Preclinical Evidence of an Allogeneic Dual CD20xCD22 CAR to Target a Broad Spectrum of Patients with B-cell Malignancies.

Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches. To address both challenges simultaneously, we generated a CD20xCD22 dual allogeneic CAR T cell. Herein, we demonstrate that allogeneic CD20x22 CAR T cells display robust, sustained and dose-dependent activity in vitro and in vivo, while efficiently targeting primary B-cell non-Hodgkin lymphoma (B-NHL) samples with heterogeneous levels of CD22 and CD20. Altogether, we provide preclinical proof-of-concept data for an allogeneic dual CAR T cell to overcome current mechanisms of resistance to CAR T-cell therapies in B-NHL, while providing a potential alternative to CD19 targeting.

Notch-mediated hepatocyte MCP-1 secretion causes liver fibrosis.

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

Preclinical evaluation of a precision medicine approach to DNA vaccination in type 1 diabetes.

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

Tissue-Engineered Stromal Reticula to Study Lymph Node Fibroblastic Reticular Cells in Type I Diabetes.

INTRODUCTION: Fibroblastic reticular cells (FRCs) support and remodel the lymph node (LN), express and present self-antigens to T cells to promote tolerance. In Type 1 diabetes (T1D), decrease in FRC frequency and in their expression of T1D-related self-antigens may hinder tolerogenic engagement of autoreactive T cells. FRC reticular organization in LNs is critical for adaptive immunity. Thus, we engineered LN-like FRC reticula to determine if FRC reticular properties were altered in T1D and to study engagement of autoreactive T cells in vitro. METHODS: We characterized FRC networks in pancreatic and skin-draining LNs of 4- and 12-week old non-obese diabetic (NOD) and diabetes resistant NOR mice by immunofluorescence. Murine FRCs isolated from NOR, NOD or human pancreatic LNs were cultured in collagen sponges for up to 21 days before immunofluorescence and flow cytometry analysis. NOD FRCs expressing T1D antigens were co-cultured with CellTrace-labeled specific T cells in 2D or in scaffolds. T cell engagement was quantified by CD25 upregulation, CellTrace dilution and by T cell tracking. RESULTS: FRC networks in both 4- and 12-week old NOD LNs displayed larger reticular pores than NOR controls. NOD FRCs had delayed scaffold remodeling compared to NOR FRCs. Expression of the gp38 FRC marker in NOD FRCs was lower than in NOR but improved in 3D. FRC reticula expressing T1D antigens promoted higher engagement of specific T cells than 2D. CONCLUSION: We engineered LN-like FRC reticula that recapitulate FRC organization and phenotype of T1D LNs for studying tolerogenic autoreactive T cell engagement in T1D.

Phenotypic alterations in pancreatic lymph node stromal cells from human donors with type 1 diabetes and NOD mice.

AIMS/HYPOTHESIS: Tolerance induction in lymph nodes can be mediated by both haematopoietic cells (e.g. specific dendritic cells subsets) and by non-haematopoietic cells (e.g. lymph node stromal cells [LNSCs]) when they present peripheral tissue antigens to autoreactive T cells. LNSCs normally regulate T cell trafficking and survival and help to maintain peripheral tolerance by exerting immunosuppressive effects. However, whether autoimmunity can be associated with defective tolerogenic functions of LNSCs is unknown and studies aimed at characterising LNSCs in humans are lacking. We hypothesised that dysregulated T cell responses in pancreatic lymph nodes (PLNs) from donors with type 1 diabetes and from NOD mice may be associated with altered LNSC function. METHODS: We analysed PLNs from donors with type 1 diabetes and NOD mice for LNSC distribution and phenotype using flow cytometry. We assessed the expression of tolerance-related genes in different subsets of LNSCs from human donors, as well as in a population of dendritic cells enriched in autoimmune regulator (AIRE)+ cells and identified as HLA-DRhigh CD45low. RESULTS: The relative frequency of different LNSC subsets was altered in both donors with type 1 diabetes and NOD mice, and both MHC class II and programmed death-ligand 1 (PD-L1) expression were upregulated in human type 1 diabetes. Tolerance-related genes showed similar expression profiles between mouse and human LNSCs at steady state but were generally upregulated in the context of human type 1 diabetes, while, at the same time, many such genes were downregulated in the AIRE-enriched dendritic cell population. CONCLUSION/INTERPRETATION: Our study shows that LNSCs are substantially altered in type 1 diabetes, but, surprisingly, they exhibit an enhanced tolerogenic phenotype along with increased antigen-presenting potential, which may indicate an attempt to offset dendritic cell-related tolerogenic defects in tolerance. Thus, LNSCs could constitute alternative therapeutic targets in which to deliver antigens to help re-establish tolerance and prevent or treat type 1 diabetes. DATA AVAILABILITY: All data generated or analysed during this study are included in the published article (and its online supplementary files). Biomark gene expression data were deposited on the Mendeley repository at https://data.mendeley.com/datasets/d9rdzdmvyf/1 . Any other raw datasets are available from the corresponding author on reasonable request. No applicable resources were generated or analysed during the current study.

