Immunotherapies for malignant melanoma are challenged by the resistance developed in a significant proportion of patients. Myeloid-derived suppressor cells (MDSC), with their ability to inhibit antitumor T-cell responses, are a major contributor to immunosuppression and resistance to immune checkpoint therapies in melanoma. Damage-associated molecular patterns S100A8, S100A9, and HMGB1, acting as toll like receptor 4 (TLR4) and receptor for advanced glycation endproducts (RAGE) ligands, are highly expressed in the tumor microenvironment and drive MDSC activation. However, the role of TLR4 and RAGE signaling in the acquisition of MDSC immunosuppressive properties remains to be better defined. Our study investigates how the signaling via TLR4 and RAGE as well as their ligands S100A9 and HMGB1, shape MDSC-mediated immunosuppression in melanoma.
MDSC were isolated from the peripheral blood of patients with advanced melanoma or generated in vitro from healthy donor-derived monocytes. Monocytes were treated with S100A9 or HMGB1 for 72 hours. The immunosuppressive capacity of treated monocytes was assessed in the inhibition of T-cell proliferation assay in the presence or absence of TLR4 and RAGE inhibitors. Plasma levels of S100A8/9 and HMGB1 were quantified by ELISA. Single-cell RNA sequencing (scRNA-seq) was performed on monocytes from patients with melanoma and healthy donors.
We showed that exposure to S100A9 and HMGB1 converted healthy donor-derived monocytes into MDSC through TLR4 signaling. Our scRNA-seq data revealed in patient monocytes enriched inflammatory genes, including S100 and those involved in NF-κB and TLR4 signaling, and a reduced major histocompatibility complex II gene expression. Furthermore, elevated plasma S100A8/9 levels correlated with shorter progression-free survival in patients with melanoma.
These findings highlight the critical role of TLR4 and, to a lesser extent, RAGE signaling in the conversion of monocytes into MDSC-like cells, underscore the potential of targeting S100A9 to prevent this conversion, and highlight the prognostic value of S100A8/9 as a plasma biomarker in melanoma.
© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.
© 2023. Springer Nature Limited.

Four antibodies that inhibit interleukin (IL)-23 are approved for the treatment of moderate-to-severe plaque psoriasis. Here, we present non-clinical data comparing ustekinumab, guselkumab, tildrakizumab and risankizumab with regard to thermostability, IL-23 binding affinity, inhibitory-binding mode, in vitro potency and in vivo efficacy. Risankizumab and guselkumab exhibited 5-fold higher affinity for IL-23 and showed more potent inhibition of IL-23 signaling than ustekinumab and tildrakizumab. Risankizumab and guselkumab completely blocked the binding of IL-23 to IL-23Rα as expected, whereas tildrakizumab did not. In vitro, risankizumab and guselkumab blocked the terminal differentiation of TH17 cells in a similar manner, while tildrakizumab had minimal impact on TH17 differentiation. In a human IL-23-induced ear-swelling mouse model, risankizumab and guselkumab were more effective than ustekinumab and tildrakizumab at reducing IL-17, IL-22, and keratinocyte gene expression. Our results indicate that the four clinically approved antibodies targeting IL-23 differ in affinity and binding epitope. These attributes contribute to differences in in vitro potency, receptor interaction inhibition mode and in vivo efficacy in preclinical studies as described in this report, and similarly may affect the clinical performance of these drugs.

Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2-rearranged (JAK2r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common; elucidating potential mechanisms of rux resistance in JAK2r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a JAK2 p.G993A mutation. Using in vitro models of JAK2r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.
© 2021. The Author(s).

Inborn errors of TLR3-dependent IFN-α/β- and IFN-λ-mediated immunity in the CNS can underlie herpes simplex virus 1 (HSV-1) encephalitis (HSE). The respective contributions of IFN-α/β and IFN-λ are unknown. We report a child homozygous for a genomic deletion of the entire coding sequence and part of the 3'-UTR of the last exon of IFNAR1, who died of HSE at the age of 2 years. An older cousin died following vaccination against measles, mumps, and rubella at 12 months of age, and another 17-year-old cousin homozygous for the same variant has had other, less severe, viral illnesses. The encoded IFNAR1 protein is expressed on the cell surface but is truncated and cannot interact with the tyrosine kinase TYK2. The patient's fibroblasts and EBV-B cells did not respond to IFN-α2b or IFN-β, in terms of STAT1, STAT2, and STAT3 phosphorylation or the genome-wide induction of IFN-stimulated genes. The patient's fibroblasts were susceptible to viruses, including HSV-1, even in the presence of exogenous IFN-α2b or IFN-β. HSE is therefore a consequence of inherited complete IFNAR1 deficiency. This viral disease occurred in natural conditions, unlike those previously reported in other patients with IFNAR1 or IFNAR2 deficiency. This experiment of nature indicates that IFN-α/β are essential for anti-HSV-1 immunity in the CNS.