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AB0135 Weak tcr signaling due to repeated immunization with antigen is the key for tcr revision to generate aicd4 t cell: implication to self-organized criticality theory
  1. S. Shiozawa1,
  2. K. Uto2,
  3. K. Tsumiyama1
  1. 1Medicine, KYUSHU UNIVERSITY BEPPU HOSPITAL, Beppu
  2. 2Laboratory Medicine, Kobe University Hospital, Kobe, Japan

Abstract

Background Most important question in elucidating the pathogenesis of autoimmunity is how autoreactive lymphocyte clones survive or emerge beyond the firewall called ‘forbidden clone’. According to the autoimmune disease theory, peripheral autoreactive lymphocytes can derive from either a few autoreactive clones that have slipped thru negative selection of thymus or the clones reactivated from tolerance. Such restricted clones apparently cannot acquire wide varieties of TCR repertoire or autoantibody of more than 100 distinct specificities as found in SLE. We instead found that autoreactive clones newly emerged via de novo TCRa but not TCRb revision at periphery in the spleen from thymus-passed non-autoreactive clones. We name this novel T cell type an autoantibody-inducing CD4 T cell (aiCD4 T cell). This aiCD4 T cell stimulates B cells to generate varieties of autoantibody and also helps final differentiation of CD8 T cell into cytotoxic T lymphocyte (CTL) via antigen cross-presentation under the help of aiCD4 T cell to induce lupus tissue injuries.

Objectives We investigated the molecular mechanism how TCR revision took place at periphery in spleen and how aiCD4 T cell was generated.

Methods After repeated immunization 8x with staphylococcus enterotoxin B (SEB), we titrated intracellular signaling molecules by using western blot and their phosphorylation states were examined by using respective antibodies.

Results The histone of all TCR promoter V and J region was not acetylated nor tri-methylated (H3K4me3), indicating that recombinases can access to this region and thus, TCR revision seemed to depend primarily on whether or not RAG1/2 recombinase complexes were activated. We then investigated the condition how RAG1/2 was activated: we found that the amount and phosphorylation level of CD3z, ZAP70, LAT, SLP-76, PLCg1, and NFAT1/2, but not ERK, were decreased in splenic Vb8+CD4 T cells upon repeated immunization 8x with SBE. While CD3z and ZAP70 were moderately recovered, the finding shows that all upper and lower signaling pathway of TCR was down-modulated, and accordingly RAG1/2 was reactivated upon repeated immunization with antigen at the peripheral lymphoid organ in spleen. The finding is compatible with the current view that expression of RAG1/2 is under the control of T cell activation signal, we next tested if increased cell signaling down-modulates vice versa the re-activation of RAG1/2 by overly stimulating TCR with PMA+ionomycin. The result showed that it was indeed the case, and the re-appearance of RAG1/2 after repeated stimulation was cancelled upon over-stimulation with PMA+ionomycin that bypass TCR signaling.

Conclusions Since the histone of TCR promoter region is acetylated and H3K4-methylated and thus open, as expected, the TCR revision taking place upon repeated immunization with antigen depends primarily on the re-expression of RAG1/2 and in particular on weak signaling thru TCR because of heavily repeated immunization is the key for the TCR revision and subsequent generation of aiCD4 T cell, which ultimately leads to SLE.

Disclosure of Interest None Declared

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