Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

CD45 pre-exclusion from the tips of T cell microvilli prior to antigen recognition.

The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but is independent of the CD45 extracellular domain. We further show that brief MV-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed antigen presenting cells (APC). We propose that the scarcity of CD45 phosphatase activity at the tips of MV enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these MV-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.

Surfing on Membrane Waves: Microvilli, Curved Membranes, and Immune Signaling.

Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.

Immune checkpoint inhibitor-associated celiac disease.

BACKGROUND: Rare cases of immune checkpoint inhibitor (ICI)-associated celiac disease (ICI-CeD) have been reported, suggesting that disruption of tolerance mechanisms by ICIs can unmask celiac disease (CeD). This study aims to characterize the clinicopathological and immunophenotypic features of ICI-CeD in comparison to ICI-associated duodenitis (ICI-Duo) and usual CeD. METHODS: A medical and pathological records search between 2015 and 2019 identified eight cases of ICI-CeD, confirmed by tTG-IgA. Nine cases of ICI-Duo, 28 cases of moderate CeD, as well as 5 normal controls were used as comparison groups. Clinical information was collected from the electronic medical records. Immunohistochemistry for CD3, CD8, T-cell receptor gamma/delta (γδ), programmed death ligand 1 (PD-L1), and programmed death 1 (PD-1) were performed, with quantification of intraepithelial lymphocyte (IEL) subsets in three well-oriented villi. CD68, PD-L1, and PD-1 were assessed as a percentage of lamina propria surface area infiltrated by positive cells. Statistical significance was calculated by the Student's t-test and Fisher's exact test. RESULTS: The eight patients with ICI-CeD (F:M=1:3) and nine patients with ICI-Duo (F:M=5:4) presented similarly with diarrhea (13/17) and abdominal pain (11/17) after a median of 1.6 months on ICI therapy. In patients with ICI-CeD, tTG-IgA ranged from 104 to >300 IU/mL. Histological findings in ICI-CeD and ICI-Duo were similar and included expansion of the lamina propria, active neutrophilic duodenitis, variably increased IELs, and villous blunting. Immunohistochemistry showed that the average number of IELs per 100 enterocytes is comparable between ICI-CeD and ICI-Duo, with increased CD3+ CD8+ T cells compared with normal duodenum but decreased γδ T cells compared with CeD. Average PD-L1 percentage was 9% in ICI-CeD and 18% in ICI-Duo, in comparison to <1% in CeD and normal duodenum; average PD-1 percentage was very low to absent in all cases (<3%). On follow-up, five patients with ICI-CeD improved on a gluten-free diet (GFD) as the sole therapeutic intervention (with down-trending tTG-IgA) while the other three required immunosuppression. All patients who developed ICI-Duo received immunosuppression with variable improvement in symptoms. CONCLUSIONS: ICI-CeD resembles ICI-Duo clinically and histologically but shares the serological features and response to gluten withdrawal with classic CeD. Immunophenotyping of IELs in ICI-CeD and ICI-Duo also shows similar CD3, CD8, γδ T cell subsets, and PD-L1 populations, all of which differed quantitatively from usual CeD. We conclude that ICI-CeD is biologically similar to ICI-Duo and is likely a variant of ICI-Duo, but treatment strategies differ, with ICI-CeD often improving with GFD alone, whereas ICI-Duo requires systemic immunosuppression.

T Cell Microvilli: Sensors or Senders?

Communication between cells is essential for multicellular life. During cognate immune interactions, T cells communicate with antigen-presenting cells (APC) via direct cell-cell contact or the release of molecules and vesicles containing T cell messages. A wide variety of mechanisms have been reported and among them a process called "trogocytosis" has traditionally been thought to be the fastest way to directly transfer membrane portions containing intact proteins from one cell to another; however, the mechanism is unverified. Trogocytosis has been distinguished from the generation of extracellular vesicles (EVs), a term that encompasses exosomes and microvesicles, as EVs are released via a contact-independent manner and are suggested to potentially send molecular messages over a distance. However, some previous reports regarding EVs in T cells may be misleading in terms of explaining their cellular origins. In addition, there is little evidence on how EVs are generated from T cells in vivo and function to regulate complex immune responses. A recent work demonstrated that T cell microvilli-thin and finger-like membrane protrusions-are highly fragile and easily separated as membrane particles by trogocytosis, forming a new class of EVs. Surprisingly, released T cell microvilli-derived particles act as vectors, transmitting T cell messages to cognate APCs. This review focuses on how T cell microvilli vesicles are connected with immune regulation mechanisms discovered previously.

