T cell activation and inhibit NK cell production of INF-
to suppress further activation of NK and T cells in SA and RA [19]. Importantly, apoptosis of immune and synovial cells results in the shedding of dif- ferent HLA variants (Face-1, Face-2, Face-3, and Face-4) into synovial fluid and then into circulation. These HLA variants, particularly Face-2, may expose amino acid sequences or epitopes previously masked by 2-microglobulin. Upon exposure, these cryptic epitopes become immunogenic and can elicit Abs [35,64]. Some of these exposed cryptic sequences are shared among different HLA-I alleles, such as AYDGKDY among all the isomers of classical HLA-Ia (HLA-A, -B, and -C) and non-classical HLA-Ib (HLA-E, -F, and –G) loci. After immunizing mice with Face-2 of HLA-E, some of the resulting monoclonal Abs (TFL- 006 and TFL-007) recognized the shared epitopes of all loci of HLA-Ia and HLA-Ib [41,42]. However, when Face-1 HLA-I are shed, the Abs may form not only against HCs, but also against 2m, which is not present in the other HLA-variants (Face-2, Face-3, and Face-4). 4.2. Unique Structural Variants of HLA-F on Activated Immune Cells in RA HLA-F is frequently expressed, without 2m (Face-2), on activated lymphocytes [65] and on proliferating lymphoid and monocyte cells [66]. DNA microarray analysis further revealed an abnormal network associated with HLA-F in bone marrow cells from patients with RA [67]. Furthermore, using comprehensive gene-expression meta-analysis, it was documented that the expression of the HLA-F gene is significantly upregulated in PBMCs of clinical RA patients [68,69]. It is well known that among the HLA-I (Face-2), HLA-F is more stable, and it has a propensity to bind to NK cell Ig-like receptor KIR3DSI [66] and different alleles of other HLA-I HCs or Face-2 molecules to form homodimers (Face-3) or heterodimers (Face-4), as diagrammatically illustrated in Figure 4. The presence of HLA-F HCs or Face-2, and Face-3, and Face-4 molecules may possibly account for the production anti-HLA-F IgG without the presence of 2m Abs in RA patients. If Abs were formed against intact or 2m-associated HLA HCs (Face-1), one can expect Abs against 2m. However, only 15 of 61 RA patients’ sera had only IgM anti- 2m Abs, and 8 had only anti- 2m IgG Abs. The rest of the 46 patients were devoid of either anti- 2m IgM or IgG. The paucity of anti- 2m Abs suggested that Abs would have been developed against 2m-free HLA HCs, as illustrated in Figure 4. With these unique variants of HLA, which are often reported on the cell surfaces of activated immune cells, it is logical to expect Abs against HCs without anti- 2m. 4.3. IgM and IgG HLA-Ib Abs with and without Anti-b2m Abs May Reflect the Phases of Immunological Progression during Immunosuppressive Therapies While intact or 2m-associated HLA HCs (Face-1) are prevalent in all immune and non- immune cells, 2m-free HLA HCs (Face-2) are found predominantly after the activation of immune cells. It is well known that in RA patients, several immune cells are hyper-activated, suggesting the prevalence of the Face-2 variant of HLA. Face-2 homo- or heterodimerization forms Face-3 or Face-4 variants, as shown in Figure 4. Abs formed against HCs in the absence of anti- 2m-IgM or IgG mark the early phases of immunological progression of RA, characterized by the infiltration of activated immune cells into the synovium. Cell death, mostly by apoptosis, is the most important event taking place in the final phase of RA. The release of 2m from intact HLA (Face-1) can be expected, which may lead to the formation of anti- 2m-IgM, followed by anti- 2m-IgG. Therefore, the patients (n = 40) with sera devoid of anti- 2m-IgM or IgG may represent those in early stages of RA, and Antibodies 2023, 12, 26 16 of 22 Antibodies 