Angélique Biancotto

The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome

Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1–blocking agents. Herein we report a de novo missense mutation (c.1009A>T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4–transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.

Horse versus Rabbit Antithymocyte Globulin in Acquired Aplastic Anemia

BACKGROUND In severe acquired aplastic anemia, hematopoietic failure is the result of immune-mediated destruction of bone marrow stem and progenitor cells. Immunosuppressive therapy with antithymocyte globulin (ATG) plus cyclosporine is an effective alternative to stem-cell transplantation and improves blood counts and survival. Although horse ATG is the standard therapy, rabbit ATG is more potent in depleting peripheral-blood lymphocytes and is preferred in other clinical circumstances. METHODS From December 2005 through July 2010, we performed a randomized trial comparing these two ATG formulations in conventional regimens. Patients were treated at a single facility. The primary outcome was hematologic response at 6 months, as determined by blood counts. The study was designed to enroll 60 patients each for the rabbit-ATG and horse-ATG groups and was powered to detect a difference of 25 percentage points in the response rate. RESULTS A large, unexpected difference was observed in the rate of hematologic response at 6 months in favor of horse ATG (68%; 95% confidence interval [CI], 56 to 80) as compared with rabbit ATG (37%; 95% CI, 24 to 49; P<0.001). Overall survival at 3 years also differed, with a survival rate of 96% (95% CI, 90 to 100) in the horse-ATG group as compared with 76% (95% CI, 61 to 95) in the rabbit-ATG group (P=0.04) when data were censored at the time of stem-cell transplantation, and 94% (95% CI, 88 to 100) as compared with 70% (95% CI, 56 to 86; P=0.008) in the respective groups when stem-cell-transplantation events were not censored. CONCLUSIONS In a randomized study, rabbit ATG was inferior to horse ATG as a first treatment for severe aplastic anemia, as indicated by hematologic response and survival. (Funded by the Intramural Research Program of the National Institutes of Health; ClinicalTrials.gov number, NCT00260689.).

Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia

BACKGROUND Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells. METHODS We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag. RESULTS Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes. CONCLUSIONS Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.).

Global Analyses of Human Immune Variation Reveal Baseline Predictors of Postvaccination Responses

A major goal of systems biology is the development of models that accurately predict responses to perturbation. Constructing such models requires the collection of dense measurements of system states, yet transformation of data into predictive constructs remains a challenge. To begin to model human immunity, we analyzed immune parameters in depth both at baseline and in response to influenza vaccination. Peripheral blood mononuclear cell transcriptomes, serum titers, cell subpopulation frequencies, and B cell responses were assessed in 63 individuals before and after vaccination and were used to develop a systematic framework to dissect inter- and intra-individual variation and build predictive models of postvaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation cell populations alone, which were validated using independent baseline time points. Most of the parameters contributing to prediction delineated temporally stable baseline differences across individuals, raising the prospect of immune monitoring before intervention.

Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues

For most viruses, there is a need for antimicrobials that target unique viral molecular properties. Acyclovir (ACV) is one such drug. It is activated into a human herpesvirus (HHV) DNA polymerase inhibitor exclusively by HHV kinases and, thus, does not suppress other viruses. Here, we show that ACV suppresses HIV-1 in HHV-coinfected human tissues, but not in HHV-free tissue or cell cultures. However, addition of HHV-6-infected cells renders these cultures sensitive to anti-HIV ACV activity. We hypothesized that such HIV suppression requires ACV phosphorylation by HHV kinases. Indeed, an ACV monophosphorylated prodrug bypasses the HHV requirement for HIV suppression. Furthermore, phosphorylated ACV directly inhibits HIV-1 reverse transcriptase (RT), terminating DNA chain elongation, and can trap RT at the termination site. These data suggest that ACV anti-HIV-1 activity may contribute to the response of HIV/HHV-coinfected patients to ACV treatment and could guide strategies for the development of new HIV-1 RT inhibitors.

