Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Pooled genetic perturbation screens with image-based phenotypes.

Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >106 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.

CloneSifter: enrichment of rare clones from heterogeneous cell populations.

BACKGROUND: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones. RESULTS: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population. CONCLUSIONS: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.

Cell Intrinsic Deregulated ß-Catenin Signaling Promotes Expansion of Bone Marrow Derived Connective Tissue Type Mast Cells, Systemic Inflammation, and Colon Cancer.

Mast cells constitutively express ß-catenin and expand in solid tumors such as colon and skin cancer. However, the role of ß-catenin signaling in mast cells and the cause or effect of mast cell expansion and tumor growth has yet to be established. In earlier studies we used mast cell depletion and protease staining approaches, to provide evidence for a causative role of mast cells in small bowel polyposis, and related specific phenotypes and distributions of tumor infiltrating mast cells to stages of tumor growth. Here we report that, stabilization of ß-catenin expands mast cells to promote high incidence of colon polyposis and infrequent small bowel polyps and skin cancer. Expression of a dominant acting ß-catenin in mast cells (5CreCAT) stimulated maturation and expression of granule stored proteases. Both mucosal and connective tissue type mast cells accumulated in colonic small bowel polyps independent of gender, and mice developed chronic systemic inflammation with splenomegaly. Reconstitution of polyposis-prone mice with bone marrow from 5CreCAT mice resulted in focal expansion of connective tissue like mast cells, which are normally rare in benign polyps and characteristically expand during adenoma-to-carcinoma transition. Our findings highlight a hitherto unknown contribution of ß-catenin signaling in mast cells to their maturation and to increased risk of colon cancer.

Bim suppresses the development of SLE by limiting myeloid inflammatory responses.

The Bcl-2 family is considered the guardian of the mitochondrial apoptotic pathway. We demonstrate that Bim acts as a molecular rheostat by controlling macrophage function not only in lymphoid organs but also in end organs, thereby preventing the break in tolerance. Mice lacking Bim in myeloid cells (LysMCreBimfl/fl) develop a systemic lupus erythematosus (SLE)-like disease that mirrors aged Bim-/- mice, including loss of marginal zone macrophages, splenomegaly, lymphadenopathy, autoantibodies (including anti-DNA IgG), and a type I interferon signature. LysMCreBimfl/fl mice exhibit increased mortality attributed to glomerulonephritis (GN). Moreover, the toll-like receptor signaling adaptor protein TRIF (TIR-domain-containing adapter-inducing interferon-ß) is essential for GN, but not systemic autoimmunity in LysMCreBimfl/fl mice. Bim-deleted kidney macrophages exhibit a novel transcriptional lupus signature that is conserved within the gene expression profiles from whole kidney biopsies of patients with SLE. Collectively, these data suggest that the Bim may be a novel therapeutic target in the treatment of SLE.

The contribution of the programmed cell death machinery in innate immune cells to lupus nephritis.

Systemic lupus erythematosus (SLE) is a chronic multi-factorial autoimmune disease initiated by genetic and environmental factors, which in combination trigger disease onset in susceptible individuals. Damage to the kidney as a consequence of lupus nephritis (LN) is one of the most prevalent and severe outcomes, as LN affects up to 60% of SLE patients and accounts for much of SLE-associated morbidity and mortality. As remarkable strides have been made in unlocking new inflammatory mechanisms associated with signaling molecules of programmed cell death pathways, this review explores the available evidence implicating the action of these pathways specifically within dendritic cells and macrophages in the control of kidney disease. Although advancements into the underlying mechanisms responsible for inducing cell death inflammatory pathways have been made, there still exist areas of unmet need. By understanding the molecular mechanisms by which dendritic cells and macrophages contribute to LN pathogenesis, we can improve their viability as potential therapeutic targets to promote remission.

ApoE deficiency exacerbates the development and sustainment of a semi-chronic K/BxN serum transfer-induced arthritis model.

BACKGROUND: The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE. METHODS: K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE(-/-) mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively. RESULTS: ApoE(-/-) mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE(-/-) mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE(-/-) mice. However, arthritic ApoE(-/-) mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE(-/-) mice and arthritic C57BL/6 mice. CONCLUSIONS: Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis.

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

INTRODUCTION: Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. METHODS: Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test. RESULTS: Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6C(high)CD11b(+)F4/80(+) splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in Cre (LysM) Casp8 (fl/fl) mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. CONCLUSIONS: These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.

Methods for the study of mast cells in cancer.

Tumor growth requires interactions of tumor cells with a receptive and inductive microenvironment. Two major populations of tumor-infiltrating cells are considered to be essential for producing such a microenvironment: (1) proinflammatory cells that nurture the tumor with growth factors and facilitate invasion and metastasis by secreting proteases and (2) immune suppressive leukocytes including T-regulatory cells (Treg) that hinder tumor-specific CD8 T-cell responses, which otherwise could potentially reject the tumor. Among the proinflammatory cells, accumulation of mast cells (MCs) in human tumors is frequently recorded and was recently linked with poor prognosis. Causative links between mast cell infiltration and tumor progression can be deduced from animal studies. There is an interesting link between mast cells and Treg. The adoptive transfer of Treg from healthy syngeneic mice to mice susceptible to colon cancer suppresses focal mastocytosis and hinders tumor progression. Furthermore, T-cell-deficient mice susceptible to colon cancer show enhanced focal mastocytosis and tumor invasion. Here, we describe methods to assess MCs in mouse models of cancer and to investigate how MCs affect tumor epithelium. Additionally, we will detail methods used to investigate how T cells influence MCs and how MCs influence T cells.

PI3K/AKT signaling is essential for communication between tissue-infiltrating mast cells, macrophages, and epithelial cells in colitis-induced cancer.

PURPOSE: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. EXPERIMENTAL DESIGN: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10(-/-) mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. RESULTS: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. CONCLUSION: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10(-/-) mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002.

The significant role of mast cells in cancer.

Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient.