Autoimmune Mouse Model Research Articles

Antigen-specific nanoparticle tolerance treatment actively induces regulatory mechanisms via the STING pathway

Abstract Antigen (Ag)-specific T cells are the underlying cause of many autoimmune diseases. T cells found at the site of tissue destruction not only in the exacerbation of disease, but contribute to the release of additiotnal self-epitopes resulting in the activation of spread eptitope-specific T cell populations perpetuating disease. Treatment with Ag-containingbiodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, i.e. tolerogenic immune-modifying particles (TIMP), has been shown to be both safe and induces Ag-specific tolerance in mouse models of autoimmunity and allergy, as well as in a celiac disease Phase I/IIa clinical trial. We are in the process of assessing if the mechanism of action between Ag-specific TIMP treatment and Ag-coupled cell treatment, to thereby identify self-tolerance mechanisms in common between the two highly efficacious therapies. The PLP 139-151TCR transgenic model system was utilized to determine the cellular and molecular mechanisms driving Ag-specific tolerance mediated by tolerogenic nanoparticle treatment. These data show Ag-specific TIMP treatment induced both FoxP3 +iTregs and IL-10 +Tr1 regulatory in both naïve mice and mice pre-primed with Ag/CFA. Additionally, the expression of STING is required for the induction of tolerance. The involvement of the STING pathway for both TIMP and Ag-coupled cell treatment was confirmed in MOG 35-55EAE. The data also show that Ag-specific tolerance induction requires both Type I IFNs and PD-1/PD-L1 expression, which are downstream of STING activation and signaling. Treatment thus activates various Ag-specific Treg subsets capable of regulating responses to disease-relevant autoepitopes via a STING/IFNAR/PD-1/PD-L1 pathway.

Decreased memory persistence in Piezo1 deficient T cells ameliorates disease progression in autoimmune mouse models

Abstract To mount a successful immune response, T cells must successfully (1) migrate from the bloodstream into secondary lymphoid organs (naïve) or inflamed tissue (effector), and (2) form an effective immunological synapse for activation. Although the molecular interactions of these processes have been well described, the emergence of novel biophysical approaches has revealed that both critical processes require mechanosensing. Previously we have demonstrated that the mechanosensitive cation channel, Piezo1, forms a complex with activated integrin LFA-1, an important regulator of T cell migration and T cell receptor-triggered T cell activation. Therefore, we hypothesized that Piezo1 deficiency in T cells would diminish T cell activation and homing.To investigate Piezo1’s role on T cell function, we have generated T cell-specific Piezo1 knockout mice (P1cKO) by crossing Piezo1-floxed mice to CD4-Cre mice. Initial studies in vitro showed that under polarizing conditions P1cKO T cells produce more Th1 and Th17 inflammatory cytokines. However, when challenged with experimental autoimmune encephalomyelitis (EAE) and Graft-vs-Host Disease (GVHD), P1cKO mice showed a significant decrease in disease phenotype. Moreover, adoptive transfer mouse models of EAE show that activated P1cKO T cell transfer fails to establish disease phenotype. Upon further investigation, we found similar numbers of P1cKO T cells at sites of inflammation early in disease, but there was a decreased persistence of effector and central memory P1cKO T cells. Taken together, our data suggests that Piezo1 plays an important in role in the development and maintenance of T cell memory pools, while potentially contributing to T cell activation and migration. This project was supported by grants from the NIH (TL1 TR002549, T32 GM007250, T32 AI089474), the St. Baldrick's Foundation Innovation Fund, the Theresia G. & Stuart F. Kline Family Foundation, and the Karen & Alan Krause Pediatric Immunotherapy Innovation and Education Fund.

MiR-21 promotes B cell autoreactivity, inflammatory signaling, and metabolism in TLR7-driven systemic autoimmunity

Abstract MicroRNA-21 (miR-21) is upregulated in SLE patients and promotes disease in multiple autoimmune mouse models; however, mechanisms by which miR-21 promote autoimmunity are unknown. Here we show that TLR7 overexpressing SLE-prone B6.Sle1b Yaamice deficient in miR-21 (Sle1b YaamiR-21 −/−) resisted autoimmunity development through reduced germinal center (GC) B cell, T follicular helper cell, and plasma cell responses. Sle1b YaamiR-21 −/−mice had reduced serum autoantibody titers and numbers of autoantibody-producing antibody forming cells (AFCs) in the spleen and bone marrow. Downstream of TLR7 signaling, PELI1, a negative regulator of the NF-κB family member c-Rel and a direct target of miR-21, was increased in Sle1b YaamiR-21 −/−B cells. Consequently, Sle1b YaamiR-21 −/−B cells had reduced c-Rel expression and proinflammatory cytokines IL-6 and IL-1β. Additionally, Sle1b YaamiR-21 −/−B cells had reduced oxidative phosphorylation, glycolysis, and mitochondrial membrane potential compared to Sle1b YaaB cells. To define the cell-intrinsic mechanisms, we generated B cell-, T cell- and DC-specific miR-21 conditional knockout (cKO) mice on the Sle1b Yaabackground. Reduced B cell autoreactivity, inflammatory signaling, and metabolism in Sle1b YaamiR-21 −/−mice were recapitulated in B cell-specific miR-21 cKO mice. T cell-specific miR-21 cKO mice had a moderate reduction in cellular responses due to reduced co-stimulatory molecules and glycolysis; however, autoimmune responses remained intact. miR-21 deficiency in DCs had no deficit in cellular or autoimmune responses. Our data substantiate a B cell-intrinsic role for miR-21 in promoting inflammatory signaling and metabolic activity in B cells, leading to autoimmunity.

Role of the adipocyte immune brain axis in Parkinson's disease: friend or foe?

Role of the adipocyte immune brain axis in Parkinson's disease: friend or foe?

Clonal relationships of memory B cell subsets in autoimmune mice.

Immunological memory protects our body from re-infection and it is composed of a cellular and a humoral arm. The B-cell branch with its memory B cells (MBCs), plasma cells and antibodies, formed either in a germinal centre (GC) -dependent or -independent manner, ensure that we can rapidly mount a recall immune response. Previous work in immunised wildtype (WT) mice have identified several subsets of MBCs whereas less is known under autoimmune conditions. Here, we have investigated the heterogeneity of the MBC compartment in autoimmune mouse models and examined the clonal relationships between MBC subsets and GC B cells in one of the models. We demonstrate the presence of at least four different MBC subsets based on their differential expression pattern of CD73, CD80 and PD-L2 in surrogate light chain-deficient (SLC-/-), MRL+/+ and MRLlpr/lpr mice, where most of the MBCs express IgM. Likewise, four MBC subsets could be identified in WT immunised mice. In SLC-/- mice, high-throughput sequencing of Ig heavy chains demonstrates that the two CD73-positive subsets are generally more mutated. Lineage tree analyses on expanded clones show overlaps between all MBC subsets and GC B cells primarily in the IgM sequences. Moreover, each of the three IgM MBC subsets could be found both as ancestor and progeny to GC B cells. This was also observed in the IgG sequences except for the CD73-negative subset. Thus, our findings demonstrate that several MBC subsets are present in autoimmune and WT mice. In SLC-/- mice, these MBC subsets are clonally related to each other and to GC B cells. Our results also indicate that different MBC subsets can seed the GC reaction.

Morphometric changes among testis of autoimmune mouse model and healthy strains

Morphometric changes among testis of autoimmune mouse model and healthy strains

The classical pathway triggers pathogenic complement activation in membranous nephropathy

Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. However, both the initiation pathways and the pathogenic significance of complement activation in MN are poorly understood. Here, we show that components from all three complement pathways (alternative, classical and lectin) are found in renal biopsies from patients with MN. Proximity ligation assays to directly visualize complement assembly in the tissue reveal dominant activation via the classical pathway, with a close correlation to the degree of glomerular C1q-binding IgG subclasses. In an antigen-specific autoimmune mouse model of MN, glomerular damage and proteinuria are reduced in complement-deficient mice compared with wild-type littermates. Severe disease with progressive ascites, accompanied by extensive loss of the integral podocyte slit diaphragm proteins, nephrin and neph1, only occur in wild-type animals. Finally, targeted silencing of C3 using RNA interference after the onset of proteinuria significantly attenuates disease. Our study shows that, in MN, complement is primarily activated via the classical pathway and targeting complement components such as C3 may represent a promising therapeutic strategy.

Precision targeting of autoantigen-specific B cells in muscle-specific tyrosine kinase myasthenia gravis with chimeric autoantibody receptor T cells.

Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.

Blocking CD40 Alleviates Th1 and Th17 Cell Responses in Elastin Peptide-Induced Murine Emphysema.

To investigate the role of the CD40-CD40 ligand (CD40L) pathway in the regulation of Th1, Th17, and regulatory T (Treg)-cell responses in an elastin peptide (EP)-induced autoimmune emphysema mouse model. BALB/c mice were transnasally treated with EP on day 0, injected intravenously with anti-CD40 antibody via the tail vein on day 33, and sacrificed on day 40. The severity of emphysema was evaluated by determining the mean linear intercept (MLI) and destructive index (DI) from lung sections. The proportions of myeloid dendritic cells (mDCs) and Th1, Th17, and Treg cells in the blood, spleen, and lungs were determined via flow cytometry. The levels of the cytokines interleukin (IL)-6, IL-17, interferon (IFN)-γ, and transforming growth factor (TGF)-β were detected via enzyme-linked immunosorbent assay. Ifnγ, IL17a, Rorγt and Foxp3 transcription levels were detected via polymerase chain reaction. CD40+ mDCs accumulated in the lungs of EP-stimulated mice. Blocking the CD40-CD40L pathway with an anti-CD40 antibody alleviated Th1 and Th17 responses; increased the proportion of Treg cells; decreased MLI and DI; reduced the levels of cytokines IL-6, IL-17, and IFN-γ as well as the transcription levels of Ifnγ, IL17a, and Rorγt; and upregulated the expression of TGF-β and Foxp3. The CD40-CD40L pathway could play a critical role in Th1, Th17 and Treg cell dysregulation in EP-mediated emphysema and could be a potential therapeutic target.

How GRAIL controls Treg function to maintain self-tolerance.

Regulatory T cells (Tregs) normally maintain self-tolerance. Tregs recognize "self" such that when they are not working properly, such as in autoimmunity, the immune system can attack and destroy one's own tissues. Current therapies for autoimmunity rely on relatively ineffective and too often toxic therapies to "treat" the destructive inflammation. Restoring defective endogenous immune regulation (self-tolerance) would represent a paradigm shift in the therapy of these diseases. One recent approach to restore self-tolerance is to use "low dose IL-2" as a therapy to increase the number of circulating Tregs. However, studies to-date have not demonstrated that low-dose IL-2 therapy can restore concomitant Treg function, and phase 2 studies in low dose IL-2 treated patients with autoimmune diseases have failed to demonstrate significant clinical benefit. We hypothesize that the defect in self-tolerance seen in autoimmunity is not due to an insufficient number of available Tregs, but rather, due to defects in second messengers downstream of the IL-2R that normally control Treg function and stability. Previous studies from our lab and others have demonstrated that GRAIL (a ubiquitin E3 ligase) is important in Treg function. GRAIL expression is markedly diminished in Tregs from patients with autoimmune diseases and allergic asthma and is also diminished in Tregs of mice that are considered autoimmune prone. In the relevant pathway in Tregs, GRAIL normally blocks cullin ring ligase activity, which inhibits IL-2R desensitization in Tregs and consequently promotes Treg function. As a result of this defect in GRAIL expression, the Tregs of patients with autoimmune diseases and allergic asthma degrade IL-2R-associated pJAK1 following activation with low dose IL-2, and thus cannot maintain pSTAT5 expression. pSTAT5 controls the transcription of genes required for Treg function. Additionally, the GRAIL-mediated defect may also allow the degradation of the mTOR inhibitor, DEP domain-containing mTOR interacting protein (Deptor). This can lead to IL-2R activation of mTOR and loss of Treg stability in autoimmune patients. Using a monoclonal antibody to the remnant di-glycine tag on ubiquitinated proteins after trypsin digestion, we identified a protein that was ubiquitinated by GRAIL that is important in Treg function, cullin5. Our data demonstrate that GRAIL acts a negative regulator of IL-2R desensitization by ubiquitinating a lysine on cullin5 that must be neddylated to allow cullin5 cullin ring ligase activity. We hypothesize that a neddylation inhibitor in combination with low dose IL-2 activation could be used to substitute for GRAIL and restore Treg function and stability in the Tregs of autoimmune and allergic asthma patients. However, the neddylation activating enzyme inhibitors (NAEi) are toxic when given systemically. By generating a protein drug conjugate (PDC) consisting of a NAEi bound, via cleavable linkers, to a fusion protein of murine IL-2 (to target the drug to Tregs), we were able to use 1000-fold less of the neddylation inhibitor drug than the amount required for therapeutically effective systemic delivery. The PDC was effective in blocking the onset or the progression of disease in several mouse models of autoimmunity (type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis) and a mouse model of allergic asthma in the absence of detectable toxicity. This PDC strategy represents targeted drug delivery at its best where the defect causing the disease was identified, a drug was designed and developed to correct the defect, and the drug was targeted and delivered only to cells that needed it, maximizing safety and efficacy.

Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Inhibition of IL-17A Protects against Thyroid Immune-Related Adverse Events while Preserving Checkpoint Inhibitor Antitumor Efficacy.

Immune checkpoint inhibitor (ICI) immunotherapy leverages the body's own immune system to attack cancer cells but leads to unwanted autoimmune side effects in up to 60% of patients. Such immune-related adverse events (IrAEs) may lead to treatment interruption, permanent organ dysfunction, hospitalization, and premature death. Thyroiditis is one of the most common IrAEs, but the cause of thyroid IrAEs remains unknown. In this study, we use a new, physiologically relevant mouse model of ICI-associated autoimmunity to identify a key role for type 3 immune cells in the development of thyroid IrAEs. Multiple lineages of IL-17A-producing T cells expand in thyroid tissue with ICI treatment. Intrathyroidal IL-17A-producing innate-like γδT17 cells were increased in tumor-free mice, whereas adaptive Th17 cells were also prominent in tumor-bearing mice, following ICI treatment. Furthermore, Ab-based inhibition of IL-17A, a clinically available therapy, significantly reduced thyroid IrAE development in ICI-treated mice with and without tumor challenge. Finally, combination of IL-17A neutralization with ICI treatment in multiple tumor models did not reduce ICI antitumor efficacy. These studies suggest that targeting Th17 and γδT17 cell function via the IL-17A axis may reduce IrAEs without impairing ICI antitumor efficacy and may be a generalizable strategy to address type 3 immune-mediated IrAEs.

Nature vs. nurture: FOXP3, genetics, and tissue environment shape Treg function.

The importance of regulatory T cells (Tregs) in preventing autoimmunity has been well established; however, the precise alterations in Treg function in autoimmune individuals and how underlying genetic associations impact the development and function of Tregs is still not well understood. Polygenetic susceptibly is a key driving factor in the development of autoimmunity, and many of the pathways implicated in genetic association studies point to a potential alteration or defect in regulatory T cell function. In this review transcriptomic control of Treg development and function is highlighted with a focus on how these pathways are altered during autoimmunity. In combination, observations from autoimmune mouse models and human patients now provide insights into epigenetic control of Treg function and stability. How tissue microenvironment influences Treg function, lineage stability, and functional plasticity is also explored. In conclusion, the current efficacy and future direction of Treg-based therapies for Type 1 Diabetes and other autoimmune diseases is discussed. In total, this review examines Treg function with focuses on genetic, epigenetic, and environmental mechanisms and how Treg functions are altered within the context of autoimmunity.

777 Overexpression of Vgll3 induces cutaneous fibrosis in a mouse model of lupus-like autoimmunity using single-cell RNA analyses

Fibrosis is characterized by collagen deposition, fibro/myofibroblast accumulation, and extracellular matrix remodeling. Fibrosis can be seen in autoimmune diseases where it may be widespread and affect organs beyond the skin, with high morbidity and mortality, and no effective treatment. In cutaneous lupus, scar formation after discoid lesion eruption may evolve from enhanced fibrotic phenotypes. Our research has shown that epidermal-directed overexpression of murine Vgll3 causes severe lupus-like skin lesions suggestive of discoid lupus erythematosus (DLE).

B Cell-Specific Deletion of CR6-Interacting Factor 1 Drives Lupus-like Autoimmunity by Activation of Interleukin-17, Interleukin-6, and Pathogenic Follicular Helper T Cells in a Mouse Model.

