Sympathetic nervous system development depends upon many factors that mediate neuron migration, differentiation and survival. Target tissue-derived nerve growth factor (NGF) signaling-induced gene expression is required for survival, differentiation and target tissue innervation of post-migratory sympathetic neurons. However, the transcriptional regulatory mechanisms mediated by NGF signaling are very poorly defined. Here, we identify Egr3, a member of the early growth response (Egr) family of transcriptional regulators, as having an important role in sympathetic nervous system development. Egr3 is regulated by NGF signaling and it is expressed in sympathetic neurons during development when they depend upon NGF for survival and target tissue innervation. Egr3-deficient mice have severe sympathetic target tissue innervation abnormalities and profound physiological dysautonomia. Unlike NGF, which is essential for sympathetic neuron survival and for axon branching within target tissues, Egr3 is required for normal terminal axon extension and branching, but not for neuron survival. The results indicate that Egr3 is a novel NGF signaling effector that regulates sympathetic neuron gene expression required for normal target tissue innervation and function. Egr3-deficient mice have a phenotype that is remarkably similar to humans with sympathetic nervous system disease, raising the possibility that it may have a role in some forms of human dysautonomia,most of which have no known cause.
Egr3 is a nerve growth factor (NGF)-induced transcriptional regulator that is essential for normal sympathetic nervous system development. Mice lacking Egr3 in the germline have sympathetic target tissue innervation abnormalities and physiologic sympathetic dysfunction similar to humans with dysautonomia. However, since Egr3 is widely expressed and has pleiotropic function, it has not been clear whether it has a role within sympathetic neurons and if so, what target genes it regulates to facilitate target tissue innervation. Here, we show that Egr3 expression within sympathetic neurons is required for their normal innervation since isolated sympathetic neurons lacking Egr3 have neurite outgrowth abnormalities when treated with NGF and mice with sympathetic neuron-restricted Egr3 ablation have target tissue innervation abnormalities similar to mice lacking Egr3 in all tissues. Microarray analysis performed on sympathetic neurons identified many target genes deregulated in the absence of Egr3, with some of the most significantly deregulated genes having roles in axonogenesis, dendritogenesis, and axon guidance. Using a novel genetic technique to visualize axons and dendrites in a subpopulation of randomly labeled sympathetic neurons, we found that Egr3 has an essential role in regulating sympathetic neuron dendrite morphology and terminal axon branching, but not in regulating sympathetic axon guidance to their targets. Together, these results indicate that Egr3 has a sympathetic neuron autonomous role in sympathetic nervous system development that involves modulating downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons.
While autologous T cell therapies can effectively treat B-cell leukemia and lymphoma, the personalized manufacturing process is difficult to scale, expensive and may fail. Even when autologous products are successfully manufactured, they are not immediately available to acutely ill patients. "Off-the-shelf" T cell products derived from healthy donors that can rapidly be administered, would improve accessibility and reduce the cost of T cell therapy. However, major obstacles to successful allogeneic T cell products include their potential for graft-versus-host disease (GVHD) and graft rejection, mediated by host and recipient alloreactive T cells respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVSTs) as our platform since they are virus specific rather than allospecific and have not produced GVHD in more than 300 allogeneic recipients. To prevent graft rejection we have introduced into these EBVSTs, a chimeric antigen receptor for CD30 (CD30.CAR). CD30 is upregulated during the activation of alloreactive T cells, which leads to them becoming targets. The CD30.CAR provides the additional advantage of targeting CD30-positive lymphoma and has proved safe and effective in prior clinical trials (NCT02917083) using autologous CAR-T cells. Hence, we expect off-the-shelf CD30.CAR EBVSTs to eliminate the alloreactive T cells they elicit in allogeneic hosts, and therefore persist for sufficient time to eliminate CD30-positive lymphoma, without causing GVHD. Here we show that CD30.CAR-EBVSTs resist fratricide by masking their own CD30 molecules expressed in cis, but are nonetheless protected from rejection when co-cultured with alloreactive T cells expressing CD30 in trans. Notably, CD30.CAR EBVSTs preserve the function of both their TCR and the CD30.CAR, with retention of EBV specificity and the ability to eliminate CD30-positive tumor cells. We have manufactured a bank of clinical grade CD30.CAR EBVSTs from donors with HLA types designed to provide a partial HLA match for our diverse recipients. Clinical grade CD30.CAR EBVST cultures readily expanded to sufficient numbers for a planned clinical trial and expressed the CD30.CAR on 77% to 99% of cells. All of the lines passed functional release criteria of having greater than 100 IFNɣ spot-forming units (SFU) per 105 cells in response to both latent and lytic EBV antigens, and greater than 20% specific cytolysis against a CD30-positive Hodgkin lymphoma cell line, HDLM2, at an effector to target ratio of 20:1. Although CD30.CAR killing is not HLA restricted, we will select the CD30.CAR EBVST product for each recipient, based on the best HLA class I and class II match. This will allow endogenous EBV to boost the in vivo activity of CD30.CAR EBVSTs, and will provide additional reactivity for patients with CD30-positive and EBV-positive tumors. The IND for the clinical trial (NCT04288726) has been approved and we will recruit patients with CD30-positive lymphomas including Hodgkin lymphoma, diffuse large B cell lymphoma and NK/T cell lymphoma. In summary, we present an approach to making an off-the-shelf T cell therapy that can rapidly translate to the clinic, requires no gene editing, and can serve as a platform for other CAR/TCRs to target a multiplicity of malignancies. Disclosures Quach: Tessa Therapeutics: Research Funding. Brenner:Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tumstone: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Heslop:Tessa Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ramos:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Kuur Therapeutics: Research Funding. Rouce:Tessa Therapeutics: Other, Research Funding; Novartis: Honoraria. Rooney:Marker Therapeutics: Current equity holder in publicly-traded company, Other: co-founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allovir: Current equity holder in publicly-traded company, Other: co-founder.
