Biology subjects

Levings, M. K.

Publications and source records attributed to Levings, M. K..

17 recordsLinked to original sources

bioRxiv2025

Human and mouse regenerative macrophages enhance beta cell survival, function, and proliferation

Aims/hypothesisType 1 diabetes is an autoimmune disease caused by immune-mediated destruction of insulin-producing pancreatic beta cells. Interestingly, individuals with long-standing type 1 diabetes have residual beta cells, suggesting the existence of regenerative mechanisms that help maintain beta cell survival. Islet-resident macrophages have an important role in type 1 diabetes, and during disease progression can adopt a tissue-regenerating phenotype which may support beta cells. However, the specific roles of macrophages in beta cell survival, function, and proliferation remains poorly defined. This study aimed to elucidate how different macrophage subtypes influence beta cell survival, function, and proliferation. MethodsMouse and human islets were isolated from the pancreas and co-cultured in vitro with macrophages. To investigate whether macrophages enhance beta cell survival and function, beta cell apoptosis was measured using flow cytometry, and insulin secretion was assessed using a glucose-stimulated insulin secretion assay. We also examined whether macrophages further increased beta cell proliferation in the presence of harmine, a DYRK1A inhibitor. Finally, we evaluated the effect of islet co-culture on macrophage phenotype by flow cytometry and cytokine secretion analysis. ResultsWe found that regenerative, but not pro-inflammatory, macrophages enhanced beta cell survival and function through mechanisms that did not require direct cell contact. Direct contact between macrophages and islets further promoted a regenerative phenotype in macrophages, characterized by increased CD206 expression and secretion of anti-inflammatory factors. Additionally, regenerative macrophages promoted beta cell proliferation in the presence of harmine. ConclusionsOur findings demonstrate that regenerative macrophages support pancreatic beta cell survival, function, and proliferation. Harnessing the regenerative properties of macrophages could offer a novel strategy to promote beta cell survival and function, thereby improving outcomes for individuals with type 1 diabetes. Research in ContextWhat is already known about this subject? O_LIMacrophages are the predominant resident immune cells within pancreatic islets in non-diabetic individuals; they contribute to tissue homeostasis and immune surveillance. C_LIO_LIDepending on their activation state, macrophages can exert either beneficial (pro-regenerative) or harmful (pro-inflammatory) effects on beta cell function. C_LI What is the key question? O_LIHow do different macrophage subtypes regulate islet survival, regeneration, and function? C_LI What are the new findings? O_LICo-culturing mouse or human islets with regenerative macrophages enhanced beta cell survival and function. When added in the presence of a DYRK1A inhibitor (harmine), they also promoted beta cell proliferation. C_LIO_LIRegenerative macrophage-derived factors promoted mouse islet function via a contact independent mechanism. C_LIO_LIMouse islets enhanced the regenerative phenotype of macrophages. C_LI How might this impact on clinical practice in the foreseeable future? O_LILeveraging the regenerative potential of macrophages represents a novel therapeutic approach to enhance beta cells, ultimately improving outcomes for individuals with type 1 diabetes. C_LI

cell biology↗

bioRxiv2025

Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through development of automated flow cytometric gating software, which had superior reproducibility compared to manual analysis. We also characterized antigen-specific Tregs, finding that gating on a combination of CD134, CD137, FOXP3 and HELIOS optimally detects antigen-specific cells. The combined methodology results in a high degree of reproducibility within and between research groups and provides a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings. MOTIVATIONReliable detection of antigen-specific T cells is critical to understand immune responses to infection and vaccination, and has translational potential to monitor T cell responses across diverse clinical settings. Activation-induced marker (AIM) assays offer a variety of advantages over methods such as ELISPOT and tetramers, but are limited by methodological heterogeneity between research groups and a lack of standardized protocols. As such, the degree to which AIM assay results are reproducible is unknown. Key variables such as cell source, media, stimulation time, marker selection for CD4+ T cells, CD8+ T cells and regulatory T cells; as well as data analysis parameters such as flow cytometric gating strategies and mathematical correction for background AIM+ frequencies in unstimulated control samples, have not been rigorously studied. To address this, we comprehensively characterized variability in AIM assays, including within and between operators and across multiple research centres, and sought to optimize a standard AIM workflow to enhance reproducibility at both the experimental and analytical levels.

immunology↗

bioRxiv2025

An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

Siglecs are immunomodulatory receptors that regulate immune cell function. A fundamental challenge in studying Siglec-ligand interactions is the low affinity of Siglecs for their ligands. Inspired by how nature uses multivalency, we developed Siglec-liposomes as a highly multivalent and versatile platform for detecting Siglec glycan ligands in which recombinant Siglecs were conjugated to liposomes using the SpyCatcher-SpyTag system. Siglec-liposomes offer tunable multivalency and a modular assembly, enabling presentation of different Siglecs on the same liposome. Using Siglec-liposomes, we profiled Siglec ligands on human leukocytes, revealing new insights into Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin-dependent manner. Given the abundance of Siglec ligands on T cells, we investigated whether Siglec-liposomes modulate T cell function and find that Siglec-7-liposomes increase T cell proliferation in a ST3Gal1-dependent and CD43-independent manner. Taken together, Siglec-liposomes are a versatile and sensitive tool for detecting Siglec ligands and immunomodulation.

biochemistry↗

bioRxiv2024

Tunable differentiation of human CD4+ and CD8+ T cells from pluripotent stem cells

SummaryAllogeneic T cell therapies are a highly desirable option to circumvent the cost and complexity of using autologous T cells to treat diseases. Allogeneic CD8+ T cells can be made from pluripotent stem cells (PSCs), but deriving CD4+ T cells from PSCs remained a significant challenge. Using feeder-and serum-free conditions, we found that CD4+ versus CD8+ T cell commitment from PSCs can be controlled by fine-tuning the dynamics of Notch and T cell receptor signaling delivered to CD4+CD8+ double positive T cells. Notch signaling negatively impacts CD4+ T cell commitment, and its timed removal allows generation of clonally-diverse and expandable CD4+ T cells from PSCs. The resulting CD4+ T cells respond to cytokine-mediated polarization by differentiating into Th1, Th2, or Th17 cells, recapitulating canonical helper cell function. These findings represent a significant step towards using PSC-derived CD4+ T cells as a low cost, off-the-shelf, cell therapy.

bioengineering↗

bioRxiv2024

TYK2 inhibition enhances Treg differentiation and function while preventing Th1 and Th17 differnetiation

Janus kinase (JAK) inhibitors are widely use to inhibit inflammatory cytokine signalling in autoimmune and inflammatory diseases but their effect on regulatory T cells (Tregs) is poorly characterized. We investigated the effect of JAK inhibition on human Treg differentiation, phenotype, and function using a JAK inhibitor, upadacitinib, in comparison to BMS-986202, a selective Tyrosine kinase 2 (TYK2) inhibitor. Both upadacitinib and BMS-986202 blocked the differentiation of naive CD4+ T cells into Th1 and Th17 cells, but only BMS-986202 spared IL-2 signalling and Treg differentiation. BMS-986202 also increased Treg suppressive function and stability under Th1- and Th17-polarizing conditions, whereas upadacitinib significantly impaired the phenotype and viability of ex vivo Tregs. Analysis of lamina propria mononuclear cells from patients with inflammatory bowel disease revealed that, under Th17 polarizing conditions, BMS-986202 redirected CD4+ T cells towards a Treg phenotype. The Treg-sparing and enhancing properties of TYK2 inhibition suggest that TYK2 inhibitors are a promising pharmacological approach for tolerance induction. eTOC SUMMARYTuomela et al. report that TYK2 inhibition does not affect human Treg induction from naive CD4+ T cells, promotes Treg differentiation in lamina propria-derived T cells, and increases blood-derived Treg stability/function. In contrast, JAK inhibition strongly impairs Treg function.

immunology↗

bioRxiv2024

Fourth generation CAR Tregs with PDCD1-driven IL-10 have enhanced suppressive function

