(Sub)clonal architecture and temporal dynamics of the TCR repertoire in patients with T-LGL leukemia
T cell large granular lymphocyte (T-LGL) leukemia is characterized by increased numbers of mono/oligoclonal cytotoxic T cells in peripheral blood. Even though chronic antigenic stimulation is known to drive LGL proliferation, to date no specific antigens that drive T cell receptor (TR)αβ CD8+ T-LGL proliferation have been established. In fact, as T-LGL proliferations arise in various disease contexts, such as auto-immune disease, hematological malignancies, solid tumors, but also in hematopoietic stem cell transplants, these might arguably be triggered by various antigens. Previously, the TR gene repertoire was determined mainly through low-resolution Sanger sequencing, precluding firm conclusions from being drawn regarding the precise impact of antigen drive. Here, we applied next generation sequencing (NGS) to characterize in high resolution the (sub)clonal architecture and temporal dynamics of patients withT-LGL proliferations with the aim to characterize in-depth the TR beta (TRB) gene repertoire in different contexts (neutropenia-associated, STAT3-mutated, malignancy-associated).
Our cohort included 27 individuals with T-LGL proliferations and 22 age-matched healthy controls. 6 cases with T-LGL proliferations were selected for longitudinal analysis. DNA of PBMCs or CD8+ T cells was amplified using the TRB BIOMED-2 protocol. After purification paired-end libraries were prepared with the NEBNEXT Ultra II library preparation kit. NGS reads were processed through a purpose-built bioinformatics pipeline and productive TRBV-TRBD-TRBJ rearrangements were further evaluated.
The TRB gene repertoire diversity score of T-LGL proliferations as a whole was lower compared with healthy controls, indicating repertoire skewing. Cross-entity analysis of T-LGL patients with neutropenia and/or STAT3 mutation and/or additional malignancies revealed no differences in TRB diversity scores. However, TRB monoclonality was noted in the majority of the neutropenic and STAT3-mutated patients, whereas patients with co-existing malignancies predominantly showed an oligoclonal TRB gene repertoire. Longitudinal analysis revealed that the TRB gene repertoire in patients with T-LGL proliferations is profoundly dynamic and context-driven. Subclonal drift was observed, with the oligoclonal background contracting and expanding over different time points whilst the monoclonal population remained stable. In the group with co-existing clonal B lymphoproliferations (MGUS) analysis of the TRB gene repertoire and clinical data suggested a complex interplay between T-LGL proliferations and the clonal B cells. Additionally, by analyzing a patient developing T-LGL proliferation after SCT, we unraveled the temporal TRB repertoire dynamics and distinguished between early EBV-reactive clonotypes and unrelated T-LGL clonotypes occurring years after SCT.
In conclusion, we demonstrate that the TRB gene repertoire of T-LGL proliferations is highly complex displaying context-dependent architectures. Moreover, based on longitudinal analyses we provide compelling evidence for pronounced context-driven, temporal dynamics of clonal T-LGL proliferations.