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Structured AbstractBackgroundHidradenitis suppurativa (HS) skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu the biology and contribution of these cells in HS pathogenesis is unclear.ObjectiveWe aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions.MethodsWe combined histological analysis, single-cell RNA-sequencing (scRNAseq), and spatial transcriptomic profiling of HS lesions to define the tissue microenvironment relative to B cell activity within this disease.ResultsOur findings identify tertiary lymphoid structures (TLS) within HS lesions and describe organized interactions between T cells, B cells, antigen presenting cells and skin stroma. We find evidence that B cells within HS TLS actively undergo maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells are primed to support the formation of TLS and facilitate B cell recruitment during HS.ConclusionOur data definitively demonstrate the presence of TLS in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed non-lymphoid tissue.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

The specific communication of multiple cell types in the tumor microenvironment plays a critical role in cancer progression. Current engineering methods have failed to adequately replicate the complexities of the tumor microenvironment (TME). In particular, generating engineered tissue-like environments with multiple TME cell types has remained challenging. Here we demonstrate the capability to pattern complex single cell circuit configurations, using a novel microfluidic bioprinting method, to study cell-cell communication in the early TME. A microfluidic dispenser (Biopixlar, Fluicell AB) was optimized to determine the delivery pressure (5 – 80 mbar), internal vacuum (0 – 80 -mbar), and external vacuum (0 – 80 -mbar) to enable highly controllable deposition of single cells suspended in complete media supplemented with polyethylene glycol (15 mg/mL, 1:1) at 1 × 106 cells/mL. Flow conditions were optimized for human cells: MDA-MB-231, MCF7, PC3, breast epithelial cells (MCF10a), fibroblasts, cancer associated fibroblasts, THP-1 derived macrophages, CD4+ T cells, CD8+ T cells, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells. As proof of concept, the optimized settings were used to replicate a 2D tumor biopsy region of interest with high spatial precision. Next, cell-cell communication circuits were fabricated with cancer cells (PC3 or MDA-MB-231) and HUVECs. Communication circuits were bioprinted as 4 by 4 cell arrays, with 100 µm spacing between each cell, equal number of HUVECs and cancer cells, and three different cellular arrangements: alternating cell types, like cell types grouped, and groups of four like cell types. The circuits were live cell imaged for up to 30 hours to observe cell migration patterns, proliferation, and morphological changes as a function of cell-cell communication circuit arrangements. Optimal printing parameters were identified as 80 mbar delivery pressure, -25 mbar internal vacuum, and -55 mbar external vacuum. These parameters maintained >99% cell viability and ±10 µm spatial precision of printed cells. Live cell imaging of circuits containing PC3s or MDA-MB-231s with HUVECs on collagen substrates revealed changes in migration patterns, proliferation, and morphology depending on the surrounding cellular arrangement. HUVECs were highly migratory throughout the duration of the experiment, frequently extended protrusions towards nearby HUVECs, but did not display the same level of interaction with PC3s as they did with MDA-MB-231s. In MDA-MB-231 circuits, irrespective of patterning, we identified clear tendencies of HUVECs to herd MDA-MB-231s, travel overtop of MDA-MB-231s, collect and carry visible particles released from MDA-MB-231s, and maintain dendritic morphology instead of undergoing the expected vascular tubulogenesis. We found that HUVECs had the best morphology when clustered in groups of four and proliferated most when surrounded by MDA-MB-231s (alternating pattern). We found that MDA-MB-231s only proliferated when surrounded by HUVECs and had the least displacement when surrounded by like cells. These results demonstrate a method to precisely bioprint single cell circuits, enabling the investigation of cellular spatial organization and composition within the tumor microenvironment as it relates to tumor initiation and progression. Citation Format: Haylie R. Helms, Alexander E. Davies, Rebekka Duhen, Joshua M. Moreau, Ellen M. Langer, Luiz E. Bertassoni. Single cell bioprinted cell circuits for the systematic assessment of cell-cell communication in the early tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB161.

Transforming growth factor–β1 (TGF-β1) is inextricably linked to regulatory T cell (T reg ) biology. However, precisely untangling the role for TGF-β1 in T reg differentiation and function is complicated by the pleiotropic and context-dependent activity of this cytokine and the multifaceted biology of T regs . Among CD4 + T cells, T regs are the major producers of latent TGF-β1 and are uniquely able to activate this cytokine via expression of cell surface docking receptor glycoprotein A repetitions predominant (GARP) and αv integrins. Although a preponderance of evidence indicates no essential roles for T reg -derived TGF-β1 in T reg immunosuppression, TGF-β1 signaling is crucial for T reg development in the thymus and periphery. Furthermore, active TGF-β1 instructs the differentiation of other T cell subsets, including T H 17 cells. Here, we will review TGF-β1 signaling in T reg development and function and discuss knowledge gaps, future research, and the TGF-β1/T reg axis in the context of cancer immunotherapy and fibrosis.

