International, multicenter standardization of acute graft-versus-host disease clinical data collection: a report from the Mount Sinai Acute GVHD International Consortium

Acute graft-versus-host disease (GVHD) remains a leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. The clinical staging of GVHD varies greatly between transplant centers and is frequently not agreed on by independent reviewers. The lack of standardized approaches to handle common sources of discrepancy in GVHD grading likely contributes to why promising GVHD treatments reported from single centers have failed to show benefit in randomized multicenter clinical trials. We developed guidelines through international expert consensus opinion to standardize the diagnosis and clinical staging of GVHD for use in a large international GVHD research consortium. During the first year of use, the guidance followed discussion of complex clinical phenotypes by experienced transplant physicians and data managers. These guidelines increase the uniformity of GVHD symptom capture, which may improve the reproducibility of GVHD clinical trials after further prospective validation

Timing Is Critical for Effective Glucocorticoid Receptor Mediated Repression of the cAMP-Induced CRH Gene

Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR) expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH) mRNA expression: cyclic AMP (cAMP) and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP-induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing ‘positive’ response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids

A Crucial Role for Primary Cilia in Cortical Morphogenesis

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain

Identification and Visualization of CD8+ T Cell Mediated IFN-γ Signaling in Target Cells during an Antiviral Immune Response in the Brain

CD8+ T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8+ T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8+ T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8+ T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4⁺ T-Cells to Recognition by Cytotoxic T-Lymphocytes

Resting CD4⁺ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8⁺ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8⁺ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8⁺ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8⁺ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8⁺ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam₃CSK₄. In contrast, we did not observe CD8⁺ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8⁺ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8⁺ T-cells in HIV eradication strategies.United States. National Institutes of Health (AI111860

Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades