Biology subjects

Blazar, B. R.

Publications and source records attributed to Blazar, B. R..

11 recordsLinked to original sources

bioRxiv2026

Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

immunology↗

bioRxiv2026

Mechanistic machine learning enables interpretable and generalizable prediction of prime editing outcomes

Although prime editing (PE) can effect virtually any specified local change to genomic DNA in living systems, its efficient application currently requires extensive optimization of prime editing guide RNA (pegRNA) sequences. We present OptiPrime, a machine learning model of PE efficiency based on our current understanding of the mechanism of prime editing. OptiPrime achieves state-of-the-art accuracy on PE efficiency prediction and also enables prediction of nicking guide RNA (PE3) and dual pegRNA (twinPE) outcomes. We validated that OptiPrime has learned the determinants of mammalian mismatch repair (MMR), and is therefore well suited for nominating MMR-evasive silent edits that improve PE efficiency. We demonstrate the utility of OptiPrime in a variety of prospective therapeutic contexts, including in primary human and mouse cells. Finally, we show how OptiPrime can be used to achieve highly streamlined and efficient in vivo correction of a pathogenic mutation in the brain of a mouse model of KIF1A-associated neurological disorder.

biochemistry↗

bioRxiv2025

Keratinocyte VISTA attenuates UV light-induced skin injury by suppressing cutaneous type I interferon (IFN-I) response

Persistent production of type I interferons (IFN-Is) is a hallmark of cutaneous lupus erythematosus (CLE). Ultraviolet (UV) light stimulates IFN-I response in the skin and exacerbates CLE. Here, we identify V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) as a negative regulator of both basal and UV-induced IFN-I responses in the skin and show that VISTA limits skin photosensitivity in an IFN-I- dependent manner, in part through Stimulator of Interferon Genes (STING). Furthermore, we demonstrate a novel role for VISTA in keratinocytes both at steady state and in response to UV light. Conditional deletion of VISTA in epidermal keratinocytes results in >10-fold increase in the basal skin IFN-I score and a heightened UV-induced skin injury score, which is dependent on IFN-I signaling. VISTA-targeting monoclonal antibodies suppress UV-induced IFN-I response in human keratinocytes and in mice expressing human VISTA in vivo, which also reduces UV-induced skin injury score. Our findings highlight VISTA as a potential therapeutic target for suppressing IFN-I responses in cutaneous lupus patients who suffer from UV-induced skin inflammation.

immunology↗

bioRxiv2025

Precision Editing of Cyclophilin A Generates Cyclosporine and Voclosporin Resistant Cellular Therapies

Recipients of allogeneic transplants or patients with autoimmune disease require immune suppression, often with calcineurin inhibitors. There is an expanding repertoire of immune effector cell therapies, including CD19 CAR-T cells and viral-specific T cells, deployed in these patients; however, ongoing calcineurin inhibition may be detrimental to cell therapy function. We developed a CRISPR/Cas9-based approach to engineer dual cyclosporine/voclosporin resistant cell therapies by targeting PPIA (encoding cyclophilin A), a critical binding partner for both drugs. Because Cyclophilin A has homeostatic functions in T cells, a complete knock-out is detrimental to cell viability. We thus targeted its C-terminus, disrupting drug binding while leaving the majority of the protein intact. C-terminal editing was stable throughout expansion and preserved Cyclophilin A expression. Edited CD19 CAR-T cells retained effector function in the presence of cyclosporine and voclosporin, including proliferation, cytokine production, and target cell killing, resulting in improved survival in murine models of CD19+ leukemia. Edited CMV-specific T cells also demonstrated preserved antigen-specific proliferation and cytokine production in the presence of these drugs. C-terminal editing of Cyclophilin A offers a promising avenue for developing next-generation cell therapies for patients receiving calcineurin inhibitors.

immunology↗

bioRxiv2024

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the {beta}-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

biochemistry↗

bioRxiv2024

Regulatory T cells crosstalk with tumor and endothelium through lymphotoxin signaling