Regulatory T cells (Tregs) reside in nonlymphoid tissues where they carry out unique functions. The molecular mechanisms responsible for Treg accumulation and maintenance in these tissues are relatively unknown. Using an unbiased discovery approach, we identified LAYN (layilin), a C-type lectin-like receptor, to be preferentially and highly expressed on a subset of activated Tregs in healthy and diseased human skin. Expression of layilin on Tregs was induced by TCR-mediated activation in the presence of IL-2 or TGF-β. Mice with a conditional deletion of layilin in Tregs had reduced accumulation of these cells in tumors. However, these animals somewhat paradoxically had enhanced immune regulation in the tumor microenvironment, resulting in increased tumor growth. Mechanistically, layilin expression on Tregs had a minimal effect on their activation and suppressive capacity in vitro. However, expression of this molecule resulted in a cumulative anchoring effect on Treg dynamic motility in vivo. Taken together, our results suggest a model whereby layilin facilitates Treg adhesion in skin and, in doing so, limits their suppressive capacity. These findings uncover a unique mechanism whereby reduced Treg motility acts to limit immune regulation in nonlymphoid organs and may help guide strategies to exploit this phenomenon for therapeutic benefit.

The diverse populations of tissue-resident and transitory T cells present in the skin share a common functional need to enter, traverse, and interact with their environment. These processes are largely dependent on the regulated expression of adhesion molecules, such as selectins and integrins, which mediate bidirectional interactions between immune cells and skin stroma. Dysregulation and engagement of adhesion pathways contribute to ectopic T-cell activity in tissues, leading to the initiation and/or exacerbation of chronic inflammation. In this paper, we review how the molecular interactions supported by adhesion pathways contribute to T-cell dynamics and function in the skin. A comprehensive understanding of the molecular mechanisms underpinning T-cell adhesion in inflammatory skin disorders will facilitate the development of novel tissue-specific therapeutic strategies.

B cell development is regulated by stromal cells (SCs) that form a supportive microenvironment. These SCs along with other cell types produce cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. BM, spleen (Sp), and the gut lamina propria (LP) constitute distinctive anatomical compartments that support B cell differentiation. In order to characterize and compare the signals necessary to generate IgA+ B cells, we developed an in vitro system to co-culture gut LP, BM, or Sp-derived SCs with B lineage cells. Using this co-culture system, we found that gut LP SCs promote IgA+ B cell accumulation through the production of soluble stimulatory factors. In contrast to gut LP SCs, BM and splenic SCs were found to impair IgA+ B cell accumulation in vitro. Taken together, these observations provide new insights into how SCs derived from different anatomical locations shape IgA+ B cell responses.

Pro- and anti-inflammatory B cell subsets that localize to unperturbed and inflamed skin are newly emerging components of the skin immune system. To test the relevance of regulatory B cells (Bregs) in the suppression of cutaneous inflammation, we asked whether impaired migration of these cells into the skin exacerbates skin inflammation. Using a mouse model with a B cell-specific tamoxifen-inducible deletion of α4β1-integrin we demonstrate that selective disruption of α4β1-integrin expression in B cells significantly decreases IL-10(+) Bregs in inflamed skin while not affecting their counterparts in lymphoid tissues. Impaired skin-homing and reduced cutaneous accumulation of IL-10(+) Bregs leads to a significant increase in clinical and histopathological parameters of inflammation in both psoriasiform skin inflammation and cutaneous delayed contact hypersensitivity. Thus, our data show a crucial function of skin-homing IL-10(+) Bregs in the suppression of skin inflammation, supporting the notion that Bregs are critical players in the cutaneous environment during inflammatory skin diseases.

Regulatory T cells (Tregs) utilize multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e. nonlymphoid) tissues have specialized functions, specifically skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-β pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation following skin barrier breach. Using a single cell discovery approach, we identified preferential expression of the integrin αvβ8 on skin Tregs. Upon skin injury, Tregs utilized this integrin to activate latent TGF-β which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvβ8 expressing Tregs in skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Layilin is a small type I transmembrane receptor thought to bridge extracellular ligands with the cytoskeleton through its intracellular interactions with the scaffolding protein talin. Recent bulk- and single-cell RNA sequencing experiments have repeatedly found layilin to be highly upregulated in key T cell sub-populations in multiple disease states, suggesting its importance to the adaptive immune response. Despite this prevalence, little is known about layilin's precise role in mediating extracellular interactions or how these interactions can be modulated in disease states. Here we take advantage of layilin's dependence on calcium ions to discover its interactions with highly glycosylated type II, IV, V, and VI collagens. Toward exploring layilin's role in disease, we exploited the Ca

