T Cell Repertoire Diversity in Lymphedema: Investigating the Antigens Driving the T Cell Response

Abstract

BACKGROUND: Lymphedema (LE) is a progressive condition that is estimated to affect as many as 1 in 1000 Americans. The disease is a major cause of decreased quality of life and is characterized by swelling, fibrosis, and recurrent infections. Activation of T-helper (Th) inflammatory responses plays a key role in lymphedema development and has been correlated with disease severity. This activation requires interaction with antigen-presenting cells (APCs), suggesting that the T-cell responses occur in response to antigens that are present in tissues after lymphatic injury. Clonal analysis of these T cell responses is significant due to its potential to uncover a common clonal architecture between patients with the disease. Identification of antigen-responsive T cell clones that are selectively expanded has important implications for developing therapies aimed at a common antigen driving the disease process. METHODS: High throughput sequencing (HTS) of clinical lymphedema and normal tissue DNA was utilized to identify unique T cell clones in lymphedematous tissue. Briefly, genomic DNA (gDNA) was isolated from lymphedematous, and control matched upper limb biopsies of six patients. Deep sequencing of the TCR repertoire was conducted by using a two-step biased controlled multiplex PCR to amplify genes of abundant T cell populations. These clones were quantified and characterized by CDR3 sequence length and TCRB Variable (V) gene usage. Predictive analysis of T cell antigen specificity was performed using the Basic Local Alignment Search Tool (BLAST) of the TCR epitope sequences and the activation of antigen-responsive clones was assessed by Flow cytometry. RESULTS: Our analysis demonstrated that the T cell responses in lymphedema are oligoclonal. TCR sequencing of the top five abundant clones in lymphedematous and normal tissue biopsies revealed six conserved amino acid sequences at the CDR3 region unique to the lymphedema group, however, no overlap of T cell repertoires was seen between patients with the disease. BLAST analysis of the corresponding TCRB epitope sequence revealed a list of probable antigens corresponding to each unique clonotype. Of the epitope sequences, unique to LE, staphylococcal enterotoxin represented the highest affinity bacterial antigen, pro-insulin corresponded to the representative self-antigen, and gluten peptide corresponded to plant antigen. CONCLUSION: Our results show that the T cell response in lymphedema is oligoclonal in nature, similar to what is seen in other inflammatory skin diseases such as atopic dermatitis and cutaneous psoriasis. In addition, clonality infers common antigen specificity of a population of T cells. Here we present representative antigens using BLAST analysis of the unique TCRB sequences of clones detected in lymphedematous tissue. Identification of these T cell populations that are selectively expanded in LE may provide a targeted approach for identifying antigenic stimuli or similarly eliminating pathogenic T cell clones as a means of treating the disease.