High-dimensional profiling reveals Tc17 cell enrichment in active Crohn's disease and identifies a potentially targetable signature.

The immune-pathology in Crohn's disease is linked to dysregulated CD4+ T cell responses biased towards pathogenic TH17 cells. However, the role of CD8+ T cells able to produce IL-17 (Tc17 cells) remains unclear. Here we characterize the peripheral blood and intestinal tissue of Crohn's disease patients (n = 61) with flow and mass cytometry and reveal a strong increase of Tc17 cells in active disease, mainly due to induction of conventional T cells. Mass cytometry shows that Tc17 cells express a distinct immune signature (CD6high, CD39, CD69, PD-1, CD27low) which was validated in an independent patient cohort. This signature stratifies patients into groups with distinct flare-free survival associated with differential CD6 expression. Targeting of CD6 in vitro reduces IL-17, IFN-γ and TNF production. These results identify a distinct Tc17 cell population in Crohn's disease with proinflammatory features linked to disease activity. The Tc17 signature informs clinical outcomes and may guide personalized treatment decisions.

Unequal sisters - Past and potential future range development of Anatolian and Hyrcanian brown frogs.

Phylogeography can reconstruct historical evolutionary processes by comparing historical patterns of gene flow, divergence among species and by using species distribution models (SDM) upon geographic distribution. We investigate the phylogeographic patterns of Anatolian brown frogs including R. macrocnemis and R. tavasensis as well as the Hyrcanian brown frog, R. pseudodalmatina, using a fragment of the mitochondrial 16S rRNA gene for 145 specimens across the entire range of these frogs. We calculate parameters of molecular diversity, such as the number of variable sites (S), the number of haplotypes (h), haplotype diversity (Hd) and nucleotide diversity (π). We generated a haplotype network and used three methods (Neutrality tests, mismatch distributions and Bayesian skyline plots) to reconstruct the demographic histories of R. macrocnemis and R. pseudodalmatina. Finally, we used SDMs to predict the habitat suitability for three periods: The Present Day, the Last Glacial Maximum (LGM) and the future until 2070 for R. macrocnemis and R. pseudodalmatina. Our phylogenetic analyses support a late Miocene origin of Anatolian and Hyrcanian lineages. Hyrcanian brown frogs were enclosed in lowlands of the southern coast of the Caspian Sea after the uplift of the Elburz range and the Armenian plateau. The formation of a salinity belt from the north Aegean corridor (the south western Turkey) to northward during the Late Tortonian led to the subdivision of ancestor of the Anatolian lineage into today isolated western and eastern populations. The salinity belt had a considerable impact on the divergence of R. tavasensis from R. macrocnemis. Combined historical demographic analyses and SDMs revealed a rapid expansion occurring during the Pleistocene in R. macrocnemis and R. pseudodalmatina. Currently, suitable habitat for R. macrocnemis has declined compared to the LGM, and the species is predicted to do even worse under future climatic conditions. In contrast, R. pseudodalmatina found suitable habitat from the LGM to present within its restricted distribution area; it is predicted to do fine even under future climate.

Refining the dermatological spectrum in primary immunodeficiency: mucosa-associated lymphoid tissue lymphoma translocation protein 1 deficiency mimicking Netherton/Omenn syndromes.

The proteinase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which forms part of the caspase recruitment domain-containing protein 11-B-cell lymphoma 10-MALT1 signalosome complex, plays a direct role in nuclear factor kappa B activation. Here, we describe the case of a female infant with severe immune dysregulation leading to recurrent systemic infections, failure to thrive and severe crises of ichthyosiform erythroderma with high levels of serum IgE. Hence, initial symptoms indicated Netherton syndrome or Omenn syndrome. Surprisingly, sequence analyses of SPINK5 and RAG1/RAG2, respectively, excluded these diseases. During the hospital stay the patient's health deteriorated, despite intensive care therapy, and she died. In order to delineate the diagnosis, whole-exome sequencing was performed. Two compound heterozygous mutations in MALT1 were found and verified by Sanger sequencing (exon 2 c.245T>C, exon 2 c.310dup), which led to a MALT1 deficiency at the protein level. Based on these results, an immunological analysis was performed, as was immunofluorescence staining of key skin proteins, to confirm a diagnosis of MALT1 deficiency. This case report provides a closer description of the clinical and histological skin phenotype of MALT1 deficiency, and we conclude that MALT1 deficiency must be considered a possible differential diagnosis of Netherton and Omenn syndromes. What's already known about this topic? Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) deficiency is a combined immunodeficiency. MALT1 is part of the caspase recruitment domain-containing protein 11-B-cell lymphoma 10-MALT1 signalosome complex, which is essential for nuclear factor kappa B activation. Current publications describe a phenotype of recurrent systemic infections; only in a few cases has an inflammatory involvement of the integument been described. What does this study add? A closer description of the cutaneous phenotype of MALT1 deficiency in a patient with two novel MALT1 mutations. Immune mapping of follicular epidermis shows lympho-epithelial Kazal-type-related inhibitor is reduced in MALT1 deficiency and absent on interfollicular staining. Clinically, MALT1 deficiency mimics Netherton syndrome and Omenn syndrome, and should be considered a differential diagnosis.

