Oligoclonal Expansion of Effector Memory CD8+CD57+ T Cells May Sustain Bone Marrow Destruction in Aplastic Anemia

The character of oligoclonal expansion of CD8+CD28- lymphocytes in aplastic anemia (AA), described by Risitano et al. (Blood, 2002 and Lancet, 2004), strongly suggests an antigen driving mechanism of T cell activation leading to destruction of hematopoietic stem and progenitor cells. In this study, we focused on a subset of CD8+CD28- T lymphocytes termed effector memory cells because of their high antigen-affinity and ability to be rapid activated after antigen stimulation. We investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD4+ and CD8+ T subsets and the T cell receptor (TCR) Vβ repertoire by flow cytometry, and next-generation sequencing (NGS). Peripheral blood mononuclear cells from 20 AA patients and 14 healthy controls were evaluated for Vβ usage by flow cytometry. At the time of sampling, none of the patients had yet received immunosuppressive therapy. CD28+CD57- and CD28-CD57+ cells in CD4+ and CD8+ populations were assessed using the IOTest Beta Mark and a LSRII Fortessa cytometer for acquisition. The mean + 3SD of each Vβ group in controls was used as a cut-off to determine Vβ skewing in patients. When the frequencies of CD28+ and CD57+ cells in CD4+ and CD8+ subsets were compared, no differences were found between patients and controls (p=0.6125). Polyclonal expansion was described in CD8+CD28+, CD4+CD28+ and CD4+CD57+ T cells in 60%, 45% and 70% of patients, respectively. Using the mean of CD8+CD57+ cell frequency in controls as a cut-off, 12 of 20 patients displayed expansion of effector memory CD8+ cells and 11 of them (55% of all patients) showed expansion in 1 to 6 Vβ families with 1 to 4 immunodominant clones, while only 4 of 14 (29%) healthy controls displayed expansion of two Vβ families in expanded CD8+CD57+ cells without polyclonal expansion of CD8+CD28+ cells. In four cases (20% of all cases) of the remaining 8 patients whose cells did not display expanded CD8+CD57+ cells, there was expansion of 1 to 3 Vβ families with 1 dominant clone. These findings suggest activation of the immune response, polyclonal expansion of effector CD28+ compartments, and oligoclonal activation of effector memory CD8+ cells, regardless of frequency in peripheral blood. The frequency of CD8+CD57+ cells modestly correlated with the mean Vβ expansion in each patient (r2=0.5831, p<0.001). Also, we analyzed VDJ combinations and complementary region 3 (CDR3) sequences in CD4+ and CD8+ cells from 8 AA patients with CD8+CD57+ expansion in the ILLumina Hiseq 2000 sequencer. For the CD4+ compartment, patients were similar to healthy control CD4+ cells: frequencies of the most abundant clones less than 6%, CDR3 size profiles with Gaussian distribution and low degree of diversity (Simpson's indexes range 0-1: 0 means infinite diversity, and 1 means no diversity). For CD8+ cells, 6 of 8 patients displayed a "skyscraper" Vβ/Jβ plot due to the presence of 1 to 3 dominant clones, with frequency greater than 10%, predominant classes in CDR3 size profile, and Simpson's index similar to the CD8+CD57+ subset (p=0.1914). Analyzing CDR3 amino acid sequences for homology, we found that all the dominant sequences from patients were present at very low frequency in a healthy donor CD8+ cell pool (range, 0.12% - 53.6% vs 0.01% - 1.64%, p=0.0037), but no matches were described for the healthy CD8+CD57+ CDR3 repertoire. Only three CDR3 sequences were shared between patients. One of these CDR3 sequences (CSARDPPVSGTRGTDTQYF) was present in all patients and controls at very low frequency (mean expression, 5.01%), and it was the dominant clone in one patient, carried by TRBV20-1/TRBJ2-3 rearrangement at a frequency of 53.6%. Shared sequences were not found in reported viral TCR repertoires, nor in T cells recognizing peptide of myelin basic protein in multiple sclerosis, or synovial T cells from rheumatoid arthritis patients, nor matches were identified in T-large granular lymphocyte leukemia, AA, cancer or melanoma-derived TCR repertoires. Our data support the hypothesis of autologous immune-mediated attack leading to bone marrow destruction, likely triggered by autoantigens. Our results highlight the role of effector memory CD8+CD57+ T cells in sustaining an aberrant immune response during active disease. CD8+CD57+ cell expansion mirrors Vβ oligoclonal expansion, and Vβ flow cytometry should be useful in diagnosis and periodic evaluation of AA patients.