2023, 12, x FOR PEER REVIEW 17 of 23 the 15 patients sera with anti- 2m-IgM or anti- 2m IgG may represent advanced phases of immunological progression of RA, as summarized in Table 11. Figure 4. Diagrammatic illustration of HLA-E, HLA-F, and HLA-G homodimers and possible Figure 4. Diagrammatic illustration of HLA-E, HLA-F, and HLA-G homodimers and possible for- formation of HLA-F heterodimers with HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F HCs. Different mation of HLA-F heterodimers with HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F HCs. Different colors of the domain refer to differences in the alleles. colors of the domain refer to differences in the alleles. 4.3. IgM and IgG HLA-Ib Abs with and without Anti-β2m Abs May Reflect the Phases of Immu- Table 11. Patterns of IgM and IgG antibodies formed against 2m and HCs of HLA-E, HLA-F, and nological Progression during Immunosuppressive Therapies HLA-G in different groups of RA patients during different phases of disease progression. While intact or β2m-associated HLA HCs (Face-1) are prevalent in all immune and Heavy Chains non-immune cells, β2m-free HLA HCs (Face-2) are found predominantly after the activa- 2M Phases of Disease HLA-E HLA-F HLA-G Groups Tables Progression tion of immune cells. It is well known that in RA patients, several immune cells are hyper- IgM IgG IgM IgG IgM IgG IgM IgG activated, suggesting the prevalence of the Face-2 variant of HLA. Face-2 homo- or heter- Present/ Present/ Present/ Present/ Present/ Present/ Group 1 Table 2 Absent Absent Phase-1 odimerization forms Face-3 or Face-4 variants, as shown in Figure 4. Abs formed against Absent Absent Absent Absent Absent Absent HCs in the absence of anti-β2m-IgM or IgG mark the early phases of immunological pro- Present/ Present/ Present/ Group 2 Table 3 Absent Absent Absent Absent Absent Phase-2 Absent Absent Absent gression of RA, characterized by the infiltration of activated immune cells into the syno- Present/ Present/ Present/ Present/ Present/ Group 3 Table 4 Present vium. Cell Absent dea Prth, most esent ly by apoptosis, is the most important event taking place in the fi Phase-3a nal Absent Absent Absent Absent Absent phase of RA. The release of β2m from intact HLA (Face-1) can be expected, which may Present/ Present/ Present/ Group 4 Table 5 Present Absent Absent Absent Absent Phase-3b Absent Absent Absent lead to the formation of anti-β2m-IgM, followed by anti-β2m-IgG. Therefore, the patients Present/ Present/ Present/ Present/ Present/ Present/ (n = 40) with sera devoid of anti-β2m-IgM or IgG may represent those in early stages of Group 5 Table 6 Absent Present Phase-3c Absent Absent Absent Absent Absent Absent RA, and the 15 patients sera with anti-β2m-IgM or anti-β2m IgG may represent advanced Present/ Present/ Present/ Group 6 Table 7 Absent Present Absent Absent Absent Phase-3d phases of immunological progression of RA, as summarized in Table 11. Absent Absent Absent Table 11. Patterns of IgM and IgG antibodies formed against β2m and HCs of HLA-E, HLA-F, and Despite the defined immunological progression of patients with RA, it is important HLA-G in different groups of RA patients during different phases of disease progression. to note that almost all the patients in our cohort were receiving a variety of immunosup- Heavy Chains Phases of Dis- pressive drugs. Most importantly, several drugs interfere with the natural immunological β2M Groups Tables HLA-E HLA-F HLA-G ease Progres- progression of RA, as presented earlier in Material and Methods. Since these drugs are IgM IgG IgM IgG IgM IgG IgM IgG sion used in combination with other drugs, it is not possible to create categories of antibody re- Present/ Present/ Present/ Present/ Present/ Present/ sponse in conjunction with the specific