Cutaneous immunosurveillance and regulation of inflammation by group 2 innate lymphoid cells.

Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.

Low Pretreatment CD4+:CD8+ T Cell Ratios and CD39+CD73+CD19+ B Cell Proportions Are Associated with Improved Relapse-Free Survival in Head and Neck Squamous Cell Carcinoma.

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV+ patients contained increased proportions of CD39-CD73-CD4+ T cells and reduced proportions of CD39-/+CD73+CD4+ T cells compared to the equivalent cells from HPV- patients. Notably, the pretreatment CD4+:CD8+ T cell ratios and CD39+CD73+CD19+ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.

Increased P2X7 expression in the gastrointestinal tract and skin in a humanised mouse model of graft-versus-host disease.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. METHODS: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. RESULTS: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. CONCLUSION: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.

Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73.

CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNß and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.

Pro- and anti-tumour effects of B cells and antibodies in cancer: a comparison of clinical studies and preclinical models.

The primary immune role of B cells is to produce antibodies, but they can also influence T cell function via antigen presentation and, in some contexts, immune regulation. Whether their roles in tumour immunity are similar to those in other chronic immune responses such as autoimmunity and chronic infection, where both pro- and anti-inflammatory roles have been described, remains controversial. Many studies have aimed to define the role of B cells in antitumor immune responses, but despite this considerable body of work, it is not yet possible to predict how they will affect immunity to any given tumour. In many human cancers, the presence of tumour-infiltrating B cells and tumour-reactive antibodies correlates with extended patient survival, and this clinical observation is supported by data from some animal models. On the other hand, T cell responses can be adversely affected by B cell production of immunoregulatory cytokines, a phenomenon that has been demonstrated in humans and in animal models. The isotype and concentration of tumour-reactive antibodies may also influence tumour progression. Recruitment of B cells into tumours may directly reflect the subtype and strength of the anti-tumour T cell response. As the response becomes chronic, B cells may attenuate T cell responses in an attempt to decrease host damage, similar to their described role in chronic infection and autoimmunity. Understanding how B cell responses in cancer are related to the effectiveness of the overall anti-tumour response is likely to aid in the development of new therapeutic interventions against cancer.

Tumour-specific CD4 T cells eradicate melanoma via indirect recognition of tumour-derived antigen.

The importance of CD4 T cells in tumour immunity has been increasingly recognised, with recent reports describing robust CD4 T cell-dependent tumour control in mice whose immune-regulatory mechanisms have been disturbed by irradiation, chemotherapy, immunomodulatory therapy and/or constitutive immunodeficiency. Tumour control in such models has been attributed in large part to direct Major Histocompatibility Complex (MHC) class II-dependent CD4 T cell killing of tumour cells. To test whether CD4 T cells can eradicate tumours without directly killing tumour cells, we developed an animal model in which tumour-derived antigen could be presented to T-cell receptor (TCR)-transgenic CD4 T cells by host but not tumour MHC class II molecules. In I-E(+) mice bearing I-E(null) tumours, naive I-E-restricted CD4 T cells proliferated locally in tumour-draining lymph nodes after recognising tumour-derived antigen on migratory dendritic cells. In lymphopaenic but not immunosufficient hosts, CD4 T cells differentiated into polarised T helper type 1 (Th1) cells expressing interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin (IL)-2 but little IL-17, and cleared established tumours. Tumour clearance was enhanced by higher TCR affinity for tumour antigen-MHC class II and was critically dependent on IFNγ, as demonstrated by early tumour escape in animals treated with an IFNγ blocking antibody. Thus, CD4 T cells and IFNγ can control tumour growth without direct T-cell killing of tumour cells, and without requiring additional adaptive immune cells such as CD8 T cells and B cells. Our results support a role for effective CD4 T cell-dependent tumour immunity against MHC class II-negative tumours.

