CD4 + Foxp3 + regulatory T cells (Treg) efficiently foster wound healing after myocardial infarction (MI). Therapeutically shifting the balance between CD4 + Foxp3 - conventional T cells (Tconv) and Treg towards Tregs enhanced survival in a mouse model of MI. Due to species-specific differences in cardiac wound healing and remodelling, it remains, however, unclear whether these findings can be translated into novel immunotherapies for human patients after MI. Therefore, we studied pigs whose cardiac wound healing after MI and the composition of the immune system are very close to humans. This includes the relevant complication of developing a cytokine release syndrome (CRS) after infusion of saturating amounts of a superagonistic anti-CD28 monoclonal antibody (mAb). To achieve the intended shift in the Treg/Tconv balance, we treated pigs three days after interventional MI induction with a non-superagonistic, i.e. conventional, anti-CD28 mAb, clone 3D11. Infusion of a saturating dose (1 mg/kg body weight) of mAb 3D11 was clinically well tolerated without signs of CRS induction or any other complications. Molecularly, mAb 3D11 infusion led to a downmodulation of CD28 expression on porcine T cells in vivo with the remaining CD28 molecules blocked from binding natural ligand proteins CD80 and CD86, as we show in this publication. Apart from modulating CD28 expression, treatment with mAb 3D11 did not induce any overt changes in the peripheral T cell compartment. However, after mAb 3D11 treatment, we observed Treg accumulation in the infarcted heart, particularly the border zone, on day 7 post-MI using immunofluorescence histology. Our findings thus suggest that even saturating doses of conventional anti-CD28 monoclonal antibodies could potentially be safely administered in patients to therapeutically shift the Treg/Tconv balance in the infarcted myocardium. This might be sufficient to enhance cardiac wound healing in patients short-term and prevent adverse remodelling long-term.

Fatty acid transport protein (FATP)4 was thought to mediate intestinal lipid absorption, which was disputed by a study using keratinocyte-Fatp4-rescued Fatp4(-/-) mice. These knockouts when fed with a Western diet showed elevated intestinal triglyceride (TG) and fatty acid levels. To investigate a possible role of FATP4 on intestinal lipid processing, ent-Fatp4 (KO) mice were generated by Villin-Cre-specific inactivation of the Fatp4 gene. We aimed to measure circulating and intestinal lipids in control and KO mice after acute or chronic fat intake or during aging. Remarkably, ent-Fatp4 mice displayed an approximately 30% decrease in ileal behenic, lignoceric, and nervonic acids, ceramides containing these FA, as well as, ileal sphingomyelin, phosphatidylcholine, and phosphatidylinositol levels. Such decreases were concomitant with an increase in jejunal cholesterol ester. After a 2-wk recovery from high lipid overload by tyloxapol and oral-lipid treatment, ent-Fatp4 mice showed an increase in plasma TG and chylomicrons. Upon overnight fasting followed by an oral fat meal, ent-Fatp4 mice showed an increase in plasma TG-rich lipoproteins and the particle number of chylomicrons and very low-density lipoproteins. During aging or after feeding with a high-fat high-cholesterol (HFHC) diet, ent-Fatp4 mice showed an increase in plasma TG, fatty acids, glycerol, and lipoproteins as well as intestinal lipids. HFHC-fed KO mice displayed an increase in body weight, the number of lipid droplets with larger sizes in the ileum, concomitant with a decrease in ileal ceramides and phosphatidylcholine. Thus, enterocyte FATP4 deficiency led to a metabolic shift from polar to neutral lipids in distal intestine rendering an increase in plasma lipids and lipoproteins. NEW & NOTEWORTHY Enterocyte-specific Fatp4 deficiency in mice increased intestinal lipid absorption with elevation of blood lipids during fasting and aging, as well as after an acute oral fat-loading or chronic HFHC feeding. Lipidomics revealed that knockout mice displayed a shift from very long-chain to long-chain fatty acids, and from polar to neutral lipids, predominantly in the ileum. Thus, FATP4 may have a physiological function in the control of blood lipids via metabolic shifts in distal intestine.

Vγ9Vδ2 T cells comprise 1-10% of human peripheral blood T cells. As multifunctional T cells with a strong antimicrobial and antitumor potential, they are of strong interest for immunotherapeutic development. Their hallmark is the eponymous Vγ9Vδ2 T-cell antigen receptor (TCR), which mediates activation by so-called "phosphoantigens" (PAg). PAg are small pyrophosphorylated intermediates of isoprenoid synthesis of microbial or host origin, with the latter elevated in some tumors and after administration of aminobisphosphonates. This review summarizes the progress in understanding PAg-recognition, with emphasis on the interaction between butyrophilins (BTN) and PAg and insights gained by phylogenetic studies on BTNs and Vγ9Vδ2 T cells, especially the comparison of human and alpaca. It proposes a composite ligand model in which BTN3A1-A2/A3-heteromers and BTN2A1 homodimers form a Vγ9Vδ2 TCR activating complex. An initiating step is the binding of PAg to the intracellular BTN3A1-B30.2 domain and formation of a complex with the B30.2 domains of BTN2A1. On the extracellular surface this results in BTN2A1-IgV binding to Vγ9-TCR framework determinants and BTN3A-IgV to additional complementarity determining regions of both TCR chains. Unresolved questions of this model are discussed, as well as questions on the structural basis and the physiological consequences of PAg-recognition.