Danazol Treatment for Telomere Diseases

BACKGROUND Genetic defects in telomere maintenance and repair cause bone marrow failure, liver cirrhosis, and pulmonary fibrosis, and they increase susceptibility to cancer. Historically, androgens have been useful as treatment for marrow failure syndromes. In tissue culture and animal models, sex hormones regulate expression of the telomerase gene. METHODS In a phase 1-2 prospective study involving patients with telomere diseases, we administered the synthetic sex hormone danazol orally at a dose of 800 mg per day for a total of 24 months. The goal of treatment was the attenuation of accelerated telomere attrition, and the primary efficacy end point was a 20% reduction in the annual rate of telomere attrition measured at 24 months. The occurrence of toxic effects of treatment was the primary safety end point. Hematologic response to treatment at various time points was the secondary efficacy end point. RESULTS After 27 patients were enrolled, the study was halted early, because telomere attrition was reduced in all 12 patients who could be evaluated for the primary end point; in the intention-to-treat analysis, 12 of 27 patients (44%; 95% confidence interval [CI], 26 to 64) met the primary efficacy end point. Unexpectedly, almost all the patients (11 of 12, 92%) had a gain in telomere length at 24 months as compared with baseline (mean increase, 386 bp [95% CI, 178 to 593]); in exploratory analyses, similar increases were observed at 6 months (16 of 21 patients; mean increase, 175 bp [95% CI, 79 to 271]) and 12 months (16 of 18 patients; mean increase, 360 bp [95% CI, 209 to 512]). Hematologic responses occurred in 19 of 24 patients (79%) who could be evaluated at 3 months and in 10 of 12 patients (83%) who could be evaluated at 24 months. Known adverse effects of danazol--elevated liver-enzyme levels and muscle cramps--of grade 2 or less occurred in 41% and 33% of the patients, respectively. CONCLUSIONS In our study, treatment with danazol led to telomere elongation in patients with telomere diseases. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01441037.).

Dual Role of Prostratin in Inhibition of Infection and Reactivation of Human Immunodeficiency Virus from Latency in Primary Blood Lymphocytes and Lymphoid Tissue

ABSTRACT To design strategies to purge latent reservoirs of human immunodeficiency virus type 1 (HIV-1), we investigated mechanisms by which a non-tumor-promoting phorbol ester, prostratin, inhibits infection of CD4+ T lymphocytes and at the same time reactivates virus from latency. CD4+ T lymphocytes from primary blood mononuclear cells (PBMC) and in blocks of human lymphoid tissue were stimulated with prostratin and infected with HIV-1 to investigate the effects of prostratin on cellular susceptibility to the virus. The capacity of prostratin to reactivate HIV from latency was tested in CD4+ T cells harboring preintegrated and integrated latent provirus. Prostratin stimulated CD4+ T cells in an aberrant way. It induced expression of the activation markers CD25 and CD69 but inhibited cell cycling. HIV-1 uptake was reduced in prostratin-stimulated CD4+ T PBMC and tissues in a manner consistent with a downregulation of CD4 and CXCR4 receptors in these systems. At the postentry level, prostratin inhibited completion of reverse transcription of the viral genome in lymphoid tissue. However, prostratin facilitated integration of the reverse-transcribed HIV-1 genome in nondividing CD4+ T cells and facilitated expression of already integrated HIV-1, including latent forms. Thus, while stimulation with prostratin restricts susceptibility of primary resting CD4+ T cells to HIV infection at the virus cell-entry level and at the reverse transcription level, it efficiently reactivates HIV-1 from pre- and postintegration latency in resting CD4+ T cells.

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

CD4+ T Cells, Including Th17 and Cycling Subsets, Are Intact in the Gut Mucosa of HIV-1-Infected Long-Term Nonprogressors

ABSTRACT During acute human immunodeficiency virus (HIV) infection, there is a massive depletion of CD4+ T cells in the gut mucosa that can be reversed to various degrees with antiretroviral therapy. Th17 cells have been implicated in mucosal immunity to extracellular bacteria, and preservation of this subset may support gut mucosal immune recovery. However, this possibility has not yet been evaluated in HIV-1-infected long-term nonprogressors (LTNPs), who maintain high CD4+ T cell counts and suppress viral replication in the absence of antiretroviral therapy. In this study, we evaluated the immunophenotype and function of CD4+ T cells in peripheral blood and gut mucosa of HIV-uninfected controls, LTNPs, and HIV-1-infected individuals treated with prolonged antiretroviral therapy (ART) (VL [viral load]<50). We found that LTNPs have intact CD4+ T cell populations, including Th17 and cycling subsets, in the gut mucosa and a preserved T cell population expressing gut homing molecules in the peripheral blood. In addition, we observed no evidence of higher monocyte activation in LTNPs than in HIV-infected (HIV−) controls. These data suggest that, similar to nonpathogenic simian immunodeficiency virus (SIV) infection, LTNPs preserve the balance of CD4+ T cell populations in blood and gut mucosa, which may contribute to the lack of disease progression observed in these patients.