Distinct Escape Mechanisms of HLA Class I Allele-Lacking Hematopoietic Stem Progenitor Cells (HSPCs) from GPI-Deficient HSPCs in Acquired Aplastic Anemia
Authors: Tsuji Noriaki , Hosokawa Kohei , Yoroidaka Takeshi , Mikoto Tanabe , Mizumaki Hiroki , Nakao Shinji 
Published: November 2019

[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<0.05). In 4 of the 9 patients on treatment and in 4 patients who had been free of therapy, the percentages of HLA(-) cells in HSPCs were similar to those of Gs (1%/1%, 1%/1%, 14%/15% and 100%/98% in the 4 treated patients; 16%/11% in the untreated patient; 13%/13%, 95%/95%, 99%/99% and 99%/99% in the 4 patients in remission after therapy). BM subsets including HSCs of 2 patients whose Gs and PB HSPCs lacked HLA-A2 almost completely, were 99% negative for HLA-A2. In contrast, in the remaining 6 patients, the percentages of HLA(-) cells in HSPCs were markedly lower than those in Gs (0%/25%, 2%/7%, 4%/65%, 6%/77% [Case 1], 9%/75% and 10%/39% [Case 2]), suggesting the persistence of CTL attack against HPCs or more differentiated myeloid cells (Figure 1). In contrast to HLA(-) PB HSPCs, there was no discordance in the percentage of GPI(-) cells between PB Gs and HSPCs (4%/4%, 49%/50%, 94%/100%, 96%/96%, 96%/99% and 99%/92%) in all 6 AA/PNH patients including 3 responding to CsA. Lastly, serial changes in the percentage of PB HLA(-) and GPI(-) Gs during CsA therapy were compared in seven patients who possessed both aberrant cells. In 4 patients who were responding to CsA, the HLA(-) G percentage gradually decreased in 3 and remained stable in the remaining one, while the GPI(-) G percentage reciprocally increased in 3 and remained stable in one. In contrast, in 3 patients who obtained sustained remission after CsA therapy, the HLA(-) G percentage increased after cessation of CsA. The GPI(-) G percentages decreased in 1 and remained unchanged in 2 (Figure 2).

[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.