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Background: Small populations of paroxysmal nocturnal hemoglobinuria (PNH)-type cells ( Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF without malignant diseases were prospectively recruited to the study between April 1 st, 2016 and December 31 st, 2019 in Japan. Participants were excluded from the study if treated with eculizumab or ravulizumab. Peripheral blood samples were obtained with informed consent and sent to the single laboratory every 12months (mos) for 36 mos. A high-resolution flow cytometry assay known as OPTIMA method (Ann Hematol 97(12):2289-2297) was used to precisely detect a small population of GPI(-) cells, which defines ≥0.003% PNH-type granulocytes (Gran) and ≥0.005% erythrocytes as an abnormal increase. The quality of life (QOL) of the pts was assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. All other lab data and clinical information were obtained at each participating institute or hospital on sample collection, and were accumulated by the Japan PNH Study Group for analysis. Results: Total 1,985 pts were registered and 1,813 pts were eligible for analysis. Median age was 67 with 50.5% male patients. PNH-type cells were positive in 50.4% (235/466) of AA, 19.7% (70/355) of MDS, 22.3% (61/273) of suspected PNH, and 33.9% (232/685) of undiagnosed BMF. PNH-type cells were increased in nearly 30% of the pts with RCUD, RCMD, MDS-U, and 5q-, but not in RARS, RAEB-1, or RAEB-2. Time-course data of the size of PNH-type cells were available in 651 pts at 12mos and in 210 pts at 36mos. Small ( Conclusion: PNH-type cells were detected exclusively in AA and low-risk MDS, supporting the hypothesis that the increase of PNH-type cells in BMF underpin the benign immune-mediated feature of the disease. The presence of PNH-type cells predicts a better response to IST in BMF, which is consistent with previous reports. Detection of subclinical PNH-type cells was associated with an improvement of QOL scores in multiple items at 36mos. Those small populations of PNH-type cells stayed subclinical in most of the cases, but caution should be exercised in monitoring the sizes as some may evolve into clinical PNH. Figure 1 Figure 1. Disclosures Ueda: Chugai Pharmaceutical: Consultancy, Honoraria, Research Funding; Alexion Pharma: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Yonemura: Alexion Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Novartis Pharma: Honoraria. Obara: Novartis Pharma: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Alexion Pharma: Honoraria, Research Funding. Ando: Novartis: Honoraria; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Celgene: Honoraria; Astellas Pharma: Honoraria; Takeda Pharmaceutical: Research Funding. Kawaguchi: Alexion Pharma: Honoraria. Nishimura: Alexion Pharma: Consultancy; Chugai Pharmaceutical: Consultancy; Novartis Pharma: Consultancy; Roche: Consultancy; apellis pharmaceuticals: Consultancy; Biocryst: Consultancy; Sanofi: Consultancy. Nakao: Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Symbio: Consultancy; Alexion Pharma: Research Funding.

[Background] Minor populations (0.003%-1.0%) of glycosylphosphatidylinositol-anchored protein-deficient granulocytes (GPI[-] Gs) are often detected in the peripheral blood (PB) of patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes and are thought to represent immune pathophysiology of bone marrow failure. We previously reported that minor GPI(-) G populations were detected in some healthy individuals (HIs) and persisted over several years at similar percentages (Katagiri T, et al. Stem Cells 2013). Several lines of evidence have suggested that small numbers of GPI(-) Gs detected in HIs are polyclonal populations mostly derived from short-lived PIGA-mutated committed progenitor cells. Minor GPI(-) G populations in AA patients may also be derived from multiple committed progenitor cells rather than from a few hematopoietic stem progenitor cells (HSPCs) with PIGA mutations. However, minor