The intraovarian cellular origins of GDF9 and BMP15 in the mouse and aspects of their biological properties

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are both members of the TGF-ß protein superfamily and are known to be essential for normal follicular development in mammals. Several studies have highlighted the species-specific effects of BMP15 and GDF9, which could be attributed, at least in part to the differences in the follicular expression patterns and to different forms of the secreted proteins. In the mouse, GDF9 is required for follicular development, whereas BMP15 appears to be only required near ovulation with contradictory reports as to the timing of BMP15 expression. However, mouse BMP15 and GDF9 are known to have the capability of acting together synergistically. The aims of this thesis were to characterise in the mouse ovary, the expression patterns (localisation and levels) of Bmp15 and Gdf9 mRNA throughout follicular development, and to determine the peri-ovulatory expression of the corresponding proteins. In situ hybridisation and quantitative PCR analyses of ovarian samples and follicular cells collected from control and superovulated mice confirmed that Gdf9 and Bmp15 mRNA are expressed exclusively in oocytes from primary and early secondary stage follicles respectively. qPCR analysis of denuded oocytes (DO) revealed a tight correlation, and therefore co-regulation, between the expression levels of Bmp15 and Gdf9 irrespective of follicular developmental stage, with steady expression until the preovulatory LH surge when down-regulation of Bmp15 and Gdf9 occurred. Throughout the follicular developmental stages examined, Gdf9 was expressed in greater abundance relative to Bmp15, with a Bmp15:Gdf9 mRNA ratio of 1:4.12. [...] In conclusion, oocyte-derived Bmp15 and Gdf9 mRNA expression is co-regulated throughout follicular development in mice, with Gdf9 being more abundant than Bmp15, which might be an important factor in determining high ovulation quota. The expression of the target genes is down-regulated as the oocyte reaches developmental competence following the preovulatory LH surge. Protein expression data provided evidence that in vivo the immature mouse oocyte is capable of secreting all BMP15 protein forms previously detected in vitro. After the preovulatory LH surge, all visible protein forms are associated with the somatic follicular cells, in particular with the expanded cumulus mass. Of particular interest is the presence of the large protein complexes in the cumulus cell lysates, which suggests a storage and activation process involving ECM proteins, similar to the mechanism reported for other TGF-ß superfamily members, such as TGF-ß1 and myostatin. The finding that the BMP15 precursor protein is biologically active with a different activity to that of the processed mature protein form suggests that the full-length precursor protein may regulate or provide at least a portion of the biological activity of BMP15 in mice

Expression of CD1a and Type-1 Polarization Are Dissociated in Human Monocyte-Derived Dendritic Cells.

Ex vivo generated monocyte-derived dendritic cell (moDC)-vaccines have long been touted as promising immunotherapeutic agents for cancer treatment, although the response rate generally remains low. The reasons for this are still unclear and confounded by the diversity in manufacturing protocols that may affect moDC function. Preclinical studies have shown that the stimulatory function of dendritic cells can be improved by engaging invariant NKT cells in vivo through the presentation of the glycolipid alpha-galactosylceramide via CD1d. However, expression of CD1d on moDC has been shown to be negatively correlated with expression of CD1a, which in turn has been suggested to be a surrogate marker for IL-12 secreting type-1 polarized moDC, the preferred functional characteristics for cancer vaccines. Here we challenge this notion by showing that plasma-derived lipids drive functional levels of CD1d expression, while CD1a expression can vary considerably in these cells without being correlated with a loss of polarization or immunogenicity

Impact of autologous plasma on expression of CD1d, phenotype and function of moDC.

<p><b>a)</b> Representative CD1d expression histograms of moDC cultured in either serum-free AIM-V or RPMI supplemented with either 2% autologous plasma (AP), 10% fetal calf serum (FCS) or 2% delipidated AP. At least three independent experiments were performed. <b>b)</b> Activation of NKT hybridoma cells by alpha-GalCer-loaded moDC generated in either AIM-V or RPMI supplemented with 0.2–1% AP. NKT cell activation was assessed using an IL-2 bioassay. Percentages are relative to the highest activation achieved using 1% AP in the culture medium. Mean and error (SD) are shown. ** p<0.01 as tested by one way ANOVA (Kruskal-Wallis test with Dunn’s post test) <b>c)</b> Proliferation of allogeneic PBMC in a mixed lymphocyte reaction. 10⁵ PBMC were incubated with increasing numbers of moDC. PBMC proliferation was measured by ³H-thymidine uptake. Percentages are relative to the highest cell proliferation observed with 12.5 x 10³ moDC^AIM-V. Mean and error (SD) are shown. <b>d)</b> Representative flow cytometry histograms depicting the expression levels of CD80, CD83, CD86 and HLA-DR on moDC matured overnight with IL-1-beta, IL-6, TNF-alpha and PGE₂. At least three independent experiments were performed. <b>e)</b> Cytokine secretion of LPS/IFN-gamma activated moDC as measured by multiplex assay. Each line represents one individual. V1-V5 denominates the five different individuals tested. Statistical significance was determined using a non-parametric Mann-Whitney test.</p

Impact of autologous plasma on expression of CD1a and CD1a-associated functional dichotomy.

<p><b>a)</b> CD1a expression histogram plots of moDC generated from two individuals with opposite response patterns to autologous plasma (AP). The left plot depicts moDC that upregulated CD1a in the presence of AP while the individual represented in the right plot downregulated CD1a in response to AP. <b>b)</b> Percentages of CD1a⁺ cells in moDC cultures generated in either AIM-V or RPMI 2% AP. Each line represents one individual. Statistical significance was determined using a non-parametric Mann-Whitney test. <b>c)</b> Cytokine secretion of LPS/IFN-gamma activated CD1a^- and CD1a⁺ moDC as measured by multiplex assay. Each line represents one individual. V1-V8 denominates the eight different individuals tested (V1-V5 identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140432#pone.0140432.g001" target="_blank">Fig 1</a>). Statistical significance was determined using a non-parametric Mann-Whitney test.</p

Third-generation anti-CD19 chimeric antigen receptor T-cells incorporating a TLR2 domain for relapsed or refractory B-cell lymphoma: a phase I clinical trial protocol (ENABLE).

INTRODUCTION: Autologous T-cells transduced to express a chimeric antigen receptor (CAR) directed against CD19 elicit high response rates in relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL). However, r/r B-NHL remissions are durable in fewer than half of recipients of second-generation CAR T-cells. Third-generation (3G) CARs employ two costimulatory domains, resulting in improved CAR T-cell efficacy in vitro and in animal models in vivo. This investigator-initiated, phase I dose escalation trial, termed ENABLE, will investigate the safety and preliminary efficacy of WZTL-002, comprising autologous T-cells expressing a 3G anti-CD19 CAR incorporating the intracellular signalling domains of CD28 and Toll-like receptor 2 (TLR2) for the treatment of r/r B-NHL. METHODS AND ANALYSIS: Eligible participants will be adults with r/r B-NHL including diffuse large B-cell lymphoma and its variants, follicular lymphoma, transformed follicular lymphoma and mantle cell lymphoma. Participants must have satisfactory organ function, and lack other curative options. Autologous T-cells will be obtained by leukapheresis. Following WZTL-002 manufacture and product release, participants will receive lymphodepleting chemotherapy comprising intravenous fludarabine and cyclophosphamide. A single dose of WZTL-002 will be administered intravenously 2 days later. Targeted assessments for cytokine release syndrome and immune cell effector-associated neurotoxicity syndrome, graded by the American Society Transplantation and Cellular Therapy criteria, will be made. A modified 3+3 dose escalation scheme is planned starting at 5×104 CAR T-cells/kg with a maximum dose of 1×106 CAR T-cells/kg. The primary outcome of this trial is safety of WZTL-002. Secondary outcomes include feasibility of WZTL-002 manufacture and preliminary measures of efficacy. ETHICS AND DISSEMINATION: Ethical approval for the study was granted by the New Zealand Health and Disability Ethics Committee (reference 19/STH/69) on 23 June 2019 for Protocol V.1.2. Trial results will be reported in a peer-reviewed journal, and results presented at scientific conferences or meetings. TRIAL REGISTRATION NUMBER: NCT04049513