IL-2 is a critical regulator of group 2 innate lymphoid cell function during pulmonary inflammation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear. OBJECTIVE: We sought to understand the role of IL-2 in the regulation of ILC2 function in the lung. METHODS: We used histology, flow cytometry, immunohistochemistry, ELISA, and quantitative PCR with knockout and reporter mice to dissect pulmonary ILC2 function in vivo. We examined the role of ILC2s in eosinophilic crystalline pneumonia, an idiopathic type 2 inflammatory lung condition of mice, and the effect of IL-2 deficiency on this disease. We determined the effect of IL-2 administration on pulmonary ILC2 numbers and function in mice in the steady state and after challenge with IL-33. RESULTS: We discovered an unexpected role for innate cell-derived IL-2 as a major cofactor of ILC2 function during pulmonary inflammation. Specifically, we found that IL-2 was essential for the development of eosinophilic crystalline pneumonia, a type 2 disease characterized by increased numbers of activated ILC2s. We show that IL-2 signaling serves 2 distinct functions in lung ILC2s, namely promoting cell survival/proliferation and serving as a cofactor for the production of type 2 cytokines. We further demonstrate that group 3 innate lymphoid cells are an innate immune source of IL-2 in the lung. CONCLUSION: Innate cell-derived IL-2 is a critical cofactor in regulating ILC2 function in pulmonary type 2 pathology.

Proliferative features of IgG4-related disease.

IgG4-related disease is an immune-mediated disease that can lead to substantial morbidity and organ damage. Capable of affecting nearly any organ system or anatomic site, and showing considerable overlap in clinical presentation with various other diseases, IgG4-related disease often poses a diagnostic challenge for clinicians. Furthermore, there are no diagnostic biomarkers with high specificity for IgG4-related disease, and histopathological examination is nuanced and requires clinical correlation for accurate diagnosis. Therefore, it is crucial for clinicians to recognise the clinical phenotypes of IgG4-related disease. The disease is generally considered to have predominantly fibrotic and proliferative (or inflammatory) manifestations, with distinct clinical, serological and histopathological findings associated with each manifestation. However, the fibrotic and proliferative manifestations of this disease frequently occur together, thereby blurring this dichotomous distinction. In this Series paper, we provide a detailed overview of the clinical manifestations typical of the proliferative features of IgG4-related disease, with an emphasis on the diagnostic evaluation and differential diagnosis of each proliferative disease manifestation. In addition, we summarise the immune mechanisms underlying IgG4-related disease, suggest a framework for how to approach management and monitoring after the diagnosis is established, and highlight current unmet needs for patient care surrounding this disease.

MHCII restriction demonstrates B cells have very limited capacity to activate tumour-specific CD4+ T cells in vivo.

There has been growing interest in the role of B cells in antitumour immunity and potential use in adoptive cellular therapies. To date, the success of such therapies is limited. The intrinsic capacity of B cells to specifically activate tumour-specific CD4+ T cells in vivo via TCR-dependent interactions remains poorly defined. We have developed an in vivo tumour model that utilizes MHCII I-E restriction which limits antigen presentation to tumour-specific CD4 T cells to either tumour-specific B cells or host myeloid antigen presenting cells (APCs) in lymphopenic RAG-/-mice. We have previously shown that these naive tumour-specific CD4+ T cells can successfully eradicate established tumours in this model when activated by host APCs. When naïve tumour-specific B cells are the only source of I-E+ APC, very limited proliferation of naïve CD4+ T cells is observed, whereas host I-E+ APCs are potent T cell activators. B cells pre-activated with an anti-CD40 agonistic antibody in vivo support increased T cell proliferation, although far less than host APCs. CD4+ T cells that have already differentiated to an effector/central memory phenotype proliferate more readily in response to naïve B cells, although still 100-fold less than in response to host APCs. This study demonstrates that even in a significantly lymphopenic environment, myeloid APCs are the dominant primary activators of tumour-specific T cells, in contrast to the very limited capacity of tumour-specific B cells. This suggests that future anti-tumour therapies that incorporate activated B cells should also include mechanisms that activate host APCs.

Sex as a predictor of clinical phenotype and determinant of immune response in IgG4-related disease: a retrospective study of patients fulfilling the American College of Rheumatology-European League Against Rheumatism classification criteria.

