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Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO+ Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO+ Tregs using single-cell images captured by the DEPArray™ system. The frequency of CD30+OX40+CD45RO+ Tregs was significantly higher in CRC patients than in healthy subjects (P +OX40+ Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30+OX40+ Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30+OX40+ Tregs as a diagnostic or prognostic biomarker in CRC.

Simple Summary Urothelial carcinoma (UC) is the most frequently diagnosed cancer of the urinary tract and is ranked the sixth most diagnosed cancer in men worldwide. About 70–75% of newly diagnosed UCs are non-invasive or low grade. Different tests such as urine cytology and cystoscopy are used to detect UC. If abnormal tissue is found during cystoscopy, then a biopsy will be performed. Cytology has low sensitivity for low-grade cancer while cystoscopy is invasive and costly. Detecting UC early improves the chances of treatment success. Therefore, many researchers have painstakingly identified urine biological markers for non-invasive UC diagnosis. In this review, we summarize some of the latest and most promising biological markers (including FDA-approved and investigational markers). We also discuss some new technologies that can aid research efforts in biological marker discovery for early UC detection. Abstract Urothelial carcinoma (UC) is the most frequent malignancy of the urinary system and is ranked the sixth most diagnosed cancer in men worldwide. Around 70–75% of newly diagnosed UC manifests as the non-muscle invasive bladder cancer (NMIBC) subtype, which can be treated by a transurethral resection of the tumor. However, patients require life-long monitoring due to its high rate of recurrence. The current gold standard for UC diagnosis, prognosis, and disease surveillance relies on a combination of cytology and cystoscopy, which is invasive, costly, and associated with comorbidities. Hence, there is considerable interest in the development of highly specific and sensitive urinary biomarkers for the non-invasive early detection of UC. In this review, we assess the performance of current diagnostic assays for UC and highlight some of the most promising biomarkers investigated to date. We also highlight some of the recent advances in single-cell technologies that may offer a paradigm shift in the field of UC biomarker discovery and precision diagnostics.

Purpose To test whether analyzing DEPArray (Menarini Silicon Biosystems) isolated single B cells from the vitreous fluid can reveal crucial genomic and clinicopathological features to distinguish patients with vitreoretinal lymphoma (VRL) from those with chronic inflammation using immunoglobulin heavy chain (IGH), disease biomarker myeloid differentiation primary response 88 (MYD88)L265P mutation, and copy number profiling. Design A single-center, retrospective study. Participants Remnant vitreous biopsies from 7 patients with VRL and 4 patients with chronic inflammation were acquired for molecular analysis. Methods Vitreous fluid samples were prefixed in PreservCyt (Hologic) and underwent cytologic analysis and immunohistochemistry examination. Single cells were isolated using the DEPArray NxT system, followed by downstream genomic analysis. Main Outcome Measures The frequencies of the dominant IGH and MYD88L265P mutation and the genome-wide copy number aberration (CNA) profiles of individual vitreous-isolated B cells were characterized. Results An average of 10 to 13 vitreous B cells were used in the single-cell IGH and MYD88 analyses. Higher frequencies of dominant IGH (88.8% ± 13.2%) and MYD88L265P mutations (35.0% ± 31.3%) were detected in patients with VRL than in patients with chronic inflammation (65.9% ± 13.4% and 1.5% ± 2.6% for IGH and MYD88L265P, respectively). In a cytology-proven VRL case, all 15 vitreous isolated B cells were derived from the same clone with 100% paired IGH: immunoglobulin light chain (IGK) sequences. Genome-wide copy number profiling revealed a high degree of similarity between B cells from the same patient with VRL, with extensive gains and losses at the same areas across the whole genome. In addition, 14 of 15 B cells showed a BCL2/JH t(14;18) translocation, confirming cellular malignancy with a clonal origin. Clustering analysis of the copy number profiles revealed that malignant B cells derived from different patients with VRL had no common genome-wide signatures. Conclusions Single B-cell genomic characterization of the IGH, MYD88L265P mutation, and copy number profile enables VRL diagnosis. Because our study involved only a small cohort, these meaningful proof-of-concept data now warrant further investigation in a larger patient cohort.

The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1 CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin–NFAT–IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine.

TO THE EDITOR: Vitreoretinal lymphoma (VRL) is a rare ocular malignancy that occurs as a primary intraocular lymphoma or secondary manifestation of primary central nervous system lymphoma (PCNSL).[1][1] Although the diagnosis is classically made by cytologic and immunohistochemical confirmation of

The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin–NFAT–IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine., Treg cells can maintain intestinal homeostasis and limit intestinal bowel disease. Here the authors use a mouse model of spontaneous colitis to show that calcineurin-NFAT-induced IL-2 production by dendritic cells regulates the balance between Treg and effector T cells in the gut lamina propria.