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

Membrane Ultrastructure and T Cell Activation.

The immune system serves as a crucial line of defense from infection and cancer, while also contributing to tissue homeostasis. Communication between immune cells is mediated by small soluble factors called cytokines, and also by direct cellular interactions. Cell-cell interactions are particularly important for T cell activation. T cells direct the adaptive immune response and therefore need to distinguish between self and foreign antigens. Even though decades have passed since the discovery of T cells, exactly why and how they are able to recognize and discriminate between antigens is still not fully understood. Early imaging of T cells was very successful in capturing the early stages of conjugate formation of T cells with antigen-presenting cells upon recognition of peptide-loaded major histocompatibility complexes by the T cell receptor (TCR). These studies lead to the discovery of a "supramolecular activation cluster" now known as the immunological synapse, followed by the identification of microclusters of TCRs formed upon receptor triggering, that eventually coalesce at the center of the synapse. New developments in light microscopy have since allowed attention to turn to the very earliest stages of T cell activation, and to resting cells, at high resolution. This includes single-molecule localization microscopy, which has been applied to the question of whether TCRs are pre-clustered on resting T cells, and lattice light-sheet microscopy that has enabled imaging of whole cells interacting with antigen-presenting cells. The utilization of lattice light-sheet microscopy has yielded important insights into structures called microvilli, which are small membrane protrusions on T cells that seem likely to have a large impact on T cell recognition and activation. Here we consider how imaging has shaped our thinking about T cell activation. We summarize recent findings obtained by applying more advanced microscopy techniques and discuss some of the limitations of these methods.

Interpreting heterogeneity in intestinal tuft cell structure and function.

Intestinal tuft cells are a morphologically unique cell type, best characterized by striking microvilli that form an apical tuft. These cells represent approximately 0.5% of gut epithelial cells depending on location. While they are known to express chemosensory receptors, their function has remained unclear. Recently, numerous groups have revealed startling insights into intestinal tuft cell biology. Here, we review the latest developments in understanding this peculiar cell type's structure and function. Recent advances in volumetric microscopy have begun to elucidate tuft cell ultrastructure with respect to its cellular neighbors. Moreover, single-cell approaches have revealed greater diversity in the tuft cell population than previously appreciated and uncovered novel markers to characterize this heterogeneity. Finally, advanced model systems have revealed tuft cells' roles in mucosal healing and orchestrating type 2 immunity against eukaryotic infection. While much remains unknown about intestinal tuft cells, these critical advances have illuminated the physiological importance of these previously understudied cells and provided experimentally tractable tools to interrogate this rare cell population. Tuft cells act as luminal sensors, linking the luminal microbiome to the host immune system, which may make them a potent clinical target for modulating host response to a variety of acute or chronic immune-driven conditions.

Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation.

T cells have a central function in mounting immune responses. However, mechanisms of their early activation by cognate antigens remain incompletely understood. Here we use live-cell multi-colour single-molecule localization microscopy to study the dynamic separation between TCRs and CD45 glycoprotein phosphatases in early cell contacts under TCR-activating and non-activating conditions. Using atomic force microscopy, we identify these cell contacts with engaged microvilli and characterize their morphology, rigidity and dynamics. Physical modelling and simulations of the imaged cell interfaces quantitatively capture the TCR-CD45 separation. Surprisingly, TCR phosphorylation negatively correlates with TCR-CD45 separation. These data support a refined kinetic-segregation model. First, kinetic-segregation occurs within seconds from TCR activation in engaged microvilli. Second, TCRs should be segregated, yet not removed too far, from CD45 for their optimal and localized activation within clusters. Our combined imaging and computational approach prove an important tool in the study of dynamic protein organization in cell interfaces.

LDL Receptor-Related Protein 2 (Megalin) as a Target Antigen in Human Kidney Anti-Brush Border Antibody Disease.

