Cell cultureGeneration of stem cell derived beta-like cells from human embryonic stem cells and from induced pluripotent stem cells MEL1 human embryonic stem cells (hESC) containing the INS GFP/W reporter 18 (referred to as pINSGFP throughout the manuscript) and sub-clones thereof 19,31 , type 1 diabetes patient induced pluripotent stem cells (T1D-iPSC) 24 (Supp. Fig 8 .) and iPSC from a healthy donor 48 (Supp. Fig. 9), as well as pNKX6.1 GFP reporter iPSC 46 were employed in this study. All iPSC lines were maintained on hESC qualified Matrigel (Corning #354277) in mTeSR+ media (STEMCELL Technologies #05826). MEL1 Subclones were only used for bulk RNA sequencing analysis experiments. Differentiation to stem cellderived beta-like cells (sBCs) was carried out in suspension-based, low attachment suspension culture plates as described 19 or in a bioreactor magnetic stirring system (Reprocell #ABBWVS03A-6, #ABBWVDW-1013, #ABBWBP03N0S-6) as follows:Confluent hESC cultures were dissociated into single-cell suspensions by incubation with TrypLE (Gibco #12-604-021) for 6 min at 37C. Detached cells were quenched with mTESR media. Live cells were counted using a MoxiGo II cell counter (Orflow), followed by seeding 0.5 × 10 6 cells per ml in mTeSR media supplemented with 10 M ROCK inhibitor (Y-27632, R&D Systems #1254-50) (cluster media). Bioreactors were placed on a magnetic stirring system set at 60 RPM in a cell culture incubator at 5 % CO2 to induce sphere formation for 48 h. To induce definitive endoderm differentiation, spheres were collected in a 50 mL Falcon tube, allowed to settle by gravity, washed once with RPMI

Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) regulates a panoply of leukocyte signaling pathways. A single nucleotide polymorphism (SNP) in PTPN22, rs2476601, is associated with increased risk of Type 1 Diabetes (T1D) and other autoimmune diseases. Over the past decade PTPN22 has been studied intensely in T cell receptor (TCR) and B cell receptor (BCR) signaling. However, the effect of the minor allele on PTPN22 function in TCR signaling is controversial with some reports concluding it has enhanced function and blunts TCR signaling and others reporting it has reduced function and increases TCR signaling. More recently, the core function of PTPN22 as well as functional derangements imparted by the autoimmunity-associated variant allele of PTPN22 have been examined in monocytes, macrophages, dendritic cells, and neutrophils. In this review we will discuss the known functions of PTPN22 in human cells, and we will elaborate on how autoimmunity-associated variants influence these functions across the panoply of immune cells that express PTPN22. Further, we consider currently unresolved questions that require clarification on the role of PTPN22 in immune cell function.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of β-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual’s collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and β-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

Infection of HIV-1 remains incurable because long-lived, latently-infected cells persist during prolonged antiretroviral therapy. Attempts to pharmacologically reactivate and purge the latent reservoir with latency reactivating agents (LRAs) such as protein kinase C (PKC) agonists (e.g. ingenol A) or histone deacetylase (HDAC) inhibitors (e.g. SAHA) have shown promising but incomplete efficacy. Using the J-Lat T cell model of HIV latency, we found that the plant-derived compound harmine enhanced the efficacy of existing PKC agonist LRAs in reactivating latently-infected cells. Treatment with harmine increased not only the number of reactivated cells but also increased HIV transcription and protein expression on a per-cell basis. Importantly, we observed a synergistic effect when harmine was used in combination with ingenol A and the HDAC inhibitor SAHA. An investigation into the mechanism revealed that harmine, when used with LRAs, increased the activity of NFκB, MAPK p38, and ERK1/2. Harmine treatment also resulted in reduced expression of HEXIM1, a negative regulator of transcriptional elongation. Thus, harmine enhanced the effects of LRAs by increasing the availability of transcription factors needed for HIV reactivation and promoting transcriptional elongation. Combination therapies with harmine and LRAs could benefit patients by achieving deeper reactivation of the latent pool of HIV provirus.