Differential Gene Expression Profile Identifies Defects and Abnormalities In Infiltrating T Cells In Patients with Follicular Lymphoma at Diagnosis

Abstract 141

Patients with follicular lymphoma (FL) have an extremely variable clinical course. Although clinical parameters can be used to define prognostic subgroups, there is a need to identify robust biomarkers that not only aid in prognosis, but also help define the underlying pathophysiology of the disease. Previous gene expression profiling studies demonstrated that among the most important prognostic markers were the molecular features of nonmalignant tumor-infiltrating immune cells present in the tumor at diagnosis (Sandeep et al. NEJM 2004). To investigate the molecular mechanisms whereby T cells are altered in the FL microenvironment we studied highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as from peripheral blood (PB) (n=10) of age matched healthy individuals. These tumor infiltrating T cells (TILs) have impaired proliferative and cytotoxic responses and impaired capacity to form immunologic synapses with antigen presenting cells. Co-culture of FL cells in contact with healthy allogeneic T cells induces similar T cell functional defects, demonstrating that it is the FL cells that drive the altered T cell function. To investigate the molecular mechanisms for this, we performed global gene expression profiling using Affymetrix U133Plus2 chips of highly purified (>95% purity) CD4 and CD8 cells. For both CD4 and CD8 cells, unsupervised analyses distinguished healthy and FL T cell subsets. Using a fold change cut off > 2 and false discovery rate of 5%, 280 genes were differentially expressed for CD4 and 717 genes for CD8, with109 genes overlapped for both subsets. The gene array data was validated for RNA using Real-Time Quantitative-PCR and for protein by flow cytometry and immunohistochemistry. Pathway analysis indicated disruption in multiple pathways including cytokine signaling, T cell differentiation, cell proliferation and, actin-based motility/cytoskeleton formation. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH and ETV1. Using Tissue Microarray (TMA) we demonstrate that the intensity of expression in TILs in FL was significantly higher for PMCH (p<0.0001) and ETV1 (p<0.0001) than that of reactive tissue. PMCH is not expressed in PB T cells, but its expression is highly induced when healthy peripheral blood T cells are cultured, either in cell contact or in transwell culture, with FL cells. Surprisingly, co-culture of healthy T cells with normal B cells also induced its expression. Taken together, these data indicate that TILs in patients with FL are abnormal in terms of their function and gene expression profile, in keeping with our hypothesis that FL induces changes in immune cells in the tumor microenvironment. We are currently further characterizing the functional consequences of the identified changes in the gene expression in tumor-infiltrating T cells of patients with FL in the biology and progression of disease. As non-malignant immune cells have a major role in the clinical course of patients with FL, understanding the nature and impact of the abnormalities induced in infiltrating T cell's in these patients is vital before any immunotherapeutic strategies can be implemented to attempt to alter the immune microenvironment in FL.