GPI(-) G populations usually persist for a long period of time at similar frequencies in AA patients, suggesting that they may instead be derived from a few HSPCs that have undergone PIGA mutations. This issue remains debated due to the inability to sequence the PIGA gene in the very few GPI(-) granulocytes available. We recently developed a sensitive method capable of detecting PIGA mutations in minor GPI(-) Gs using amplicon sequencing of GPI(-) Gs that were enriched with magnetic microbeads followed by FACS sorting. Using this method, we addressed whether minor GPI(-) G populations in AA patients and HIs are oligoclonal or polyclonal as well as which cell population they are derived from HSPCs or committed progenitor cells. [Methods] Five AA patients possessing 0.025%-0.898% GPI(-) Gs, 3 HIs who were found to have ≥0.003% (0.006%, 0.051%, and 0.059%) GPI(-) Gs during a screening of more than 200 HIs for GPI(-) Gs, 30 HIs (median: 37 years old, male/female:17/13) with 0% to 0.002% GPI(-) Gs, and 8 cord blood (CB) samples were subjected to enrichment of GPI(-) Gs for PIGA sequencing. Their leukocytes were treated with PE-labelled anti-CD55 monoclonal antibodies (mAbs) and anti-CD59 mAbs, and CD55 +CD59 + granulocytes were removed using magnetic microbeads labelled with anti-PE mAbs. CD11b +FLAER-negative granulocytes were sorted from the remaining granulocytes using FACSAria Fusion. DNA from sorted GPI(-) Gs was amplified using primers covering all exons of PIGA. Nucleotide sequences of the PIGA gene in GPI(-) Gs were determined using a next-generation sequencer. [Results] This novel enrichment method enabled the detection of only 1-4 different PIGA mutations in all 5 female AA patients (AA 1-5) with the total of different VAFs in each case reaching nearly 50% (Table 1). Limited kinds of PIGA mutations were also detected in three HIs (two males [HI 1 and 3] and one female [HI 2]). For HI 1 and HI 3, the VAFs of predominant PIGA-mutated sequences were longitudinally measurable using whole-blood DNA samples with droplet digital PCR, which showed no apparent changes in the VAF (0.020%-0.027% for HI 1 and 0.012%-0.025% for HI 3 over 4 and 6 years, respectively). The presence of mono or oligoclonal GPI(-) Gs in the 3 HIs prompted us to study 30 HIs who had been judged to be negative for minor GPI(-) G populations by a high-sensitivity flow cytometry method. The enrichment method unexpectedly identified clear CD11b highFLAER - GPI(-) G clusters in granulocytes from 24 of the 30 HIs (Figure 1a). The median number of GPI(-) Gs contained in 7 ml of PB was 31 (range, 1-136 cells). Sufficient amounts of DNA for NGS were obtained from sorted GPI(-) Gs of six subjects, and PIGA amplicon sequencing revealed 1-3 different PIGA mutations in four of the six subjects. The examination of fresh CB also revealed clear GPI(-) G clusters in four of eight samples (Figure 1b). PIGA amplicon sequencing of 79 GPI(-) Gs obtained from 1 male CB sample (CB 1) showed a sole PIGA mutation with VAFs of 95% (Figure 1c). [Conclusion] Minor GPI(-) G populations detectable in patients with AA and HIs are derived from a few PIGA-mutated HSPCs, not from committed myeloid progenitor cells, a finding that negates a hypothesis that a few PIGA-mutated HSPCs are selected from polyclonal PIGA-mutated HSPCs during transition from AA to florid PNH. Very small numbers of GPI(-) Gs are present much more frequently in HIs than previously thought and may also be derived from a few HSPCs with PIGA mutations that occur in HSPCs during the fetal stage. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Research Funding; Kyowa Kirin: Honoraria. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

