Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

A novel immature natural killer cell subpopulation predicts relapse after cord blood transplantation.

Natural killer (NK) cells are highly heterogeneous, with vast phenotypic and functional diversity at the single-cell level. They are involved in the innate immune response against malignant and virus-infected cells. To understand the effect of NK diversity during immune recovery on the antitumor response after cord blood transplantation (CBT), we used high-dimensional mass cytometry and the metrics of NK cell diversity to study the NK cell repertoire in serial samples from 43 CBT recipients. A higher-diversity index based on single-cell combinatorial phenotypes was significantly associated with a lower risk for relapse after CBT (P = .005). Cytomegalovirus reactivation was a major factor in the development of a more diverse NK repertoire after CBT. Notably, we identified a group of patients whose CB-derived NK cells after transplantation possessed an immature phenotype (CB-NKim), characterized by poor effector function and a low diversity index. Frequencies of CB-NKim of 11.8% or higher during the early post-CBT recovery phase were highly predictive for relapse (area under the curve [AUC], 0.979), a finding that was validated in a second independent cohort of patients (n = 25; AUC, 0.977). Moreover, we showed that the maturation, diversity, and acquisition of effector function by CB-NKim early after CBT were driven by interleukin 15. Our data indicate that the diversity of the NK cell repertoire after CBT contributes importantly to the risk for subsequent relapse. We suggest that the use of diversity metrics and high-dimensional mass cytometry may be useful tools in predicting clinical outcomes and informing the design of therapeutic strategies to prevent relapse after CBT.

Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses.

The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.

The CXCR4-STAT3-IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide.

Chronic lymphocytic leukemia (CLL) cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12)-CXCR4-STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12-CXCR4-S727-STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

Biallelic mutations in IRF8 impair human NK cell maturation and function.

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

Urine proteome analysis in murine nephrotoxic serum nephritis.

BACKGROUND: Urine contains serum proteins filtered by the glomerulus or secreted by the renal tubules and proteins produced locally by the urinary tract. Proteomic analysis of urine holds the potential as a noninvasive means of studying or monitoring disease activity. In mice, large concentrations of albumin and lipocalins have complicated the ability to identify urinary biomarkers in disease models. METHODS: Passive nephrotoxic serum nephritis was induced in mice. Urine proteins were identified and quantified by iTRAQ and MALDI-TOF mass spectrometry. Results were compared to Western blotting and multiplex immunoassays. RESULTS: Large concentrations of major urinary proteins dominate the urine proteome of mice even in the context of acute nephritis. Increased proteinuria caused by nephrotoxic serum nephritis is transient and includes increased albumin excretion. There were no alterations in chemokine excretion. Altered hepcidin excretion was identified, most likely reflecting local production and renal retention. CONCLUSION: Proteomic analysis of mouse urine remains challenging due to the abundance of a limited subset of proteins. iTRAQ analysis does not circumvent these challenges, but can provide information on post-translational processing of some proteins. Hepcidin is identified as a potential urinary marker of nephritis and its role in disease pathogenesis warrants further study.

CD8+ T cells are not required for vaccine-induced immunity against Leishmania amazonensis in IL-12/23P40(-/-) C57BL/6 mice.

Vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated type 1 response and IL-12-driven IFN-gamma production. Surprisingly, our previous data showed that IL-12/23p40(-/-) mice could be vaccinated against L. amazonensis and were able to produce limited amounts of IFN-gamma. Since the role of CD8+ T in immunization against L. amazonensis is obscure, the aim of this study was to evaluate the effects of CD8+ cells in protection against L. amazonensis in IL-12/23p40(-/-) mice. In order to deplete CD8+ cells, one group of vaccinated animals was treated with anti-CD8 mAb. Infection was followed for 8 weeks. The vaccinated CD8+ -depleted group developed smaller lesions than the non-depleted group. CD8 depletion did not affect tissue parasitism or antibody response against the parasite, and treated animals displayed milder inflammation and better tissue integrity. IFN-gamma production in spleen and draining lymph node was impaired in the depleted group, suggesting that CD8+ cells produced this cytokine in IL-12-independent vaccination. Such results suggest that this T cell subset contributes to augmented pathology in IL12/23p40(-/-) mice vaccinated and challenged with L. amazonensis. Although these cells could produce some IFN-gamma the in the absence of IL-12, they do not affect the parasite tissue load.