The Role of ARID1a in T Cell Differentiation & Lymphomagenesis

Introduction: Peripheral T cell lymphomas (TCL) are aggressive neoplasms that exhibit clinical & genetic heterogeneity. The mechanisms underlying the pathogenesis of these disorders are not completely understood with limited options for precision therapies. We & others have demonstrated high frequency (40.3%) genetic alterations including loss of function mutations & deletions targeting ARID1a in patients with Sézary syndrome & several mature T cell lymphomas. ARID1a protein is a subunit of the SWI/SNF chromatin remodeling complex, however its role in T cell function, differentiation & lymphomagenesis is not well understood.

Hypothesis: We hypothesized that loss of function mutations in ARID1a modulates T cell function, differentiation & contributes to lymphomagenesis. Our studies aim to investigate the function of ARID1a in T cell differentiation & lymphomagenesis using an in vivo conditional mouse model.

Methods: To investigate the invivo function of ARID1a, we established a conditional murine model of Arid1a deficiency in CD4+ T cells using a cre-loxp recombination strategy to create heterozygous (CD4creArid1a^fl/wt) & homozygous (CD4cre Arid1a^fl/fl) mice. T-cell specific loss of Arid1a was validated by RT-PCR & immunoblotting of Arid1A in purified CD4 +T cells. Thymus, spleen, & lymph nodes were analyzed grossly, histologically & immunophenotypically at steady state & after sheep red blood cell stimulation. Functional characterization of T cell subsets (CD4, CD8, γ/δ, memory CD4, naïve CD4, Tregs & activated CD4 T cells), B cells & myeloid cells was performed using multiparametric flow cytometry. Lymphomagenesis was evaluated by monitoring CD4creArid1a^fl/wt & CD4cre Arid1a^fl/fl mice along with the littermate controls over an 18-month period & analyzed by histologic & immunohistochemical studies.

Results: Gross examination of adult CD4creArid1a^fl/fl mice demonstrated splenomegaly (85% increase in the spleen weight) at the steady state. Histopathological analysis demonstrated abnormal shape of lymphoid follicles in spleen, disrupted lymph node architecture (abnormal follicles) & disrupted thymic architecture (condensed medulla & expand thymic cortex) in all the mice studied at steady state (n=6). Flow cytometric analysis demonstrated significant decrease in number (2-fold; p<0.05) & proportion (120% decrease; p<0.01) of both CD4+ & CD8+ T cells in the spleen, lymph nodes & thymus in CD4cre Arid1a^fl/fl mice compared to the littermate controls, while an increase in the frequency (2-fold, p<0.01 in CD4cre Arid1a^fl/wt &12-fold, p<0.01 in CD4cre Arid1a^fl/fl) & number of CD4-CD8- double negative T cells, identified as γ/δ T cells, was noted.A 300% increase in both the number & frequency of γ/δ T cells was seen in the CD4cre Arid1a^fl/fl mice as compared to the CD4cre Arid1a^wt/wt. In contrast, a 70% decrease in CD4+ T cells expressing T cell receptor β was observed in the thymus (p<0.05). Analysis of T cell subsets demonstrates a 3.5-fold increase in "Memory phenotype” CD4+ T cells & a subsequent decrease in "naïve phenotype” CD4+ T cells in CD4cre Arid1a^fl/flmice as compared to CD4cre Arid1a^wt/wt (P<0.01). Importantly, in comparison to the littermate control, CD4+ T cells of Arid1a^fl/fl mice displayed an increase (100-200%) in activation as represented by CD25, CD69 & CD44 expression (P<0.01). Additionally, a dose dependent expansion of peripheral Tregs (38% increase in the CD4cre Arid1a^fl/wt & 150% increase in the CD4cre Arid1a^fl/fl mice) was noted in the spleen & lymph nodes. Additionally, we observed development of mature T cell lymphoma (CD3+,CD4+) in CD4cre Arid1a^fl/wt involving spleen & lung.

Conclusions: Our novel conditional mouse model of Arid1a deficiency in CD4+ T cells demonstrates that Arid1a loss results in expansion of double negative γ/δ T cells in the spleen, lymph nodes & thymus. We observed an increase in memory CD4+ T cells with a decrease in naïve CD4+ T cells in the spleen & lymph nodes. CD4+ T cells demonstrated major perturbation of T cell function & differentiation in mice lacking Arid1a. Our study has identified the role of a chromatin remodeler, Arid1a in T cell function, differentiation & lymphomagenesis.

Lim:EUSA Pharma: Honoraria.