kind of drug received. However, the immunological Group 1 Table 2 Absent Absent Phase-1 Absent Absent Absent Absent Absent Absent responses can be classified broadly as follows: Present/ Present/ Present/ Group 2 Table 3 Absent Absent Absent Absent Absent Phase-2 Absent Absent Absent Present/ Present/ Present/ Present/ Present/ Group 3 Table 4 Present Absent Present Phase-3a Absent Absent Absent Absent Absent Antibodies 2023, 12, 26 17 of 22 (1) Drugs that suppress cell proliferation of activated immune cells. Methotrexate, lefluno- mide, and azathioprine belong to this category. (2) Drugs that inhibit IgM and IgG production. Leflunomide, prednisone, and azulfidine (sulfasalazine) belong to this category. (3) Drugs that promote apoptosis of activated human T cells and immune cells. Azulfidine (sulfasalazine) and azathioprine belong to this category. (4) Most of the drugs listed in Table 1 suppress pro-inflammatory cytokines. Therefore, our results on anti-HLA HC Abs not only reflect immunological progression of the disease but also the impacts of the drug treatments the patients received. For example, Azulfidine and azathioprine promote the apoptosis of activated T cells and macrophages. Consequently, intact HLA molecules with 2m may be released into the circulation, which would have promoted production of anti- 2m IgM, as in patients Alb-RA-051F35 and 017F64 (Table 4). These anti- 2m IgM may be azulfidine-dependent. However, the rest of the 5 RA patients who had anti- 2m IgM Abs (Table 4) were not treated with drugs promoting apoptosis. Therefore, they may represent the last phase of the progression of natural events, characterized by cell death (apoptosis) of immune cells in the synovium. The sera without the presence of anti- 2m-IgM or IgG in 47 patients could have been a consequence of immunosuppressive treatments. A higher prevalence of anti-HLA-F HC Abs was found compared to other HLA-Ib loci. Once again, the high prevalence of anti- HLA-F IgG (64/69), in contrast to the low prevalence (8/76) of anti- 2m IgG, suggests that HLA-F variants (Face-2, Face-3, and Face-4; Figure 4) are indeed the major immunogenic antigens in RA patients. This highlights the need to characterize the expression of variants of HLA-F in immune and in non-immune cells of RA patients, which may shed a brighter light on the immunodynamics of HLA-F specifically, and HLA-Ib Abs in general, in RA patients. 5. HLA-Ib Antibody Profiles in the Normal Control Group Naturally occurring IgM and IgG antibodies in normal and healthy volunteers were studied earlier [63]. Comparatively, the incidences of MFI of IgM and IgG antibodies against HLA-E, HLA-F, and HLA-G in normal males and females are considerably higher than those observed in RA patients. Evidently, the antibodies levels stay lower in RA patients, confirming the impact of the immunosuppressive drugs received by the patients. The most striking feature is that only 5 females of the 69 patients examined had both IgM and IgG serum Abs at comparable level to the normal cohort. It is possible they were yet to be impacted by the immunosuppressive drugs. Therefore, studying both IgM and IgG antibodies against HLA-Ib before and during treatment of immunosuppressive drugs, at different doses, may reveal progressive and possibly tolerable drug mediated immunosuppression. It is under these circumstances that the high prevalences of anti-HLA- F IgG antibodies in contrast to those of anti-HLA-E IgG and anti-HLA-G IgG in the majority of the RA patients is noteworthy. These findings strongly favor monitoring anti-HLA-F IgG in RA patients while they receive treatment protocols and may serve a biomarker to regulate dosage and combination drugs during the course of the disease until a total loss of antibody response, as observed in Group 7. 