Title : “ A role for PTP 1 B in dendritic cell maturation , migration and T cell activation ” Short title : PTP 1 B regulation of dendritic cell function

Dendritic cells (DCs) are the major antigen-presenting cells bridging innate and adaptive immune responses, a function they perform by converting a quiescent DC to an active, mature DC able to activate naïve T cells. Here, we demonstrate that myeloid-cell specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. Mature DCs migrate and home to secondary lymphoid tissues to activate T cells. We demonstrate that myeloid-cell PTP1B deletion causes decreased migratory capacity of epidermal DC and reduced mature BMDC expression of the lymph node homing chemokine receptor CCR7 and BMDC chemotaxis to CCL19. Further, immature LysM PTP1B BMDCs display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527 and loss of Src localisation to podosome puncta. In addition, we show decreased numbers of contacts and contact duration in BMDC:T cell co-cultures from LysM PTP1B mice. Finally, we show that LysM PTP1B BMDCs fail to present antigen to T cells as efficiently as control BMDCs. In summary, we provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation. Introduction DCs are the most potent professional antigen-presenting cells (APC) connecting the innate immune response with the initiation of adaptive immunity (Steinman et al., 2003; Steinman, 2012). DCs are generated primarily in the bone marrow whence they enter the blood stream and populate the peripheral tissues where their default function is to promote tolerance (Steinman et al., 2003; Alvarez et al., 2008). In their immature state, DCs reside in peripheral tissues and traffic to the secondary lymphoid organs (SLO) in a slow “trickle” to promote immunological homeostasis by regulating responses to self antigens. Tissue-resident DCs respond to microbial challenge particularly in barrier tissues such as the skin and intestine where they are specialized in uptake, processing, and presentation of foreign antigens to T cells. Antigen processing induces a program of DC maturation characterised by upregulation of the expression of chemokine receptors particularly CCR7, co-stimulatory molecules (CD80, CD86, CD40) and MHC class I and II molecules on their surface. This process facilitates the migration of DCs from the peripheral tissues via lymph and blood to SLO (Alvarez et al., 2008; Randolph et al., 2008), and the presentation of antigen to naïve T cells. The ability of DCs to migrate in and out of these various compartments is key to their functions. During migration, the DC navigates through the tissues to enter the afferent lymphatics. In this process it must release its relatively firm adhesive attachment to tissue cells (Van den Bossche et al., 2012) and invade the extracellular matrix. This is achieved by formation of protrusive/adhesive plasma membrane structures (podosomes) a general cellular phenomenon used by migrating immune cells to traverse dense tissues such as vascular basement membranes (Carman et al., 2007; Gawden-Bone et al., 2010) and is critical for immune cell function. For instance, lymphocytes use Src kinase-dependent podosomes and extended “invasive podosomes” to sense the surface of the endothelium and ultimately form transcellular pores (Carman et al., 2007). In vitro studies have shown that podosomes are small cylindrical structures containing a central branched-actin bundle core and an adhesive surrounding ring. They are initially formed as adhesive points of contact (“puncta”), partially comprised of trans-membrane αβ integrin cytoskeletal connections, involving “inside-out signalling” via Srccontaining intracellular protein complexes (Hoshino et al., 2013). PTP1B (protein tyrosine phosphatase 1B) is a non-receptor tyrosine phosphatase that has multiple substrates, which, in turn, are involved in a wide range of fundamental cellular processes (Mertins et al., 2008). PTP1B is an attractive drug target for cancer and diabetes due to its regulatory role in cell and metabolic signalling (Elchebly et al., 1999; Bence et al., 2006; Delibegovic et al., 2009; Grant et al., 2014). PTP1B deficient mice are protected against the effects of high-fat diet, ageinduced obesity and insulin resistance associated with chronic inflammation in white adipose tissue (Elchebly et al., 1999; Gonzalez-Rodriguez et al., 2012). Moreover, we recently demonstrated that myeloid-cell specific PTP1B knockout mice were protected against LPS-induced endotoxaemia and liver damage, which correlated with low levels of proinflammatory cytokine secretion in vivo (Grant et al., 2014). Furthermore, p38 mitogen-activated protein kinase is a substrate of PTP1B in B cell and loss of PTP1B in B cells is associated with autoimmunity in mice and humans (Medgyesi et al., 2014). Despite the increasing number of reports exploring the roles of PTP1B in immune cells, the role of PTP1B in dendritic cells is unknown. Here we demonstrate that genetic deletion of PTP1B in LysM PTP1B mice leads to decreased migratory capacity of epidermal DCs from split skin explants, as well as a marked reduction in the ability of mature BMDCs to respond to the lymph node homing chemokine CCL19 in vitro. We also show that LysM PTP1B immature BMDCs display decreased ability to develop podosomes while simultaneously forming increased numbers of focal contacts. Interestingly, these phenotypes are consistent with increased levels of phosphorylated Src at Tyr527, which renders Src inactive and leads to loss of Src localisation to podosome puncta. Furthermore, we show decreased numbers and duration of BMDC:T cell contacts in co-cultures from LysM PTP1B mice, compared with co-cultures of control mice. Notably, these defects caused by loss of PTP1B in BMDCs correlate with lowered expression of molecules which contribute to the immunological synapse. Finally, we demonstrate that LysM PTP1B DC exhibit reduced ability to present antigen to T cells. In summary, we provide first evidence for a key regulatory role of PTP1B in DC physiology specifically in the process of DC maturation, a process central to the initiation of adaptive immunity. Hence, our studies highlight the potential of DC-specific targeting of PTP1B for intervention in regulation of the immune response.