Summary A central challenge in medicine is translating from observational understanding to mechanistic understanding, where some observations are recognized as causes for the others. This can lead not only to new treatments and understanding, but also to recognition of novel phenotypes. Here, we apply a collection of mathematical techniques (empirical dynamics), which infer mechanistic networks in a model-free manner from longitudinal data, to hematopoiesis. Our study consists of three subjects with markers for cyclic thrombocytopenia, in which multiple cells and proteins undergo abnormal oscillations. One subject has atypical markers and may represent a rare phenotype. Our analyses support this contention, and also lend new evidence to a theory for the cause of this disorder. Simulations of an intervention yield encouraging results, even when applied to patient data outside our three subjects. These successes suggest that this blueprint has broader applicability in understanding and treating complex disorders., Graphical Abstract, Highlights • Many disorders are “complex” and include multiple dysfunctional elements • Empirical dynamics can infer networks of interactions from longitudinal data • Subnetworks around focal points of interest can explain phenotypes • Dimensional reduction narrows the search for therapeutic interventions, The Bigger Picture While some diseases have known root causes, many consist of multiple abnormal behaviors connected by unknown biology. These complex disorders require new tools that are able to leverage data to sketch out the equivalent of a “food web” in ecology, i.e., a network of interactions between various cells and protein signals. Constructing these networks allows us to examine differences between healthy and afflicted individuals and identify potential therapies. Discerning meaningful differences in interaction networks also gives a paradigm for delineating phenotypes. Here, we integrate recent techniques for empirical network inference and apply them to cyclic thrombocytopenia, a complex blood disease characterized by platelet oscillations. The agreement between the techniques builds confidence in the networks we have inferred. We validated a plausible therapeutic intervention in silico, suggesting that interactions along the axis we identify as the problem may be clinically viable., Many disorders are characterized by more than one aberrant biomarker. When the number of such biomarkers increases, it becomes difficult to define meaningful variation within patients as well as to identify targets for therapeutics. With such complexity and biological uncertainty, traditional techniques are ineffective. We suggest an empirical-dynamical toolbox for inferring networks of biomarker interactions. We infer and validate networks in three subjects with cyclic thrombocytopenia, confirming clinical suspicions of a novel phenotype and identifying a new therapeutic axis.

The C-type lectin layilin has been observed in several human cancers, but its function on immune cells is unknown. This study demonstrates that layilin is highly expressed on clonally expanded CD8+ T cells in human melanoma and augments integrin-mediated cellular adhesion to enhance antitumor immunity., Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain–containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLβ2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1–dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or “dysfunctional” CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential.

At the center of fibrosing diseases is the aberrant activation of tissue fibroblasts. The cellular and molecular mechanisms of how the immune system augments fibroblast activation have been described; however, little is known about how the immune system controls fibroblast function in tissues. Here, we identify regulatory T cells (Tregs) as important regulators of fibroblast activation in skin. Bulk cell and single cell analysis of Tregs in murine skin and lungs revealed that Tregs in skin are transcriptionally distinct and skewed towards T(H)2 differentiation. When compared to Tregs in lung, skin Tregs preferentially expressed high levels of GATA3, the master T(H)2 transcription factor. Genes regulated by GATA3 were highly enriched in skin ‘T(H)2 Treg’ subsets. In functional experiments, Treg depletion resulted in a preferential increase in T(H)2 cytokine production in skin. Both acute depletion and chronic reduction of Tregs resulted in spontaneous skin fibroblast activation, profibrotic gene expression and dermal fibrosis, all of which were exacerbated in a bleomycin-induced murine model of skin sclerosis. Lineage-specific deletion of Gata3 in Tregs resulted in an exacerbation of T(H)2 cytokine secretion that was preferential to skin, resulting in enhanced fibroblast activation and dermal fibrosis. Taken together, we demonstrate that Tregs play a critical role in regulating fibroblast activation in skin and do so by expressing a unique tissue-restricted transcriptional program that is mediated, at least in part, by GATA3.

Commensal-specific T cells dually possess type-2 and type-17 effector potential, allowing plasticity in orchestrating tissue immunity.