Claudins belong to a large family of transmembrane proteins that localize at tight junctions (TJs) where they play a central role in regulating paracellular transport of solutes and nutrients across epithelial monolayers. Their ability to regulate the paracellular pathway is highly influenced by changes in extracellular pH. However, the effect of changes in pH on the strength and kinetics of claudin mediated adhesion is poorly understood. Using atomic force microscopy, we characterized the kinetic properties of homophilic trans-interactions between full length recombinant GST tagged Claudin-2 (Cldn2) under different pH conditions. In measurements covering three orders of magnitude change in force loading rate of 10(2)-10(4) pN/s, the Cldn2/Cldn2 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier throughout pH range of 4.5-8. Comparing to the neutral condition (pH 6.9), differences in the inner energy barriers for the dissociation of Cldn2/Cldn2 mediated interactions at acidic and alkaline environments were found to be0.65 k(B)T, which is much lower than the outer dissociation energy barrier (1.37 k(B)T). The relatively stable interaction of Cldn2/Cldn2 in neutral environment suggests that electrostatic interactions may contribute to the overall adhesion strength of Cldn2 interactions. Our results provide an insight into the changes in the inter-molecular forces and adhesion kinetics of Cldn2 mediated interactions in acidic, neutral and alkaline environments.

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.

Cellular adhesion and barriers formed by intercellular adhesion proteins [tight junctions (occludin and claudins) and adherens junction (E-cadherin)] are important in maintaining tissue homeostasis. However, disruption of these junction proteins is associated with diseases in the organ systems such as multiple sclerosis, diarrhea, asthma, and gastro-intestinal tract carcinomas among others. In this paper, the separation force needed to separate two cells expressing some of these proteins was measured using the dual micropipette assay. Results show that L-fibroblasts transfected with claudin-1 and claudin-2 exhibit higher separation force (~2.8 nN and 2.3 nN, respectively) as compared to control cells or cells transfected with occludin (~1 nN). Furthermore, the separation force was not affected on addition of calcium chelating agent (ethylene diamine tetra acetic acid, EDTA). The separation force was, however, significantly decreased on treating cells with the actin disrupting agent Cytochalasin-D. These results show that the dual micropipette assay is a simple and useful experimental technique for quantifying cell–cell adhesion.

Nectins are Ca2+ independent cell adhesion molecules localizing at the cadherin based adherens junctions. In this study, we have used atomic force microscopy to study interaction of a chimera of extra cellular fragment of nectin-1 and Fc of human IgG (nef-1) with wild type L-fibroblasts that express endogenous nectin-1 to elucidate the biophysical characteristics of homophilic nectin-1 trans-interactions at the level of single molecule. Bond strength distribution revealed three distinct bound states (or configurations) of trans-interactions between paired nectins, where each bound state has a unique unstressed off-rate and reactive compliance. Kinetic analysis of force-dependent off-rate of the bound state involving trans-interacting V-V domains between paired nectin-1 (unstressed off-rate approximately 1.465+/-0.779 s(-1), reactive compliance approximately 0.143+/-0.072 nm) was found to be closest to E-cadherin, indicating that V-V domain trans-interactions are probably necessary to initiate and promote adhesions of E-cadherin at adherens junctions (AJs).

Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.

Claudins are a family of tetraspan membrane proteins that localize at tight junctions in an epithelial monolayer forming a selective barrier to diffusion of solutes via the intercellular spaces. It is widely accepted that the interaction between the extracellular loops of claudin molecules from adjacent cells is critical for this function. Though previous experiments utilizing traditional biological, biochemical, morphological, and electrophysiological approaches have provided significant insights into the role of claudins in regulating ion permeability, the interaction kinetics between these molecules has not been characterized. In this chapter, we describe two experimental procedures to study the adhesion forces imparted by claudins: (a) dual micropipette assay to quantify the adhesion forces at the cellular level and (b) single molecule force spectroscopy using atomic force microscopy to characterize the interaction kinetics at the molecular level. Though the experimental procedures are described for claudins, they can be easily modified for studying the interaction properties of a wide variety of other proteins.

Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC–T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC–T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.

Claudins are a family of tetraspan membrane proteins that localize at tight junctions in an epithelial monolayer forming a selective barrier to diffusion of solutes via the intercellular spaces. It is widely accepted that the interaction between the extracellular loops of claudin molecules from adjacent cells is critical for this function. Though previous experiments utilizing traditional biological, biochemical, morphological, and electrophysiological approaches have provided significant insights into the role of claudins in regulating ion permeability, the interaction kinetics between these molecules has not been characterized. In this chapter, we describe two experimental procedures to study the adhesion forces imparted by claudins: (a) dual micropipette assay to quantify the adhesion forces at the cellular level and (b) single molecule force spectroscopy using atomic force microscopy to characterize the interaction kinetics at the molecular level. Though the experimental procedures are described for claudins, they can be easily modified for studying the interaction properties of a wide variety of other proteins.