The potency of regulatory T cell (Treg) therapy has been transformed through use of chimeric antigen receptors (CAR). However, to date, CAR Treg therapy has not achieved long-lasting tolerance in mouse models, suggesting that additional engineering is required to unlock the full potential of these cells. We previously found that human Tregs produce minimal amounts of IL-10 and have a limited capacity to control innate immunity in comparison to type I regulatory (Tr1) cells. Seeking to create "hybrid" CAR Tregs that were engineered with Tr1-like properties, we examined whether the PDCD1 locus could be exploited to endow Tregs with the ability to secrete high levels of IL-10 in a CAR-regulated manner. CRISPR-mediated PD1-deletion increased the activation potential of CAR Tregs without compromising in vivo stability. Knock-in of IL10 under control of the PD1 promoter facilitated CAR-mediated secretion of IL-10 in large quantities, and improved CAR Treg function, as determined by significant inhibition of dendritic cell antigen presentation and enhanced suppression of alloantigen- and islet autoantigen-specific T cells. Overall, CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance suppressive mechanisms is a new approach to enhance the therapeutic potency of CAR Tregs.

immunology↗

bioRxiv2024

Efficacy and limitations of combined A2.CAR Treg and anti-CD154 therapy in a mouse model of haplo-mismatched heart transplantation

Successful allograft specific tolerance induction would eliminate the need for daily immunosuppression and improve post-transplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific Chimeric Antigen Receptors (CAR-Tregs) is a promising strategy, but as monotherapy, cannot prolong the survival with allografts with multiple MHC mismatches. Using an HLA-A2-transgenic haplo-mismatched heart transplantation model in immunocompetent C57Bl/6 recipients, we show that HLA-A2-specific (A2) CAR Tregs was able to synergize with low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited endogenous non-A2 donor- specific T cell, B cell and antibody responses, and promoted a significant increase in endogenous FoxP3+Tregs with indirect donor-specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FoxP3neg A2.CAR T cells preferentially accumulated in rejecting allografts and endogenous donor-specific responses were not controlled. This study therefore provides the first evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity towards allograft tolerance.

immunology↗

bioRxiv2024

CAR Tregs mediate linked suppression and infectious tolerance in islet transplantation

Regulatory T cells (Tregs) have potential as a cell-based therapy to prevent or treat transplant rejection and autoimmunity. Using an HLA-A2-specific chimeric antigen receptor (A2-CAR), we previously showed that adoptive transfer of A2-CAR Tregs limited anti-HLA-A2 alloimmunity. However, it was unknown if A2-CAR Tregs could also limit immunity to autoantigens. Using a model of HLA-A2+ islet transplantation into immunodeficient non-obese diabetic mice, we investigated if A2-CAR Tregs could control diabetes induced by islet-autoreactive (BDC2.5) T cells. In mice transplanted with HLA-A2+ islets, A2-CAR Tregs reduced BDC2.5 T cell engraftment, proliferation and cytokine production, and protected mice from diabetes. Tolerance to islets was systemic, including protection of the HLA-A2negative endogenous pancreas. In tolerant mice, a significant proportion of BDC2.5 T cells gained FOXP3 expression suggesting that long-term tolerance is maintained by de novo Treg generation. Thus, A2-CAR Tregs mediate linked suppression and infectious tolerance and have potential therapeutic use to simultaneously control both allo- and autoimmunity in islet transplantation. One Sentence SummaryAlloreactive chimeric antigen receptor-engineered regulatory T cells limit diabetogenic T cell engraftment and function to prevent type 1 diabetes.

immunology↗

bioRxiv2023

Sex biased human thymic architecture guides T cell development through spatially defined niches

