Identification of putative antigens in Systemic Sclerosis utilizing in vivo clonally expanded T cells

Abstract

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. Immune system dysregulation, excessive deposition of collagen and microvascular damage in the skin and multiple internal organs are the main pathologic characteristics of the disease. Little is known about the mechanisms that are responsible for the pathogenesis of SSc. However, evidence has been accumulated demonstrating that T cells play a key role in the initiation and propagation of the disease. Previous studies in our laboratory have identified the presence of high proportions of identical β–chains TCR transcripts, demonstrating the presence of clonal expansion of T cells in skin biopsies from patients with SSc of recent onset. These T cells have undergone proliferation and clonal expansion in response to as yet unidentified antigen(s). The hypothesis that has been tested in this study is whether clonally expanded T cells in skin biopsies of patients with SSc of recent onset recognize self or non–self (possibly viral) putative SSc antigens, including DNA topoisomerase I, cytomegalovirus (CMV) and parvovirus. With the objective to identify the antigens recognized by clonally expanded T cells in skin biopsies of patients with SSc, we examined the presence of α– and β–chain TCR transcripts. Amplification of α–chain TCR transcripts by the non–palindromic adaptor PCR (NPA–PCR)/Vα specific PCR followed by cloning and sequencing revealed the presence of several clonally expanded α–chain TCR transcripts in skin biopsies from four patients with SSc and peripheral blood from one of these patients. Additionally, several clonally expanded β–chain TCR transcripts were identified in skin biopsies from all three of these patients with SSc examined, after NPA–PCR/Vβ specific amplification followed by cloning and sequencing. To identify the antigens recognized by these in vivo clonally expanded α– and β–chain TCR clones, full length α– and β– chain TCR transcripts containing the identified CDR3 regions from the clonally expanded TCR clones from the patients SSc–21 and SSc–22 were constructed. Pairs of clonally expanded, full length α– and β–chain TCR transcripts and appropriate controls were expressed in mutant TCR negative cells of the Jurkat T cell line (J.RT3–T3.5) by using a retroviral gene transfer and expression system. Each clonally expanded α–chain TCR transcript was combined with each clonally expanded β–chain TCR transcript from the same patient, generating T cells lines containing all pairing combinations of the clonally expanded TCR transcripts for each SSc patient. A total of 52 T cell lines were generated, including 10 control T cell lines. The surface expression of the TCR complex on these T cell lines was verified by flow cytometric analysis using antibodies against the α/β TCR and CD3epsilon. We employed an intracellular calcium mobilization assay to examine whether the Jurkat T cell lines transduced with the clonally expanded TCR transcripts from skin biopsies from patients with SSc (SSc–21 and SSc–22) recognize putative SSc antigens or their peptides presented by autologous EBV–transformed B cell lines. The putative SSc antigens that were tested are the self–antigen, DNA topoisomerase I and the viral antigens, cytomegalovirus and parvovirus which have been previously suggested to be involved in the pathogenesis of SSc. Significant intracellular calcium mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines transduced with clonally expanded α– and β–chain TCR transcripts from patients SSc–21 and SSc–22.