Summary Members of the CD28 family play important roles in regulating T cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and regulatory T cells. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3 and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA4-CD80-CD86 network. In the same way that soluble CTLA4 can be used to block T cell responses, we show that soluble Vstm3 attenuates T cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T cell responses.

IL-31 signals through the heterodimeric receptor IL-31RA and oncostatin M receptor (OSMR), and has been linked with the development of atopic dermatitis, a Th2 cytokine-associated disease in humans. However, recent studies of IL-31RA knockout (KO) mice have suggested that IL-31 signaling may be required to negatively regulate Th2 type responses rather than exacerbate them. Because those studies were performed on genetically modified mice, we examined whether neutralizing IL-31 with a specific mAb would give similar results to IL-31RA KO mice in two Th2 cytokine-associated immune models. We report no difference in lymphocyte Th2-type cytokine production after Ag immunization between IL-31RA KO mice, mice treated with the IL-31 mAb, or control animals. Second, we tested whether the absence of the IL-31RA subunit in IL-31RA KO mice may allow for increased pairing of the OSMR subunit with another cytokine receptor, gp130, resulting in overrepresentation of the heterodimeric receptor for OSM and increased responsiveness to OSM protein. We found that intranasal OSM challenge of IL-31RA KO mice resulted in increased IL-6 and vascular endothelial growth factor production in the lung compared with wild-type littermate control animals. Moreover, PBS-challenged IL-31RA KO mice already had increased levels of vascular endothelial growth factor, which were further increased by OSM challenge. These data imply that IL-31RA–deficient mice produce increased levels of OSM-inducible cytokines during airway sensitization and challenge, which may be the driving force behind the apparent exacerbation of Th2-type inflammatory responses previously observed in these mice.

-Airlift pumps use buoyant forces to displace liquid in vertical pipes. In this paper, the performance of a new design of airlift pump has been investigated. This new design uses both axial and radial injectors to improve the pump performance. The behaviour of two-phase flow under different operating conditions of the pump is discussed. These include the effect of submergence ratio, air flow rate and void fraction on the pump efficiency. Flow visualization images are used to describe the two-phase flow patterns and their effect on the pump performance. It was clear from the present experiments that the pump operating at higher submergence ratios found to deliver higher water flow rates and better efficiencies. The void fraction was found to increase throughout the different flow patterns observed in the pipe riser while the water flow rate remained approximately constant.

155 Background: Sipuleucel-T (Provenge) is an autologous cellular immunotherapy designed to stimulate an immune response in men diagnosed with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer. This study examined the role of GM-CSF in T cell activation and cytokine production in sipuleucel-T from men enrolled in phase III clinical trials (D9902B – IMPACT and P07-2 - PROACT). Methods: Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) isolated every two weeks by leukapheresis. PBMCs are cultured with PA2024, a recombinant human antigen consisting of prostatic acid phosphatase (PAP) and granulocyte macrophage-colony stimulating factor (GM-CSF), for two days and cultured cells are then infused into patients. After each leukapheresis, a proportion of the PMBCs were cultured ex vivo for two days with GM-CSF (sargromostim). The GM-CSF cultures were compared to sipuleucel-T for their T cell activation profiles, cytokine production, and upregulation of CD54 and CD86 using flow cytometry and MesoScale Discovery technologies. Results: Sipuleucel-T had increased APC activation-associated cytokines (IL-1a, IL-10, IL-12p70, IFNg and TNFa) and T cell activation- associated cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFNg and TNFa) with each treatment. In contrast, GM-CSF-cultured cells had lower levels of these cytokines. Both PA2024- and GM-CSF-cultured cells had upregulated CD54 and CD86. T cell (CD4+ and CD8+) activation-markers were increased upon PA2024 restimulation, as measured by CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), and CD279 (PD-1) expression. Cells from PA2024-cultures, but not GM-CSF-cultures, displayed a generalized pattern of enhanced expression of the T cell activation markers at the second infusion compared to the first and third. Conclusions: These data indicate that T cell activation and enhanced cytokine expression are a consequence of priming after the first infusion. These effects are not driven by GM-CSF, as T cell activation and enhanced cytokine production were only observed in PA2024 cultures. [Table: see text]

Background: Adoptive T-cell therapy with engineered T-cell receptors (eTCR) has demonstrated promising, yet modest clinical benefit to date. Key obstacles for this technology are 1) competition with the endogenous TCR for CD3 components that are required for surface expression of a functional TCR complex and 2) mispairing of endogenous and exogenous TCRα and TCRβ chains. Substituting murine TCR constant domains has been shown to enhance eTCR expression, but increases the risk of immunologic rejection. Knockout or knockdown of the endogenous TCR has also been shown to improve eTCR expression. Current eTCR delivery approaches use semi-randomly integrating lentivirus or retrovirus to generate an eTCR expressing T-cell product. An alternative approach is to combine TCR knockout with targeted integration of eTCRs into the T-cell receptor alpha constant (TRAC) locus. In this study, we evaluate newly discovered eTCRs specific for human papillomavirus (HPV) type 16 oncogenic protein E7, expressed via these various gene engineering approaches, to optimize engineered T-cell functionality. Methods: HPV E7-specific eTCRs were introduced into primary human T-cells by lentiviral transduction or a dual CRISPR-Cas9/AAV eTCR delivery platform for targeted insertion into the TRAC locus using homology directed repair (HDR). Comparisons were made among TCR sufficient and TCR knockouT-cells with gene delivery by lentivirus or HDR. Engineered T-cell function was assessed both in vitro and in vivo against HPV-16+ head and neck squamous cell carcinoma cell lines. Results: CRISPR/Cas9-mediated TRAC editing eliminated endogenous TCR expression in >85% of T-cells. The impact of TRAC editing on eTCR expression and engineered T-cell function was variable across multiple eTCRαβ sequences. With many eTCRs, TRAC editing in lentivirus-derived populations resulted in increased expression and improved cytokine and killing responses. Targeted insertion of these eTCRs into the TRAC locus by HDR increased engineered cell product homogeneity and enhanced function compared to lentiviral integration. However, there were also examples of eTCRs that were minimally impacted by TRAC locus engineering. Conclusions: Our results demonstrate that elimination of the endogenous TCR, alone or combined with targeted insertion at the TRAC locus, improves eTCR expression and engineered T-cell function for the majority of eTCRs tested. While infrequent, some eTCRs were less impacted by TRAC locus engineering, suggesting a sequence-specific property that enables these eTCRs to out-compete the endogenous TCR for CD3 componentry. The cause for this variable response to TRAC locus engineering is still being explored. Citation Format: Alexandra Croft, Cameron Brandt, Stephen Burleigh, Eric Chadwick, Melissa Chin, Dean Toy, Bailey Donahue, Clay Patton, Stephen Goldfless, Brian Belmont, Ruth Salmon, Grant Welstead, Blythe D. Sather, David J. Huss. Targeted insertion of an HPV-16 E7-specific engineered T-cell receptor into the TRAC locus [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A027.