Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.

Erratum: PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis.

This corrects the article DOI: 10.1038/nature24649.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Tissue clearing and 3D imaging - putting immune cells into context.

A better understanding of cell-cell and cell-niche interactions is crucial to comprehend the complexity of inflammatory or pathophysiological scenarios such as tissue damage during viral infections, the tumour microenvironment and neuroinflammation. Optical clearing and 3D volumetric imaging of large tissue pieces or whole organs is a rapidly developing methodology that holds great promise for the in-depth study of cells in their natural surroundings. These methods have mostly been applied to image structural components such as endothelial cells and neuronal architecture. Recent work now highlights the possibility of studying immune cells in detail within their respective immune niches. This Review summarizes recent developments in tissue clearing methods and 3D imaging, with a focus on the localization and quantification of immune cells. We first provide background to the optical challenges involved and their solutions before discussing published protocols for tissue clearing, the limitations of 3D imaging of immune cells and image analysis. Furthermore, we highlight possible applications for tissue clearing and propose future developments for the analysis of immune cells within homeostatic or inflammatory immune niches.

PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis.

T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules. Although these oncogenic alterations are thought to drive TCR pathways to induce chronic proliferation and cell survival programmes, it remains unclear whether T cells contain tumour suppressors that can counteract these events. Here we show that the acute enforcement of oncogenic TCR signalling in lymphocytes in a mouse model of human T cell lymphoma drives the strong expansion of these cells in vivo. However, this response is short-lived and robustly counteracted by cell-intrinsic mechanisms. A subsequent genome-wide in vivo screen using T cell-specific transposon mutagenesis identified PDCD1, which encodes the inhibitory receptor programmed death-1 (PD-1), as a master gene that suppresses oncogenic T cell signalling. Mono- and bi-allelic deletions of PDCD1 are also recurrently observed in human T cell lymphomas with frequencies that can exceed 30%, indicating high clinical relevance. Mechanistically, the activity of PD-1 enhances levels of the tumour suppressor PTEN and attenuates signalling by the kinases AKT and PKC in pre-malignant cells. By contrast, a homo- or heterozygous deletion of PD-1 allows unrestricted T cell growth after an oncogenic insult and leads to the rapid development of highly aggressive lymphomas in vivo that are readily transplantable to recipients. Thus, the inhibitory PD-1 receptor is a potent haploinsufficient tumour suppressor in T cell lymphomas that is frequently altered in human disease. These findings extend the known physiological functions of PD-1 beyond the prevention of immunopathology after antigen-induced T cell activation, and have implications for T cell lymphoma therapies and for current strategies that target PD-1 in the broader context of immuno-oncology.

Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain-associated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

Foxp1 controls mature B cell survival and the development of follicular and B-1 B cells.

The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1 Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma.

The Small Rho GTPase TC10 Modulates B Cell Immune Responses.

Rho family GTPases regulate diverse cellular events, such as cell motility, polarity, and vesicle traffic. Although a wealth of data exists on the canonical Rho GTPases RhoA, Rac1, and Cdc42, several other family members remain poorly studied. In B cells, we recently demonstrated a critical role for Cdc42 in plasma cell differentiation. In this study, we focus on a close homolog of Cdc42, TC10 (also known as RhoQ), and investigate its physiological role in B cells. By generating a TC10-deficient mouse model, we show that despite reduced total B cell numbers, B cell development in these mice occurs normally through distinct developmental stages. Upon immunization, IgM levels were reduced and, upon viral infection, germinal center responses were defective in TC10-deficient mice. BCR signaling was mildly affected, whereas cell migration remained normal in TC10-deficient B cells. Furthermore, by generating a TC10/Cdc42 double knockout mouse model, we found that TC10 can compensate for the lack of Cdc42 in TLR-induced cell activation and proliferation, so the two proteins play partly redundant roles. Taken together, by combining in vivo and in vitro analysis using TC10-deficient mice, we define the poorly studied Rho GTPase TC10 as an immunomodulatory molecule playing a role in physiological B cell responses.

B cell-mediated CD4 T-cell costimulation via CD86 exacerbates pro-inflammatory cytokine production during autoimmune intestinal inflammation.

Dysregulated B cell responses have been described in inflammatory bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely understood. We here provide evidence for the detrimental role of activated B cells during the onset of autoimmune intestinal inflammation. Using Wiskott-Aldrich Syndrome interacting protein deficient (Wipf1-/-) mice as a mouse model of chronic colitis, we identified clusters of differentiation (CD)86 expression on activated B cells as a crucial factor exacerbating pro-inflammatory cytokine production of intestinal CD4 T cells. Depleting B cells through anti-CD20 antibody treatment or blocking costimulatory signals mediated by CD86 through cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) diminished intestinal inflammation in our mouse model of chronic IBD at the onset of disease. This was due to a reduction in aberrant humoral immune responses and reduced CD4 T cell pro-inflammatory cytokine production, especially interferon-g (IFN-g) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, in addition to B cells isolated from the inflamed colon of Wipf1-/- mice, we also found CD86 mRNA and protein expression upregulated on activated B cells isolated from inflamed tissue of human patients with IBD. B cell activation and CD86 expression were boosted by soluble CD40L in vitro, which we found in the serum of mice and human patients with IBD. In summary, our data provides detailed insight into the contribution of B cells to intestinal inflammation, with implications for the treatment of IBD.

Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Fast histological assessment of adipose tissue inflammation by label-free mid-infrared optoacoustic microscopy.

Conventional histology, as well as immunohistochemistry or immunofluorescence, enables the study of morphological and phenotypical changes during tissue inflammation with single-cell accuracy. However, although highly specific, such techniques require multiple time-consuming steps to apply exogenous labels, which might result in morphological deviations from native tissue structures. Unlike these techniques, mid-infrared (mid-IR) microspectroscopy is a label-free optical imaging method that retrieves endogenous biomolecular contrast without altering the native composition of the samples. Nevertheless, due to the strong optical absorption of water in biological tissues, conventional mid-IR microspectroscopy has been limited to dried thin (5-10 µm) tissue preparations and, thus, it also requires time-consuming steps-comparable to conventional imaging techniques. Here, as a step towards label-free analytical histology of unprocessed tissues, we applied mid-IR optoacoustic microscopy (MiROM) to retrieve intrinsic molecular contrast by vibrational excitation and, simultaneously, to overcome water-tissue opacity of conventional mid-IR imaging in thick (mm range) tissues. In this proof-of-concept study, we demonstrated application of MiROM for the fast, label-free, non-destructive assessment of the hallmarks of inflammation in excised white adipose tissue; i.e., formation of crown-like structures and changes in adipocyte morphology.

Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen.

The relevance of direct inflammatory signals (signal 3) for the activation of memory CD8(+) T cells during recall responses is so far unknown. We therefore investigated the direct impact of IL-12 and type I IFN on the formation, recall potential and protective capacity of memory T cells. Using CD8(+) T cells deficient for IL-12 or type I IFN receptors in an adoptive transfer system, we generated memory populations after infection with vaccinia virus, lymphocytic choriomeningitis virus or Listeria monocytogenes. The results demonstrate that in the absence of signal 3 cytokines during primary infection, functional memory T cells were formed. After retransfer into naïve mice, signal 3-deficient memory T cells were able to specifically lyse target cells in vivo under non-infectious conditions. However, after reinfection, secondary effector CD8(+) T cells lacking signal 3 were impaired in expansion and protective capacity dependent on the nature of the pathogen. We conclude that memory CD8(+) T cells depend on a signal 3 for expansion, independent of signals obtained during priming, thereby being influenced by the pathogen-induced inflammatory milieu during secondary infection. In summary, our results reveal an essential role for direct inflammatory cytokine signaling in secondary T-cell responses.

Partial-Brain Radiation-Induced Microvascular Cognitive Impairment in Juvenile Murine Unilateral Hippocampal Synaptic Plasticity.

PURPOSE: Radiation-induced cognitive deficits have a severe negative impact on pediatric brain tumor patients. The severity of cognitive symptoms is related to the age of the child when radiation was applied, with the most severe effects seen in the youngest. Previous studies using whole-brain irradiation in mice confirmed these findings. To understand ipsilateral and contralateral changes in the hippocampus after partial-brain radiation therapy (PBRT) of the left hemisphere, we assessed the neuroplasticity and changes in the microvasculature of the irradiated and nonirradiated hippocampus in juvenile mice. METHODS AND MATERIALS: The left hemispheres of 5-week-old mice were irradiated with 2, 8, and 20 Gy and a fractionated dose of 8 Gy in 2 fractions using a computed tomography image guided small animal radiation research platform. Long-term potentiation (LTP) has been monitored ex vivo in the hippocampal cornu ammonis 1 (CA1) region and was assessed 3 days and 5 and 10 weeks after PBRT in both hemispheres and compared to a sham group. Irradiation effects on the hippocampus microvasculature were quantified by efficient tissue clearing and multiorgan volumetric imaging. RESULTS: LTP in irradiated hippocampal slices of juvenile mice declines 3 days after radiation, lasts up to 10 weeks in the irradiated part of the hippocampus, and correlates with a significantly reduced microvasculature length. Specifically, LTP inhibition is sustained in the irradiated (20 Gy, 8 Gy in 2 fractions, 8 Gy, 2 Gy) hippocampus, whereas the contralateral hippocampus remains unaffected after PBRT. LTP inhibition in the irradiated hemisphere after PBRT might be associated with an impaired microvascular network. CONCLUSION: PBRT induces a long-lasting impairment in neuroplasticity and the microvessel network of the irradiated hippocampus, whereas the contralateral hippocampus remains unaffected. These findings provide insight into the design of PBRT strategies to better protect the young developing brain from cognitive decline.

T cells acquire cell surface determinants of APC via in vivo trogocytosis during viral infections.

