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Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer 1 , 2 . Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies 3 . However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. V-domain immunoglobulin suppressor of T cell activation (VISTA) selectively engages P-selectin glycoprotein ligand-1 (PSGL-1) and suppresses T cells at acidic pH similar to those in tumour microenvironments, thereby mediating resistance to anti-tumour immune responses.

The accuracy of protein synthesis and its relation to ageing has been of long-standing interest. To study whether spontaneous changes in the rate of ribosomal error occur as a function of age, we first determined that stop-codon readthrough is a more sensitive read-out of mistranslation due to codon-anticodon mispairing than missense amino acid incorporation. Subsequently, we developed knock-in mice for in-vivo detection of stop-codon readthrough using a gain-of-function Kat2-TGA-Fluc readthrough reporter which combines fluorescent and sensitive bioluminescent imaging techniques. We followed expression of reporter proteins in-vivo over time, and assessed Kat2 and Fluc expression in tissue extracts and by whole organ ex-vivo imaging. Collectively, our results provide evidence for an organ-dependent, age-related increase in translational error: stop-codon readthrough increases with age in muscle (+ 75%, p  < 0.001) and brain (+ 50%, p  < 0.01), but not in liver ( p  > 0.5). Together with recent data demonstrating premature ageing in mice with an error-prone ram mutation, our findings highlight age-related decline of translation fidelity as a possible contributor to ageing. In this paper, translation fidelity was assessed across aging in knock-in mice with a mistranslation reporter which determines stop codon readthrough. Age-related increases in translational errors were observed in muscle and brain, but not in liver, highlighting organ-dependent declines in translation fidelity as a possible contributor to aging.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens. VISTA is a pH-dependent inhibitory checkpoint for T-cells that is abundant on myeloid lineage cells and antagonists of VISTA may successfully reinvigorate anti-tumour immunity. Here, the authors show that the antibody SNS-101, which is currently being investigated in humans in a clinical trial, is characterized by pH-sensitivity that endows it with favorable pharmacokinetic and safety profiles, and enhanced therapeutic effect when combined with PD-1 checkpoint inhibitors.

In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need. Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A receptor (PLA R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker. BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig and T cells. , the BiAATE successfully induced T cell-dependent depletion of PLA R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA R antibody levels following active immunization with PLA R. Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

[This corrects the article DOI: 10.3389/fimmu.2024.1335998.].

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9’s efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. BRGSF mice reconstituted with human umbilical cord blood CD34 cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Preclinical models that accurately recapitulate the human immune response, particularly within the tumor microenvironment (TME), are needed for the translational and predictive testing of new therapies. Here, we examine whether the genO-BRGSF-HIS model-characterized by robust reconstitution of both human lymphoid and myeloid cells following engraftment with CD34 cord blood cells-could be a translatable mouse model for human tumor biology and a relevant platform for evaluating novel immunotherapies. genO-BRGSF mice were reconstituted with human CD34 cord blood cells (genO-BRGSF-HIS) and treated with exogenous human Flt3 ligand (hFlt3L). Myeloid and dendritic cell functionality was analyzed following treatment with different compounds (TLR agonists, TREM1 agonist, STING agonist, or T-cell engagers) and following the implantation of different tumor cell lines (MDA-MB-231, A549, HPAF-II). We show that myeloid, dendritic and lymphoid cells (including NK and γδ T cells) are functional and recruited into the TME in genO-BRGSF-HIS mice implanted with different tumor cell lines, and that different immune cell populations are activated and get polarized within the TME. The composition of the TME is dependent on tumor type and tumor burden, demonstrating plasticity in the crosstalk between the human immune system and the tumor cells. Furthermore, we observed polarization of the cells recruited to the TME, as well as a wide diversity of recruited cell populations, suggesting that this model reproduces human physiopathology in the context of cancer. Based on the recruitment of the different cell populations according to tumor type, we also demonstrate that this model can be used for testing new therapies targeting lymphoid cells, such as T-cell engagers. genO-BRGSF-HIS mice do not exhibit adverse effects associated with the development of human lymphoid and myeloid cells following CD34 cord blood cell reconstitution, and their extended lifespan allows for longer experimental study windows. Overall, we show that this model develops functional myeloid and lymphoid cells which are recruited to the TME, making it a valuable tool for testing new immunotherapies that modulate the interaction between the tumor and the immune system.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64 Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.