Primary renal tubulointerstitial disease resulting from proximal tubule antigen-specific antibodies and immune complex formation has not been well characterized in humans. We report a cohort of patients with a distinct, underappreciated kidney disease characterized by kidney antibrush border antibodies and renal failure (ABBA disease). We identified ten patients with ABBA disease who had a combination of proximal tubule damage, IgG-positive immune deposits in the tubular basement membrane, and circulating antibodies reactive with normal human kidney proximal tubular brush border. All but one of the patients also had segmental glomerular deposits on renal biopsy specimen. Patients with ABBA disease were elderly and presented with AKI and subnephrotic proteinuria. Serum from all patients but not controls recognized a high molecular weight protein in renal tubular protein extracts that we identified as LDL receptor-related protein 2 (LRP2), also known as megalin, by immunoprecipitation and mass spectrometry. Immunostaining revealed that LRP2 specifically colocalized with IgG in the tubular immune deposits on the ABBA biopsy specimen but not the control specimen analyzed. Finally, ABBA serum samples but not control samples showed reactivity against recombinantly expressed N-terminal LRP2 fragments on Western blots and immunoprecipitated the recombinantly expressed N-terminal region of LRP2. This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is LRP2. Future studies are needed to determine the disease prevalence, stimulus for ABBA, and optimal treatment.

How tetraspanins shape endothelial and leukocyte nano-architecture during inflammation.

Tetraspanins are ubiquitous membrane proteins that induce local membrane curvature and hence co-ordinate cell-to-cell contacts. This review highlights their role in inflammation, which requires control of the nano-architecture of attachment sites between endothelial cells and leukocytes. The active role of endothelial cells in preparing for transmigration of leukocytes and determining the severity of an inflammation is often underscored. A clear hint to endothelial pre-activation is their ability to protrude clustered adhesion proteins upward prior to leukocyte contact. The elevation of molecular adhesive platforms toward the blood stream is crucially dependent on tetraspanins. In addition, leukocytes require tetraspanins for their activation. The example of the B-cell receptor is referenced in some detail here, since it provides deeper insights into the receptor-coreceptor interplay. To lift the role of tetraspanins from an abstract model of inflammation toward a player of clinical significance, two pathologies are analyzed for the known contributions of tetraspanins. The recent publication of the first crystal structure of a full-length tetraspanin revealed a cholesterol-binding site, which provides a strong link to the pathophysiological condition of atherosclerosis. Dysregulation of the inflammatory cascade in autoimmune diseases by endothelial cells is exemplified by the involvement of tetraspanins in multiple sclerosis.

Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine.

Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we highlight recent advances with regard to the molecular mechanisms of cell polarity-controlled epithelial homeostasis and immunity in the human intestine.

The clinical and phenotypical assessment of seronegative villous atrophy; a prospective UK centre experience evaluating 200 adult cases over a 15-year period (2000-2015).

BACKGROUND: Seronegative villous atrophy (SNVA) is commonly attributed to coeliac disease (CD). However, there are other causes of SNVA. More recently angiotensin-2-receptor-blockers (A2RBs) have been reported as an association but data on SNVA have been limited to centres evaluating complex case referrals and not SNVA in general. OBJECTIVES: To provide clinical outcomes and associations in a large prospective study overseeing all newcomers with SNVA. DESIGN: Over a 15-year period (2000-2015) we evaluated 200 adult patients with SNVA at a UK centre. A diagnosis of either seronegative CD (SNCD) or seronegative non-CD (SN-non-CD) was reached. Baseline comparisons were made between the groups, with 343 seropositive CD subjects serving as controls. RESULTS: Of the 200 SNVA cases, SNCD represented 31% (n=62) and SN-non-CD 69% (n=138). The human leucocyte antigen (HLA)-DQ2 and/or DQ8 genotype was present in 61%, with a 51% positive predictive value for SNCD. The breakdown of identifiable causes in the SN-non-CD group comprised infections (27%, n=54), inflammatory/immune-mediated disorders (17.5%, n=35) and drugs (6.5%, n=13; two cases related to A2RBs). However, no cause was found in 18% (n=36) and of these 72% (n=26/36) spontaneously normalised duodenal histology while consuming a gluten-enriched diet. Following multivariable logistic regression analysis an independent factor associated with SN-non-CD was non-white ethnicity (OR 10.8, 95% CI 2.2 to 52.8); in fact, 66% of non-whites had GI infections. On immunohistochemistry all groups stained positive for CD8-T-cytotoxic intraepithelial lymphocytes. However, additional CD4-T helper intraepithelial lymphocytes were occasionally seen in SN-non-CD mimicking the changes associated with refractory CD. CONCLUSIONS: Most patients with SNVA do not have CD, in particular those who are not white. Furthermore, a subgroup with no obvious aetiology will show spontaneous histological resolution while consuming gluten. These findings suggest caution in empirically prescribing a gluten-free diet without investigation.

