Indirect suppression of CD4 T cell activation through LAG-3-mediated trans-endocytosis of MHC class II.

Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) has already been recognized as the next efficacious treatment, but there is still limited understanding of the mechanism of LAG-3-mediated immune suppression. Here, utilizing high-resolution molecular imaging, we find a mechanism of CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) gather at the central region of an immunological synapse and are trans-endocytosed by T cell receptor-driven internalization motility toward CD4 and CD8 T cells expressing LAG-3. Downregulation of MHC class II molecules on APCs thus results in the attenuation of their antigen-presentation function and impairment of CD4 T cell activation. From these data, anti-LAG-3 treatment is suggested to have potency to directly block the inhibitory signaling via LAG-3 and simultaneously reduce MHC class II expression on APCs by LAG-3-mediated trans-endocytosis for recovery from T cell exhaustion.

Molecular Imaging of PD-1 Unveils Unknown Characteristics of PD-1 Itself by Visualizing "PD-1 Microclusters".

Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.

Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters.

With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.

PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment.

The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1-PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale.

Strong TCR stimulation promotes the stabilization of Foxp3 expression in regulatory T cells induced in vitro through increasing the demethylation of Foxp3 CNS2.

Foxp3 is the master transcriptional regulator of regulatory T cells (Tregs), and the stabilization of Foxp3 expression is regulated by the demethylation of conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Recent studies have shown that TCR stimulation is required for the demethylation of Foxp3 CNS2 during Treg development. However, the relationship between the strength of TCR stimulation and the demethylation of Foxp3 CNS2 remains unclear. To address this issue, we compared the frequency of demethylation of the Foxp3 CNS2 among in vitro-induced Tregs (iTreg) that had received a range of TCR stimulation during their development. We found that the frequency of demethylation of the Foxp3 CNS2 was increased with increased TCR stimulation strength, whereas CD28 stimulation had only a limited effect. Mechanistically, the binding of Tet2, a member of the TET family of enzymes involved in DNA demethylation, on the Foxp3 CNS2 was increased by strong TCR stimulation. Furthermore, compared with iTreg induced by weak TCR stimulation, iTreg induced by strong TCR stimulation maintained Foxp3 expression both in vitro and in vivo. These data indicate that the strength of TCR stimulation is a key factor for induction of the demethylation of Foxp3 CNS2 and the generation of stable Tregs.

Defects in regulatory T cells due to CD28 deficiency induce a qualitative change of allogeneic immune response in chronic graft-versus-host disease.

In patients receiving allogeneic hematopoietic cell transplantation, chronic graft-versus-host disease (cGVHD) remains a frequent complication and resembles autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. Our previous work demonstrated the critical role of CD28 costimulation of donor T cells for GVHD induction. In this study, we investigate the role of CD28 costimulation of host T cells in cGVHD. CD28-intact mice as hosts showed systemic lupus erythematosus-type cGVHD, whereas CD28-deficient mice developed a distinct phenotype of cGVHD, with fibrotic damage in skin and internal organs, resembling systemic sclerosis. This phenotype was due to a lack of signaling through the C-terminal proline-rich motif within host CD28's cytoplasmic tail, a motif previously shown to be required for development of regulatory T cells (Tregs) and function of conventional T cells. Adoptive transfer experiments demonstrated that a defect in host CD4(+)CD25(+) Tregs, but not in conventional T cells, was responsible for disease phenotype. Host Treg deficiency altered the cytokine pattern of donor CD4(+) T cells and the Ag specificity of autoantibodies, and these might lead to phenotypic change. Thus, host CD28 signaling controlled the pathogenesis of cGVHD through effects on host Tregs, whose status impacts qualitatively on the allogeneic immune responses.

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation.

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit's kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation.

Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis.

The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögren's syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)→Rag1(-/-)) mice. M3R(-/-)→Rag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)→Rag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.

Effects of infliximab therapy on gene expression levels of tumor necrosis factor alpha, tristetraprolin, T cell intracellular antigen 1, and Hu antigen R in patients with rheumatoid arthritis.

OBJECTIVE: Tristetraprolin (TTP), T cell intracellular antigen 1 (TIA-1), and Hu antigen R (HuR) are adenine/uridine-rich element binding proteins (ABPs) that affect the production of tumor necrosis factor alpha (TNFalpha) by binding to TNF messenger RNA (mRNA). TTP promotes deadenylation, TIA-1 inhibits translation, and HuR stabilizes TNFalpha mRNA. The aims of this study were to understand the posttranscriptional control of TNFalpha production in patients with rheumatoid arthritis (RA), and to identify parameters that may predict the efficacy of anti-TNFalpha therapy. METHODS: Peripheral blood mononuclear cells from 38 patients with RA were obtained before therapy and 2 weeks and 54 weeks after administration of the first dose of infliximab, and from 20 healthy control subjects. TNFalpha, TTP, TIA-1, and HuR gene expression levels were analyzed by real-time polymerase chain reaction. RESULTS: At baseline, TTP and HuR gene expression levels, as well as the TTP:TNFalpha, TTP:HuR, and TIA-1:TNFalpha gene expression ratios were lower in patients with RA than in control subjects, while expression of TNFalpha, TIA-1, and TIA-1:HuR was higher in patients with RA. The TTP:HuR expression ratio decreased significantly after administration of infliximab. Positive correlations were observed between TNFalpha and TTP, TNFalpha and TIA-1, TIA-1 and HuR, and TNFalpha and HuR gene expression in both healthy control subjects and patients with RA. At baseline, the TIA-1:HuR ratio tended to be higher in patients who achieved 50% improvement according to the American College of Rheumatology criteria (ACR50) at week 54 than in those who did not achieve at least an ACR20 response. CONCLUSION: Differences in ABP gene expression may affect TNFalpha gene expression. A higher TIA-1:HuR expression ratio might correlate with the response to infliximab therapy.

Efficacy prediction of cevimeline in patients with Sjögren's syndrome.

The objective of this study was to examine the clinical and immunological factors influencing the efficacy of cevimeline hydrochloride hydrate (cevimeline) for the treatment of xerostomia in patients with Sjögren's syndrome (SS). Thirty primary SS patients who were medicated with cevimeline were enrolled in this study. Whole stimulated sialometry (WSS) was compared between pre- and posttreatment points (4 weeks after oral cevimeline administration) and the increment rate of WSS was calculated. Multiple regression was employed to examine the relative contributions of the clinical and immunological factors, including age, pretreatment WSS, duration of disease, sialography, minor salivary gland biopsy, anti-Ro/SS-A antibodies, anti-La/SS-B antibodies, and antibodies to muscarinic type 3 receptors to the posttreatment WSS. Patients with normal sialography findings, negative minor salivary gland biopsy, and absence of anti-La/SS-B antibodies had significantly higher increment rates of WSS compared with those with positive findings (p=0.042, 0.002, and 0.018, respectively). Results of the multiple regression analysis showed that sialography (coefficient=-0.867, p=0.004) and minor salivary gland biopsy (coefficient=-0.869, p=0.003) had significant associations with the posttreatment WSS. Our preliminary results demonstrated the relationship between the effect of cevimeline on saliva secretion and the degree of salivary gland destruction evaluated by sialography and histopathological findings in the labial minor salivary glands. These diagnostic approaches could provide useful prognostic information on the efficacy of cevimeline in SS patients.

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys.

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.