In immune-mediated acquired aplastic anemia (AA), the presence of an HLA allele, which is highly overrepresented or lost due to somatic mutations, may represent a specific immune pathophysiology and a clinical manifestation. HLA-B*14:02 is one of the most overrepresented class I alleles in AA and is also frequently affected by a somatic loss of expression; the inherited B*14:02 genotype was correlated with high-risk clonal evolution in two independent cohorts in the U.S. (Babushok DV et al. Blood Adv 2017; Zaimoku Y et al. manuscript in preparation). In contrast, HLA-B*14:02 is virtually absent in Japanese, in whom somatic mutations of AA have frequently been detected in HLA-B*40:02, B*54:01, and A*02:06, and occasionally in A*02:01, A*02:07, A*31:01, B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, and B*56:01 (Mizumaki H et al. Haematologica 2021). A class II allele HLA-DRB1*15 is highly overrepresented in AA across various ethnic groups, including those in the U.S. and Japanese. This retrospective study in the Japanese population aimed to explore the clinical significance of disease-associated non-B*14:02 HLA class I and II alleles in AA. A total of 423 enrolled patients with AA (very severe [n = 81], severe [n = 266], transfusion dependent non-severe [n = 76]; median age 60 [range, 1-86] years) had undergone genotyping for HLA-A, HLA-B, HLA-C, and HLA-DRB1 at 2-field resolution. The HLA allele frequencies in these patients were compared to those in a Japanese HLA haplotype dataset (n = 19183; Ikeda N et al. Tissue Antigens 2016). The most overrepresented allele in AA was HLA-DRB1*15:02, followed by DRB1*15:01, B*40:02, and A*02:06 (Table); DRB1*13:02 and B*44:03, which are in linkage disequilibrium, were markedly underrepresented, consistent with a well-known protective role of DRB1*13 against autoimmune diseases. HLA-DRB1*15:02 was also significantly correlated with age and its frequency among patients aged The overall response rate to anti-thymocyte globulin-based immunosuppressive therapy at 6 months was 63% (139 of 220 treated and evaluable patients). A trend for a higher response was observed in patients harboring mutation-related HLA-B alleles (except for minor alleles B*13:01, B*40:03, and B*55:02) and the highly overrepresented or protective HLA-DRB1 alleles, but not in the HLA-A alleles (Figure D). A multivariate logistic regression revealed that the combination of the presence of any favorable alleles in HLA-B (odds ratio 3.6, P < 0.0001) or in HLA-DRB1 (odds ratio 2.3, P = 0.00085) was significantly and independently associated with a hematologic response; the tendencies for a lower or higher response in very severe disease and the presence of paroxysmal nocturnal hemoglobinuria clone did not reach statistical significance. Further, there was likely an additive effect when two favorable alleles coexisted in HLA-B or HLA-DRB1 (Figure E); the copy number of the favorable HLA-B and HLA-DRB1 alleles stratified the response rate to four groups: three or four copies, 95% (19 of 20); two copies, 72% (61 of 85); one copy, 59% (50 of 85); and zero copy, 30% (9 of 30). Only eight patients displayed clonal evolution to monosomy 7, myelodysplastic syndrome, or acute myeloid leukemia after immunosuppression without significant overrepresentation or underrepresentation of the pathogenic HLA alleles. Using a large dataset of homogeneous Japanese population with high-resolution HLA typing, we revealed, for the first time, a strong relationship between disease-associated (overrepresented, inactivated, or protecting) HLA alleles and the responsiveness to immunosuppressive therapy. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Adaptive Biotechnologies, Eisai: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Honoraria; Kyowa Kirin: Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