Trogocytosis describes the transfer of surface determinants between immune cells and has been implicated in immune regulation. Most findings are based on in vitro studies since in vivo trogocytosis of immune cells is difficult to detect under physiological conditions. We used low frequencies of memory P14 T cells to demonstrate that T cells perform trogocytosis in vivo if in contact with APC pulsed with GP33-peptide or expressing the antigen endogenously. Furthermore, in vivo trogocytosis of T cells is demonstrated during infections with lymphocytic choriomeningitis virus and vaccinia virus. Trogocytosis-positive T cells revealed higher expression of activation marker and cytokines, showing a more activated phenotype compared to trogocytosis-negative T cells.

The Wanderings of Gut-Derived IgA Plasma Cells: Impact on Systemic Immune Responses.

Humoral immunity is mainly mediated by a B cell population highly specialized to synthesize and secrete large quantities of antibodies - the antibody-secreting cells (ASC). In the gastrointestinal environment, a mixture of foreign antigens from the diet, commensal microbiota as well as occasional harmful pathogens lead to a constant differentiation of B cells into ASC. Due to this permanent immune response, more than 80% of mammalian ASC reside in the gut, of which most express immunoglobulin A (IgA). IgA antibodies contribute to intestinal homeostasis and can mediate protective immunity. Recent evidence points at a role for gut-derived ASC in modulating immune responses also outside of mucosal tissues. We here summarize recent evidence for wandering ASC, their antibodies and their involvement in systemic immune responses.

Effector T-cell differentiation during viral and bacterial infections: Role of direct IL-12 signals for cell fate decision of CD8(+) T cells.

To study the role of IL-12 as a third signal for T-cell activation and differentiation in vivo, direct IL-12 signaling to CD8(+) T cells was analyzed in bacterial and viral infections using the P14 T-cell adoptive transfer model with CD8(+) T cells that lack the IL-12 receptor. Results indicate that CD8(+) T cells deficient in IL-12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8(+) T cells deficient in IL-12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN-gamma and TNF-alpha. However, CD8(+) T cells lacking IL-12 signaling failed to up-regulate KLRG1 and to down-regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL-12 signaling to CD8(+) T cells determines the cell fate decision between short-lived effector cells and memory precursor effector cells, which is dependent on pathogen-induced local cytokine milieu.

Efficient Tissue Clearing and Multi-Organ Volumetric Imaging Enable Quantitative Visualization of Sparse Immune Cell Populations During Inflammation.

Spatial information of cells in their tissue microenvironment is necessary to understand the complexity of pathophysiological processes. Volumetric imaging of cleared organs provides this information; however, current protocols are often elaborate, expensive, and organ specific. We developed a simplified, cost-effective, non-hazardous approach for efficient tissue clearing and multi-organ volumetric imaging (EMOVI). EMOVI enabled multiplexed antibody-based immunolabeling, provided adequate tissue transparency, maintained cellular morphology and preserved fluorochromes. Exemplarily, EMOVI allowed the detection and quantification of scarce cell populations during pneumonitis. EMOVI also permitted histo-cytometric analysis of MHC-II expressing cells, revealing distinct populations surrounding or infiltrating glomeruli of nephritic kidneys. Using EMOVI, we found widefield microscopy with real-time computational clearing as a valuable option for rapid image acquisition and detection of rare cellular events in cleared organs. EMOVI has the potential to make tissue clearing and volumetric imaging of immune cells applicable for a broad audience by facilitating flexibility in organ, fluorochrome and microscopy usage.

Shaping the humoral immune response: Actin regulators modulate antigen presentation and influence B-T interactions.

B cells are an integral part of the adaptive immune system. During an immune response, the actin cytoskeleton plays a central role in regulating B cell antigen uptake, polarization and presentation as well as B cell migration and interaction with T cells. Genetic defects affecting actin regulators can result in reduced B cell activation, limited antibody production and hence cause disease. In this review, we discuss molecular mechanisms of actin regulation and their involvement in antigen polarisation and presentation, as well as their role in influencing interactions between B and T cells. Improved understanding of these mechanisms is necessary for the development of new therapeutic options modulating humoral immune responses.

The Lack of WIP Binding to Actin Results in Impaired B Cell Migration and Altered Humoral Immune Responses.

Wiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation. We found an altered differentiation of WIPΔABD B cells and diminished antibody affinity maturation after immunization. Mechanistically, WIPΔABD B cells showed impaired B cell receptor (BCR)-induced PI3K signaling and actin reorganization, likely caused by diminished CD81 expression and altered CD19 dynamics on the B cell surface. WIPΔABD B cells displayed reduced in vivo motility, concomitantly with impaired chemotaxis and defective F-actin polarization, HS1 phosphorylation, and polarization of HS1 to F-actin-rich structures after CXCL12 stimulation in vitro. We thus concluded that WIP binding to actin, independent of its binding to WASp, is critical for actin cytoskeleton plasticity in B cells.