Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Here we present APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds upon the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states, and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-Seq yields high-confidence 5hmC profiles with at least 1000-fold less DNA input than conventional methods. Applying ACE-Seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we find that 5hmC is almost entirely confined to CG dinucleotides. The map permits cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and reveals genomic features that diverge from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods and expands the scope of epigenome profiling to include scarce samples and open new lines of inquiry regarding the role of cytosine modifications in genome biology.

SUMMARY Combination antiretroviral therapy (ART) is able to suppress HIV-1 replication to undetectable levels. However, the persistence of latent viral reservoirs allows for a rebound of viral load upon cessation of therapy. Thus, therapeutic strategies to eradicate the viral latent reservoir are critically needed. Employing a targeted RNAi screen, we identified the ubiquitin ligase BIRC2 (cIAP1), a repressor of the noncanonical NF-κB pathway, as a potent negative regulator of LTR-dependent HIV-1 transcription. Depletion of BIRC2 through treatment with small molecule antagonists known as Smac mimetics enhanced HIV-1 transcription, leading to a reversal of latency in a JLat latency model system. Critically, treatment of resting CD4+ T cells isolated from ART-suppressed patients with the histone deacetylase inhibitor (HDACi) panobinostat together with Smac mimetics resulted in synergistic activation of the latent reservoir. These data implicate Smac mimetics as useful agents for shock-and-kill strategies to eliminate the latent HIV reservoir.

The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.

Whole-genome expression profiling in postmortem brain tissue has recently provided insight into the pathophysiology of schizophrenia. Previous microarray and RNA-Seq studies identified several biological processes including synaptic function, mitochondrial function and immune/inflammation response as altered in the cortex of subjects with schizophrenia. Now using RNA-Seq data from the hippocampus, we have identified 144 differentially expressed genes in schizophrenia cases as compared with unaffected controls. Immune/inflammation response was the main biological process over-represented in these genes. The upregulation of several of these genes, IFITM1, IFITM2, IFITM3, APOL1 (Apolipoprotein L1), ADORA2A (adenosine receptor 2A), IGFBP4 and CD163 were validated in the schizophrenia subjects using data from the SNCID database and with quantitative RT-PCR. We identified a co-expression module associated with schizophrenia that includes the majority of differentially expressed genes related to immune/inflammation response as well as with the density of parvalbumin-containing neurons in the hippocampus. The results indicate that abnormal immune/inflammation response in the hippocampus may underlie the pathophysiology of schizophrenia and may be associated with abnormalities in the parvalbumin-containing neurons that lead to the cognitive deficits of the disease.