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.

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.

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.