24?hours later, BMDCs were harvested, washed, blocked with a non-fluorescent FcRII/III antibody (clone 2

24?hours later, BMDCs were harvested, washed, blocked with a non-fluorescent FcRII/III antibody (clone 2.4G2, BioLegend) and stained for cell surface expression of CD54, CD80, CD86 and MHCII I-Ab (clones YN1/1.7.4, 16-10A1, GL-1 and AF6-120.1 respectively, BioLegend). BMDCs have an enhanced capability to induce T cell proliferation. This was associated with an increased capability of BMDCs to present immune complex derived antigens and to form ova IC dependent DC-T cell conjugates. These findings highlight PTPN22 as a regulator of FcR mediated responses and provide a link between the association of PTPN22R620W with autoantibody associated autoimmune diseases. Introduction The C1858T single nucleotide polymorphism in the human protein tyrosine phosphatase non-receptor type 22 (mice were subsequently reported to display enhanced TCR signalling that results in expansion of CD4+ effector T cells10. PTPN22 also regulates signalling downstream of additional receptors in various cell subsets, including the B cell receptor11, the L2 integrin LFA-112, Toll-like receptors (TLRs)13 and dectin-114. Furthermore, PTPN22 functions to alter Src and Syk family kinase impartial signalling events by regulating TRAF ubiquitination15. The R620W mutation is located in the P1 domain name of PTPN22, which causes diminshed binding to the inhibitory tyrosine kinase Csk16,17. How the expression of PTPN22R620W affects the functions of different immune cells is not straight forward. Both gain- and loss-of-phosphatase function effects have been observed when investigating different signalling pathways in different cell types11,17C20. Autoantibodies have long been implicated in the aetiology of autoimmune diseases including RA, type 1 diabetes, Graves disease and SLE; diseases for which is also a susceptibility risk allele21. Autoantibodies bind to self-antigens forming immune complexes which are recognised by Fc receptors (FcRs), thus inducing FcR mediated antigen uptake and cell activation. FcRs are expressed on the surface of most innate immune cells and are members of the immunoglobulin superfamily of receptors. FcRs recognise the Fc region of immunoglobulins, with FcRs specifically recognising the Fc Myelin Basic Protein (87-99) regions of Myelin Basic Protein (87-99) IgGs. Mice express four cell surface FcRs: FcRI, IIb, III and IV. FcRI, III and IV are activatory receptors, whereas FcRIIb is usually inhibitory22. Most innate immune cells express both activatory and inhibitory FcRs, allowing for the modulation of downstream signalling. Activatory FcR crosslinking induces Src family kinase activation, which in turn phosphorylates two tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM), located in the associated common chain. Syk is usually then recruited via its tandem SH2 domains to the phosphorylated tyrosines. This initiates downstream signalling involving ERK, p38 and JNK, activating a range of cellular processes including DC maturation and cytokine production23. For the inhibitory receptor FcRIIb, phosphatases such as SH2-domain-containing protein tyrosine phosphatase 1 (SHP1) and SH2-domain-containing inositol polyphosphate 5 phosphatase (SHIP1) are recruited to the immunoreceptor tyrosine-based inhibition motif (ITIM), located in the cytoplasmic tail of the receptor. Co-ligation of an activatory FcR with an inhibitory FcR reduces activatory signalling by dephosphorylation of signalling intermediates. Therefore, the cellular response to FcR signalling is dependent on the balance between the positive and negative signals. The necessity for appropriate regulation of FcR signalling is usually demonstrated by the presence of polymorphisms in human genes which are linked to autoimmune diseases such as SLE, RA and multiple sclerosis24. Furthermore, mice lacking expression of the activatory FcRs are resistant to a variety of autoimmune disease models such as collagen-induced arthritis25, but are susceptible to infections including and autoantibody associated autoimmune diseases, and the regulation of FcRs by Src and Syk family kinases, we set out to investigate Myelin Basic Protein (87-99) if PTPN22 regulates Myelin Basic Protein (87-99) FcR dependent immune complex uptake and activation in DCs and whether this can alter T cell effector responses. Results Immune complex pulsed BMDCs cause enhanced T cell proliferation In view of its known substrates, we hypothesised that PTPN22 should negatively regulate FcR dependent immune responses. To determine whether PTPN22 modulates the capability of DCs to present immune complex derived peptides and in turn activate T cells, we carried out co-culture assays. Wild type (WT) and BMDCs were pulsed with ovalbumin (ova) and ova immune complexes (ICs) and co-cultured with ova specific WT CD4+ OT-II T FLJ20285 cells. After 6 days, T cell proliferation was assessed by CellTrace Violet (CTV) dilution. We have previously shown that ova and ova323C339 peptide.