Escape Hematopoiesis By HLA-B5401-Lacking Hematopoietic Stem Progenitor Cells in Male Patients with Acquired Aplastic Anemia

Abstract

[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<0.001). Notably, the 11 patients who lost HLA-B*54:01-containing haplotypes were all male. A flow cytometry analysis revealed the presence of leukocytes that lacked Bw6 (B5401) alone in one (KANA6) of the five 6pLOH(+) patients heterozygous for HLA-Bw (Bw4/Bw6). Deep sequencing of HLA class I alleles in granulocytes from the 11 6pLOH(+) patients revealed a mutation (stop mutation) only in HLA-B*54:01 of KANA6. Reprograming of KANA6's monocytes generated three different iPSCs, including wild-type (WT), 6pLOH(+), and B*54:01-mutated clones, and CD34+ cells that were induced from the three different iPSC clones reconstituted human hematopoiesis in immunodeficient mice to a similar degree, suggesting that B5401-lacking HSPCs without proliferative advantage support hematopoiesis of KANA6. The CAG repeat lengths in male AA patients possessing B*54:01 tented to be shorter than those in the healthy controls (median value 21 vs 23). [Conclusions] Our results suggest that HLA-B*54:01 plays an important role in the autoantigen presentation by HSPCs to T cells in male patients with AA. The shorter CAG repeat lengths of the AR gene that correlate with higher expression levels of AR may explain the male predominance in 6pLOH(+) patients possessing HLA-B*54:01. DisclosuresYoshida: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.