BACKGROUND: IgG4-related disease is a multiorgan fibroinflammatory disease considered to have an autoimmune origin. Case series describing individual organ involvement have suggested differences in phenotypic expression between males and females. We aimed to characterise differences in IgG4-related disease manifestations between male and female patients in a large single-centre cohort. METHODS: In this retrospective, single-centre cohort study, patients were recruited from the Massachusetts General Hospital Rheumatology Clinic (Boston, MA, USA) and classified according to the American College of Rheumatology-European Alliance of Associations for Rheumatology (ACR-EULAR) classification criteria. Only patients satisfying the ACR-EULAR classification criteria were included in the study. Data on age at diagnosis, organ involvement at baseline, treatment status, and pre-treatment laboratory values were collected. Circulating plasmablasts and B-cell subsets were quantitated by flow cytometry. Active disease was defined by an IgG4-related disease Responder Index score of more than 0. Laboratory values were analysed for patients who were untreated at baseline and had active IgG4-related disease. The main outcomes were assessed in all participants with available data. FINDINGS: Of the 564 participants enrolled in the Massachusetts General Hospital Rheumatology Clinic IgG4-related disease Registry, 328 fulfilled ACR-EULAR classification criteria and were included between January, 2008, and May, 2023. There was a strong male predominance (male:female ratio 2·2:1) with 226 (69%) males and 102 (31%) females, which contrasted markedly with our general rheumatology clinic population (0·4:1; p<0·001). The male predominance increased with each decade of life starting at age 40 years. On average, male patients were 5·5 years older at diagnosis than female patients (63·7 years vs 58·2 years; p=0·0031). We observed male patients to have higher ACR-EULAR classification criteria scores at baseline with a median score of 35·0 (IQR 28·0-46·0), compared with 29·5 (25·0-39·0) for females (p=0·0010). The proportion of male patients with pancreatic and renal involvement was almost double the proportion observed in female patients (50% of the male patients had pancreatic involvement, compared with about 26% of the female patients; p<0·0001). Male patients were more likely to have serological abnormalities at baseline. The distribution of IgG4 values differed significantly between male an female sexes, favouring higher values in males. We found that male patients with IgG4-related disease were more likely to have active B-cell responses in the blood as defined by plasmablast expansions. INTERPRETATION: IgG4-related disease is unusual among autoimmune diseases in that it is more likely to affect males than females and to present with a striking sex-dependent organ distribution and degree of B-cell response. These findings highlight important variation between IgG4-related disease and other conditions generally believed to have an autoimmune basis. Most autoimmune diseases, by contrast to IgG4-related disease, demonstrate pronounced predilections for affecting females more frequently than males. Hypotheses surrounding the cause and pathophysiology of this condition need to consider this unusual sex distribution among patients with IgG4-related disease. FUNDING: National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rheumatology Research Foundation, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

Human circulating CD24hi marginal zone B cells produce IgM targeting atherogenic antigens and confer protection from vascular disease.

IgMs that inactivate oxidation-specific epitopes (IgMOSE), which are secondary products of lipid peroxidization, protect against inflammatory diseases, including diet-induced atherosclerosis. However, the human B cell subtype that produces IgMOSE remains unknown. In this study, we used single-cell mass cytometry and adoptive transfer of B cell subtypes to NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice to identify B27+IgM+CD24hi cells as the major producers of IgMOSE in humans. Notably, these cells have characteristics of human circulatory marginal zone B (MZB) cells, which are known to be atheoroprotective IgM producers in mice. CD24 antibody treatment to reduce MZB cells and IgM in a hyperlipidemic humanized mouse model provides the evidence that MZB cells protect against vascular inflammation. Consistent with these findings, the frequency of B27+IgM+CD24hi cells (MZB) in patients inversely correlates with coronary artery disease severity.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

The depth of mutational agony and the exuberance of tumoral T cell ecstasy predict checkpoint salvation.

Genomic biomarkers will help to elucidate which cancer patients will benefit from PD-1 blockade immunotherapy.

Collaboration between tumor-specific CD4+ T cells and B cells in anti-cancer immunity.