Hematopoiesis is a dynamic process involving the up- and down-regulation of genes, as well as feed-back loops that stimulate or suppress circulating cytokine concentrations. More complete pictures of the gene regulatory networks that control the production of the blood system have emerged with the advent of single-cell sequencing techniques and refinements to the capabilities of immunoassays. However, information about the regulatory networks of cytokines is still lacking. A novel mathematical technique (convergent cross-mapping, or CCM) allows for the extraction of causal relationships from data, which is of crucial importance for understanding these networks. To reconstruct the cytokine networks within the hematopoietic system we measured the concentrations of 62 cytokines, platelets, and thrombopoietin from an individual with cyclic thrombocytopenia (regular oscillations in the megakaryocytes and platelets) over 84 days. Using CCM, we identified 61 previously unreported cytokine relationships. Our approach is the first broad-scale investigation into causal relationships between cytokines in the blood and suggests a new paradigm for understanding how dynamic regulation occurs during hematopoiesis.

Systemic inflammation perturbs the bone marrow environment by evicting resident B cells and favoring granulopoiesis over lymphopoiesis. Despite these conditions, a subset of marrow B cell remains to become activated and produce potent acute immunoglobulin M (IgM) responses. This discrepancy is currently unresolved and a complete characterization of early perturbations in the B-cell niche has not been undertaken. Here, we show that within a few hours of challenging mice with adjuvant or cecal puncture, B cells accumulate in the bone marrow redistributed away from sinusoid vessels. This response correlates with enhanced sensitivity to CXC chemokine ligand 12 (CXCL12) but not CXCL13 or CC chemokine ligand 21. Concurrently, a number of B-cell survival and differentiation factors are elevated to produce a transiently supportive milieu. Disrupting homing dynamics with a CXC chemokine receptor 4 inhibitor reduced the formation of IgM-secreting cells. These data highlight the rapidity with which peripheral inflammation modifies the marrow compartment, and demonstrate that such modifications regulate acute IgM production within this organ. Furthermore, our study indicates that conversion to a state of emergency granulopoiesis is temporally delayed, allowing B cells opportunity to respond to antigen.

Acid ceramidase (ACDase) is ubiquitous and catalyzes the degradation of ceramide. ACDase and ceramides have been implicated in many disorders, including cancer, obesity, diabetes, inflammation, and neurodegenerative diseases.[1][1]–[3][2] Deficiencies in ACDase activity lead to Farber disease, but

B cell development is regulated by stromal cells (SCs) that form a supportive microenvironment. These SCs along with other cell types produce cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. BM, spleen (Sp), and the gut lamina propria (LP) constitute distinctive anatomical compartments that support B cell differentiation. In order to characterize and compare the signals necessary to generate IgA

Summary: Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease. : Shikatani et al. demonstrate that the nuclear transcription factor c-Myb exacerbates experimental atherosclerosis directly through its effects on B lymphocytes. Paradoxically, c-Myb promotes B2 cell development yet limits numbers of IgM-producing antibody-secreting cells and levels of atheroprotective OxLDL-specific IgM antibodies.

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions.

Growing cancers are known to modify immune responses through suppressive mechanisms manifested within the local tumor microenvironment. Accumulating evidence indicates that secreted tumor products can also influence on distant immunological compartments, including myelopoiesis in the bone marrow. However, it is unknown if a similar effect can occur to regulate B-cell lymphopoiesis in breast cancer. Examining the MMTV-PyMT murine model of breast cancer, we show a complete block in bone marrow B-cell lymphopoiesis, which is dependent on tumor burden. We also observed an increase in the total number of splenic B cells and an elevated frequency of marginal zone B cells. By using in vitro assays of B-cell lymphopoiesis, we show that tumor-secreted molecules directly inhibit B-cell progenitor proliferation and favor maturation. These data demonstrate a profound sensitivity of B-cell lymphopoiesis to the accumulation of ectopically produced molecules during tumor growth in PyMT.

Inciting the cellular arm of adaptive immunity has been the fundamental goal of cancer immunotherapy strategies, specifically focusing on inducing tumor antigen–specific responses by CD8+ cytotoxic T lymphocytes (CTL). However, there is an emerging appreciation that the cytotoxic function of CD4+ T cells can be effective in a clinical setting. Harnessing this potential will require an understanding of how such cells arise. In this study, we use an IL12-transduced variant of the 70Z/3 leukemia cell line in a B6D2F1 (BDF1) murine model system to reveal a novel cascade of cells and soluble factors that activate anticancer CD4+ killer cells. We show that natural killer T cells play a pivotal role by activating dendritic cells in a contact-dependent manner; soluble products of this interaction, including MCP-1, propagate the activation signal, culminating in the development of CD4+ CTLs that directly mediate an antileukemia response while also orchestrating a multipronged attack by other effector cells. A more complete picture of the conditions that induce such a robust response will allow us to capitalize on CD4+ T-cell plasticity for maximum therapeutic effect. Cancer Immunol Res; 2(11); 1113–24. ©2014 AACR.