Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia

Abstract

Oligoclonal expansion of CD8⁺ CD28^- lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8⁺ CD28^- cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8⁺CD28^-CD57⁺ cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8⁺CD57⁺ cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8⁺ cells from aplastic anemia patients with CD8⁺CD57⁺ cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8⁺CD57⁺ cells, which also showed decreased diversity compared to total CD4⁺ and CD8⁺ cell pools. From analysis of complementarity-determining region 3 sequences in the CD8⁺ cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8⁺ T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064).

Trial registration: ClinicalTrials.gov NCT00001620 NCT01623167 NCT00001397 NCT00071045 NCT00081523 NCT00961064.

Figure 1.

Immunophenotyping and flow cytometry analysis of Vβ usage in severe aplastic anemia (SAA) patients and healthy subjects. (A) Percentages of CD28⁺ and CD57⁺ cells were calculated in both CD4⁺ and CD8⁺ compartments for healthy controls and SAA patients. Data are shown as mean±Standard Deviation (SD). Unpaired t-test was performed. *P<0.05; **P<0.01. (B) Vβ usage was studied in T-cell compartments (by row), and percentages of each Vβ family were reported as total CD4⁺ or CD8⁺ cell percentage. For Vβ usage in healthy subjects, data are shown as mean+SD, combining the results from all 34 healthy donors. For SAA patients, 2 representative cases are shown.

Figure 2.

Vβ usage at diagnosis and during treatment. (A) Percentages of Vβ family in CD8⁺CD57⁺ cells were calculated on total CD8⁺ cells, and Vβ skewing in severe aplastic anemia (SAA) patients was defined using the mean+3Standard Deviation (SD) of a given Vβ group in healthy donors. Relative expansion of each Vβ subgroup is shown in the bar graph. Patients were divided based on the absence or presence of expanded CD8⁺CD57⁺ cells, using the mean in healthy donors (13.3%). Skewing of one Vβ family is reported as an orange bar. (B) Progression-free survival rate of SAA patients with CD8⁺CD57⁺ cells ≤13.3% (n=7) or >13.3% (n=17) prior to treatment. Log-rank (Mantel-Cox) test was performed. (C) Vβ usage was performed in Patient 22 at diagnosis, at 10 days of treatment, and at 6 and 9 months (relapse). Perturbations during treatment and relapse are reported as percentage of positive CD8⁺CD57⁺ and Vβ2⁺ cells (left), or absolute lymphocyte and Vβ2 clone count (right).

Figure 3.

Characterization of Vβ/Jβ plot, CDR3 size and DJ length profiles in healthy donors by deep sequencing. (A) T-cell receptor β variable (TRBV)/T-cell receptor β joining (TRBJ) plots showed a “citylike” landscape for total CD4⁺ and CD8⁺ cell populations in healthy subjects (HC). (B) The size of the complementarity region 3 (CDR3) and DJ length profiles were also defined, showing similar features in CD4⁺ and CD8⁺ cells.

Figure 4.

The TCR repertoire by deep sequencing analysis from total CD8⁺ cells in severe aplastic anemia (SAA) patients with CD8⁺CD57⁺ cell expansion. In contrast to healthy CD8⁺ profiles, most SAA patients (AA) displayed oligoclonal features in a TRBV/TRBJ rearrangement plot (A) and CDR3 size and DJ length profiles (B). CD4⁺ and CD8⁺ profiles are shown for each SAA patient. In AA1 and AA6, only CD8⁺ cells were sufficient for deep sequencing.

Figure 5.

The TCR repertoire by deep sequencing of enriched CD8+CD57+ cells in severe aplastic anemia (SAA) patients. (A) The enrichment of the clone in effector memory CD8⁺ T cells, comparing TRBV/TRBJ rearrangement in total CD8⁺ cells (left) with those in CD8⁺CD57⁺ cells (right) from the same patients. (B) CDR3 size and DJ length profiles from CD8⁺CD57⁺ cells also overlapped with those in CD4⁺ and CD8⁺ profiles.

Figure 6.

Homology assessment. (A) CDR3 sequence pools were analyzed among patients (AA) and healthy subjects (HC) for the presence of homology. Shared and immunodominant sequences were reported as a heatmap based on their relative expression: in the same row, from lowest (gray; <0.01%) to highest (red; >5%) values. (B) Structural analysis was performed comparing the sequences for common pattern, using both alignments at the N-terminal of Vβ gene (left) or at the C-terminal of Jβ gene (right).