The title of Zhou et al. (1) claims that “Up-regulation of BTN3A1 on CD14+ cells promotes V γ 9V δ 2 T cell activation in psoriasis.” The study nicely confirms in quite some detail that V γ 9V δ 2 T cells from psoriatic patients increasingly home to skin, produce higher amounts of IFN γ , TNF α

Vγ9Vδ2 T cells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor," and BTN2A1, a direct ligand for T cell receptor (TCR) Vγ9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vγ9Vδ2 TCR and BTN3A1. Nuclear magnetic resonance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV structural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis indicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the γδ-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vγ9Vδ2 TCR triggering.

FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4 M−/− mice displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. In response to HFHC feeding, male mutants were prone to hepatic steatosis, whereas female mutants showed exaggerated fibrosis. Our study provides insights into a sex-dimorphic susceptibility to NASH by myeloid-FATP4 deficiency.

Evidence from mice with global deletion of fatty-acid transport protein4 (FATP4) indicates its role on β-oxidation and triglycerides (TG) metabolism. We reported that plasma glycerol and free fatty acids (FA) were increased in liver-specific Fatp4 deficient (L-FATP4-/-) mice under dietary stress. We hypothesized that FATP4 may mediate hepatocellular TG lipolysis. Here, we demonstrated that L-FATP4-/- mice showed an increase in these blood lipids, liver TG, and subcutaneous fat weights. We therefore studied TG metabolism in response to oleate treatment in two experimental models using FATP4-knockout HepG2 (HepKO) cells and L-FATP4-/- hepatocytes. Both FATP4-deificient liver cells showed a significant decrease in β-oxidation products by ∼30-35% concomitant with marked upregulation of CD36, FATP2, and FATP5 as well as lipoprotein microsomal-triglyceride-transfer protein genes. By using 13C3D5-glycerol, HepKO cells displayed an increase in metabolically labelled TG species which were further increased with oleate treatment. This increase was concomitant with a step-wise elevation of TG in cells and supernatants as well as the secretion of cholesterol very low-density and high-density lipoproteins. Upon analyzing TG lipolytic enzymes, both mutant liver cells showed marked upregulated expression of hepatic lipase, while that of hormone-sensitive lipase and adipose-triglyceride lipase was downregulated. Lipolysis measured by extracellular glycerol and free FA was indeed increased in mutant cells, and this event was exacerbated by oleate treatment. Taken together, FATP4 deficiency in liver cells led to a metabolic shift from β-oxidation towards lipolysis-directed TG and lipoprotein secretion, which is in line with an association of FATP4 polymorphisms with blood lipids.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.

Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.

Butyrophilins (BTN) are relatives of the B7 family (e.g., CD80, PD-L1). They fulfill a wide range of functions including immunomodulation and bind to various receptors such as the γδ T cell receptor (γδTCR) and small molecules. One intensively studied molecule is BTN3A1, which binds via its cytoplasmic B30.2 domain, metabolites of isoprenoid synthesis, designated as phosphoantigen (PAg), The enrichment of PAgs in tumors or infected cells is sensed by Vγ9Vδ2 T cells, leading to the proliferation and execution of effector functions to remove these cells. This article discusses the contribution of BTNs, the related BTNL molecules and SKINT1 to the development, activation, and homeostasis of γδ T cells and their immunomodulatory potential, which makes them interesting targets for therapeutic intervention.

Butyrophiline (BTN) sind Verwandte der als Immunmodulatoren bekannten B7-Molekülfamilie (z. B. CD80, PD-L1). Sie steuern die Entwicklung und Aktivierung von γδ-T-Zellen. Manche ihrer Mitglieder binden direkt an γδ-T-Zellantigenrezeptoren (γδTZR), andere aber auch an intrazelluläre Liganden wie die Phosphoantigene (PAg), die als Metabolite in Tumorzellen oder infizierten Zellen angereichert werden. Diese PAg-anreichernden Zellen werden schließlich von PAg-reaktiven γδ-T-Zellen (Vγ9Vδ2-T-Zellen) erkannt und eliminiert. Der Beitrag von BTN zur Aktivierung und Homöostase von γδ-T-Zellen wie auch deren immunmodulatorische Wirkung auf andere Zelltypen lenken den Fokus von Grundlagenimmunologie und immuntherapeutischer Forschung zunehmend auf die BTN als wichtigen Forschungsschwerpunkt.

The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

Summary Human Vγ9/Vδ2 T cells, mucosal-associated invariant T (MAIT) cells, and other unconventional T cells are specialised in detecting microbial metabolic pathway intermediates that are absent in humans. The recognition by such semi-invariant innate-like T cells of compounds like (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), the penultimate metabolite in the MEP isoprenoid biosynthesis pathway, and intermediates of the riboflavin biosynthesis pathway and their metabolites allows the immune system to rapidly sense pathogen-associated molecular patterns that are shared by a wide range of micro-organisms. Given the essential nature of these metabolic pathways for microbial viability, they have emerged as promising targets for the development of novel antibiotics. Here, we review recent findings that link enzymatic inhibition of microbial metabolism with alterations in the levels of unconventional T cell ligands produced by treated micro-organisms that have given rise to the concept of ‘immuno-antibiotics’: combining direct antimicrobial activity with an immunotherapeutic effect via modulation of unconventional T cell responses.

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8+ and CD4+ αβ T cells. Among γδ T cells, CD28 expression was restricted to a small CD2+ subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3+ regulatory T cells among CD4+ T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4+ regulatory T cells in humans.

EDITORIAL article Front. Immunol., 14 May 2020Sec. T Cell Biology Volume 11 - 2020 | https://doi.org/10.3389/fimmu.2020.00921