David Swan

Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells

Promising immunotherapeutic tools for T cell‐mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of naïve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1α,25‐dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by naïve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Naïve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN‐γ/high IL‐10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL‐2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL‐12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in naïve T cells, indicating that the lack of IL‐12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate naïve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.

LPS activation is required for migratory activity and antigen presentation by tolerogenic dendritic cells

Autoimmune pathologies are caused by a breakdown in self‐tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self‐tolerance in an antigen‐specific manner. Studies about tolDC have focused largely on generating stable maturation‐resistant DC, but few have fully addressed questions about the antigen‐presenting and migratory capacities of these cells, prerequisites for successful immunotherapy. Here, we investigated whether human tolDC, generated with dexamethasone and the active form of vitamin D3, maintained their tolerogenic function upon activation with LPS (LPS‐tolDC), while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS‐tolDC, but not tolDC, expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore, LPS‐tolDC were superior to tolDC in their ability to present type II collagen, a candidate autoantigen in rheumatoid arthritis. tolDC and LPS‐tolDC had low stimulatory capacity for allogeneic, naïve T cells and skewed T cell polarization toward an anti‐inflammatory phenotype, although LPS‐tolDC induced significantly higher levels of IL‐10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen‐presenting activity in tolDC is important for optimizing their therapeutic potential.

Vascular biology: the role of sphingosine 1-phosphate in both the resting state and inflammation.

•  Introduction •  Biochemistry of sphingosine 1‐phosphate ‐  Generation ‐  Degradation ‐  Transport •  S1P receptor biology ‐  S1PR1 ‐  S1PR3 •  The vascular gradient of sphingosine 1‐phosphate ‐  Compartmentalization ‐  T‐cell trafficking •  Role of S1P on vascular endothelium ‐  Maintenance of endothelial barrier integrity in the resting state ‐  Disruption of barrier integrity during inflammation •  Crosstalk between S1PR and other receptors ‐  Crosstalk with growth factor receptors ‐  Cross‐talk with other GPCR •  Therapies targeting the S1P signalling axis ‐  FTY720 ‐  Monoclonal antibody therapy •  Summary and future directions

Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4+ T cells partly via transforming growth factor-β1

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte‐derived DC. These tolDC exert potent pro‐tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)−12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)‐β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte‐derived DC. By inhibiting TGF‐β1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF‐βRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF‐β1 than healthy control CD4+ T cells [reduced TGF‐β‐induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)‐γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF‐β1‐dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.

Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4(+) T-cells partly via TGF-β1.

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte‐derived DC. These tolDC exert potent pro‐tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)−12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)‐β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte‐derived DC. By inhibiting TGF‐β1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF‐βRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF‐β1 than healthy control CD4+ T cells [reduced TGF‐β‐induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)‐γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF‐β1‐dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.

Identification of Heterozygous Single- and Multi-exon Deletions in IL7R by Whole Exome Sequencing.

PurposeWe aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions.MethodsOf a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs.ResultsWe found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions.ConclusionsWe show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs.

Post-Transplant Immunosuppression: Regulation of the Efflux of Allospecific Effector T Cells from Lymphoid Tissues

A functional sphingosine-1-phosphate (S1P) receptor antagonist specifically inhibited the egress of activated allospecific T cells from draining popliteal lymph nodes in alloantigen-sensitised mice. The level of S1P receptor 1 (S1PR1) mRNA was similarly reduced 1 and 3 days after mitogenic activation of T cells. However, the response of these cells to the S1PR1-specific agonist SEW2871 was only reduced on the first day after T cell activation with normal receptor-mediated Akt-phosphorylation restored by day 3. Longitudinal analysis of CD69 expression showed that almost all T cells expressed this antigen on days 1 and 3 after activation. However, the absolute level of cell-surface expression of CD69 peaked on undivided T cells and was then halved by each of the first 3 cycles of mitosis. CD69-specific small interfering RNA (siRNA) reduced the maximal level of CD69 expression by undivided, mitogen-stimulated T cells. These cells retained their capacity to phosphorylate Akt in response to stimulation with SEW2871. These data show that S1P receptors are involved in controlling the egress of activated T cells from lymph nodes, and that S1PR1 function is regulated by the level of T cell surface CD69. They suggest a potential for augmentation of this process to deplete alloreactive effector cells after organ transplantation.

