Functions of mucosal associated invariant T cells in eye diseases.

Mucosal-associated invariant T (MAIT) cells are a unique subset of T cells that recognizes metabolites derived from the vitamin B2 biosynthetic pathway. Since the identification of cognate antigens for MAIT cells, knowledge of the functions of MAIT cells in cancer, autoimmunity, and infectious diseases has been rapidly expanding. Recently, MAIT cells have been found to contribute to visual protection against autoimmunity in the eye. The protective functions of MAIT cells are induced by T-cell receptor (TCR)-mediated activation. However, the underlying mechanisms remain unclear. Thus, this mini-review aims to discuss our findings and the complexity of MAIT cell-mediated immune regulation in the eye.

Sulfated bile acid is a host-derived ligand for MAIT cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function.

Corrigendum to "Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes".

[This corrects the article DOI: 10.1016/j.bbrep.2022.101365.].

Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8+T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.

Symbiotic bacteria-dependent expansion of MR1-reactive T cells causes autoimmunity in the absence of Bcl11b.

MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αß T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential.

Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at a late stage and becomes resistant to several treatments. Significant clinical effects have been reported for cancer immunotherapies on a subset of patients diagnosed with epithelial cancers. Cancer organoid co-culture with autologous peripheral blood lymphocytes offers an innovative immunotherapeutic approach that is increasingly being tested, although there is a lack of cutting-edge platforms enabling the investigation of cancer-T cell interactions for individual patients. In this study, a pancreatic cancer organoid culture from a genetically engineered pancreatic cancer murine model was established and co-cultured with autologous peripheral blood lymphocytes to induce a tumour-specific T cell response to pancreatic cancer. Co-culturing autologous peripheral blood lymphocytes with cancer organoids can be an effective strategy to enrich tumour-reactive T cells from the peripheral blood of murine models; this approach could potentially be transferred to humans. Co-culture of peripheral blood lymphocytes and cancer organoids could provide an unbiased approach to evaluating the sensitivity of tumour cells to T cell-mediated priming on an individual patient level.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.

Circulating inflammatory monocytes oppose microglia and contribute to cone cell death in retinitis pigmentosa.

Retinitis pigmentosa (RP) is an intractable inherited disease that primarily affects the rods through gene mutations followed by secondary cone degeneration. This cone-related dysfunction can lead to impairment of daily life activities, and ultimately blindness in patients with RP. Paradoxically, microglial neuroinflammation contributes to both protection against and progression of RP, but it is unclear which population(s) - tissue-resident microglia and/or peripheral monocyte-derived macrophages (mφ) - are implicated in the progression of the disease. Here we show that circulating blood inflammatory monocytes (IMo) are key effector cells that mediate cone cell death in RP. Attenuation of IMo and peripherally engrafted mφ by Ccl2 deficiency or immune modulation via intravenous nano-particle treatment suppressed cone cell death in rd10 mice, an animal model of RP. In contrast, the depletion of resident microglia by a colony-stimulating factor 1 receptor inhibitor exacerbated cone cell death in the same model. In human patients with RP, IMo was increased and correlated with disease progression. These results suggest that peripheral IMo is a potential target to delay cone cell death and prevent blindness in RP.

Corrigendum: Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

[This corrects the article DOI: 10.3389/fimmu.2022.1008220.].

Mucosal-associated invariant T cells have therapeutic potential against ocular autoimmunity.

Autoimmune uveitis is a sight-threatening disease induced by pathogenic T cells that recognize retinal antigens; it is observed in disorders including Vogt-Koyanagi-Harada disease (VKH). The roles of specific T cell subsets and their therapeutic potential against autoimmune uveitis are not fully understood. Here we conducted multi-parametric single-cell protein quantification which shows that the frequency of CD161highTRAV1-2+ mucosal-associated invariant T (MAIT) cells that recognize vitamin B2 metabolite-based antigens is decreased in relapsing VKH patients compared to individuals without active ocular inflammation. An experimental autoimmune uveitis (EAU) mouse model revealed that genetic depletion of MAIT cells reduced the expression of interleukin (Il) 22 and exacerbated retinal pathology. Reduced IL-22 levels were commonly observed in patients with relapsing VKH compared to individuals without active ocular inflammation. Both mouse and human MAIT cells produced IL-22 upon stimulation with their antigenic metabolite in vitro. An intravitreal administration of the antigenic metabolite into EAU mice induced retinal MAIT cell expansion and enhanced the expressions of Il22, as well as its downstream genes related to anti-inflammatory and neuroprotective effects, leading to an improvement in both retinal pathology and visual function. Taken together, we demonstrate that a metabolite-driven approach targeting MAIT cells has therapeutic potential against autoimmune uveitis.

Agonistic or antagonistic mucosal-associated invariant T (MAIT) cell activity is determined by the 6-alkylamino substituent on uracil MR1 ligands.

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified.

Claudin-5 Redistribution Induced by Inflammation Leads to Anti-VEGF-Resistant Diabetic Macular Edema.

Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti-vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b+ Ly6C+ CCR2+ cells), macrophage/microglia activation (CD11b+ CD80+ cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGF-overexpressed retinas revealed rho-associated coiled-coil-containing protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine-stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCK-mediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.

In Staphylococcus aureus, the Particulate State of the Cell Envelope Is Required for the Efficient Induction of Host Defense Responses.