Regulatory T cells (Tregs) are suppressors of anti-tumor immunity that exert multifaceted functions by signaling surrounding cells. We revealed Tregs use their high-level surface lymphotoxin (LT)1{beta}2 to preferentially stimulate LT{beta} receptor (LT{beta}R) nonclassical NF{kappa}B signaling on both tumor and lymphatic endothelial cells (LECs) to accelerate tumor growth and metastasis. Selectively targeting LT{beta}R nonclassical NF{kappa}B pathways on both tumors and LECs cocultured with Tregs, inhibited tumor growth and migration in vitro. Further, we identified protumorigenic chemokines and interferon-stimulated response genes selectively driven by LT{beta}R nonclassical NF{kappa}B in melanoma cells. Endothelial specific genes related to oncogenic process such as SOX18 and FLRT2 were identified to be driven under LT{beta}R nonclassical NF{kappa}B in LECs. Leveraging in vivo Treg LT1{beta}2 interactions with LT{beta}R on tumor and LECs, transfer of WT but not LT-deficient Tregs promoted transplanted WT B16F10 growth and tumor cell-derived CXCL1 and CXCL10 secretion in LT{beta}R-deficient host mice, and increased endothelial specific genes related to tumor angiogenesis and lymphangiogenesis, in WT mice bearing LT{beta}R-depleted melanoma. Selectively blocking LT{beta}R nonclassical NF{kappa}B pathways remarkably suppressed tumor growth and lymphatic metastasis by reducing tumor cell and LEC-derived CXCL1 and CXCL10 production, restricting Treg and myeloid-derived suppressor cell (MDSC) recruitment to tumor. It also retained intratumoral effector T cells, especially IFN{gamma}+ CD8 T cells by restraining Treg facilitated lymphatic vessel permeability. Our data revealed that Treg LT1{beta}2 promotes LT{beta}R nonclassical NF{kappa}B signaling in tumor cells and LECs providing a rational strategy to modulate Treg-mediated protumorigenic molecules to prevent tumor growth and metastasis.

cancer biology↗

bioRxiv2022

Evolutionarily conserved effects of Notch signaling drive intestinal graft-versus-host disease in mice and non-human primates

Notch signaling promotes T-cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like ligand DLL4. To assess if Notchs effects are evolutionarily conserved and identify key mechanisms, we studied antibody-mediated DLL4 blockade in a non-human primate model similar to human allo-HCT. Short-term DLL4 blockade improved post-transplant survival with striking, durable protection from gastrointestinal GVHD, out of proportion to other disease sites. Unlike prior immunosuppressive strategies, anti-DLL4 interfered with a T-cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin a4b7 in conventional T-cells via b1 competition for a4 binding, while preserving a4b7 in regulatory T-cells. Thereby, DLL4/Notch blockade decreased effector T-cell infiltration into the gut, with increased regulatory to conventional T-cell ratios early after allo-HCT. Our results identify a conserved, biologically unique and targetable role of DLL4/Notch signaling in GVHD. One Sentence SummaryNotch signaling promotes pathogenic effector T cell infiltration of the intestine during acute graft-versus-host disease.

immunology↗

bioRxiv2020

Restructuring of the Immune Contexture Improves Checkpoint Blockade Efficacy in Murine Lung Cancer

Therapeutic options for non-small cell lung cancer (NSCLC) treatment have changed dramatically in recent years with the advent of novel immunotherapeutic approaches. Among these, immune checkpoint blockade (ICB), using monoclonal antibodies, has shown tremendous promise in a small proportion of patients. In order to better predict patients that will respond to ICB treatment, biomarkers such as tumor-associated CD8+ T cell frequency, tumor checkpoint protein status and mutational burden have been utilized, however, with mixed success. In this study, we hypothesized that significantly altering the suppressive tumor immune landscape in NSCLC could potentially improve ICB efficacy. Using sub-therapeutic doses of our Salmonella typhimurium-based therapy targeting the suppressive molecule indoleamine 2,3-dioxygenase (shIDO-ST) in tumor-bearing mice, we observed dramatic changes in immune subset phenotypes that included increases in antigen presentation markers, decreased regulatory T cell frequency and overall reduced checkpoint protein expression. Combination shIDO-ST treatment with anti-PD-1/CTLA-4 antibodies enhanced tumor growth control, compared to either treatment alone, which was associated with a significant intratumoral influx of CD8+ and CD4+ T lymphocytes. These results suggest that the success of ICB therapy may be more accurately predicted by taking into account multiple factors such as potential for antigen presentation and frequency of suppressive immune subsets in addition to markers already being considered. Alternatively, combination treatment with agents such as shIDO-ST could be used to create a more conducive tumor microenvironment for improving response rates to immunotherapy.

cancer biology↗

bioRxiv2020

Spatiotemporal single-cell profiling of gastrointestinal GVHD reveals invasive and resident memory T cell states.