Treatment of moderate-to-severe atopic eczema in adults within the UK: results of a national survey

P01 Subungual squamous cell carcinoma masquerading as onycholysis C. Kwok and A. Ong Royal Bolton Hospital, Bolton, U.K. A 69-year-old man presented with discoloration of the left index fingernail for the last 6 months. He said that the problem had first started 4 years ago and resolved with a cream given by his general practitioner (GP). He could not recall the name of this cream. The affected nail was associated with a foul odour. There was no associated pain. He denied any extension or change to the discoloration. It was treated as onychomycosis by the GP, without improvement with oral terbinafine after 6 months. On examination there was an area of onycholysis affecting the medial aspect of the nail, with a small area of dark grey discoloration in the centre of the onycholysis. The lateral and proximal nail folds were unremarkable. Underneath the nail, the skin appeared macerated. Nail clippings were negative for tinea. The nail was avulsed and a biopsy of the nail bed under the onycholysis was carried out. Histology showed irregular islands of markedly dysplastic squamous epithelium within a heavily inflamed dermis. The features were those of an invasive, moderately differentiated squamous cell carcinoma (SCC). The patient is awaiting distal phalanx amputation. Onycholysis can be classified as primary (idiopathic) or secondary (e.g. psoriasis, trauma onychomycosis). Subungual SCC is rare but is considered to be the most frequent subungual tumour. Subungual SCC usually affects a single digit in the thumb or great toe. Multiple nail involvement has been described in the literature. The incidence is higher in men, most commonly aged 40–70 years. The possible causes of subungual SCC include chronic infection, chemical/physical microtrauma, sun exposure, immunosuppression, previous human papillomavirus infection and radiation. The clinical presentation of subungual SCC is varied and includes nail dystrophy, chronic pain, swelling, onycholysis and wartlike lesions involving the nail bed and periungual areas. Therefore, subungual SCC may be mistaken for onychomycosis, chronic paronychia and viral wart. This can lead to a delay in diagnosis and invasion of the bone. Excision of the subungual SCC is the treatment of choice for those without bone involvement. Amputation of the distal phalanx is recommended for those with bone involvement. Radiotherapy has also been reported as effective. Risk of recurrence is higher in the nail unit than at other sites, hence long-term follow-up is recommended. A high index of suspicion is required for subungual SCC. Therefore, patients with chronic or recurrent nail lesions should be considered for a biopsy, as early diagnosis has more favourable outcomes. P02 Cutaneous involvement as a late manifestation of adult-onset Langerhans cell histiocytosis K.H. Kuet, N. Tiffin, J. Wright and A. McDonagh Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, U.K. A 53-year-old woman was diagnosed 12 months earlier with Langerhans cell histiocytosis (LCH) after an initial presentation with unilateral hearing loss and diabetes insipidus. She had failed to respond to initial treatment with prednisolone, vinblastine and methotrexate. She then received a 12-month course of cytarabine for extensive disease involving the pituitary, bile duct, thyroid and long bones. Her disease appeared stable during this time with no signs of any significant progression. However, soon after completing the cytarabine course she deteriorated acutely with fevers, weight loss, loose stools, increasing bone pain and an extensive rash involving the flexures and mouth. By the time of referral to dermatology, the rash had been present for several months and had spread despite treatment for suspected Candida infection. However, previous skin swabs had shown a moderate growth of anaerobes only. She also reported loss of appetite and soreness of the mouth. Examination revealed an extensive eruption of brown/haemorrhagic papules affecting the inframammary region and groins, along with areas of erosions and maceration. Over the abdomen and axillae there was also evidence of discrete scaly brown lesions. Examination of the mouth revealed numerous haemorrhagic macules affecting the buccal mucosa, hard palate and tongue. In view of the clinical history, cutaneous LCH was strongly suspected, and this was confirmed with a skin biopsy. Histological features were supported by immunohistochemistry, which showed strong staining with CD1a and S100. Subsequently she was started on vemurafenib and the skin lesions responded within 1 week; however, she died later from complications of advanced disease after a prolonged hospital admission. LCH is a rare histiocytic neoplasm characterized by clonal proliferation of Langerhans cells. LCH occurs mainly in children, and LCH in adults is extremely rare. Multiple organ systems can be involved, and the severity of visceral lesions reflects disease activity. Bone is most frequently involved followed by skin, while skin-limited involvement is usually associated with an indolent clinical course. This was a striking case demonstrating the cutaneous manifestations of an extremely rare condition. In this case cutaneous involvement was a late manifestation despite the advanced stage of this patient’s disease at initial presentation. Furthermore there was a delay in recognition and diagnosis of the cutaneous signs. However, due to the poor prognostic indicators in this case (extensive multiorgan involvement and poor response to chemotherapy), it was

Differential effects of S1P on responses to homeostatic versus inflammatory chemokines

2008 The Authors Journal Compilation 2008 Blackwell Publishing Ltd, Immunology, 125, Supplement 1, 1 144 Metchnikoff Centenary – Macrophages and Immunity IS2 Heterogeneity of mouse and human monocytesThis paper looks at the effects of dust allergen and LPS concentrations on biomarkers of asthma in adults with house dust mite-sensitive asthma.