Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes

Abstract

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

Graphical abstract

Figure 1.

TRB gene repertoire diversity in patients with T-LGL lymphoproliferations. (A) Clonotype frequencies of patients with T-LGL lymphoproliferations and % of immunodominant clones. (B) Clonotype frequencies of healthy controls and % of top 10 clonotypes (B). Shannon diversity scores of patients with T-LGL lymphoproliferations and healthy controls. (C) Graphs indicate the mean with SD. Statistical significance was tested using the Mann Whitney U test. Level of significance indicated in the plots: ***P < 0.001; ****P < 0.0001. T-LGL = T cell large granular lymphocyte; TRB = T-cell receptor beta.

Figure 2.

Context-dependent TRB gene repertoire of patients with T-LGL lymphoproliferations. (A) Shannon diversity index of subgroups of patients with T-LGL lymphoproliferations (red dots indicate STAT3-mutated cases). (B) Clonotype frequencies of neutropenic patients with T-LGL lymphoproliferations, patients with T-LGL proliferations harboring STAT3 mutations and patients with T-LGL lymphoproliferations with associated malignancies. Comparison of % immunodominant clonotypes and top 2–5 clonotypes of different T-LGL subgroups. (C) Graphs indicate the mean with SD. Statistical significance was tested using the Mann Whitney U test. Level of significance indicated in the plots: *P < 0.05; **P < 0.001. **Patients with STAT3 mutations are depicted in red (STAT3 mutated patients without clinical associations are indicated with an X), neutropenic patients are depicted in grey, patients with associated malignancies are depicted in gold, and patients with autoimmune phenomena (especially RA) are depicted in blue. T-LGL = T cell large granular lymphocyte; TRB = T-cell receptor beta.

Figure 3.

Patients with T-LGL lymphoproliferations display a context-dependent (sub)clonal TRB gene repertoire architecture that may shift over time. A. Longitudinal analysis of the subclonal TRB gene architecture of a patient showing a STAT3-mutated T-LGL leukemia. (B) Clonal dynamics of the TRB gene repertoire of a neutropenic patient with T-LGL leukemia under therapy. (C) Complex interplay between the TRB gene repertoire and M-protein levels in a case with T-LGL lymphoproliferation and an associated plasma cell malignancy. (D) Dynamics of the TRB gene repertoire in a patient developing a T-LGL lymphoproliferation post allo-HSCT. Allo-HSCT = allogeneic hematopoietic stem cell transplantation; T-LGL = T cell large granular lymphocyte; TRB = T-cell receptor beta..

Figure 4.

Connectivity networks of the dominant clonotype of patients with T-LGL lymphoproliferations. (A and B) Graphs depicting the connectivity networks formed between the dominant clonotype and other clonotypes of the same length of 2 representative cases with T-LGL lymphoproliferations taking into account the highly similar clonotypes present in each sample. The sequence logo represents the total repertoire of highly similar clonotypes identified. The sequence logo of the CDR3 region of one representative case and the graph depicting low-level connectivity of the dominant clonotype. (C and D) The sequence logo of the CDR3 region of one representative case and the graph depicting high-level connectivity of the dominant clonotype. The large circle depicts the dominant clonotype of the sample and only the CDR3s with one difference are connected. (E) Overtime kinetics and clonal dynamics of highly similar clonotypes in a representative case. T-LGL = T cell large granular lymphocyte.

Figure 5.

Clusters of cases with restricted TRB CDR3. Two representative clusters are depicted including a sequence logo of the CDR3 regions that clustered together, the TRB gene usage and the presumed antigen specificity when a clonotype of those clustered was also found in the VDJdb. One representative cluster including 23 clonotypes with relative frequency ranging between 0.1% and 5.6% characterized by homogeneity regarding the TRBV gene usage. The clustered clonotypes derived from 18 different cases (T-LGL, n = 6; EN, n = 1; CIN, n = 3; CLL, n = 2; MBL, n = 4; healthy, n = 2). The clustered clonotypes were identified in the VDJdb and different antigenic specificities can be speculated based on the literature (A). One representative cluster including 20 clonotypes (reactive frequency, 0.1%–3.7%) with heterogeneity regarding the TRBV genes used. The clustered clonotypes derived from 17 different cases (T-LGL, n = 7; EN, n = 2; CIN, n = 2; CLL, n = 2; MBL, n = 2; healthy, n = 2). No hits with the VDJdb were found. In brackets the number of clonotypes using the particular gene is indicated (B). CIN = chronic idiopathic neutropenia; CLL = chronic lymphocytic leukemia; EN = benign ethnic neutropenia; MBL = monoclonal B lymphocytosis; T-LGL = T cell large granular lymphocyte; TRB = T-cell receptor beta.