IgG trafficking in the adult pig small intestine: one- or bidirectional transfer across the enterocyte brush border?

Immunoglobulin G (IgG) transfer in opposite directions across the small intestinal brush border serves different purposes in early life and in adulthood. In the neonate, maternal IgG is taken up from the gut lumen into the blood, conferring passive immunity to the offspring, whereas in the adult immunoglobulins, including IgG made by plasma cells in the lamina propria, are secreted via the brush border to the lumen as part of the mucosal defense. Here, IgG has been proposed to perform a luminal immune surveillance which eventually includes a reuptake through the brush border as pathogen-containing immune complexes. In the present work, we studied luminal uptake of FITC-conjugated and gold-conjugated IgG in cultured pig jejunal mucosal explants. After 1 h, binding to the brush border was seen in upper crypts and lower parts of the villi. However, no endocytotic uptake into EEA-1-positive compartments was detected, neither at neutral nor acidic pH, despite an ongoing constitutive endocytosis from the brush border, visualized by the polar tracer CF594. The 40-kDa neonatal Fc receptor, FcRn, was present in the microvillus fraction, but noteworthy, a 37 kDa band, most likely a proteolytic cleavage product, bound IgG in a pH-dependent manner more efficiently than did the full-length FcRn. In conclusion, our work does not support the theory that bidirectional transfer of IgG across the intestinal brush border is part of the luminal immune surveillance in the adult.

Pathological damage and immunomodulatory effects of zebrafish exposed to microcystin-LR.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs), especially microcystin-LR (MC-LR), released during cyanobacterial blooms exert great toxicity on fish and even lead to massive death. The present study mainly investigated the pathological damage and immune response of spleen, gut and gill in zebrafish exposed to MC-LR. Fish were exposed to 0, 1, 5 and 20 µg/L of MC-LR for 30 d. In zebrafish exposed to 5 and 20 µg/L MC-LR, edematous mitochondria, deformation of the nucleus and compaction of chromatin were observed in lymphocyte of spleen; frayed gut villi, exfoliation of epithelial cells and widespread cell lyses were observed in intestines; hyperemia in gill lamellae, epithelial tissue edema and uplift and lamellar fusion were observed in gill. Varied changed gene expression was observed in spleen, intestine and gill of zebrafish. The transcriptional levels of IFN-1 and IL-8 in spleen significantly up-regulated in 20 µg/L group, and the transcription of IL-1ß and TNFα in spleen increased in 1 µg/L MC-LR treated fish. In addition, the mRNA levels of IFN-1, IL-1ß, IL-8, TGF-ß and TNF-α dramatically increased in intestine and gill in all MC-LR treated groups. The present studies indicated that MC-LR exposure caused marked pathological damage, however, fish could adjust actively the expression of innate immune-related genes to resist the tissue damage. Our findings provided strong evidence of the recovery potential of fish exposed to microcystins.

Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.

We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine diagnostics.

Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella.

The role of enterocyte defects in the pathogenesis of congenital diarrheal disorders.

Congenital diarrheal disorders are rare, often fatal, diseases that are difficult to diagnose (often requiring biopsies) and that manifest in the first few weeks of life as chronic diarrhea and the malabsorption of nutrients. The etiology of congenital diarrheal disorders is diverse, but several are associated with defects in the predominant intestinal epithelial cell type, enterocytes. These particular congenital diarrheal disorders (CDD(ENT)) include microvillus inclusion disease and congenital tufting enteropathy, and can feature in other diseases, such as hemophagocytic lymphohistiocytosis type 5 and trichohepatoenteric syndrome. Treatment options for most of these disorders are limited and an improved understanding of their molecular bases could help to drive the development of better therapies. Recently, mutations in genes that are involved in normal intestinal epithelial physiology have been associated with different CDD(ENT). Here, we review recent progress in understanding the cellular mechanisms of CDD(ENT). We highlight the potential of animal models and patient-specific stem-cell-based organoid cultures, as well as patient registries, to integrate basic and clinical research, with the aim of clarifying the pathogenesis of CDD(ENT) and expediting the discovery of novel therapeutic strategies.