A multi-epitope DNA vaccine enables a broad engagement of diabetogenic T cells for tolerance in Type 1 diabetes.

Type 1 diabetes (T1D) is caused by diabetogenic T cells that evaded tolerance mechanisms and react against multiple ß-cell antigens. Antigen-specific therapy to reinstate tolerance (typically using a single ß-cell antigen) has so far proved unsuccessful in T1D patients. Plasmid DNA (pDNA)-mediated expression of proinsulin has demonstrated transient protection in clinical trials, but long-lasting tolerance is yet to be achieved. We aimed to address whether pDNA delivery of multiple epitopes/mimotopes from several ß-cell antigens efficiently presented to CD4+ and CD8+ T cells could also induce tolerance. This approach significantly delayed T1D development, while co-delivery of pDNA vectors expressing four full antigens protected more mice. Delivery of multiple epitopes resulted in a broad engagement of specific T cells, eliciting a response distinct from endogenous epitopes draining from islets. T-cell phenotypes also varied with antigen specificity. Unexpectedly, the repertoire of T cells reactive to the same epitope was highly polyclonal. Despite induction of some CD25+ Foxp3+ regulatory T cells, protection from disease did not persist after treatment discontinuation. These data demonstrate that epitope-based tolerogenic DNA vaccines constitute effective precision medicine tools to target a broad range of specific CD4+ and CD8+ diabetogenic T-cell populations for prevention or treatment of T1D.

Altered Function of Antigen-Presenting Cells in Type 1 Diabetes: A Challenge for Antigen-Specific Immunotherapy?

Type 1 diabetes (T1D) arises from a failure to maintain tolerance to specific ß-cell antigens. Antigen-specific immunotherapy (ASIT) aims to reestablish immune tolerance through the supply of pertinent antigens to specific cell types or environments that are suitable for eliciting tolerogenic responses. However, antigen-presenting cells (APCs) in T1D patients and in animal models of T1D are affected by a number of alterations, some due to genetic polymorphism. Combination of these alterations, impacting the number, phenotype, and function of APC subsets, may account for both the underlying tolerance deficiency and for the limited efficacy of ASITs so far. In this comprehensive review, we examine different aspects of APC function that are pertinent to tolerance induction and summarize how they are altered in the context of T1D. We attempt to reconcile 25 years of studies on this topic, highlighting genetic, phenotypic, and functional features that are common or distinct between humans and animal models. Finally, we discuss the implications of these defects and the challenges they might pose for the use of ASITs to treat T1D. Better understanding of these APC alterations will help us design more efficient ways to induce tolerance.

PPARγ deacetylation dissociates thiazolidinedione's metabolic benefits from its adverse effects.

Thiazolidinediones (TZDs) are PPARγ agonists with potent insulin-sensitizing effects. However, their use has been curtailed by substantial adverse effects on weight, bone, heart, and hemodynamic balance. TZDs induce the deacetylation of PPARγ on K268 and K293 to cause the browning of white adipocytes. Here, we show that targeted PPARγ mutations resulting in constitutive deacetylation (K268R/K293R, 2KR) increased energy expenditure and protected from visceral adiposity and diet-induced obesity by augmenting brown remodeling of white adipose tissues. Strikingly, when 2KR mice were treated with rosiglitazone, they maintained the insulin-sensitizing, glucose-lowering response to TZDs, while displaying little, if any, adverse effects on fat deposition, bone density, fluid retention, and cardiac hypertrophy. Thus, deacetylation appears to fulfill the goal of dissociating the metabolic benefits of PPARγ activation from its adverse effects. Strategies to leverage PPARγ deacetylation may lead to the design of safer, more effective agonists of this nuclear receptor in the treatment of metabolic diseases.

Efficient Presentation of Multiple Endogenous Epitopes to Both CD4+ and CD8+ Diabetogenic T Cells for Tolerance.

Antigen-specific immunotherapy of type 1 diabetes, typically via delivery of a single native ß cell antigen, has had little clinical benefit to date. With increasing evidence that diabetogenic T cells react against multiple ß cell antigens, including previously unappreciated neo-antigens that can be emulated by mimotopes, a shift from protein- to epitope-based therapy is warranted. To this end, we aimed to achieve efficient co-presentation of multiple major epitopes targeting both CD4+ and CD8+ diabetogenic T cells. We have compared native epitopes versus mimotopes as well as various targeting signals in an effort to optimize recognition by both types of T cells in vitro. Optimal engagement of all T cells was achieved with segregation of CD8 and CD4 epitopes, the latter containing mimotopes and driven by endosome-targeting signals, after delivery into either dendritic or stromal cells. The CD4+ T cell responses elicited by the endogenously delivered epitopes were comparable with high concentrations of soluble peptide and included functional regulatory T cells. This work has important implications for the improvement of antigen-specific therapies using an epitope-based approach to restore tolerance in type 1 diabetes and in a variety of other diseases requiring concomitant targeting of CD4+ and CD8+ T cells.