Immunometabolism is critical in the regulation of immunity and inflammation; however, the mechanism of preventing aberrant activation-induced immunopathology remains largely unclear. Here, we report that glyoxalase II (GLO2) in the glycolysis branching pathway is specifically downregulated by NF-κB signaling during innate immune activation via tristetraprolin (TTP)-mediated mRNA decay. As a result, its substrate S-D-lactoylglutathione (SLG) accumulates in the cytosol and directly induces D-lactyllysine modification of proteins. This nonenzymatic lactylation by SLG is greatly facilitated by a nearby cysteine residue, as it initially reacts with SLG to form a reversible S-lactylated thiol intermediate, followed by SN-transfer of the lactyl moiety to a proximal lysine. Lactylome profiling identifies 2255 lactylation sites mostly in cytosolic proteins of activated macrophages, and global protein structure analysis suggests that proximity to a cysteine residue determines the susceptibility of lysine to SLG-mediated D-lactylation. Furthermore, lactylation is preferentially enriched in proteins involved in immune activation and inflammatory pathways, and D-lactylation at lysine 310 (K310) of RelA attenuates inflammatory signaling and NF-κB transcriptional activity to restore immune homeostasis. Accordingly, TTP-binding site mutation or overexpression of GLO2 in vivo blocks this feedback lactylation in innate immune cells and promotes inflammation, whereas genetic deficiency or pharmacological inhibition of GLO2 restricts immune activation and attenuates inflammatory immunopathology both in vitro and in vivo. Importantly, dysregulation of the GLO2/SLG/D-lactylation regulatory axis is closely associated with human inflammatory phenotypes. Overall, our findings uncover an immunometabolic feedback loop of SLG-induced nonenzymatic D-lactylation and implicate GLO2 as a promising target for combating clinical inflammatory disorders.
© 2024. The Author(s).

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.
Copyright © 2024 Lagassé, Ou, Sauna and Golding.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Retinoid X receptor (RXR) heterodimerizes with the PPAR nuclear hormone receptor and regulates its downstream events. We investigated the effects of RXR agonists (LG100754, bexarotene, AGN194204, and LG101506) on lenalidomide's anti-myeloma activity, T cell functions, and the level of glucose and lipids in vivo. Genetic overexpression and CRISPR/Cas9 knockout experiments were conducted in multiple myeloma (MM) cell lines and Jurkat T cell lines to determine the roles of CRBN in RXR-agonist mediated effects. A xenograft mouse model of MM was established to determine the combination effect of LG100754 and lenalidomide. The combination of RXR agonists and lenalidomide demonstrated synergistic activity in increasing CRBN expression and killing myeloma cells. Mechanistically, the RXR agonists reduced the binding of PPARs to the CRBN promoter, thereby relieving the repressor effect of PPARs on CRBN transcription. RXR agonists downregulated the exhaustion markers and increased the activation markers of Jurkat T cells and primary human T cells. Co-administration of LG100754 and lenalidomide showed enhanced anti-tumor activity in vivo. LG100754 retained its glucose- and lipid-lowering effects. RXR agonists demonstrate potential utility in enhancing drug sensitivity and T-cell function in the treatment of myeloma.

Immune cell therapies, such as adoptive T cell therapies, are an innovative and powerful treatment option for previously non-treatable diseases. Although immune cell therapies are thought to be very specific, there is still the danger of developing severe to life-threatening side effects due to the unspecific distribution of the cells throughout the body (on-target/off-tumor effects). A possible solution for the reduction of these side effects and the improvement of tumor infiltration is the specific targeting of the effector cells (e.g., T cells) to the desired destination (e.g., tumor region). This can be achieved by the magnetization of cells with superparamagnetic iron oxide nanoparticles (SPIONs) for spatial guidance via external magnetic fields. A prerequisite for the use of SPION-loaded T cells in adoptive T cell therapies is that cell viability and functionality after nanoparticle loading are preserved. Here, we demonstrate a protocol to analyze cell viability and functionality such as activation, proliferation, cytokine release, and differentiation at a single cell level using flow cytometry.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.