The Role of CMV Antigenic Stimulation in the Pathogenesis of T Cell Large Granular Lymphocytic Leukemia (T-LGL)

Background and Hypothesis: T cell large granular lymphocytic leukemia (T-LGL) is characterized by the clonal expansion of CD8+ cytotoxic T cells. Most cases have a gain of function mutation in either the STAT3 or STAT5B transcription factor gene ,The transformation of single CD8+ T cells results in a distinct rearranged T-cell receptor gene (TCR) clonotype at the time T-LGL presents. The clonality and activating STAT mutations are virtually pathognomonic for the disease. One open question in the field is whether the clonal TCR plays a role in the pathogenesis of T-LGL or the disease process. For example, patients often present with autoimmune manifestations such as rheumatoid arthritis and Sjögren syndrome. Additionally, prominent cytopenias such as anemia and neutropenia may also be autoimmune effects of T-LGL bone marrow infiltration. Here, we present the case of a 67-year-old male with T-LGL and severe neutropenia, hospitalized for two months with recurrent febrile episodes and a perianal abscess. We hypothesize that the unique TCR of the transformed CD8+ T-LGL plays a role in the pathogenesis of disease.

Methods: We present patient blood counts and bone marrow aspirate and biopsy. Patient's PBMCs were single-cell sorted for CD3+ T cells and TCR-clonotypes were identified and analyzed by targeted single-cell RNA next generationsequencing. TCR CDR3 sequences were searched in the VDJdb database to find candidate corresponding antigenic T-cell epitopes. Epitope candidates were analyzed with the TCR Model web server to predict epitope binding to the patient's MHC and TCR. In order to identify the patient's precise pp65 epitope variant patient PBMC gDNA was amplified by nested PCR withspecific primers. Tetramers corresponding to the patient's HLA were used to analyze the specificity of T-LGL cells. Cytotoxicity assays were performed with patient CD3+ T cells and pp65-pulsed T2 cells (ATCC CRL-1992).

Results: After cloning and sequencing the V-alpha and V-beta regions of the most abundant TCR clonotype of the patient's T-LGL, the VDJdb database predicted the TCR to be specific for CMV pp65 and IE1 epitopes and an autoimmune epitope, BST2. Structural modelling with the TCR model II server predicted epitope binding to the patient's MHC and TCR at high confidence levels. Nested PCR detected the CMV pp65 gene in the patient's PBMCs and CD3-sorted T-cells.

Conclusion and Potential Impact: Our data suggest a model for T-LGL pathogenesis where a CMV-reactive T cell immune response plays a role in T-LGL initiation. Reactivation of latent CMV in bone marrow could lead to catastrophic depletion of myeloid cells by cytotoxic T-LGL memory cells resulting in severe neutropenia. A similar paradigmmay be applied to other T-LGL cases, which may present with anemia or other autoimmune disorders, that may have been initiated by CMV or other chronic viral pathogens. Further, the identification of precise TCR sequences and their cognate antigens in T-LGL will guide development of targeted therapies and facilitate the development of clonotype-specific immunotherapeutic agents.

No relevant conflicts of interest to declare.