CR6-interacting factor 1 (CRIF1) is a nuclear transcriptional regulator and a mitochondrial inner membrane protein; however, its functions in B lymphocytes have been poorly defined. This study was undertaken to investigate the effects of CRIF1 on B cell metabolic regulation, cell function, and autoimmune diseases. Using mice with B cell-specific deletion of CRIF1 (Crif1ΔCD19 mice), we assessed the relevance of CRIF1 function for lupus disease parameters, including anti-double-stranded DNA (anti-dsDNA), cytokines, and kidney pathology. RNA sequencing was performed on B cells from Crif1ΔCD19 mice. The phenotypic and metabolic changes in immune cells were evaluated in Crif1ΔCD19 mice. Roquinsan/+ mice crossed with Crif1ΔCD19 mice were monitored to assess the functionality of CRIF1-deficient B cells in lupus development. Crif1ΔCD19 mice showed an autoimmune lupus-like phenotype, including high levels of autoantibodies to dsDNA and severe lupus nephritis with increased mesangial hypercellularity. While loss of CRIF1 in B cells showed impaired mitochondrial oxidative function, CRIF1-deficient B cells promoted the production of interleukin-17 (IL-17) and IL-6 and was more potent in helping T cells develop into follicular helper T cells. In a mouse model of autoimmune lupus, depletion of CRIF1 in B cells exacerbated lupus severity, and CRIF1 overexpression prevented lupus development in roquinsan/san mice. These results demonstrated that CRIF1 negatively correlates with disease severity and that overexpression of CRIF1 ameliorates disease development. Our findings suggest that CRIF1 is essential for preventing lupus development by maintaining B cell self tolerance.

Genetic Variations Controlling Regulatory T Cell Development and Activity in Mouse Models of Lupus-Like Autoimmunity.

Immune homeostasis is a constant balancing act between effector T cells and regulatory T cells defined by Foxp3 expression, the transcription factor that drives their differentiation and immunosuppressive activity. Immune homeostasis is altered when Treg cells are not generated or maintained in sufficient numbers. Treg cells rendered unstable by loss of Foxp3 expression, known as ex-Treg cells, gain pro-inflammatory functions. Treg cells may also become dysfunctional and lose their suppressive capabilities. These alterations can cause an imbalance between effector and regulatory subsets, which may ultimately lead to autoimmunity. This review discusses recent studies that identified genetic factors that maintain Treg cell stability as well as preserve their suppressive function. We focus on studies associated with systemic lupus erythematosus and highlight their findings in the context of potential therapeutic gene targeting in Treg cells to reverse the phenotypic changes and functional dysregulation inducing autoimmunity.

Antigen-specific nanoparticle tolerance treatment actively induces both FoxP3- and IL-10-dependent regulatory mechanisms

Abstract There are many autoimmune diseases where pathologic self T cells are the underlying cause of disease symptoms and progression. T cells are found at the site of tissue destruction, resulting not only in the exacerbation of disease, but also the release of self-epitopes in the context of inflammation resulting in the activation of additional T cell populations perpetuating disease. Published data have shown treatment with antigen-containing biodegradablepoly(lactide-co-glycolide) (PLGA) nanoparticles, i.e. tolerogenic immune-modifying particles (TIMP), is both safe and induces antigen-specific tolerance in mouse models of autoimmunity and allergy, as well as in a celiac disease Phase I/IIa clinical trial. This study further investigated the role and mechanism of TIMP-induced tolerance. The present data show that TIMP treatment modulated T cells specific for spread epitopes associated with disease progression, but not encapsulated within the TIMP, i.e. tissue-specific bystander suppression. The PLP139–151 TCR transgenic model systems was utilized to determine the cellular and molecular mechanisms driving antigen specific tolerance mediated by tolerogenic nanoparticle treatment. These data show antigen-specific TIMP treatment induced both FoxP3+ iTregs and IL-10+ Tr1 regulatory phenotypes within the antigen-specific T cell populations in both naïve mice and mice pre-primed with antigen/CFA. Additionally, both functional Tregs and IL-10 are required for TIMP-induced tolerance. Treatment thus activates various antigen-specific Treg subsets capable of regulating responses to disease-relevant autoepitopes not encapsulated within the TIMP via release of immunoregulatory cytokines within the inflammatory site. Supported by funding provided by Cour Pharmaceuticals Development, Inc.