[Background] HLA-DR15 (DR15) has been implicated in the susceptibility to immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA), wherein the hematopoietic function depends on cyclosporine (CsA), paroxysmal nocturnal hemoglobinuria (PNH) with BM failure, and low-risk myelodysplastic syndrome responsive to immunosuppressive therapy. However, how DR15 contributes to the development of such immune-mediated BM failure remains unclear. Although the copy-number neutral loss of heterozygosity in chromosome 6p (6pLOH) of hematopoietic stem progenitor cells (HSPCs) in AA patients sometimes involves the HLA-DRB1 region, the frequency of the resultant DR15 loss is low, suggesting little involvement of this DR allele in the escape of HSPCs from cytotoxic T-cell attack. Several studies have recently reported that acute myeloid leukemia cells that relapse after allogeneic hematopoietic stem cell transplantation often lack HLA class II expression through an epigenetic mechanism and thereby escape the graft-versus-leukemia effect. The epigenetic loss of HLA class II expression may also occur in HSPCs that survive the immune attack in AA patients in remission. [Objectives/Methods] To test this hypothesis, we determined the HLA-DR expression on HSPCs defined by lineage-CD45dimCD34+CD38+ cells as well as their subpopulations, including common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs), and granulocyte-monocyte progenitors (GMPs), in the peripheral blood of 52 AA patients (34 with DR15 and 18 without DR15) and 20 healthy individuals using flow cytometry (FCM) with anti-pan-HLA-DR antibodies. All patients were in remission after ATG-based therapy (ATG+CsA±thrombopoietin receptor agonists [TPO-RA] or anabolic steroids [AS], n=19), CsA-based therapy (CsA±TPO-RA or AS, n=27), and others (n=6), and 18 required low-dose CsA to maintain remission. Eighteen (35%) had HLA-class I allele-lacking (HLA-class I[-]) leukocytes due to 6pLOH and/or allelic mutations while 33 (63%) had 0.003-83.8% (median 0.194%) GPI-anchored protein-deficient (GPI[-]) granulocytes. HLA-DR(-) HSPCs detected in some patients were sorted together with their HLA-DR(+) counterparts and subjected to incubation in the presence of interferon gamma (IFN-γ) to see whether or not the DR expression was restored; in addition, they were subjected to RNA sequencing to compare the gene expression profiles between DR(-) and DR(+) HSPCs. [Results] Five (9.6%) of the 52 AA patients had 28.6% to 42.3% (median 34.0%) DR(-) cell populations in HSPCs, which were not detected in either monocytes or B lymphocytes of the same patients or in HSPCs of any healthy individuals (Figure 1a). All 5 patients possessed either HLA-DRB1*15:01 (n=3), DRB1*15:02 (n=1), or DRB1*15:01/15:02 (n=1), with the other DRB1 alleles differing among individuals, and their hematopoietic function depended on CsA, except for 1 patient (Case 5) whose HSPCs consisted of 69% GPI(+) and 31% GPI(-) cells. Of particular interest, Case 5's DR(-) cells were detected in GPI(+) HSPCs but not in GPI(-) HSPCs (Figure 1b). None of the 5 patients possessed HLA-class I(-) leukocytes, which were detected in 18 (38%) of 47 patients not possessing DR(-) HSPCs. In contrast to the patients possessing DR(-) HSPCs, CsA dependency was only observed in 13 (28%) of the 47 AA patients without DR(-) HSPCs. Incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in full restoration of the DR expression in all HSPC subpopulations (Figure 2). A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed that the signature of differentially expressed genes was enriched in immune response-related genes. [Conclusions] HSPCs that lacked DR due to an epigenetic mechanism were frequently detected in AA patients with DR15 characterized by CsA dependency. Although the loss of expression occurred in both DR alleles, the fact that only DRB1*15:01 or 15:02 was an allele shared by the five patients indicates that the DR loss phenomenon targeted DR15. The DR15(-) HSPCs may escape from antigen-specific CD4+ T-cell attack, which cannot be completely abolished by CsA. As demonstrated by findings of Case 5 showing the presence of a DR(-) cell population only in GPI(+) HSPCs, the lack of GPI may be a mechanism underlying substitution for the DR15 loss. Disclosures Takamatsu: Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Yamazaki:Kyowa Kirin: Honoraria, Research Funding; Novartis: Honoraria. Nakao:Alexion: Research Funding; Novartis: Honoraria; Kyowa Kirin: Honoraria; Symbio: Consultancy.