Biochemical studies have determined that reoviruses attach to cells by combining attachment protein sigma1 to the binding interface of its receptor protein junctional adhesion molecule A (JAM-A), and the interface normally takes care of the homodimerization of JAM-A. Tighter binding and slower dissociation of for the sigma1-JAM complex than for the JAM-JAM complex have been probed by both biological and atomic force microscopy experiments; however, the mechanism of the binding preference of the attachment protein for JAM-A still remains unclear. With the help of classical and steered molecular dynamics and energy calculations, the unbinding forces and kinetic properties of the complexes are investigated, together with detailed structural information analyses. A multireceptor mechanism is proposed for the binding preference, which can be helpful for future viral infection and vector targeting studies.

Claudins (Cldns) comprise a large family of important transmembrane proteins that localize at tight junctions where they play a central role in regulating paracellular transportation of solutes across epithelia. However, molecular interactions occurring between the extracellular domains of these proteins are poorly understood. Here, using atomic force microscopy, the adhesion strength and kinetic properties of the homophilic interactions between the two extracellular loops of Cldn2 (C2E1or C2E2) and full-length Cldn2 were characterized at the level of single molecule. Results show that while the first extracellular loop is sufficient for Cldn2/Cldn2 trans-interaction, the second extracellular loop does not interact with the full-length Cldn2, with the first extracellular loop, or with itself. Furthermore, within the range of loading rates probed (102–104 pN/s), dissociation of Cldn2/Cldn2 and C2E1/C2E1 complexes follows a two-step energy barrier model. The difference in activation energy for the inner and outer barriers of Cldn2/Cldn2 and C2E1/C2E1 dissociation was found to be 0.26 and 1.66 kBT, respectively. Comparison of adhesion kinetics further revealed that Cldn2/Cldn2 dissociates at a much faster rate than C2E1/C2E1, indicating that the second extracellular loop probably has an antagonistic effect on the kinetic stability of Cldn2-mediated interactions. These results provide an insight into the importance of the first extracellular loop in trans-interaction of Cldn2-mediated adhesion.

Claudins are proteins that are selectively expressed at tight junctions (TJs) of epithelial cells where they play a central role in regulating paracellular permeability of solutes across epithelia. However, the role of claudins in intercellular adhesion and the mechanism by which they regulate the diffusion of solutes are poorly understood. Here, using single molecule force spectroscopy, the kinetic properties and adhesion strength of homophilic claudin-1 interactions were probed at the single-molecule level. Within the range of tested loading rates (10(3)-10(5) pN/s), our results showed that homophilic claudin-1 interactions have a reactive compliance of 0.363 +/- 0.061 nm and an unstressed dissociation rate of 1.351 +/- 1.312 s-1. This is more than 100-fold greater than that of E-cadherin. The weak and short-lived interactions between claudin-1 molecules make them highly unstable and dynamic in nature. Such a dynamic interaction is consistent with a model where breaking and resealing of TJ strands regulate the paracellular diffusion of solutes.

Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8+ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c+ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8+ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.

Antigen recognition and discrimination by T lymphocyte are essential in initiating appropriate immune responses. The mechanisms underlying exquisite sensitivity and specificity of antigen discrimination are not fully elucidated but involved physical intercellular interactions between T cell and antigen-presenting cell (APC) including dendritic cells (DCs) and B cells. Using single cell force spectroscopy (SCFS), we have successfully probe mechanical interactions between DC and T cells in response to a panel of altered peptide ligands (APLs). In the presence of different type of APLs, DC:T conjugate displays different ranges of mechanical forces that match their corresponding T cell responsiveness. Strong cell-cell interaction forces are contributed not only by surface adhesion molecules including TCR:pMHC, CD28:B7 and ICAM-1:LFA-1 interaction pairs, but also dependent on the integrity of membrane cholesterol and cytoskeleton dynamics. As compared to B:T interactions, stronger DC:T cell-cell interactions are likely to provide a mechanically stable environment that induces potent functional T cell activation, suggesting that the measurement of mechanical forces could be a significant predictor of T cell functional activation efficacy. Our SCFS studies provide mechanical insights into the adhesive roles of cell surface proteins in regulating APC:T cell-cell interactions. Functional roles of TLR ligands in regulating APC:T interactions and T-cell responses will be discussed.

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the ‘holder’ for providing stable binding.