Within the thymus, regulation of the cellular cross-talk directing T cell development is dependent on spatial interactions within specialized niches. To create a holistic, spatially defined map of tissue niches guiding postnatal T cell development we employed the multidimensional imaging platform CO-detection by indEXing (CODEX), as well as CITE-seq and ATAC-seq. We generated age-matched 4-5-month-old postnatal thymus datasets for male and female donors, and identify significant sex differences in both T cell and thymus biology. We demonstrate a crucial role for JAG ligands in directing thymic-like dendritic cell development, reveal important functions of a novel population of ECM- fibroblasts, and characterize the medullary niches surrounding Hassalls corpuscles. Together, these data represent a unique age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and T cell development arise, and provide an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

immunology↗

bioRxiv2023

Insulin B peptide-MHC class II-specific chimeric antigen 1 receptor-Tregs prevent autoimmune diabetes

Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs stably expressed Foxp3 and prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes. Brief SummaryChimeric antigen receptor Tregs specific for an insulin B-chain peptide presented by MHC class II prevent autoimmune diabetes.

immunology↗

bioRxiv2023

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease

BackgroundType 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). MethodsWe expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. ResultsThe speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. ConclusionsInjection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

immunology↗

bioRxiv2022

Integration of exogenous and endogenous co-stimulatory signals by CAR-Tregs

Regulatory T cells (Tregs) expressing chimeric antigen receptors (CAR) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs) and donor-specific antibodies. We generated mouse Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo. In vitro assays revealed the CD28-encoding CAR had superior antigen-specific suppression, proliferation and cytokine production. In contrast, in vivo protection from skin allograft rejection and alloantibody production was similar between Tregs expressing CARs encoding CD28, ICOS or PD1, but not GITR, 41BB or OX40, co-stimulatory domains. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3{zeta} but no co-stimulatory domain. These data revealed that exogenous co-stimulation via APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo. This study reveals a new dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

immunology↗

bioRxiv2022

PTEN is required for human Treg suppression of costimulation

Regulatory T cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signalling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernable defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signalling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

immunology↗

bioRxiv2021

Helios is a marker, not a driver, of human Treg stability

Regulatory T cell (Treg) therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T cells and thus exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Tregs lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid)-expressing population correlated with Treg destabilization. We then used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-knockout and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T cells.

immunology↗

bioRxiv2021

Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

immunology↗

bioRxiv2020

Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how two methods which induce conventional T cell exhaustion – repetitive stimulation or expression of a tonic-signaling chimeric antigen receptor (CAR) – affect human Tregs. With each repetitive polyclonal stimulation Tregs progressively acquired an exhausted phenotype, and became less suppressive in vitro. Tregs expressing a tonic-signaling CAR rapidly acquired an exhausted phenotype and had major changes in their transcriptome and metabolism. Although tonic-signaling CAR-Tregs remained stable and suppressive in vitro, they lost in vivo function, as tested in a model of xenogeneic graft-versus-host disease. The finding that human Tregs are susceptible to exhaustion has important implications for the design of Treg adoptive immunotherapy strategies.Competing Interest StatementMKL received research funding from TxCell, Bristol-Myers Squibb, Pfizer, Takeda and CRISPR Therapeutics for work unrelated to this report. CLM holds several patent applications in the area of CAR based immunotherapy, and is a founder of, holds equity in, and receives consulting fees from Lyell Immunopharma which develops CAR based therapies. EWW holds several patent applications in the area of CAR based immunotherapy and receives consulting fees from Lyell Immunopharma. CLM is a member of the Parker Institute for Cancer Immunotherapy, which supports the Stanford University Cancer Immunotherapy Program.AbbreviationsCARChimeric antigen receptorECARextracellular acidification rateOCARoxygen consumption rateTconvconventional T cellTregRegulatory T cellTStonic signalingTSDRRegulatory T-cell-Specific Demethylated RegionView Full Text

immunology↗

bioRxiv2019

Functional effects of chimeric antigen receptor co-receptor signaling domains in human Tregs

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptor (CARs) are a potent immunosuppressive cellular therapy in multiple disease models. To date the majority of CAR Treg studies employed second generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3{zeta}, but it was not known if this CAR design was optimal for Tregs. Using an HLA-A2-specific CAR platform and human Tregs, we compared ten CARs with different co-receptor signaling domains and systematically tested their function. Tregs expressing a CAR encoding wild-type CD28 were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and ability to suppress CD80 expression on DCs as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling-domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

immunology↗