Background The use of “off-the-shelf” cellular therapy products derived from healthy donors addresses many of the challenges associated with customized cell products. However, the potential of allogeneic cell products to produce graft-versus-host disease (GVHD), and their likely rejection by host alloreactive T-cells are major barriers to their clinical safety and efficacy. We have developed a molecule that when expressed in T-cells, can eliminate alloreactive T-cells and hence can be used to protect cell therapy products from allospecific rejection. Further, expression of this molecule in virus-specific T-cells (VSTs) should virtually eliminate the potential for recipients to develop GVHD. Methods To generate a molecule that can mediate killing of cognate alloreactive T-cells, we fused beta-2 microglobulin (B2M), a universal component of all human leukocyte antigen (HLA) class I molecules, to the cytolytic endodomain of the T cell receptor ζ chain, to create a chimeric HLA accessory receptor (CHAR). To determine if CHAR-modified human VSTs could eliminate alloreactive T-cells, we co-cultured them with allogeneic peripheral blood mononuclear cells (PBMC), and assessed proliferation of PBMC-derived alloreactive T-cells and the survival of CHAR-modified VSTs by flow cytometry. Results The CHAR was able to transport HLA molecules to the cell surface of Daudi cells, that lack HLA class I expression due to defective B2M expression, illustrating its ability to complex with human HLA class I molecules. Furthermore, VSTs expressing CHAR were protected from allospecific elimination in co-cultures with allogeneic PBMCs compared to unmodified VSTs, and mediated killing of alloreactive T-cells. Unexpectedly, CHAR-modified VSTs eliminated not only alloreactive HLA class I restricted CD8 T-cells, but also alloreactive CD4 T-cells. This beneficial effect resulted from non-specific elimination of activated T-cells. Of note, we confirmed that CHAR-modified VSTs did not affect pathogen-specific T-cells which are essential for protective immunity. Conclusions Human T-cells can be genetically modified to eliminate alloreactive T-cells, providing a unique strategy to protect off-the-shelf cell therapy products. Allogeneic cell therapies have already proved effective in treating viral infections in the stem cell transplant setting, and have potential in other fields such as regenerative medicine. A strategy to prevent allograft rejection would greatly increase their efficacy and commercial viability. Electronic supplementary material The online version of this article (10.1186/s12967-019-1988-y) contains supplementary material, which is available to authorized users.
Nerve Growth Factor (NGF) is a target tissue derived neurotrophin required for normal sympathetic neuron survival and target tissue innervation. NGF signaling regulates gene expression in sympathetic neurons, which in turn mediates critical aspects of neuron survival, axon extension and terminal axon branching during sympathetic nervous system (SNS) development. Egr3 is a transcription factor regulated by NGF signaling in sympathetic neurons that is essential for normal SNS development. Germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal innervation to many target tissues. The extent to which sympathetic innervation abnormalities in the absence of Egr3 is caused by altered innervation or by neuron death during development is unknown. Using Bax-deficient mice to abrogate apoptotic sympathetic neuron death in vivo, we show that Egr3 has an essential role in target tissue innervation in the absence of neuron death. Sympathetic target tissue innervation is abnormal in many target tissues in the absence of neuron death, and like NGF, Egr3 also appears to effect target tissue innervation heterogeneously. In some tissues, such as heart, spleen, bowel, kidney, pineal gland and the eye, Egr3 is essential for normal innervation, whereas in other tissues such as lung, stomach, pancreas and liver, Egr3 appears to have little role in innervation. Moreover, in salivary glands and heart, two tissues where Egr3 has an essential role in sympathetic innervation, NGF and NT-3 are expressed normally in the absence of Egr3 indicating that abnormal target tissue innervation is not due to deregulation of these neurotrophins in target tissues. Taken together, these results clearly demonstrate a role for Egr3 in mediating sympathetic target tissue innervation that is independent of neuron survival or neurotrophin deregulation.
There was an error in the ePress version of this article published on 24 July 2008. David Quach's middle initial was incorrectly given as 'M'.The correct author list appears above, and the print and final online versions are correct.The authors apologise to readers for this mistake.
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