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

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates

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

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

Precise spatiotemporal regulation of O2 --generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67phox and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2 --generating system consisting of isolated membranes and recombinant p47phox and p67phox proteins. Addition of soluble RacGAPs decreased O 2 - production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2 - production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2 - production by Nox2, allowing selective regulation via different signaling pathways. © 2013 Elsevier Inc

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

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

Functional Inactivation of EBV-Specific T-Lymphocytes in Nasopharyngeal Carcinoma: Implications for Tumor Immunotherapy

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy with high prevalence in Southern Chinese. In order to assess whether defects of EBV-specific immunity may contribute to the tumor, the phenotype and function of circulating T-cells and tumor infiltrating lymphocytes (TILs) were investigated in untreated NPC patients. Circulating naïve CD3+CD45RA+ and CD4+CD25− cells were decreased, while activated CD4+CD25+ T-cells and CD3−CD16+ NK-cells were increased in patients compared to healthy donors. The frequency of T-cells recognizing seven HLA-A2 restricted epitopes in LMP1 and LMP2 was lower in the patients and remained low after stimulation with autologous EBV-carrying cells. TILs expanded in low doses of IL-2 exhibited an increase of CD3+CD4+, CD3+CD45RO+ and CD4+CD25+ cells and 2 to 5 fold higher frequency of LMP1 and LMP2 tetramer positive cells compared to peripheral blood. EBV-specific cytotoxicity could be reactivated from the blood of most patients, whereas the TILs lacked cytotoxic activity and failed to produce IFNγ upon specific stimulation. Thus, EBV-specific rejection responses appear to be functionally inactivated at the tumor site in NPC

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

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