One of the central challenges in the field of allo-immunity is deciphering the mechanisms driving T cells to infiltrate and subsequently occupy target organs to cause disease. The act of CD8-dominated T cell infiltration is critical to acute graft-versus-host disease (aGVHD), wherein donor T cells become activated, tissue-infiltrating and highly cytotoxic, causing wide-spread tissue damage after allogeneic hematopoietic stem cell transplant (allo-HCT). However, in human and non-human primate studies, deconvolving the transcriptional programs of newly recruited relative to resident memory T cells in the gastrointestinal (GI) tract has remained a challenge. In this study, we combined the novel technique of Serial Intravascular Staining (SIVS) with single-cell RNA-Seq (scRNA-seq) to enable detailed dissection of the tightly connected processes by which T cells first infiltrate tissues and then establish a pathogenic tissue residency program after allo-HCT in non-human primates. Our results have enabled the creation of a spatiotemporal map of the transcriptional drivers of CD8 T cell infiltration into the primary aGVHD target-organ, the GI tract. We identify the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphdepletion-driven T cell infiltration. The donor CD8 T cells that infiltrate the GI tract demonstrate a highly activated, cytotoxic phenotype while simultaneously rapidly developing canonical tissue-resident memory (TRM) protein expression and transcriptional signatures, driven by IL-15/IL-21 signaling. Moreover, by combining SIVS and transcriptomic analysis, we have been able to work backwards from this pathogenic TRM programing, and, for the first time, identify a cluster of genes directly associated with tissue invasiveness, prominently including specific chemokines and adhesion molecules and their receptors, as well as a central cytoskeletal transcriptional node. The clinical relevance of this new tissue invasion signature was validated by its ability to discriminate the CD8 T cell transcriptome of patients with GI aGVHD. These results provide new insights into the mechanisms controlling tissue infiltration and pathogenic CD8 TRM transcriptional programing, uncovering critical transitions in allo-immune tissue invasion and destruction. One sentence summaryFlow cytometric and transcriptomic analysis reveals coordinated tissue-infiltration and tissue-residency programs driving gastrointestinal aGVHD.

immunology↗

bioRxiv2019

Ribosome-targeting antibiotics impair T cell effector function and ameliorate autoimmunity by blocking mitochondrial protein synthesis

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive, for reasons that remain unclear. Here we show that linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of Th17 effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromises the integrity of the electron transport chain (ETC). Ultimately, this leads to loss of mitochondrial metabolism and cytokine production in differentiating Th cells. In accordance, mice lacking Gfm1 in T cells are protected from EAE, demonstrating that this pathway plays a key role in maintaining T cell function and pathogenicity.

immunology↗

bioRxiv2018

TRAF6 directs Foxp3 localization and facilitates Treg function through K63-type ubiquitination

Regulatory T cells (Treg) are crucial mediators of immune control. The characteristic gene expression and suppressive function of Treg depend considerably on the stable expression and activity of the transcription factor Foxp3. While transcriptional regulation of the Foxp3 gene has been studied in depth, both the expression and function of Foxp3 are also modulated at the protein level. However, the molecular players involved in posttranslational Foxp3 regulation are just beginning to be elucidated. Here we found TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to B16 melanomas and displayed enhanced anti-tumor immunity. We further determined that Foxp3 undergoes lysine-63 chain (K63) ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, Foxp3 displayed aberrant, perinuclear accumulation, disrupted function. Thus, Foxp3 ubiquitination by TRAF6 ensures proper localization of Foxp3 and facilitates Foxp3s gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing force that may be targeted in novel tolerance-breaking therapies.

immunology↗