[Background] HLA-class I allele-lacking (HLA[-]) leukocytes are detected in approximately 30% of patients with acquired aplastic anemia (AA), and are thought to represent the involvement of cytotoxic T lymphocyte attack against hematopoietic stem cells (HSCs) in the development of AA, based on the high response rate to immunosuppressive therapy (IST) in patients with such aberrant leukocytes. Similar to glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient (GPI[-]) leukocytes in patients with paroxysmal nocturnal hemoglobinuria (PNH), HLA(-) leukocytes in AA patients are often clonal or oligoclonal and expand to account for more than 50% of the total leukocytes. Despite such overwhelming proliferation, somatic mutations in driver genes as well as telomere shortening that portend clonal evolution are rarely detected in HLA(-) granulocytes, suggesting the genetic stability of HLA(-) HSCs and the persistence of the immune pressure on HSCs that favors expansion of HLA(-) HSCs (Imi, et al. Blood Adv). However, recent studies from the United States have shown a higher incidence of clonal evolution to secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in AA patients with HLA(-) leukocytes than in those without such leukocytes, a finding inconsistent with the results of our previous study. Given the high prevalence of HLA(-) leukocytes in AA patients, it is critical to determine whether or not the presence of the aberrant leukocytes is associated with clonal evolution. We therefore addressed this issue by studying the prognosis of a large number of AA patients with or without HLA(-) leukocytes who had been followed for a long term period. We also studied the clonal composition of granulocytes in AA patients with HLA(-) cells, wherein aberrant clones other than HLA(-) cells might be responsible for clonal evolution to MDS/AML. [Methods] We retrospectively analyzed the clinical characteristics of 633 AA patients and peripheral blood samples were examined for the presence of HLA(-) leukocytes using a high-sensitivity flow cytometry (FCM) assay, droplet digital PCR, single-nucleotide polymorphism arrays, or next generation sequencing (NGS) between 2010 and 2020. GPI(-) cells were detected using a high-sensitivity FCM assay as previously described. [Results] HLA(-) granulocytes were detected in 127 (20.1%) of the 633 patients with a median clone size of 16.9% (range, 0.04%-100%); the aberrant granulocytes accounted for greater than 50% of the total granulocytes in 29 (22.8%) of 127 patients. Eighty-nine (70.0%) of the 127 patients possessed aberrant clones other than HLA(-) clones, which included 0.005% to 91.6% GPI(-) cells (n=86), del(13q) cells (n=3), t(1;10) cells (n=1), t(9;13) cells (n=1), inv12 cells (n=1), and trisomy 8 cells (n=1). The prevalence of GPI(-) cells was not significantly different between patients with and without HLA(-) cells (67.7% vs 65.4%). Eighty-five of 102 (83.3%) patients with HLA(-) cells responded to IST, whereas 231 of 318 (72.6%) without HLA(-) cells responded (p90% of granulocytes, suggesting that these few escape clones were enough to sustain the hematopoietic function of the patients. The prognosis survey revealed no clonal evolution to MDS/AML in any of the 127 AA patients with HLA(-) leukocytes after a follow-up period of the median 5 years. In contrast, 15 of 234 (6.4%) patients without HLA(-) cells who were trackable evolved to MDS/AML during a median 5 year follow-up. [ Conclusions] The presence of HLA(-) leukocytes and concomitant aberrant clones was not associated with clonal evolution to MDS/AML in Japanese AA patients, even in those possessing a large (>50% of the total granulocyte) HLA(-) cell population. The discrepancy between our results and the data from the United States may be due to the difference in the race and mechanism underlying HLA loss. These data suggest that HSC clones that escape immune attack, such as HLA(-) and GPI(-) clones, are healthy enough to support hematopoiesis for a long term in AA patients. Disclosures Ishiyama: Novartis: Honoraria; Alexion: Research Funding. Yamazaki:Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding. Ogawa:Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nakao:Alexion: Research Funding; Kyowa Kirin: Honoraria; Novartis: Honoraria; Symbio: Consultancy.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