6. Limitations of This Investigation Since this investigation was carried out on sera obtained from a large cohort of patients visiting different clinical centers in Mexico, the following detailed information could not be obtained: (i) the dosages received for individual drugs for each patient, (ii) the time interval between date of sera collection and the duration of initiation of the drug administration prior to sera collection, (iii) the disease severity (DAS28, CDAI, etc.), and (iv) the disease duration after serum collection. It would be worthwhile if every patient were to be tested for antibodies against not only rheumatoid factor, but also cyclic citrullinated peptide to assess correlations between these classical seropositive biomarkers and anti-HLA-Ib antibodies. In addition, analysis of serum anti-HLA-Ia (HLA-A, HLA-B, and HLA-C) IgM and IgG antibodies would be valuable, since HLA-Ia and HLA-Ib share several amino acid Antibodies 2023, 12, 26 18 of 22 141 147 152 159 sequences (such as AYDGKDY and EDLARSWTA ). There is a compelling need to undertake a detailed investigation on the analysis of IgM and IgG antibodies reacting to all isomers of HLA-Ia and HLA-Ib, rheumatoid factor, and cyclic citrullinated peptide during the well-defined course of RA disease progression, to validate the hypothesis proposed in this investigation. 7. Summary This investigation reported the differences in the strengths (MFI) of IgM and IgG Abs against 2m and HCs of HLA-E, HLA-F, and HLA-G in 74 RA patients. Twenty-nine of seventy-six patients’ sera had anti- 2m Abs. Of these, 21 had anti- 2m IgM, and 8 had only anti- 2m IgG. One can reasonably expect that the Abs against 2m were generated against intact or 2m-associated HLA HCs (Face-1). The vast majority of the remaining patients were devoid of either anti- 2m IgM or IgG but had Abs against HCs of different HLA-Ib molecules. This suggests that Abs against 2m-free HLA HCs, such as Face-2, Face-3, and Face-4, have been developed. Most strikingly, anti-HLA-F IgG Abs were observed in 92.7% of RA patients examined. Both the nature and the immunogenicity of variants of HLA-F on immune and non-immune cells of RA patients deserve further in-depth correlative investigation. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antib12020026/s1, Table S1: Details regarding demographic data and individual treatment protocols. Author Contributions: All authors jointly studied the literature and formulated the concept of the article, after discussions. Conceptualization, M.H.R.; formal analysis, M.H.R., N.M.R., C.J.A.-M., F.E.H. and E.J.F.; investigation, M.H.R.; methodology, M.H.R. and L.E.M.-B.; resources, L.E.M.-B.; validation, M.H.R., N.M.R., S.R.S. and E.J.F.; writing—original draft, M.H.R.; writing—review and editing, N.M.R., C.J.A.-M., F.E.H., S.R.S., E.J.F. and L.E.M.-B.; N.M.R. provided institutional resources. All authors have extensively revised the final version from the clinical perspective. All authors have read and agreed to the published version of the manuscript. Funding: This research utilizes the funds received from Mark Terasaki, first son of the late Professor Paul Terasaki and Terasaki Foundation Laboratory (TFL). Institutional Review Board Statement: Sera of patients were provided by Luis Eduardo Morales- Buenrostro, a visiting professor from MEXICO to TFL. The sera were collected after obtaining written informed consent approved by the institutional Review Board by Morales-Buenrostro. The experiments were conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Terasaki Foundation laboratory. TFL is not affiliated with any universities or hospitals, and it is a private research institute. Informed Consent Statement: Informed consent was obtained by the team of clinicians under the