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The number of people being diagnosed with heart failure in the UK continues to grow, and the poorest people are significantly more likely to be affected by the condition, new research from The George Institute for Global Health at the University of Oxford has found. The research was co-led by Professor Kazem Rahimi, and RDM Principal Investigator.
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Sideways lipid presentation by the antigen-presenting molecule CD1c.
For the MHC, MR1 and CD1 systems, antigen recognition involves contact of the membrane distal surfaces of both the αβ T cell receptor (TCR) and the antigen-presenting molecule. Whether other antigen display mechanisms by antigen-presenting molecules operate remains unknown. Here, we report mass spectrometry analyses of endogenous lipids captured by CD1c when bound to an autoreactive αβ TCR. CD1c binds twenty-six lipid species with bulky headgroups that cannot fit within the tight TCR-CD1c interface. We determined the crystal structures of CD1c presenting several gangliosides, revealing a general mechanism whereby two lipids, rather than one, are bound in the CD1c cleft. Bulky lipids are oriented sideways so that their polar headgroups protrude laterally through a side portal of the CD1c molecule - an evolutionarily conserved structural feature. The sideways-presented ganglioside headgroups do not hinder TCR binding and so represent a mechanism that allows autoreactive TCR recognition of CD1c. In addition, ex vivo studies showed that the sideways-presented gangliosides can also represent TCR recognition determinants. These findings reveal that CD1c simultaneously presents two lipid antigens from the top and side of its cleft, a general mechanism that differs markedly from other antigen-presenting molecules.
Bridging science and accessibility: a tactile journey from gluten through to coeliac disease.
As part of the Monash Sensory Science Exhibition, our team guided participants through a multisensory journey unraveling coeliac disease development and pathology. Through tactile and sensory exhibits, we showed how benign dietary gluten can be transformed into a harmful entity for the 1 in 70 Australians with this illness. In contrast to the common misconception of coeliac disease as a food allergy, our exhibits revealed its closer association with autoimmune diseases such as type 1 diabetes, involving genetic susceptibility linked to specific human leukocyte antigens, crucial antigen-specific T- and B-cell responses and autoantibody production. Tactile models underscored the severe consequences of the proinflammatory immune response to gluten on patient health and quality of life. This educational event affirmed to us the value and importance of fostering inclusivity in science education.
Access all areas: multisensory science exhibitions tailored toward blind, low-vision and diverse-needs communities.
Monash Sensory Science is a scientific outreach initiative specifically tailored to members of the community who are blind, have low vision and have diverse needs. The purpose of this initiative is to showcase Australian science and encourage greater participation in science from these often-overlooked communities. This article presents our experience in establishing Monash Sensory Science at Monash University and inspiring other institutions to launch similar outreach events.
Conveying the pathogenesis of type 1 diabetes to the blind, low-vision and diverse needs communities through sensory stimulation.
To educate members of the blind, low-vision and diverse needs communities on the pathogenesis of the chronic autoimmune disease, type 1 diabetes, members of our team with research expertise in immune-mediated diseases, participated in the 2023 Monash Sensory Science (MSS) Exhibition. Using QR code linked audio commentary, participants were guided through tactile displays demonstrating normal insulin action in the regulation of blood glucose levels and its vital role in providing energy to tissues, followed by displays describing the various stages of the immune system's aberrant attack and the eventual complete destruction of the insulin producing beta-cells of the pancreatic islets in type 1 diabetes. These models conveyed to the participants the huge effect that this autoimmune-mediated disease has on the quality of life of affected individuals including the subsequent lifelong reliance on insulin injections to maintain glucose homeostasis. This MSS Exhibition provided a unique opportunity for our researchers to engage with under-represented members of the community and to raise awareness about such a debilitating and common autoimmune disease.
Structural bases of T cell antigen receptor recognition in celiac disease.
Celiac disease (CeD) is a human leukocyte antigen (HLA)-linked autoimmune-like disorder that is triggered by the ingestion of gluten or related storage proteins. The majority of CeD patients are HLA-DQ2.5+, with the remainder being either HLA-DQ8+ or HLA-DQ2.2+. Structural studies have shown how deamidation of gluten epitopes engenders binding to HLA-DQ2.5/8, which then triggers an aberrant CD4+ T cell response. HLA tetramer studies, combined with structural investigations, have demonstrated that repeated patterns of TCR usage underpins the immune response to some HLADQ2.5/8 restricted gluten epitopes, with distinct TCR motifs representing common landing pads atop the HLA-gluten complexes. Structural studies have provided insight into TCR specificity and cross-reactivity towards gluten epitopes, as well as cross-reactivity to bacterial homologues of gluten epitopes, suggesting that environmental factors may directly play a role in CeD pathogenesis. Collectively, structural immunology-based studies in the CeD axis may lead to new therapeutics/diagnostics to treat CeD, and also serve as an exemplar for other T cell mediated autoimmune diseases.
Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease.
Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3β loop of the TCR and Arg70β from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.
T cell receptor cross-reactivity between gliadin and bacterial peptides in celiac disease.
The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD.
Discriminative T-cell receptor recognition of highly homologous HLA-DQ2-bound gluten epitopes.
Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5-restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5-DQ2.5-glia-α1a and HLA-DQ2.5-DQ2.5-glia-ω1 tetramers and single-cell αβ T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide-HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a- and DQ2.5-glia-ω1-reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5-peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide-HLA-II landscape in a human disease setting.
Phenoscaping Reveals Multimodal γδ T-cell Cytotoxicity as a Strategy to Overcome Cancer Cell-Mediated Immunomodulation.
UNLABELLED: γδ T cells can kill cancer cells via antibody-independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC). A better understanding of how these cytotoxic mechanisms are affected by different cancer cells and different T-cell donors could help identify improved immunotherapeutic strategies. To test the combinatorial interactions among T cell interdonor heterogeneity, cancer cell intertumor heterogeneity (ITH), and multimodal γδ T-cell killing, we performed a systematic single-cell phenoscaping analysis of more than 1,000 γδ T-cell and colorectal cancer patient-derived organoid cultures. Single-cell analysis of posttranslational modification (PTM) signaling, cell cycle, apoptosis, and T-cell immunophenotypes revealed that whereas unmodified γδ T cells have limited antitumor activity, IL15Rα-IL15 fusion protein [stabilized IL15 (stIL15)]-engineered γδ T cells can kill patient-derived organoids via AIC without exogenous cytokine support. However, when stIL15 γδ T cells only killed via AIC, cancer cells reciprocally rewired γδ T-cell PTM signal networks in an ITH-specific manner to suppress anticancer cytotoxicity. stIL15 γδ T cells could overcome this cancer cell immunomodulation by also engaging B7-H3-targeted ADCC independent of B7-H3 checkpoint activity. Combined AIC and ADCC rescued γδ T-cell PTM signaling flux and enabled γδ T cells to kill chemorefactory revival colon cancer stem cells. Together, these results demonstrate that multimodal γδ T-cell cytoxicity mechanisms can overcome ITH-specific immunomodulation to kill chemorefractory cancer cells. SIGNIFICANCE: Single-cell phenoscaping of more than 1,000 γδ T-cell and patient-derived organoid cultures shows that cancer cells suppress anticancer γδ T-cell cytotoxicity but γδ T cells can use multimodal killing to overcome immunomodulation.
Oncogenic PIK3CA corrupts growth factor signaling specificity.
Technical limitations have prevented understanding of how growth factor signals are encoded in distinct activity patterns of the phosphoinositide 3-kinase (PI3K)/AKT pathway, and how this is altered by oncogenic pathway mutations. We introduce a kinetic, single-cell framework for precise calculations of PI3K-specific information transfer for different growth factors. This features live-cell imaging of PI3K/AKT activity reporters and multiplexed CyTOF measurements of PI3K/AKT and RAS/ERK signaling markers over time. Using this framework, we found that the PIK3CAH1047R oncogene was not a simple, constitutive activator of the pathway as often presented. Dose-dependent expression of PIK3CAH1047R in human cervical cancer and induced pluripotent stem cells corrupted the fidelity of growth factor-induced information transfer, with preferential amplification of epidermal growth factor receptor (EGFR) signaling responses compared to insulin-like growth factor 1 (IGF1) and insulin receptor signaling. PIK3CAH1047R did not only shift these responses to a higher mean but also enhanced signaling heterogeneity. We conclude that oncogenic PIK3CAH1047R corrupts information transfer in a growth factor-dependent manner and suggest new opportunities for tuning of receptor-specific PI3K pathway outputs for therapeutic benefit.
Functional analysis of cell plasticity using single-cell technologies.
Metazoan organisms are heterocellular systems composed of hundreds of different cell types, which arise from an isogenic genome through differentiation. Cellular 'plasticity' further enables cells to alter their fate in response to exogenous cues and is involved in a variety of processes, such as wound healing, infection, and cancer. Recent advances in cellular model systems, high-dimensional single-cell technologies, and lineage tracing have sparked a renaissance in plasticity research. Here, we discuss the definition of cell plasticity, evaluate state-of-the-art model systems and techniques to study cell-fate dynamics, and explore the application of single-cell technologies to obtain functional insights into cell plasticity in healthy and diseased tissues. The integration of advanced biomimetic model systems, single-cell technologies, and high-throughput perturbation studies is enabling a new era of research into non-genetic plasticity in metazoan systems.
Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.
Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.
An oncogenic phenoscape of colonic stem cell polarization.
Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell fate, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-β-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs and disrupt stromal-epithelial communication-trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity.
Multiplexed single-cell analysis of organoid signaling networks.
Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type-specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type-specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run.
Deciphering Organoids: High-Dimensional Analysis of Biomimetic Cultures.
Organoids are self-organising stem cell-derived ex vivo cultures widely adopted as biomimetic models of healthy and diseased tissues. Traditional low-dimensional experimental methods such as microscopy and bulk molecular analysis have generated remarkable biological insights from organoids. However, as complex heterocellular systems, organoids are especially well-positioned to take advantage of emerging high-dimensional technologies. In particular, single-cell methods offer considerable opportunities to analyse organoids at unprecedented scale and depth, enabling comprehensive characterisation of cellular processes and spatial organisation underpinning organoid heterogeneity. This review evaluates state-of-the-art analytical methods applied to organoids, discusses the latest advances in single-cell technologies, and speculates on the integration of these two rapidly developing fields.