Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.

Late-onset cytomegalovirus (CMV) disease commonly occurs after discontinuation of antiviral prophylaxis. We determined the utility of testing CD8+ T-cell response against CMV as a predictor of late-onset CMV disease after a standard course of antiviral prophylaxis. Transplant patients at high-risk for CMV disease were enrolled. CD8+ T-cell-mediated immunity (CMI) was tested using the QuantiFERON-CMV assay at baseline, 1, 2 and 3 months posttransplant by measurement of interferon-gamma response to whole blood stimulation with a 21-peptide pool. The primary outcome was the ability of CMI testing to predict CMV disease in the first 6 months posttransplant. There were 108 evaluable patients (D+/R+ n = 39; D-/R+ n = 34; D+/R- n = 35) of whom 18 (16.7%) developed symptomatic CMV disease. At the end of prophylaxis, CMI was detectable in 38/108 (35.2%) patients (cutoff 0.1 IU/mL interferon-gamma). CMV disease occurred in 2/38 (5.3%) patients with a detectable interferon-gamma response versus 16/70 (22.9%) patients with a negative response; p = 0.038. In the subgroup of D+/R- patients, CMV disease occurred in 1/10 (10.0%) patients with a detectable interferon-gamma response (cutoff 0.1 IU/mL) versus 10/25 (40.0%) patients with a negative CMI, p = 0.12. Monitoring of CMI may be useful for predicting late-onset CMV disease.

The primary in vivo immune response to Mls-1 (Mtv-7 sag). Route of injection determines the immune response pattern.

Injection of cells expressing the retroviral superantigen Mls-1 (Mtv-7 sag) into adult Mls-1- mice induces a strong immune response including both T- and B-cell activation. This model was used for studying qualitative aspects of the immune response in normal mice with a defined antigen-presenting cell (the B cell) and without the use of adjuvant. BALB/c mice were injected locally or systemically with Mls-1-expressing spleen cells from Mls-1-congenic BALB.D2 mice. Intravenous injection led to an initially strong expansion of Mls-1-reactive V beta 6+ CD4+ cells mainly in the spleen, to a large degree explained by the trapping of reactive cells, and a rapid down-regulation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production, consistent with the proposed tolerogenic property of B cells as antigen-presenting cells. However, these mice developed a slowly appearing but persistent B-cell response dominated by IgG1-producing cells, suggesting a shift in lymphokines produced rather than complete unresponsiveness. Subcutaneous injection into the hind footpad with the same number of cells led to a strong local response in the draining lymph node, characterized by a dramatic increase of V beta 6+ CD4+ T cells, local production of IL-2 and IFN-gamma and a strong but short-lived antibody response dominated by IgG2a-producing cells, characteristic of a T-helper type 1 (Th1) type of response. Both routes of injection led ultimately to deletion of reactive T cells and anergy, as defined by the inability to produce IL-2 upon in vitro stimulation with Mls-1. It is concluded that Mls-1 presented by B cells induces qualitatively different responses in vivo dependent on the route of injection. We propose that the different responses result from the migration of the injected cells to different micro-anatomical sites in the lymphoid tissue. Furthermore, these results suggest that B cells may function as professional antigen-presenting cells in vivo present in an appropriate environment.

The role of macrophage migration inhibitory factor in mouse islet transplantation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. METHODS: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. RESULTS: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. CONCLUSION: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

QuantiFERON-TB Gold assay for the diagnosis of latent tuberculosis infection.

Tuberculin skin test (TST) has been used for 100 years for the diagnosis of latent tuberculosis (TB) infection (LTBI). In recent years, increasing interest in the diagnosis of TB has led to the development of new assays. QuantiFERON-TB Gold (QFT-G) is an IFN-gamma-release assay that measures the release of interferon after stimulation in vitro by Mycobacterium tuberculosis antigens. The main advantage of this assay with respect to TST is the lack of crossreaction with bacillus Calmette-Guérin and most nontuberculous mycobacteria. QFT-G also eliminates the need for the patient to return for test reading in 48-72 h. In the immunocompromised host and in pediatric populations, studies suggest that the QFT-G better correlates with the risk of TB than the TST, but data remain inconclusive. In contrast to TST, there are no prospective studies regarding the association of the QFT-G result and the risk for development of TB. Given its advantages, the QFT-G may become the standard test for the diagnosis of LTBI.