Upon microbial infection, host immune cells recognize bacterial cell envelope components through cognate receptors. Although bacterial cell envelope components function as innate immune molecules, the role of the physical state of the bacterial cell envelope (i.e., particulate versus soluble) in host immune activation has not been clearly defined. Here, using two different forms of the staphylococcal cell envelope of Staphylococcus aureus RN4220 and USA300 LAC strains, we provide biochemical and immunological evidence that the particulate state is required for the effective activation of host innate immune responses. In a murine model of peritoneal infection, the particulate form of the staphylococcal cell envelope (PCE) induced the production of chemokine (C-X-C motif) ligand 1 (CXCL1) and CC chemokine ligand 2 (CCL2), the chemotactic cytokines for neutrophils and monocytes, respectively, resulting in a strong influx of the phagocytes into the peritoneal cavity. In contrast, compared with PCE, the soluble form of cell envelope (SCE), which was derived from PCE by treatment with cell wall-hydrolyzing enzymes, showed minimal activity. PCE also induced the secretion of calprotectin (myeloid-related protein 8/14 [MRP8/14] complex), a phagocyte-derived antimicrobial protein, into the peritoneal cavity at a much higher level than did SCE. The injected PCE particles were phagocytosed by the infiltrated neutrophils and monocytes and then delivered to mediastinal draining lymph nodes. More importantly, intraperitoneally (i.p.) injected PCE efficiently protected mice from S. aureus infection, which was abolished by the depletion of either monocytes/macrophages or neutrophils. This study demonstrated that the physical state of bacterial cells is a critical factor for efficient host immune activation and the protection of hosts from staphylococcal infections.

The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation.

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2'-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2'-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.

In vivo blockade of T cell development reveals alternative pathways for generation of intraepithelial lymphocytes in mice.

Intraepithelial lymphocytes (IELs) are resident cells localized within the intestinal epithelia and play an important role in regulating gut inflammations and host defense against pathogens. CD8α+ TCRαß+ IELs are heterogeneous populations that are generated from T cell precursors including CD4- CD8α- double-negative (DN) cells and CD4+ CD8α+ double-positive (DP) cells. However, developmental pathways of TCRαß+ IELs remained unclear. To gain insight into the mechanisms, we generated mice (Bcl11bΔDN2 mice) that lack thymic precursors (DN CD5+ TCRß+ cells) for CD4- CD8αα+ TCRαß+ IELs. Unexpectedly, we found that, in the absence of the precursors in thymi of Bcl11bΔDN2 mice, CD4- CD8αα+ TCRαß+ IELs were still present in the intestine though the number was reduced. Adoptive transfer experiment showed that their precursors were highly enriched in CD8α+ TCRß- thymocytes. The CD4- CD8αα+ TCRαß+ IELs in Bcl11bΔDN2 mice are distinguished by Thy1.2 expression and are indeed present in WT mice. Taken together, our study reveal a novel developmental pathway for CD8αα+ TCRαß+ IELs.

Synthesis and Biological Evaluation of O-Methylated Glycolipids Related to PGLs via Direct Stereoselective Glycosidation and Sequential Suzuki-Miyaura Coupling using Boracyclane.

Synthesis of O-methylated glycolipids via direct stereoselective glycosidation whose sugar moieties are related to those in phenolic glycolipids (PGLs) is reported. Treatment of 2-O-methyl-rhamnosyl imidates with I2 and nBu4 NOTf resulted in their activation under low temperature and provided the α-rhamnosides with excellent α-selectivity. nBu4 NOTf enhanced the electorophilicity of iodine. This methodology improved the efficiency of the synthesis of both PGL-1 and PGL-tb1 sugars. The process involved the formation of 2-O-naphthylmethyl-α-rhamnoside and 2-O-methyl-α-fucoside. Sequential Suzuki-Miyaura coupling using synthetic glycosides, boracyclane, and aryl bromides provided glycolipids related to PGL sugars, and was accomplished with a one-pot process. Finally, we elucidated the immunosuppressive activities of all these synthetic compounds and found that a phenyl 3-O-α-rhamnosyl-2-O-methyl-α-rhamnoside possessing a 6-(2-naphthyl)hexyl group exhibited the strongest inhibitory effect.

C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein ß-Glucuronidase on Dendritic Cells.

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. ß-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2.

Evolution of Green Plants Accompanied Changes in Light-Harvesting Systems.

Photosynthetic organisms have various pigments enabling them to adapt to various light environments. Green plants are divided into two groups: streptophytes and chlorophytes. Streptophytes include some freshwater green algae and land plants, while chlorophytes comprise the other freshwater green algae and seawater green algae. The environmental conditions driving the divergence of green plants into these two groups and the changes in photosynthetic properties accompanying their evolution remain unknown. Here, we separated the core antennae of PSI and the peripheral antennae [light-harvesting complexes (LHCs)] in green plants by green-native gel electrophoresis and determined their pigment compositions. Freshwater green algae and land plants have high Chl a/b ratios, with most Chl b existing in LHCs. In contrast, seawater green algae have low Chl a/b ratios. In addition, Chl b exists not only in LHCs but also in PSI core antennae in these organisms, a situation beneficial for survival in deep seawater, where blue-green light is the dominant light source. Finally, low-energy Chl (red Chl) of PSI was detected in freshwater green algae and land plants, but not in seawater green algae. We thus conclude that the different level of Chl b accumulation in core antennae and differences in PSI red Chl between freshwater and seawater green algae are evolutionary adaptations of these algae to their habitats, especially to high- or low-light environments.