supervision of Prof. Dr. Luis Eduardo Morales-Buenrostro, a visiting scientist at TFL. Data Availability Statement: Data are available from the first author. Acknowledgments: The experiments were carried out at Terasaki Foundation Laboratory (TFL) in Santa Monica, California, with the guidance of the late Paul Ichiro Terasaki, and with the active and enthusiastic involvement of several dynamic research associates. Luminex Single Antigen Bead immunoassays for monitoring HLA-I reactivity were assisted by the following research associates: Tho Pham and Vadim Jucaud. Senthamil R. Selvan contributed to this article in his personal capacity. The views expressed are his own and do not necessarily represent the views of the Food and Drug Administration or the United States Government. Conflicts of Interest: The authors declare no conflict of interest. Dedication: The first author (M.H.R.) and the last author, Luis Eduardo Morales-Buenrostro, ded- icate this article to our mentor, the late Paul Ichiro Terasaki, whose constant encouragements and discussions held at his office and at weekly seminars molded the thought process for this article. Antibodies 2023, 12, 26 19 of 22 Abbreviations ACEI: Angiotensin Convertase Enzyme inhibitor; Azat: Azathioprine; Azulf: Azulfidine; 2m: 2-Microglobulin; Chlrqn: Chloroquine; diabet: Diabetics; Dslipid: Dyslipidemia; Folic: Folic acid; HCs: Heavy Chains; HLA: Human Leukocyte Antigens; Hypertns: Hypertension; Hypothyr: Hy- pothyroidism; Met: Methotrexate; MFI: Mean Fluorescence Intensity; NFB: Nuclear Factor B; Ompr: Omeprazole; PBMC: Peripheral blood monocytes; Predns: Prednisone; RA: Rheumatoid Arthritis; Ren Dis: Renal disease; SLE: Systemic Lupus Erythematosus; Sys Vasc: Systemic Vasculitis; Thrmbss: Thrombosis. References 1. Firestein, G.S.; McInnes, I.B. Immunopathogenesis of rheumatoid arthritis. Immunity 2017, 46, 183–196. [CrossRef] [PubMed] 2. Malmstrom, V.; Catrina, A.I.; Klareskog, L. The immunopathogenesis of seropositive rheumatoid arthritis: From triggering to targeting. Nat. Rev. Immunol. 2017, 17, 60. [CrossRef] [PubMed] 3. Weyand, C.M.; Goronzy, J.J. The immunology of rheumatoid arthritis. Nat. Immunol. 2021, 22, 10–18. [CrossRef] [PubMed] 4. McInnes, I.B.; Schett, G. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 2011, 365, 2205–2219. [CrossRef] [PubMed] 5. Mellado, M.; Martínez-Muñoz, L.; Cascio, G.; Lucas, P.; Pablos, J.L.; Rodríguez-Frade, J.M. T Cell Migration in Rheumatoid Arthritis. Front. Immunol. 2015, 6, 384. [CrossRef] [PubMed] 6. Jang, S.; Kwon, E.-J.; Lee, J.J. Rheumatoid Arthritis: Pathogenic roles of diverse Immune cells. Int. J. Mol. Sci. 2022, 23, 905. [CrossRef] 7. Onishi, R.M.; Gaffen, S.L. Interleukin-17 and its target genes: Mechanisms of interleukin-17 function in disease. Immunology 2010, 129, 311–321. [CrossRef] 8. Shen, F.; Gaffen, S.L. Structure-function relationships in the IL-17 receptor: Implications for signal transduction and therapy. Cytokine 2008, 41, 92–104. [CrossRef] 9. Kinne, R.W.; Brauer, R.; Stuhlmutler, B.; Palombo-Kinne, E.; Burmester, G.R. Macrophages in rheumatoid arthritis. 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Ther. 2011, 13, R89. [CrossRef] Antibodies 2023, 12, 26 22 of 22 68. Afroz, A.S.; Giddaluru, J.; Vishwakarma, S.; Naz, S.; Khan, A.A.; Khan, N. A Comprehensive Gene Expression Meta-analysis Identifies Novel Immune Signatures in Rheumatoid Arthritis Patients. Front. Immunol. 2017, 8, 74. [CrossRef] 69. Xia, W.; Wu, J.; Deng, F.Y.; Wu, L.F.; Zhang, Y.H.; Guo, Y.F.; Lei, S.F. Integrative analysis for identification of shared markers from various functional cells/tissues for rheumatoid arthritis. Immunogenetics 2017, 69, 77–86. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
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