T Large Granular Lymphocyte (LGL) Leukemia Characterized by Expansion of Terminal CD3+/CD8+/CD62L−/CD45RA+ Effector Memory T Cells.

Abstract

Background: Clonal proliferation by mature Large Granular Lymphocytes is associated with LGL leukemia. This expansion of CD3− NK cells or CD3+ T cells may be the result of chronic antigen stimulation by autoantigens or viral antigens. In association with T cell lymphoproliferation, approximately 45% of patients with LGL leukemia have severe neutropenia (absolute neutrophil count <0.5×109/L) and 20% of patients have transfusion-dependent anemia. Homeostatic mechanisms normally modulate the generation of naïve and memory T cell pools and regulate the T cell repertoire; however, the pathways elicited during T memory differentiation, maintenance and expansion are not fully characterized. The goal of this work was to characterize the homeostatic mechanisms that regulate LGL leukemia.Methods: Peripheral blood mononuclear cells were isolated from patients with LGL leukemia and normal controls. We performed multiplex TCR-Vβ (CDR3) PCR on cells from 16 LGL patients to identify clonal T cell proliferation. The percentage of CD3+ T cells that expressed each of the TCR-Vβ families was determined in 20 healthy donors to establish the mean and standard deviation (S.D.) of the control population. Naïve and memory CD4 and CD8 T cell sub-populations were segregated by expression of CD45RA and CD62L expression by flow cytometry and T cell proliferation was assessed by Brdu incorporation in CD4+ and CD8+ T cells.Results: The absolute number of CD4+ T cells was reduced in LGL patients compared to normal donors and T cell clones were characterized by a CD8+ phenotype. By flow cytometry, expansion of a single Vβ clonal population occurred in 8 of 16 patients (50%), two clones were present in 4 of 16 patients (25%), and three clones in 4 of 16 patients (25%). The immunophenotype of TCR Vβ+ clonal T cells was CD8 positive, CD57 positive, CD28 negative, CD25 negative, and NKG2D (NKG2-family) positive and CD244 (2B4) positive. Three patients examined expressed Killer-Immunoglobulin-like (KIR) receptors. Further phenotype analysis showed that there were fewer than normal CD4+ naïve (CD4+/CD45RA+/CD62L+) T cells (23%±16 vs. 41%± 15, P=0.04 by a t test) in LGL patients. CD4+ T cells from patients had reduced proliferation in response to antigen stimulation. The reduction in CD4+ naïve T cells was associated with increased percentages of CD4+/CD45RA−/CD62L+ central memory T cells (P<0.05). Reduced percentage of naive CD8+ T cells in detected in LGL leukemia patients. In addition, CD4+ central memory cells were also significantly reduced in patients. CD8+ T cells were primarily characterized by a CD45RA+/CD62L− terminal effector memory phenotype that was significantly increased compared to normal donors (mean 75% ± 13 in patients vs 30% ± 13 in normal controls, P<0.0001). In the presence of a skewed repertoire and terminal effector memory cell accumulation, antigen-induced proliferation of CD8+ T cells in LGL did not differ from normal controls (13% ± 11 in patients vs. 9% ± 3 in normal controls, P=0.3).Conclusions: These results suggest that leukemic LGL represent the accumulation of CD8+ terminal effector memory cells with the capacity for increased proliferation. Our findings suggest that normal homeostatic signals are impaired in LGL leukemia that limits the terminal CD8 differentiation phase of an immune response.