Tolerogenic Artificial Antigen Presenting Cells for Selective Tolerance in Autoimmune Disease

Abstract Autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes develop when autoreactive immune cells attack healthy cells and tissues. Current treatments are non-curative and leave patients immunocompromised. As an alternative, therapies that expand regulatory T cells (Tregs) are being actively explored to establish antigen-specific tolerance. We recently showed that an IL-2 immunocytokine (IL-2_IC), a fusion of an antibody to IL-2, preferentially engages and expands Tregs over effector T cells. As a more targeted approach to tolerize the immune system towards a specific autoantigen, we explored the design and therapeutic efficacy of a microparticle bearing an IL-2_IC and an MHC II/peptide complex on its surface (TolAPC). Microparticles composed of a blend of PLGA and PBAE were loaded with rapamycin, and then chemically conjugated with IL-2_IC and MHC II I-A(b) tetramers loaded with a peptide derived from myelin oligodendrocyte glycoprotein (MOG). Characterized TolAPC were tested in a mouse model of MS, Experimental Autoimmune Encephalomyelitis (EAE). When administered prophylactically, TolAPC delayed EAE development and reduced severity compared to unconjugated controls, with 36.4% of mice remaining asymptomatic for the duration of the study. When administered therapeutically, TolAPC reversed symptomatic EAE. Clinical improvements correlated with a nearly two-fold increase in Tregs in the brain and spinal cord of TolAPC-treated mice compared to controls six days after treatment. Furthermore, administration of additional booster doses led to sustained amelioration of the disease. Overall, TolAPC can selectively expand Tregs and provide both protection and treatment in an autoimmune mouse model. Supported by grants from DoD (W81XWH-18-1-0735; W81XWH-21-1-0892), NIH (R01EB029455), and JDRF (1-INO-2020-923-A-N)

Evaluating potential toxicities of cancer immunotherapies using autoimmune animal models.

Abstract During the past few decades, cancer immunotherapy has recorded breakthroughs involving therapeutic agents that mediate immune response against several tumor types. However, an important consideration with immunotherapy treatment in the clinic has been the emergence of immune related adverse events (irAEs), that could lead to frequent hospitalization, premature therapy cessation and fatalities, thus limiting the efficacy of these therapies. Additionally, despite the clinical benefits of combining therapies, it has been observed that severity of toxicity increases with combination of therapy. Although, these irAEs are commonly seen in patients receiving immunotherapy, such events have been rarely observed in preclinical mouse models due to their nature: inbred, resistant, and short study duration. Here, we report the use of EAE autoimmune mouse model to evaluate potential toxicities of a novel cancer immunotherapy: anti-CD3ɛ mono-Fab, that is effective as a single therapy and could synergize with checkpoint inhibitors for superior anti-metastatic tumor response. EAE model groups treated with combination therapies showed more disease severity recapitulating the higher grade of irAEs seen in patients receiving combination therapies. The data suggest that autoimmune mouse models could serve as an effective tool to study the severity of toxicity associated with immunotherapeutic agents, and to determine optimized combinatorial regimens for best therapeutic performance. Supported by NCI U01CA244314

Development of uveitis in a mouse model of spontaneous autoimmunity correlates with frequency of autoantigen-specific regulatory T cells

Abstract B6 AireGW/+Lyn−/− mice are a model of spontaneously autoimmunity in which 50% of mice develop uveitis by 9 weeks of age, and the remaining mice do not develop autoimmunity. Uveitis results from CD4+ T cells responding to the retina protein interphotoreceptor retinoid-binding protein (IRBP). To define cellular mechanisms that determine whether mice developed autoimmunity or not, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+ T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+ T cells included distinct subsets that were Ly6c1hi, Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. The two types of effector CD4+ T cells of mice with and without disease largely clustered together relative to their gene expression. Similar analysis of P2-specific CD4+ T cells from retinas with inflammation indicated a progression from proliferative (Mki67hi) cells to a Lag3hi subset to a Lag3hi/Pdcd1hi subset. In mice without uveitis, depletion of Treg by treatment of AireGW/+Lyn−/− Foxp3DTR+/Y mice with diphtheria toxin resulted in rapid expansion of P2–specific CD4+T cells in the draining LN, development of inflammation in the retina, and immune activation in other locations leading to lethality. Ongoing experiments are further testing the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells determines whether or not these genetically susceptible mice develop eye-specific autoimmunity. Supported by AI138479