The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group ...including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0(SLC1A5). Both RDR and ATB0serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.
The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on ...the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.
•Lentiviral vectors displaying T-cell activation and costimulatory molecules (VivoVec) generate CAR T cells in vivo without lymphodepletion.•VivoVec administration in nonhuman primates generates ...anti-CD20 CAR T cells in vivo, leading to prolonged complete B-cell depletion.
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Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.
Abstract
Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively ...little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro‐survival BCL‐2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post‐translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK‐dependent apoptosis and the subsequent NLRP3 inflammasome‐dependent and inflammasome‐independent maturation of the inflammatory cytokine IL‐1β. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL‐1β activation triggered by
Neisseria gonorrhoeae
‐derived outer membrane vesicles (NOMVs). Consequently, A1‐deficient mice exhibit heightened IL‐1β production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host‐directed antimicrobial therapeutics.
Synopsis
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Cell death and inflammatory signalling crosstalk is critical for innate immune responses to pathogens. This study identifies the BCL‐2 family member A1 as a temporal regulator of these processes in myeloid cells upon bacterial recognition.
LPS‐induced A1 expression in macrophages and monocytes delays apoptosis and NLRP3 inflammasome activation.
Monocyte survival upon pathogen sensing is regulated by the short‐lived pro‐survival proteins MCL‐1 and A1.
A1‐deficient myeloid cells can engage necroptotic signalling when apoptotic caspase activity is compromised.
A1‐deficiency enhances IL‐1β responses to
Neisseria gonorrhoeae
‐derived outer membrane vesicles (NOMVs).
IntroductionThe immunosuppressive tumor microenvironment (TME) is a major barrier to the efficacy of chimeric antigen receptor T cells (CAR-T cells) in glioblastoma (GBM). Transgenic expression of ...IL15 is one attractive strategy to modulate the TME. However, at present, it is unclear if IL15 could be used to directly target myeloid-derived suppressor cells (MDSCs), a major cellular component of the GBM TME. Here, we explored if MDSC express IL15Rα and the feasibility of exploiting its expression as an immunotherapeutic target.MethodsRNA-seq, RT-qPCR, and flow cytometry were used to determine IL15Rα expression in paired peripheral and tumor-infiltrating immune cells of GBM patients and two syngeneic murine GBM models. We generated murine T cells expressing IL13Rα2-CARs and secretory IL15 (CAR.IL15s) or IL13Rα2-CARs in which IL15 was fused to the CAR to serve as an IL15Rα-targeting moiety (CAR.IL15f), and characterized their effector function in vitro and in syngeneic IL13Rα2+glioma models.ResultsIL15Rα was preferentially expressed in myeloid, B, and dendritic cells in patients’ and syngeneic GBMs. In vitro, CAR.IL15s and CAR.IL15f T cells depleted MDSC and decreased their secretion of immunosuppressive molecules with CAR.IL15f T cells being more efficacious. Similarly, CAR.IL15f T cells significantly improved the survival of mice in two GBM models. TME analysis showed that treatment with CAR.IL15f T cells resulted in higher frequencies of CD8+T cells, NK, and B cells, but a decrease in CD11b+cells in tumors compared with therapy with CAR T cells.ConclusionsWe demonstrate that MDSC of the glioma TME express IL15Ra and that these cells can be targeted with secretory IL15 or an IL15Rα-targeting moiety incorporated into the CAR. Thus, IL15-modified CAR T cells act as a dual targeting agent against tumor cells and MDSC in GBM, warranting their future evaluation in early-phase clinical studies.
The leukocyte‐restricted tetraspanin CD53 has been shown to promote lymphocyte homing to lymph nodes (LNs) and myeloid cell recruitment to acutely inflamed peripheral organs, and accelerate the onset ...of immune‐mediated disease. However, its contribution in the setting of chronic systemic autoimmunity has not been investigated. We made use of the Lyn−/− autoimmune model, generating Cd53−/−Lyn−/− mice, and compared trafficking of immune cells into secondary lymphoid organs and systemic autoimmune disease development with mice lacking either gene alone. Consistent with previous observations, absence of CD53 led to reduced LN cellularity via reductions in both B and T cells, a phenotype also observed in Cd53−/−Lyn−/− mice. In some settings, Cd53−/−Lyn−/− lymphocytes showed greater loss of surface L‐selectin and CD69 upregulation above that imparted by Lyn deficiency alone, indicating that absence of these two proteins can mediate additive effects in the immune system. Conversely, prototypical effects of Lyn deficiency including splenomegaly, plasma cell expansion, elevated serum immunoglobulin M and anti‐nuclear antibodies were unaffected by CD53 deficiency. Furthermore, while Lyn−/− mice developed glomerular injury and showed elevated glomerular neutrophil retention above than that in wild‐type mice, absence of CD53 in Lyn−/− mice did not alter these responses. Together, these findings demonstrate that while tetraspanin CD53 promotes lymphocyte trafficking into LNs independent of Lyn, it does not make an important contribution to development of autoimmunity, plasma cell dysfunction or glomerular injury in the Lyn−/− model of systemic autoimmunity.