A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

The German national registry for primary immunodeficiencies (PID).

In 2009, a federally funded clinical and research consortium (PID-NET, http://www.pid-net.org) established the first national registry for primary immunodeficiencies (PID) in Germany. The registry contains clinical and genetic information on PID patients and is set up within the framework of the existing European Database for Primary Immunodeficiencies, run by the European Society for Primary Immunodeficiencies. Following the example of other national registries, a central data entry clerk has been employed to support data entry at the participating centres. Regulations for ethics approvals have presented a major challenge for participation of individual centres and have led to a delay in data entry in some cases. Data on 630 patients, entered into the European registry between 2004 and 2009, were incorporated into the national registry. From April 2009 to March 2012, the number of contributing centres increased from seven to 21 and 738 additional patients were reported, leading to a total number of 1368 patients, of whom 1232 were alive. The age distribution of living patients differs significantly by gender, with twice as many males than females among children, but 15% more women than men in the age group 30 years and older. The diagnostic delay between onset of symptoms and diagnosis has decreased for some PID over the past 20 years, but remains particularly high at a median of 4 years in common variable immunodeficiency (CVID), the most prevalent PID.

The European internet-based patient and research database for primary immunodeficiencies: update 2011.

In order to build a common data pool and estimate the disease burden of primary immunodeficiencies (PID) in Europe, the European Society for Immunodeficiencies (ESID) has developed an internet-based database for clinical and research data on patients with PID. This database is a platform for epidemiological analyses as well as the development of new diagnostic and therapeutic strategies and the identification of novel disease-associated genes. Since its start in 2004, 13,708 patients from 41 countries have been documented in the ESID database. Common variable immunodeficiency (CVID) represents the most common entity with 2880 patients or 21% of all entries, followed by selective immunoglobulin A (sIgA) deficiency (1424 patients, 10·4%). The total documented prevalence of PID is highest in France, with five patients per 100,000 inhabitants. The highest documented prevalence for a single disease is 1·3 per 100,000 inhabitants for sIgA deficiency in Hungary. The highest reported incidence of PID per 100,000 live births was 16·2 for the period 1999-2002 in France. The highest reported incidence rate for a single disease was 6·7 for sIgA deficiency in Spain for the period 1999-2002. The genetic cause was known in 36·2% of all registered patients. Consanguinity was reported in 8·8%, and 18·5% of patients were reported to be familial cases; 27·9% of patients were diagnosed after the age of 16. We did not observe a significant decrease in the diagnostic delay for most diseases between 1987 and 2010. The most frequently reported long-term medication is immunoglobulin replacement.

[Interdisciplinary AWMF guideline for the diagnostics of primary immunodeficiency]. / Interdisziplinäre AWMF-Leitlinie zur Diagnostik von primären Immundefekten (S2k).

BACKGROUND: Primary immunodeficiencies are potentially life-threatening diseases. Over the last years, the clinical phenotype and the molecular basis of an increasing number of immunological defects have been characterized. However, in daily practice primary immunodeficiencies are still often diagnosed too late. Considering that an early diagnosis may reduce morbidity and mortality of affected patients, an interdisciplinary guideline for the diagnosis of primary immunodeficiencies was developed on behalf of the Arbeitsgemeinschaft Pädiatrische Immunologie (API) and the Deutsche Gesellschaft für Immunologie (DGfI). METHODS: The guideline is based on expert opinion and on knowledge from other guidelines and recommendations from Germany and other countries, supplemented by data from studies that support the postulated key messages (level of evidence III). With the contribution of 20 representatives, belonging to 14 different medical societies and associations, a consensus-based guideline with a representative group of developers and a structured consensus process was created (S2k). Under the moderation of a representative of the Association of the Scientific Medical Societies in Germany (AWMF) the nominal group process took place in April 2011. RESULTS: The postulated key messages were discussed and voted on following a structured consensus procedure. In particular, modified warning signs for primary immunodeficiencies were formulated and immunological emergency situations were defined.

Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency.

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαß+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

Reduced memory B cells in patients with hyper IgE syndrome.

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Polymyositis--an unusual presentation of cGvHD in children.

Polymyositis is a rare manifestation of cGvHD in adult patients following allogeneic BMT. Here, we report on a 2.1-yr-old girl who presented with a facial swelling and rapidly developing respiratory failure eight months after BMT for severe combined immunodeficiency. Possible infectious agents and autoimmune origin other than cGvHD were excluded. The girl responded promptly to steroid therapy and remains well without other signs of cGvHD four months later.

Development of granulomatous common variable immunodeficiency subsequent to infection with Toxoplasma gondii.

Common variable immunodeficiency (CVID) is a heterogeneous immunodeficiency that is accompanied by granulomatous lesions in 5-10% of cases. Why some patients develop granulomatous disease remains unclear. Here we describe a 12-year-old previously healthy girl who presented with pancytopenia and granulomatous lymphoproliferation subsequent to infection with Toxoplasma gondii. Loosely arranged non-fibrosing granulomas were observed in the liver, lymph nodes and lung, but no Toxoplasma tachyzoites could be demonstrated and polymerase chain reaction (PCR) and culture were negative for Toxoplasma and a wide range of other pathogens. While the patient had a normal peripheral B cell status at presentation, the development of CVID could be observed during the following months, leading to a loss of memory B cells. This was accompanied by an increasingly activated CD4(+) T cell compartment and high serum levels of angiotensin-converting enzyme (ACE), tumour necrosis factor (TNF) and sCD25. Steroid therapy reduced pancytopenia, granulomatous lymphoproliferation and cytokine elevations, but did not improve the B cell status. This is the first report of an association of Toxoplasma infection with granulomatous CVID and provides one of the rare examples where the onset of CVID could be documented subsequent to an infectious disease.

An improved protocol for measuring cytotoxic T cell activity in anatomic compartments with low cell numbers.

The study of target cell lysis and cytokine production are valuable tools to characterize antigen-specific T and NK cell function during virus infections. After localized infections in compartments such as the lung or the brain, however, cell numbers isolated from these organs are too low to perform standard assays with individual mice. Here, we report a few simple modifications of the classical 51Cr release assay allowing reduction of the number of required effector cells by a factor of 10 without loosing sensitivity or specificity. Using not more than 4x10(5) effector cells, we were able to study ex vivo virus-specific CTL or NK activity from the lungs of individual mice after infection with respiratory syncytial virus (RSV) and from the brains of mice infected with Borna disease virus (BDV). Flow cytometric analysis of interferon-gamma production by virus-specific T cells including appropriate controls was achieved with as few as 10(5) effector cells.

Low level viral persistence after infection with LCMV: a quantitative insight through numerical bifurcation analysis.

Many important viruses persist at very low levels in the body in the face of host immunity, and may influence the maintenance of this state of 'infection immunity'. To analyse low level viral persistence in quantitative terms, we use a mathematical model of antiviral cytotoxic T lymphocyte (CTL) response to lymphocytic choriomeningitis virus (LCMV). This model, described by a non-linear system of delay differential equations (DDEs), is studied using numerical bifurcation analysis techniques for DDEs. Domains where low level LCMV coexistence with CTL memory is possible, either as an equilibrium state or an oscillatory pattern, are identified in spaces of the model parameters characterising the interaction between virus and CTL populations. Our analysis suggests that the coexistence of replication competent virus below the conventional detection limit (of about 100 pfu per spleen) in the immune host as an equilibrium state requires the per day relative growth rate of the virus population to decrease at least 5-fold compared to the acute phase of infection. Oscillatory patterns in the dynamics of persisting LCMV and CTL memory, with virus population varying between 1 and 100 pfu per spleen, are possible within quite narrow intervals of the rates of virus growth and precursor CTL population death. Whereas the virus replication rate appears to determine the stability of the low level virus persistence, it does not affect the steady-state level of the viral population, except for very low values.

Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus.

In mice acutely infected with respiratory syncytial virus (RSV), more than 20% of pulmonary CD8(+) T cells, but only 2-3% of CD8(+) T cells in the draining lymph node secreted interferon-gamma in response to a single peptide. Surprisingly, the percentage of virus-specific T cells in the lung remained at these high levels long after the acute infection. Pulmonary memory T cells were further studied in a sensitive adoptive transfer system, which allows visualizing polyclonal CD4(+) and CD8(+) virus-specific memory T cell responses. Fifty days after infection, persisting RSV-specific pulmonary T cells remained CD69(hi) CD62L(lo), but had returned to a resting memory state according to functional criteria. In the absence of neutralizing antibodies reinfection first induced cell division among virus-specific memory T cells 3 days after infection predominantly in the local lymph node. However, divided cells then rapidly accumulated in the lung without significantly increasing in the lymph node. These results suggest rapid export of reactivated cells from the lymph node to the target organ. Thus, although memory T cells can be maintained in the infected organ after a localized virus infection, amplification of a recall response appears to be most effective in organized lymphoid tissue.

Predicting the dynamics of antiviral cytotoxic T-cell memory in response to different stimuli: cell population structure and protective function.

This paper examines the numerical and functional consequences of various stimuli on antiviral CD8+ T-cell memory using a mathematical model. The model is based upon biological evidence from the murine model of infection with lymphocytic choriomeningitis virus (LCMV) that the phenotype of immunological memory represents low-level responses driven by various stimuli, and the memory CTL population is partitioned between resting, cycling and effector cells. These subpopulations differ in their lifespan, their potential to mediate antiviral protection and in the stimuli needed for their maintenance. Three types of maintenance stimuli are examined: non-antigen-specific (bystander) stimulation, persisting antigen stimulation and reinfection-mediated stimulation. The modelling predicts that: (i) stable persistence of CTL memory requires the presence of either bystander or antigen-specific stimulation above a certain threshold depending on the sensitivity of memory CTL to stimulation and their life-span; (ii) a relatively low level of stimuli (approximately 10(4) fold less on a per CTL basis compared to acute infection) is needed to stabilize the expanded memory CTL population; (iii) the presence of CTL subsets in the memory pool of different activation states and lifespans ensures the robustness of memory persistence in the face of temporal variation in the low-level stimuli and; (iv) an 'optimal' population structure of the memory CTL pool, in terms of immediate protection, requires the presence of both activated cycling and effector CTL. For this, persisting antigen alone or synergistically with bystander signals provide the appropriate stimulation, so that the stimuli equivalent to approximately 30 p.f.u. of LCMV in the spleen are sufficient to maintain approximately 10(5)-10(6) specific CTL in the memory pool. These observations are relevant both to our understanding of natural protective immunity and to vaccine design.

Direct quantitation of rapid elimination of viral antigen-positive lymphocytes by antiviral CD8(+) T cells in vivo.

Lysis of infected cells by CD8(+) T cells is an important mechanism for the control of virus infections, but remains difficult to quantify in vivo. Here, we study the elimination kinetics of viral antigen-positive lymphocytes by antiviral CD8(+) T cells using flow cytometry and mathematical analysis. In mice acutely infected with lymphocytic choriomeningitis virus, more than 99.99 % of target cells were eliminated each day, corresponding to a half-life of 1.4 h. Even in mice exposed to virus 300 days previously, and with no ex vivo killing activity, 84 % of the target cells were eliminated per day. Unexpectedly, the elimination kinetics of antigen-positive lymphocytes was not significantly impaired in mice deficient in either perforin-, CD95 ligand- or TNF-mediated cytotoxicity. For viruses with a particular tropism for lymphocytes, such as Epstein-Barr virus or HIV, our results illustrate how effectively CD8(+) T cell-mediated elimination of target cells can potentially contribute to virus control and immunosuppression.

Donor cell persistence and activation-induced unresponsiveness of peripheral CD8+ T cells.

We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen-expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus-derived cytotoxic T lymphocyte (CTL) epitope (gp33-41) either alone or in combination with the male H-Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen-expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen-expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen-presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.