The role of B cells and antibodies in anti-tumor immunity is controversial, with both positive and negative effects reported in animal models and clinical studies. We developed a murine B16.F10 melanoma model to study the effects of collaboration between tumor-specific CD4+ T cells and B cells on tumor control. By incorporating T cell receptor transgenic T cells and B cell receptor isotype switching B cells, we were able to track the responses of tumor-reactive T and B cells and the development of anti-tumor antibodies in vivo. In the presence of tumor-specific B cells, the number of tumor-reactive CD4+ T cells was reduced in lymphoid tissues and the tumor itself, and this correlated with poor tumor control. B cells had little effect on the Th1 bias of the CD4+ T cell response, and the number of induced FoxP3+ regulatory cells (iTregs) generated from within the original naive CD4+ T cell inoculum was unrelated to the degree of B cell expansion. In response to CD4+ T cell help, B cells produced a range of isotype-switched anti-tumor antibodies, principally IgG1, IgG2a/c and IgG2b. In the absence of CD4+ T cells, B cells responded to agonistic anti-CD40 administration by switching to production of IgG2a/c and, to a lesser extent, IgG1, IgG3, IgA and IgE, which reduced the number of lung metastases after i.v. tumor inoculation but had no effect on the growth of subcutaneous tumors.

Selective Treg reconstitution during lymphopenia normalizes DC costimulation and prevents graft-versus-host disease.

Regulatory T cells (Tregs) have been shown to enhance immune reconstitution and prevent graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation; however, it is unclear how Tregs mediate these effects. Here, we developed a model to examine the mechanism of Treg-dependent regulation of immune reconstitution. Lymphopenic mice were selectively reconstituted with Tregs prior to transfer of conventional CD4+ T cells. Full Treg reconstitution prevented the rapid oligoclonal proliferation that gives rise to pathogenic CD4 effector T cells, while preserving the slow homeostatic form of lymphopenia-induced peripheral expansion that repopulates a diverse peripheral T cell pool. Treg-mediated CTLA-4-dependent downregulation of CD80/CD86 on DCs was critical for inhibition of rapid proliferation and was a function of the Treg/DC ratio achieved by reconstitution. In an allogeneic BM transplant model, selective Treg reconstitution before T cell transfer also normalized DC costimulation and provided complete protection against GVHD. In contrast, cotransfer of Tregs was not protective. Our results indicate that achieving optimal recovery from lymphopenia should aim to improve early Treg reconstitution in order to increase the relative number of Tregs to DCs and thereby inhibit spontaneous oligoclonal T cell proliferation.

A systems biology approach to the analysis of subset-specific responses to lipopolysaccharide in dendritic cells.

Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 commitment, generating inducible Tregs, and mediating tolerance. It is believed that distinct DC subsets have evolved to control these different immune outcomes. However, how DC subsets mount different responses to inflammatory and/or tolerogenic signals in order to accomplish their divergent functions remains unclear. Lipopolysaccharide (LPS) provides an excellent model for investigating responses in closely related splenic DC subsets, as all subsets express the LPS receptor TLR4 and respond to LPS in vitro. However, previous studies of the LPS-induced DC transcriptome have been performed only on mixed DC populations. Moreover, comparisons of the in vivo response of two closely related DC subsets to LPS stimulation have not been reported in the literature to date. We compared the transcriptomes of murine splenic CD8 and CD11b DC subsets after in vivo LPS stimulation, using RNA-Seq and systems biology approaches. We identified subset-specific gene signatures, which included multiple functional immune mediators unique to each subset. To explain the observed subset-specific differences, we used a network analysis approach. While both DC subsets used a conserved set of transcription factors and major signalling pathways, the subsets showed differential regulation of sets of genes that 'fine-tune' the network Hubs expressed in common. We propose a model in which signalling through common pathway components is 'fine-tuned' by transcriptional control of subset-specific modulators, thus allowing for distinct functional outcomes in closely related DC subsets. We extend this analysis to comparable datasets from the literature and confirm that our model can account for cell subset-specific responses to LPS stimulation in multiple subpopulations in mouse and man.