TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration.

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia.

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. Herein, we investigated the relationship between these two processes in normal and leukemic B cells. We found that leukemic B cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B cells. Furthermore, ligation of either surface CD79B, a B-cell receptor component required for downstream signaling, or surface Immunoglobulin M (IgM) by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to the dissociation of these two molecules in leukemic cells. A comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node- and peripheral blood-derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton's tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization.

KSHV-encoded miRNAs target MAF to induce endothelial cell reprogramming.

Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.

Human immunodeficiency virus Tat associates with a specific set of cellular RNAs.

BACKGROUND: Human Immunodeficiency Virus 1 (HIV-1) exhibits a wide range of interactions with the host cell but whether viral proteins interact with cellular RNA is not clear. A candidate interacting factor is the trans-activator of transcription (Tat) protein. Tat is required for expression of virus genes but activates transcription through an unusual mechanism; binding to an RNA stem-loop, the transactivation response element (TAR), with the host elongation factor P-TEFb. HIV-1 Tat has also been shown to alter the expression of host genes during infection, contributing to viral pathogenesis but, whether Tat also interacts with cellular RNAs is unknown. RESULTS: Using RNA immunoprecipitation coupled with microarray analysis, we have discovered that HIV-1 Tat is associated with a specific set of human mRNAs in T cells. mRNAs bound by Tat share a stem-loop structural element and encode proteins with common biological roles. In contrast, we do not find evidence that Tat associates with microRNAs or the RNA-induced silencing complex (RISC). The interaction of Tat with cellular RNA requires an intact RNA binding domain and Tat RNA binding is linked to an increase in RNA abundance in cell lines and during infection of primary CD4+ T cells by HIV. CONCLUSIONS: We conclude that Tat interacts with a specific set of human mRNAs in T cells, many of which show changes in abundance in response to Tat and HIV infection. This work uncovers a previously unrecognised interaction between HIV and its host that may contribute to viral alteration of the host cellular environment.

Defining an Optimized Workflow for Enriching and Analyzing Residual Tumor Populations Using Intracellular Markers.

Tumor relapse is well recognized to arise from treatment-resistant residual populations. Strategies enriching such populations for in-depth downstream analyses focus on tumor-specific surface markers; however, enrichment using intracellular biomarkers remains challenging. Using B-cell lymphoma as an exemplar, we demonstrate feasibility to enrich B-cell lymphoma 2 (BCL2)high populations, a surrogate marker for t(14;18)+ lymphomas, for use in downstream applications. Different fixation protocols were assessed for impact on antibody expression and RNA integrity; glyoxal fixation demonstrated superior results regarding minimal effects on surface and intracellular expression, and RNA quality, compared with alternative fixatives evaluated. Furthermore, t(14;18)+ B cells were effectively detected using intracellular BCL2 overexpression to facilitate tumor cell enrichment. Tumor cell populations were enriched using the cellenONE F1.4 single-cell sorting platform, which detected and dispensed BCL2high-expressing cells directly into library preparation reagents for transcriptome analyses. Sorted glyoxal-fixed cells generated good quality sequencing libraries, with high concordance between live and fixed single-cell transcriptomic profiles, discriminating cell populations predominantly on B-cell biology. Overall, we successfully developed a proof-of-concept workflow employing a robust cell preparation protocol for intracellular markers combined with cell enrichment using the cellenONE platform, providing an alternative to droplet-based technologies when cellular input is low or requires prior enrichment to detect rare populations. This workflow has wider prognostic and therapeutic potential to study residual cells in a pan-cancer setting.

Identification of proliferative and non-proliferative subpopulations of leukemic cells in CLL.