Efficient transduction of dendritic cells and induction of a T-cell response by third-generation lentivectors.

In order to induce a therapeutic T lymphocyte response, recombinant viral vaccines are designed to target professional antigen-presenting cells (APC) such as dendritic cells (DC). A key requirement for their use in humans is safe and efficient gene delivery. The present study assesses third-generation lentivectors with respect to their ability to transduce human and mouse DC and to induce antigen-specific CD8+ T-cell responses. We demonstrate that third-generation lentivectors transduce DC with a superior efficiency compared to adenovectors. The transfer of DC transduced with a recombinant lentivector encoding an antigenic epitope resulted in a strong specific CD8+ T-cell response in mice. The occurrence of lower proportions of nonspecifically activated CD8+ cells suggests a lower antivector immunity of lentivector compared to adenovector. Thus, lentivectors, in addition to their promise for gene therapy of brain disorders might also be suitable for immunotherapy.

Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7-27+28+ and RA+CCR7-27+28-, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon gamma, and tumor necrosis factor alpha. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.

Recombinant human tumor necrosis factor: an efficient agent for cancer treatment.

Recombinant human TNF (rhTNF) has a selective effect on endothelial cells in tumour angiogenic vessels. Its clinical use has been limited because of its property to induce vascular collapsus. TNF administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs was shown to be safe and efficient. When combined to the alkylating agent melphalan, a single ILP produces a very high objective response rate. ILP with TNF and melphalan provided the proof of concept that a vasculotoxic strategy combined to chemotherapy may produce a strong anti-tumour effect. The registered indication of TNF-based ILP is a regional therapy for regionally spread tumours. In soft tissue sarcomas, it is a limb sparing neoadjuvant treatment and, in melanoma in-transit metastases, a curative treatment. Despite its demonstrated regional efficiency TNF-based ILP is unlikely to have any impact on survival. High TNF dosages induce endothelial cells apoptosis, leading to vascular destruction. However, lower TNF dosage produces a very strong effect that is to increase the drug penetration into the tumour, presumably by decreasing the intratumoural hypertension resulting in better tumour uptake. TNF-ILP allowed the identification of the role of alphaVbeta3 integrin deactivation as an important mechanism of antiangiogenesis. Several recent studies have shown that TNF targeting is possible, paving the way to a new opportunity to administer TNF systemically for improving cancer drug penetration. TNF was the first agent registered for the treatment of cancer that improves drug penetration in tumours and selectively destroys angiogenic vessels.

Indeterminate test results of T-SPOT.TB performed under routine field conditions

Interferon-gamma release assays for the diagnosis of tuberculosis (TB) can give indeterminate results. The prevalence of indeterminate test results (ITRs) among T-SPOT.TB tests was assessed. A retrospective analysis of samples processed in 2005 was performed. ITRs were assessed by age, sex, immunosuppression, distance to the laboratory and season. A subgroup of tests performed for specific indications (contact tracing, migrants with positive tuberculin skin test, TB suspects and immunosuppression) were analysed separately. Of a total of 1,429 tests, 49 (3.4%) were indeterminate. ITRs were significantly associated with old age (>75 versus 5-75 yrs; odds ratio (OR) 7.97, 95% confidence interval (CI) 3.968-15.438) and the season during which samples were transported (autumn and winter versus spring and summer; OR 3.47, 95% CI 1.753-7.514). The incidence of ITR was 302 (2.0%) among TB contacts, 75 (1.6%) among immigrants, 156 (3.0%) in TB suspects and 32 (3.0%) among immunosuppressed patients. Sex, young age and distance to the laboratory were not associated with the rate of ITR. Of the 13 tests with ITR that were repeated, 10 gave a clear positive or negative result. Indeterminate test results with T-SPOT.TB under routine conditions were infrequent and more common in individuals aged >75 yrs than in children and younger adults. The incidence of indeterminate test results was low and similar among healthy tuberculosis contacts, immigrants with a positive tuberculin skin test, tuberculosis suspects and the immunosuppressed. The conditions of transportation may influence the incidence of indeterminate test results.