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V. Giudice et al.
26 Each chain is the result of a complex gene locus rearrangement, known as VDJ recombination.23 On a rearranged VDJ segment, a terminal deoxynucleotidyl transferase enzyme increases TCR variability through insertions/deletions within a hypervariable region, the CDR3, generating a unique potential antigen-specific sequence.23 Early in infection, CD28+ CTLs with different antigen affinity are selected and expanded (polyclonal phase).27 In late stages, high antigen-affinity CD28– T cells are in resting state as memory T cells.28-29 The expression of CD57 on CD8+CD28– T cells identifies a subset of memory T cells termed effector memory because of their high antigen-affinity and ability to be rapidly activated after antigen stimulation, as “tissue-guards”.29,30 Direct evi- dence of their high antigen-affinity is decreased diversity in the CD8+CD57+ TCR repertoire (oligoclonality) due to the presence of only a few selected clones of memory cells.27
In this work, we investigated the frequency and oligo- clonal expansion of effector CD28–CD57+ memory cells in CD4+ and CD8+ T cells and the TCR Vβ repertoire in AA patients by flow cytometry and deep sequencing tech- nologies, to provide additional evidence for the immune hypothesis in AA pathophysiology.
Methods
Human samples
Heparinized whole PB was collected from patients and healthy subjects after informed consent, in accordance with the Declaration of Helsinki31 and protocols approved by the National Heart, Lung, and Blood Institute Institutional Review Board (National Institutes of Health, Bethesda, MD, USA) (see Online Supplementary Table S1 for clinical characteristics). HLA haplotypes are reported in Online Supplementary Table S2. All patients received a diagnosis of severe AA (SAA) according to the International Study of Aplastic Anemia and Agranulocytosis32 and to the criteria of Camitta.33 At the time of blood sampling, none of the patients had received therapy. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation (MP Biomedicals, LLC, Santa Ana, CA, USA) according to the manu- facturer’s instructions.
Flow cytometry
A minimum of 4x106 PBMCs from each subject were stained for TCR Vβ repertoire analysis (Online Supplementary Figure S1). The manufacturer’s instructions for the IOTest Beta Mark (Beckman Coulter, Miami, FL, USA) were optimized to avoid incorrect com- pensation due to the existence of FITC/PE double positive anti- bodies in the kit, as described in Online Supplementary Methods. Expansion of CD8+CD57+ cells was defined using as a threshold the mean frequency calculated in healthy subjects. Vβ skewing in SAA patients was described when the frequency of one Vβ family was higher than the mean+3Standard Deviation (SD) in healthy subjects.16 The term “immunodominant clone” was defined as the Vβ+ population by flow cytometry or the DNA sequence by deep sequencing present in the highest abundance.17
TCR repertoire deep sequencing
For VDJ combination and CDR3 sequence profiling,34 DNA was isolated from FACS- or beads- (Miltenyi Biotec Inc., San Diego, CA, USA) sorted CD4+ and CD8+ T cells from 12 SAA patients with CD8+CD57+ cell expansion and 9 healthy subjects (mean: 3.2 μg of DNA; range: 0.2-27.4 μg) (Online Supplementary
Table S2). DNA was also isolated from beads-sorted CD8+CD57+ cells from 2 of the 12 SAA patients with enough cells for further analysis (mean: 1.6 μg of DNA). TCR repertoire sequencing was performed with an Illumina HiSeq 2000 sequencer (Illumina Inc., San Diego, CA, USA). Detailed information is provided in the Online Supplementary Methods. Data have been deposited in the NCBI GEO database (accession n. GSE101660).
Statistical analysis
Data were analyzed using R (RStudio, Boston, MA, USA) and Prism (v.7.02; GraphPad software Inc., La Jolla, CA, USA). Mann- Whitney U test, Wilcoxon signed rank sum test, pair and un- paired t-tests, or χ2 test were used for data with abnormal distribu- tions. Bonferroni and Dunn’s corrections were used for multiple comparisons. P≤0.05 was considered statistically significant, after adjustment with Bonferroni and false discovery rate (FDR).35 Linear regression was performed for correlations. Log-rank (Mantel-Cox) test was used for progression-free survival data analysis. Simpson's diversity index was calculated according to the following formula:
in which ni represents the clone size as the number of copies of each clonotype (i) or the total number of CDR3 sequences belong- ing to each i clonotype, and n is the total number of different clonotypes in the sample or the total number of sequences for each sample.
Results
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Effector memory CD8+CD57+ T cells frequently show oligoclonal expansion of TCR Vβ repertoire by flow cytometry
Immunophenotyping and flow-cytometry Vβ usage were performed in 24 SAA patients. Clinical characteris- tics are reported in Online Supplementary Table S1. A group of 34 healthy subjects was studied in order to define nor- mal ranges of T-cell populations and Vβ family expression. SAA patients showed higher frequencies of CD8+CD57+ cells (25.6±17.3% vs. 13.3±12.6% in healthy individuals; P=0.003), and decreased frequency of CD8+CD28+ cells (56.8±25.7% vs. 68.8±19.1%; P=0.046). A negative corre- lation between CD57 and CD28 expression was also pres- ent (r2=0.601, P<0.0001). No differences were found for CD28+ and CD57+ cells within the CD4+ subset (P=0.974 and P=0.250, respectively) (Figure 1A).
By Vβ usage, polyclonal expansion was observed in total CD4+, CD4+CD28+, CD4+CD57+ and CD8+CD28+ cells in both healthy subjects and SAA patients (Figure 1B and Online Supplementary Figure S2). Oligoclonal expan- sion of CD8+CD57+ cells was present in 92% of SAA patients with 1-3 immunodominant clones, while in total CD8+ cells oligoclonality was reported only in 33% of cases (Online Supplementary Figure S3). Patients did not show expansion of a shared Vβ family, as each subject car- ried a different TCR Vβ rearrangement in effector memo- ry CD8+ T cells (Figure 2A). None of the 7 patients with- out CD8+CD57+ cell expansion showed Vβ skewing in any subgroup, with mean frequency of the immunodominant clone of 3.8% (range: 0.21-6.01%). Conversely, all 17 patients with effector memory CD8+ cell expansion
haematologica | 2018; 103(5)