Pathogenesis in chronic lymphocytic leukemia (CLL) is strongly linked to the potential for leukemic cells to migrate to and proliferate within lymph-nodes. Previous in vivo studies suggest that all leukemic cells participate in cycles of migration and proliferation. In vitro studies, however, have shown heterogeneous migration patterns.To investigate tumor subpopulation kinetics, we performed in vivo isotope-labeling studies in ten patients with IgVH-mutated CLL (M-CLL). Using deuterium-labeled glucose, we investigated proliferation in sub-populations defined by CXCR4/CD5 and surface (sIgM) expression. Mathematical modeling was performed to test the likelihood that leukemic cells exist as distinct sub-populations or as a single population with the same proliferative capacity. Further labeling studies in two patients with M-CLL commencing idelalisib investigated the effect of B-cell receptor (BCR) antagonists on sub-population kinetics.Modeling revealed that data were more consistent with a model comprising distinct sub-populations (p = 0.008) with contrasting, characteristic kinetics. Following idelalisib therapy, similar labeling suppression across all sub-populations suggested that the most proliferative subset is the most sensitive to treatment. As the quiescent sub-population precedes treatment, selection likely explains the persistence of such residual non-proliferating populations during BCR-antagonist therapy. These findings have clinical implications for discontinuation of long-term BCR-antagonist treatment in selected patients.

Viral population analysis and minority-variant detection using short read next-generation sequencing.

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.

Measurement of CD4+ and CD8+ T-lymphocyte cytokine secretion and gene expression changes in p-phenylenediamine allergic patients and tolerant individuals.

Factors predisposing to individual susceptibility to contact allergic dermatitis are ill defined. This study was designed to characterize the response of allergic and tolerant individuals' T-lymphocytes after exposure to p-phenylenediamine (PPD). Peripheral blood mononuclear cells (PBMCs) from allergic patients proliferated when treated with PPD and Bandrowski's base (BB) and secreted IL-1alpha, -1beta, -4, -5, -6, -8, -10, and -13; IFN-gamma; tumor necrosis factor-alpha; MIP-1alpha/beta; MCP-1 (monocyte chemotactic protein-1); and RANTES. PBMCs from tolerant individuals were stimulated to proliferate only with BB, and they secreted significantly lower levels of Th2 cytokines. Principal component analysis showed that genes are differentially expressed between the patient groups. A network-based analysis of microarray data showed upregulation of T helper type 2 (Th2) gene pathways, including IL-9, in allergic patients, but a regulatory gene profile in tolerant individuals. Real-time PCR confirmed the observed increase in Th2 cytokine gene transcription in allergic patients. Purified CD4+ and CD8+ T cells from allergic patients were stimulated to proliferate and secrete Th2 cytokines following antigen exposure. Only CD4+ T cells from tolerant individuals were stimulated by BB, and levels of Th2 cytokines were 80% lower. The nature of the antigenic determinant stimulating PBMCs and levels of Th2 cytokines, including IL-9, was confirmed in a validation cohort. These studies show increased activity of Th2 cytokines in CD4+ and CD8+ T cells from individuals with allergic contact dermatitis.

Activation of T-cells from allergic patients and volunteers by p-phenylenediamine and Bandrowski's base.

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.

Activation of human dendritic cells by p-phenylenediamine.

Exposure to p-phenylenediamine (pPD), a primary intermediate in hair dye formulations, is often associated with the development of allergic contact dermatitis. Such reactions involve activation of the subject's immune system. The aim of these studies was to explore the relationship between pPD oxidation and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells were incubated with pPD and Bandrowski's base (BB) for 16 h, and expression of the costimulatory receptors CD40, CD80, CD83, CD86, and major histocompatibility complex class II intracellular glutathione levels and cell viability were measured. In certain experiments, glutathione (1 mM) was added to culture medium. Liquid chromatography-mass spectrometry (LC-MS) analysis and exhaustive solvent extraction were used to monitor the rate of [(14)C]pPD oxidation and the extent of pPD binding to cellular and serum protein, respectively. Proliferation of allogeneic lymphocytes was determined by incorporation of [(3)H]thymidine. Exposure of dendritic cells to pPD (5-50 microM), but not BB, was associated with an increase in CD40 and MHC class II expression and proliferation of allogeneic lymphocytes. Dendritic cell activation with pPD was not associated with apoptotic or necrotic cell death or depletion of glutathione. Neither pPD nor BB altered dendritic cell expression of CD80, CD83, or CD86. LC-MS analysis revealed pPD was rapidly oxidized in cell culture media to BB. Addition of glutathione inhibited BB formation but did not prevent covalent binding of pPD to dendritic cell protein or dendritic cell activation. Collectively, these studies show that pPD, but not BB, selectively activates human dendritic cells in vitro.