[Background] In cases of immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA) and AA/PNH, aberrant hematopoietic stem progenitor cells (HSPCs) that acquire resistance to immune attack are thought to survive and support hematopoiesis in convalescent patients. Two representative progenies of such "escape" HSPC clones are HLA class I allele-lacking (HLA[-]) leukocytes and glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells. The mechanism underlying the immune selection of HLA(-) HSPCs is the failure of cytotoxic T lymphocyte (CTL) to recognize target antigens that are presented by particular HLA class I alleles of HSPCs. However, the mechanisms underlying the immune selection of GPI(-) HSPCs remain unclear. In addition, whether or not immune pressure that persists after immunosuppressive therapy (IST) contributes to the development and maintenance of clonal hematopoiesis by HLA(-) or GPI(-) HSPCs that are often seen in patients in long-term remission is also unknown. Phenotypical analyses of HSPCs that can be obtained from peripheral blood (PB) of AA patients who possess HLA(-) or GPI(-) leukocytes may provide a hint to elucidate these unsolved issues. [Objectives/Methods] We analyzed PB lineage-CD45dimCD34+CD38+ HSPCs of 15 AA patients who had 1%-99% HLA-A2(-) or HLA-A24(-) granulocytes (Gs) using flow cytometry (FCM). PB samples from 1 patient with severe AA were obtained before IST while the other 14 patients were in remission at the time of sampling; 10 were on cyclosporin (CsA) and eltrombopag (EPAG) (n=1), CsA and anabolic steroids (n=3), CyA (n=4), anabolic steroids (n=1) and EPAG alone (n=1); and 4 were free of therapy. We also determined the percentages of HLA(-) cells in different CD34+ subsets of BM, including HSCs (CD38-CD90+CD45RA-), MPPs, CMPs, GMPs, MEPs and CLPs for patients whose BM cells were available. Six AA/PNH patients whose GPI(-) Gs were 4-99% of the total Gs were subjected to the same PB HSPC analysis. For a separate group of seven AA patients who responded to CsA and had both HLA(-) and GPI(-) G populations, the percentages of each population were serially determined over one to 12 years. [Results] FCM identified 0.01%-0.4% (median 0.01%) CD45dimCD34+CD38+ HSPCs in the mononuclear cell population of the 15 AA patients, values that were significantly lower than those of seven healthy volunteers (0.19-0.78%, median 0.58%, P [Conclusions] Immune selection that favors the survival of HLA(-) HSPCs or HPCs takes place even in AA patients who have been in remission for many years after successful IST and may contribute to clonal hematopoiesis by HLA(-) HSPCs. Given no signs of selection at the HSPC stage and the reciprocal increases in the percentage of GPI(-) Gs in AA/PNH patients responding to CsA therapy, the proliferation of GPI(-) HSPCs may not be affected by immune pressure in convalescent patients. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Novartis Pharma K.K: Honoraria; SynBio Pharmaceuticals: Consultancy; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Kyowa Kirin: Honoraria.

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that CTLs are involved in the development of AA, but the precise mechanisms underlying HLA missing and clonal hematopoiesis by such HLA(-) HSPCs are unknown. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in Japanese AA patients. The study also showed that B*54:01 was one of three HLA-alleles that were most likely to be possessed by 6pLOH(+) patients (29% [5/17]) when only patients not carrying HLA-B*40:02 were analyzed. These results prompted us to study the role of HLA-B*54:01 in the pathogenesis of AA in a larger number of patients. [Method] To identify HLA class I alleles other than HLA-B*40:02 that are closely involved in the auto-antigen presentation in AA, we studied leukocytes of 549 AA patients for the presence of 6pLOH as well as HLA alleles that are lost due to 6pLOH. To gain insight into the mechanism underlying clonal hematopoiesis by HLA-B*54:01-lacking HSPCs, we studied HSPCs derived from induced pluripotent stem cells (iPSCs) that were generated from an AA patient possessing B5401-lacking monocytes. We also investigated the association between male AA patients possessing B*54:01 and CAG microsatellites of androgen receptor (AR) gene which are related to transactivation of the AR gene. [Results] 6pLOH was detected in 91 (16.6%) of the total patients and in 48 (10.4%) of the 462 patients not possessing B*40:02. Among the HLA alleles possessed by the 48 patients, B*54:01 was the most frequent (23%). 6pLOH was detected in 17 (34%) of 50 patients possessing B*54:01, and the incidence was markedly higher in males (15/24, 62.5%) than in female patients (2/26, 7.7%, P Disclosures Yoshida: Nihon-shinyaku: Research Funding. Akashi:Novartis pharma: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MSD: Research Funding; Eisai: Research Funding; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding; Pfizer: Research Funding; Celgene: Research Funding, Speakers Bureau; sanofi: Research Funding; Chugai Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. Disclosures Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

[Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs. [Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing. [Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2). [Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. Disclosures Nakao: Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

[Background] Acquired aplastic anemia (AA) is a rare syndrome characterized by pancytopenia and bone marrow hypoplasia. The cytotoxic T lymphocyte (CTL) attack against autologous hematopoietic stem progenitor cells (HSPCs) is thought to be responsible for bone marrow failure in the majority of AA cases; however, little is known about the target antigens of the CTLs. HLA class I-allele lacking leukocytes (HLA-LL) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or somatic loss-of-function mutations in HLA class I genes are detected in approximately 20% of patients with newly diagnosed AA, and the presence of HLA-LL represents compelling evidence to support that CTLs specific to HSPCs are involved in the development of AA. Our recent studies using single nucleotide polymorphism array (SNP-A) genotyping and droplet digital polymerase chain reaction (ddPCR) revealed that HLA-B*40:02 is the most frequently lost among all class I alleles that are lost as a result of 6pLOH (Zaimoku, et al. Blood 2017). Various somatic loss-of-function mutations in B*40:02 revealed by deep sequencing in the study substantiated the important role of HLA-B4002 in the autoantigen presentation of AA. However, in the other 6pLOH(+) AA patients who did not possess HLA-B4002, which accounted for 20% of the total AA cases involving patients possessing HLA-LL, the allele in the missing haplotype that was responsible for the autoantigen presentation was largely unknown because the lost fragment of chromosome 6p usually contained 2 or more HLA class I alleles. [Objectives/Methods] To identify class I alleles other than HLA-B*40:02 that are critically involved in the auto-antigen presentation of AA, we screened a total of 624 patients for the presence of HLA-LL using monoclonal antibodies specific to class I HLA alleles, SNP-A, and ddPCR, and performed targeted deep sequencing of HLA class I genes by using SeqCap EZ Choice pobes (Roche) and MiSeq sequencer (Illumina). The paired fractions, including granulocytes that lacked an HLA-A allele and granulocytes that retained the HLA-A allele, as well as CD3+ T cells, were sorted using monoclonal antibodies specific to HLA-A alleles with a BD FACSAria Fusion system (BD Biosciences), and were subjected to DNA extraction. All DNA samples of granulocytes and control cells (CD3+ T cells or buccal mucosa cells) were prepared for targeted deep sequencing. [Results] One hundred and fourteen patients were found to be positive for HLA-LL and 62 (54.4%) of the 114 HLA-LL(+) patients did not carry B*40:02 (severe, n=30; non-severe, n=32; male, n=38; female, n=24; median age, 62 [range, 6-93] years). Apart from B*40:02 (45.6%), A*02:06 (24.6%) was the second-most frequent HLA class I allele in the lost haplotype. The targeted deep sequencing of 20 patients with HLA-LL revealed 6pLOH alone in 11 patients, and somatic loss-of-function mutations plus 6pLOH in 9 patients; none of the patients were positive for somatic loss-of-function mutations alone. Of note, somatic loss-of-function mutations were found in only 5 alleles (A*02:06 in four, B*40:01 in two, B*40:03, A*31:01, and B*54:01 in one each) out of 27 different alleles contained in the lost haplotype. Among the 9 patients with somatic loss-of-function mutations, the median number of mutations per patient was 1 (range, 1-2); these included a missense mutation (n=1), frameshift deletions (n=3) and nonsense mutations (n=7) (Figure). Four patients had a breakpoint of 6pLOH in between the HLA-A and C loci; their lost alleles were A*02:06 (n=2) and A*31:01 (n=2), and the occurrence of 6pLOH in the four patients was therefore attributed to the two HLA-A alleles. Sixty-six percent of the HLA-LL(+) B*40:02(-) patients had at least one of the five alleles in the lost haplotypes. The frequencies of each "high risk" allele found in patients possessing HLA-LL are summarized in Table. [Conclusions] In addition to B*40:02, five class I alleles including HLA-A*02:06, A*31:01, B*54:01, B*40:03 and B*40:01 are thought to play an essential role in the auto-antigen presentation by the HSPCs of Japanese AA patients. The frequencies of the six class I alleles in general Japanese population are much higher than those in the general Caucasian populations but similar to the frequencies in East Asian populations. The higher frequencies of the six alleles in comparison to Caucasian countries may account for the higher incidence of AA in East Asia. Disclosures Takamatsu: Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Nakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with Kdvalues of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation.