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TCR repertoire of effector memory T cells in AA
cell pool of AA3 and AA4 showed the same “skyscraper” landscape, but more enriched (Figure 5A), as well as CDR3 size and DJ length profiles that overlapped those in total CD8+ cells, although these were again more enriched (Figure 5B).
TCR repertoire diversity and shared CDR3 sequences in SAA patients and healthy subjects
Simpson’s index of diversity was calculated for each healthy and each SAA subject. This index is used in ecol- ogy for in-depth assessment of the degree of diversity of a system, related to the richness (or number of species pres- ent) and evenness (or relative abundance of each).38 Thus, this index assesses the probability that 2 randomly select- ed individuals from a system belong to the same species. For a TCR repertoire, the index measures the probability that 2 CDR3 sequences randomly selected from CD4+ or CD8+ pools of one subject could be identical.17,38 A value close to 0 means infinite diversity, and a value close to 1 no diversity.36 In our cohort, Simpson’s indexes were sim- ilar in healthy controls and SAA patients for CD4+ (0.990±0.005 vs. 0.991±0.005, respectively; P=0.537) and CD8+ cells (0.983±0.013 vs. 0.927±0.084, respectively; P=0.060). By paired t-test, Simpson’s indexes in SAA sub- jects were significantly different between CD4+ and CD8+ cells (P=0.047) (Online Supplementary Figure S9A). When compared to those indices in CD8+CD57+ cells, decreased diversity in the CD8+ effector memory compartment was described (total CD4+ cells vs. CD8+CD57+ cells, P<0.0001; total CD8+ cells vs. CD8+CD57+ cells, P=0.0003) (Online Supplementary Figure S9B).
In order to test the hypothesis that an autoreactive clone is triggered by autoantigens, CDR3 amino acid sequences from healthy subjects and patients were screened for homology and then compared to public and private TCR repertoires reported in literature,39 as described in the Online Supplementary Methods. From analysis of sequences in the CD8+ cell pool, a total of 29 CDR3 sequences were shared between patients and controls, but these sequences were highly expressed in SAA patients and also present in their immunodominant clones (Figure 6A). When we searched for these common sequences in the whole CDR3 sequence repertoire from CD8+CD57+ cells in AA subjects, CD8+CD57+ cell pools from AA3 and AA4 showed enrich- ment in frequencies of the immunodominant clones (from 53.61% to 79.12% for AA3 patient and from 23.45% to 38.14% for AA4 patient). Structural analysis of shared and immunodominant sequences did not show a pattern of common charged residue among patients (Figure 6B and Online Supplementary Methods). Immunodominant and shared CDR3 sequences were compared with CDR3 β repertoires reported in literature for infectious, autoim- mune and malignant diseases.39 For confirmation, the VDJdb database, a database of known antigen specific sequences (https://vdjdb.cdr3.net), was also used. No match- es were found for infectious diseases, while 2 sequences present in AA9 were reported previously in AA and parox- ysmal nocturnal hemoglobinuria (PNH).8,40 Lastly, we sought to perform homology assessment for reported PNH-related clonotypes on the entire TCR repertoire without frequency restriction, as small PNH clones could be present in healthy individuals.40 Two of the reported 12 CDR3 sequences (CATSRTGGETQYF and CATSRV- VAGETQYF) were found in our TCR repertoires with a
mean frequency of 0.1±0.2% and 4.7±9.4% in SAA patients, and 0.0003±0.0001% and 0.01±0.002% in healthy subjects. No matches were found in the enriched CD8+CD57+ T-cell pool (Online Supplementary Table S3).
Discussion
The character of oligoclonal expansion of CD8+CD28– lymphocytes in AA, described by Risitano et al.,8,41 strong- ly suggests an antigen driven mechanism of T-cell activa- tion, ultimately leading to destruction of hematopoietic stem and progenitor cells. In this study, we focused on a subset of CD8+CD28–CD57+ T lymphocytes, termed effec- tor memory cells because of their high antigen-affinity and their ability to undergo activation after antigen stimu- lation.27,36 Others have reported the expansion of effector memory CD8+ T cells in the tumor microenvironment and in PB from patients with solid tumors, hematologic malig- nancies, chronic infections and autoimmune disorders.36,41- 44 In cancers, effector memory T cells appear to have an important role in immune surveillance; for example, their increase after interferon (IFN)a treatment correlates to better prognosis in melanoma patients,43 and expanded CD8+CD57+ T cells reach normal levels after removal of head and neck cancer.36 Higher frequencies of CD8+CD57+ cells have also been described in SAA and MDS patients, which decrease in responders after anti-thymocyte globu- lin treatment.44 The expansion of effector memory cells could also occur in older healthy subjects as a result of life- long exposure to common pathogens, but it is related to reduced overall immune responsiveness to novel antigens.45,46 Consistent with previous studies, the SAA patients in our cohort had higher frequencies of CD8+CD57+ cells at diagnosis (25.6% in SAA patients vs. 13.3% in healthy subjects). Moreover, patients with CD8+ effector memory T-cell expansion at diagnosis experi- enced shorter PFS. No age-effects were seen for clone size in either patients or healthy subjects.
There is indirect evidence of immune pathophysiology of AA, including oligoclonal expansion of effector CD8+ T lymphocytes.8,41 Clonality of T-cell subsets can be studied by flow cytometry analysis of Vβ usage, CDR3 size spec- tratyping or deep sequencing of VDJ combinations, and CDR3 nucleotide and amino acid sequences.17-20,47 In SAA patients, expansion of at least one Vβ family in both CD4+ and CD8+ effector CD28dim cells by flow cytometry with polyclonal features in CD4+ and oligoclonal characteristics in CD8+ cells has been described.1,8,15,41,44 The CDR3 size skewing by spectratyping in the CD8+ population but not in CD4+ cells confirmed clonality in CD8+ cells.8,21,41 In our current work, we demonstrate by flow cytometry and deep sequencing that oligoclonal expansion occurs mainly in effector memory CD8+ cell compartment, as the immunodominant clones were highly enriched in CD8+CD57+ cells and had decreased diversity on deep sequencing. Effector memory T cells are a circulating T- cell population that can migrate to the BM under different types of stimulation.30 Vβ usage of peripheral CD8+CD57+ T cells may mirror that in the BM, as suggested by the high concordance between PB and BM in our AA patients. In our cohort, 75% of SAA patients with effector memory cell expansion also showed oligoclonal features by deep sequencing of the total CD8+ cell compartment: TRBV/TRBJ rearrangement plots with a “skyscraper” pro-
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