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V. Giudice et al.
in serial samples available for Patients 4, 22, and 34 in order to understand the correlation of Vβ usage with clin- ical course (Figure 2C and Online Supplementary Figure S4A). For Patient 22, at baseline, Vβ was the immunodom- inant clone and mostly enriched in CD8+CD57+ cell popu- lation, rather than in CD4+CD28+, CD4+CD57+, and CD8+CD28+ cells. After ten days of IST, the size of the CD8+CD57+ clone was greatly reduced. The clone was detected again at six months (3 months before clinical relapse) and further increased at relapse (Figure 2C), sug- gesting association of Vβ expansion with clinical status. Patients 4 and 34 were non-responders at three months, and non- and minimal partial-responders at the 6-month time point. However, no significant changes in immun- odominant clone size were observed (Online
Supplementary Figure S4A). Vβ usage was also investigated at baseline in the BM of these 2 patients (Online Supplementary Figure S4B), and high concordance with Vβ usage of PB CD8+CD57+ cells was described.
Increased expansion of effector memory CD8+ T cells with age has been reported;36 therefore, we used a pool of age-matched healthy controls to assess the effect of age. There was no correlation between the size of the immun- odominant clone in CD8+CD57+ cells and age in healthy subjects (r2=0.0003, P=0.919) or in SAA patients (r2=0.140, P=0.079) (Online Supplementary Figure S5). However, a cor- relation was found between CD57 expression and age in SAA patients (r2=0.552, P<0.0001).
We then assessed the effect of transfusion history on oligoclonal expansion of effector memory T cells, as trans-
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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 treat- ment. 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).
haematologica | 2018; 103(5)