Proinflammatory and antigen-specific CD4+ T cells in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease primarily affecting peripheral joints. In this thesis work I aimed to increase the knowledge about proinflammatory and antigen-specific CD4+ T cell subsets involved in RA pathogenesis and I also investigated the effect of T cell directed therapy (abatacept) on CD4+ T cell subsets. Abatacept (CTLA4-Ig) is a biologic therapy that blocks T cell co-stimulation by interfering with the binding of CD28 to CD80/86. Treatment of abatacept leads to reduced TH1 and TH17 cytokine production in ACPA-positive RA patients and diminished frequencies of several Treg subsets. This was also confirmed by in vitro studies where abatacept was added in vitro to cell cultures. Approximately 60 % of RA patients have antibodies against citrullinated proteins (ACPA) and their presence is associated to HLA-DRB1*04, one of the strongest genetic risk factors associated with RA. It is believed that T cells recognizing citrullinated epitopes presented by HLA-DRB1*04 alleles may drive the development of ACPA and disease. We have used MHC class II tetramer technology in order to identify and enumerate autoantigen-specific T cells in blood and synovial fluid of RA patients. Several citrullinated epitopes of CILP, α-enolase, fibrinogen and vimentin were identified. The frequency of citrulline-specific T cells was higher in the blood of RA patients compared to HLA-matched healthy controls and the citrulline-specific T cells in RA patients were of a memory TH1 phenotype. Furthermore, we enumerated and characterized α-enolase native and citrulline-specific T cells in blood and synovial fluid of HLA-DRB1*04:01 RA patients. Higher frequencies of citrullinated α-enolase specific T cells were present in synovial fluid compared to blood and T cells recognizing the citrullinated variant of α-enolase were also more often of a memory phenotype (i.e. had encountered their cognate antigen in vivo), than those recognizing the native α-enolase epitope. Some T cells showed cross-reactivity between the two investigated epitopes and HLA-DRB1*04:01-IE transgenic mice were used to substantiate our findings. Another potentially contributing T cell subset to RA pathology is an expanded T cell subset that lacks the co-stimulatory molecule CD28, often referred to as CD4+CD28null T cells. These proinflammatory cells are present in approximately 1/3 of RA patients. Their frequency can be up to 50 % of all CD4+ T cells in blood, but they are infrequent in synovial fluid. CD28null T cells differ from conventional CD4+ T cells in several aspects, but we demonstrate here that even within the CD28null subset there are differences due to their localization. CD28null cells from synovial fluid expressed more CXCR3 and CCR6 than those form the circulation and CD28null cells from synovial fluid were able to produce IL-17 even though they displayed a hypomethylated IFNG promoter. During my thesis studies, several novel HLA-DRB1*04:01 restricted citrullinated T cell epitopes have been identified. The auto-reactive T cells did not overlap with the CD28null phenotype. We have demonstrated the proof of principle that auto-reactive T cells can be identified by MHC class II tetramer technology in an assay not dependent on in vitro stimulation. LIST OF PUBLICATIONS I. CTLA4-Ig (abatacept) therapy modulates T cell effector functions in autoantibody-positive rheumatoid arthritis patients Jennifer Pieper, Jessica Herrath, Sukanya Raghavan, Khalid Muhammad, Ronald van Vollenhoven and Vivianne Malmström BMC Immunology, 2013 Aug 5;14:34 II. Citrulline specific CD4+ T cells exhibit a TH1 memory phenotype in rheumatoid arthritis subjects and their ex vivo frequency is influenced by both disease duration and biologic therapy Eddie James*, Mary Rieck*, Jennifer Pieper, John A. Gebe, Betty B. Yue, Megan Tatum, Melissa Peda, Charlotta Sandin, Lars Klareskog, Vivianne Malmström, Jane H.