Tetraspanin CD53 controls immune cell trafficking in health and inflammatory disease, although its role in systemic autoimmunity remains unknown. Here we examined the role of CD53 in the Lyn‐deficient model of systemic autoimmune disease by generating CD53‐deficient Lyn−/− mice. The role of CD53 in controlling lymphocyte homing to lymph nodes persisted in the context of Lyn deficiency, although absence of CD53 did not alter development of systemic autoimmunity or renal inflammatory disease in Lyn−/− mice.
CD4
CD25
CD127
FOXP3
regulatory T cells (T
) play a key role in preventing autoimmunity. In autoimmune type 1 diabetes (T1D), adoptive transfer of autologous polyclonal T
has been shown to be safe in ...adults in phase 1 clinical trials. We explored factors contributing to efficacy of autologous polyclonal expanded T
(expT
) in a randomized phase 2 multi-center, double-blind, clinical trial (Sanford/Lisata Therapeutics T-Rex phase 2 trial, ClinicalTrials.gov NCT02691247). One hundred ten treated children and adolescents with new-onset T1D were randomized 1:1:1 to high-dose (20 × 10
cells/kilogram) or low-dose (1 × 10
cells/kilogram) treatments or to matching placebo. Cytometry as well as bulk and single-cell RNA sequencing were performed on selected expT
and peripheral blood samples from participants. The single doses of expT
were safe but did not prevent decline in residual β cell function over 1 year compared to placebo (
= 0.94 low dose,
= 0.21 high dose), regardless of age or baseline C-peptide. ExpT
were highly activated and suppressive in vitro. A transient increase of activated memory T
was detectable 1 week after infusion in the high-dose cohort, suggesting effective transfer of expT
. However, the in vitro fold expansion of expT
varied across participants, even when accounting for age, and lower fold expansion and its associated gene signature were linked with better C-peptide preservation regardless of T
dose. These results suggest that a single dose of polyclonal expT
does not alter progression in T1D; instead, T
quality may be an important factor.
The bottom water conditions in the Central South Pacific (CSP) and associated changes in the Lower Circumpolar Deep Water (LCDW) and Antarctic Bottom Water (AABW) under warmer-than-present conditions ...need to be better understood. These water masses transfer their properties to the major ocean basins. We analyzed Late Miocene to Early Pliocene (5.6–3.6 Ma) marine sediment core sections from the CSP for benthic foraminifera, ice rafted debris (IRD), Ostracoda, planktic foraminifera Orbulina universa abundance, and organic geochemical proxies to assess the bottom water characteristics under warmer-than-present day conditions. A significant increase in IRD abundance between 5.3 and 4.9 Ma marks the Early Pliocene warm phase. The benthic foraminiferal assemblages indicate shifts in bottom water conditions over time in the CSP region. Between 5.6 and 5.3 Ma, predominantly oxygenated bottom water with moderate organic matter flux prevailed. This shifted to suboxic conditions with increased organic matter flux from 5.3 to 4.9 Ma. Subsequently, between 4.9 and 4.4 Ma, bottom water conditions alternated frequently between oxic and suboxic states. Enhanced bottom water formation and inflow of LCDW and AABW in the CSP during 4.4–4.0 Ma promoted oxygenated conditions, accompanied by low organic export flux. However, sluggish bottom water circulation from 4.0 to 3.6 Ma reverted to suboxic conditions, associated with increased carbon burial. Notably, productivity peaked intermittently between 5.3 and 3.6 Ma, as indicated by the occurrence of suboxic species assemblages and increase in the abundance of Orbulina universa, benthic microfauna (ostracods), and other paleoproductivity indicators.
•The first Early Pliocene benthic foraminiferal record from the Central South Pacific (CSP).•Our data suggests a periodic switch between oxic and suboxic conditions and productivity.•Circumpolar Deepwater (CDW) was stronger between 4.4 and 4.0 Ma.•Extension of West Antarctic Ice Sheet supported stronger CDW and ventilated CSP.
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