Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell–cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ~100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.

Key Points• We elucidate a molecular mechanism by which thyroid hormones sustain TCL survival.• We demonstrate that the membrane receptor of THs, integrin avb3, constitutes a potential target for TCL.The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin avb3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin avb3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated avb3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin avb3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin avb3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients. (Blood. 2015;125(5):841-851)

Non-Hodgkin lymphomas are a heterogeneous group of lymphoproliferative disorders of B and T cell origin that are treated with chemotherapy drugs with variable success rate that has virtually not changed over decades. Although new classes of chemotherapy-free epigenetic and metabolic drugs have emerged, durable responses to these conventional and new therapies are achieved in a fraction of cancer patients, with many individuals experiencing resistance to the drugs. The paucity in our understanding of what regulates the drug resistance phenotype and establishing a predictive indicator is, in great part, due to the lack of adequate ex vivo lymphoma models to accurately study the effect of microenvironmental cues in which malignant B and T cell lymphoma cells arise and reside. Unlike many other tumors, lymphomas have been neglected from biomaterials-based microenvironment engineering standpoint. In this study, we demonstrate that B and T cell lymphomas have different pro-survival integrin signaling requirements (αvβ3 and α4β1) and the presence of supporting follicular dendritic cells are critical for enhanced proliferation in three-dimensional (3D) microenvironments. We engineered adaptable 3D tumor organoids presenting adhesive peptides with distinct integrin specificities to B and T cell lymphoma cells that resulted in enhanced proliferation, clustering, and drug resistance to the chemotherapeutics and a new class of histone deacetylase inhibitor (HDACi), Panobinostat. In Diffuse Large B cell Lymphomas, the 3D microenvironment upregulated the expression level of B cell receptor (BCR), which supported the survival of B cell lymphomas through a tyrosine kinase Syk in the upstream BCR pathway. Our integrin specific ligand functionalized 3D organoids mimic a lymphoid neoplasm-like heterogeneous microenvironment that could, in the long term, change the understanding of the initiation and progression of hematological tumors, allow primary biospecimen analysis, provide prognostic values, and importantly, allow a faster and more rational screening and translation of therapeutic regimens.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4 + T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4 + T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4 + T cells when compared with naive, central, and transitional memory CD4 + T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Several lines of evidence link the canonical oncogene BCL6 to stress response. Here we demonstrate that BCL6 evolved in vertebrates as a component of the HSF1driven stress response, which has been co-opted by the immune system to support germinal center formation and may have been decisive in the convergent evolution of humoral immunity in jawless and jawed vertebrates. We fi nd that the highly conserved BTB corepressor binding site of BCL6 mediates stress adaptation across vertebrates. We demonstrate that pan-cancer cells hijack this stress tolerance mechanism to aberrantly express BCL6. Targeting the BCL6 BTB domain in cancer cells induces apoptosis and increases susceptibility to repeated doses of cytotoxic therapy. The chemosensitization effect upon BCL6 BTB inhibition is dependent on the derepression of TOX , implicating modulation of DNA repair as a downstream mechanism. Collectively, these data suggest a form of adaptive nononcogene addiction rooted in the natural selection of BCL6 during vertebrate evolution. SIGNIFICANCE:We demonstrate that HSF1 drives BCL6 expression to enable stress tolerance in vertebrates. We identify an HSF1-BCL6-TOX stress axis that is required by cancer cells to tolerate exposure to cytotoxic agents and points toward BCL6-targeted therapy as a way to more effectively kill a wide variety of solid tumors.

Background Despite early antiretroviral therapy (ART), ART-suppressed people with HIV (PWH) remain at higher risk for infections and infection-related malignancies than the general population. The immunologic pathways that remain abnormal in this setting, potentially contributing to these complications, are unclear. Methods ART-suppressed PWH and HIV-negative controls, all CMV-seropositive and enriched for HIV risk factors, were sampled from an influenza vaccine responsiveness study. PWH were stratified by timing of ART initiation (within 6 months of infection [Early ART] vs. later) and nadir CD4 count among later initiators. Between-group differences in kynurenine/tryptophan (KT) ratio, IP-10, sCD14, sCD163, sTNFR2, IL-6, and suPAR were assessed after adjustment for confounders. Results Most participants (92%) were male, reflecting demographics of early ART initiators in San Francisco. Most biomarkers were higher among later ART initiators. Early ART participants achieved near-normal sTNFR2, IL-6, and suPAR levels, but substantially higher KT ratio than those without HIV after adjustment for confounders (P=0.008). sCD14, sCD163, and IP-10 followed a similar trend. Conclusions While early ART initiators restore near-normal levels of many inflammatory markers, the kynurenine pathway of tryptophan catabolism remains abnormally high. As this pathway confers adaptive immune defects and predicts tuberculosis and cancer progression, this pathway may contribute to persistent risks of these complications in this setting.

BackgroundRefractory and/or relapsed diffuse large B cell lymphoma (RR-DLBCL) patients are incurable with conventional chemotherapy due to the aggressiveness and the chemorefractory state of these tumors. DNA hypermethylation and histone deacetylation are two major epigenetic modifications by which aggressive DLBCL maintain their oncogenic state. We have previously reported that DNA methyltransferase inhibitors (DNMTI) affect RR-DLBCL growth and improve chemosensitivity. Here, we hypothesized that the combination of DNMTI with histone deacetylase inhibitor (HDI) would be an active and feasible therapeutic strategy in RR-DLBCL. Thus, we evaluated the anti-lymphoma activity of the HDI vorinostat (VST) in combination with the DNMTI azacitidine (AZA) or decitabine (DAC) in pre-clinical models of RR-DLBCL, and we determined the feasibility of the combination by conducting a phase Ib trial in RR-DLBCL patients.ResultsConcurrent combination of DNMTI and HDI resulted in synergistic anti-lymphoma effect toward RR-DLBCL cells in vitro and in vivo, with no significant toxicity increase. In a phase Ib trial, a total of 18 patients with a median of three prior therapies were treated with four different dose levels of AZA and VST. The most common toxicities were hematological, followed by gastrointestinal and metabolic. The clinical benefit was low as only one subject had a partial response and three subjects had stable disease. Interestingly, two of the seven patients that received additional chemotherapy post-study achieved a complete response and three others had a significant clinical benefit. These observations suggested that the combination might have a delayed chemosensitization effect that we were able to confirm by using in vitro and in vivo models. These studies also demonstrated that the addition of VST does not improve the chemosensitizing effect of DAC alone.ConclusionsOur data supports the strategy of epigenetic priming by employing DNMTI in RR-DLBCL patients in order to overcome resistance and improve their outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0245-y) contains supplementary material, which is available to authorized users.