Multiple recurrences of tuberculosis in an HIV infected individual.

Correspondenc

Alveolar Epithelial Type II Cells Activate Alveolar Macrophages and Mitigate P. Aeruginosa Infection

Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1−/− mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1−/− mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1−/− mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFκB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn−/− mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions

Abstract 544: Follicular Dendritic Cells promote tumor B cell survival through BAFF mediated anti-apoptotic signaling in the tumor microenvironment of Follicular Lymphoma

Abstract Follicular lymphoma (FL) is the second most common B-cell non-Hodgkin's lymphoma that responds well to initial therapy but has a continuous pattern of relapses and remissions and eventually leads to a fatal outcome in the majority of the patients. Preliminary meta-analysis of microarray data from various gene expression studies led to the identification of unique features of FL such as up regulation in genes associated with TNFSF family (BAFF, APRIL), BMP signaling pathway, 14-3-3-mediated signaling, NFκB signaling, NFAT signaling and IL-4 signaling. The results indicate that some of the gene expression patterns are possibly derived from infiltrating T cells, lymphoma associated macrophages (LAM) or follicular dendritic cells (FDC). FDCs play a vital role in the maturation of normal B cells in the germinal center. FDCs have been found to be the common source for both BAFF and APRIL. Three main receptors TACI, BCMA and BAFF-R that recognize these factors are known to be expressed by many immune cell types including GC B cells. Whether the levels of expression of BAFF-R and TACI are important for tumor cell survival is not clear. From immunohistochemistry based studies BAFF-R has been demonstrated to be up regulated in tumor B cells from FL patients but absent in grade 3 FL and DLBCL. We have detected elevated serum levels of BAFF in FL patients (n=10) compared with normal donors (n=10). Further cell surface staining for BAFF-R was significantly higher in CD20+ tumor B cells from FL compared to normal donor tonsil derived B cells. We employed HK cell line (human FDC phenotype) to study the cell to cell interactions in a co-culture system with either FL tumor or normal donor tonsil derived B cells. HK cells behave like typical FDCs and secrete BAFF and APRIL into the conditioned medium that was detected and confirmed by ELISA. We found that FL derived tumor B cells survived significantly longer in co-culture with HK cells (10 to 14 days) compared to when cultured alone in vitro (5 to 7 days) based on Annexin-V FITC analysis by flowcytometry. BrdU cell proliferation assay showed that there was sustainable cell growth and mitosis over this two week period. In conclusion we determined a positive correlation between levels of BAFF in the conditioned medium and tumor B cell survival suggesting a role for BAFF mediated anti-apoptotic signaling. Future experiments will determine other factors that may have a role in contributing to tumor B cell survival and further study the mechanism of cell survival in this co-culture system. This study holds much promise for developing newer therapeutic targets like monoclonal antibodies to cell surface receptors that can block the anti-apoptotic signaling in vivo thereby negating the supportive role of tumor microenvironment. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 544.</jats:p

Impact of Human Exome Sequencing on Clinical Research

Recent advances in human exome sequencing and the associated advantages have made it a technology of choice in various domains. The savings in time, cost and data storage compared with whole genome sequencing make this technology a potential game changer in clinical research settings. Recent advances in NGS have made it feasible to use exome sequencing in clinical research for identifying novel and rare variants that can lead to change in protein structure and function which may finally culminate into a totally different phenotype. If whole exome is not desired the same technology can be used for studying target exonic regions to investigate causative genes for a specific phenotype associated with disease. Exome sequencing has emerged as an effective and efficient tool for the translational and clinical research. There is a demand for systematically storing variant information in large databanks. Meaningful information from the exome-seq data can be combined with other data. This can be correlated with clinical findings within a clinical trial setting for a better study outcome.</jats:p

Cross-Talk Between Tumor B Cells, Follicular Dendritic Cells, and Follicular Helper T Cells: Implications for Follicular Lymphoma Pathogenesis and Therapy,

Abstract Abstract 3696 Background: The stromal microenvironment is thought to play a critical role in supporting the survival and growth of follicular lymphoma (FL) tumor cells. However, the key stromal factors involved are not well characterized. We hypothesized that FL tumor cells hijack the survival and growth mechanisms that are usually used by normal B cells in the lymph node microenvironment. For example, follicular dendritic cells (FDC) residing in germinal centers of lymph nodes play a vital role in supporting normal B cell survival and proliferation through adhesion molecules and by secreting chemokines and cytokines. In normal lymph nodes, lymphotoxin-alpha (LTA, TNFSF1) secreted by B cells activates FDC through LTbR and induces secretion of BAFF (TNFSF13B) and CXCL13. CXCL13 recruits follicular helper T cells (Tfh) into the B cell zone and CD40L (expressed by Tfh) together with BAFF prevents apoptosis and promotes proliferation of B cells. Here, we determined whether a similar cross-talk exists between FL tumor B cells, FDC, and Tfh. Methods: FL tumor samples were processed into single-cell suspension and cryopreserved in aliquots for later use. Primary tumor B cells were isolated by magnetic cell separation from single-cell suspensions of FL. The FDC-like cell line HK derived from human tonsil was used in place of primary FDC. Levels of BAFF, CXCL13, and LTA in culture supernatants were determined by ELISA. Apoptosis of tumor cells was determined by Annexin V assay and proliferation by tritiated thymidine incorporation assay. Activation of NFkB was determined by the TransAM NFkB p52 ELISA kit. Results: We developed a co-culture model to mimic the FL tumor microenvironment to study the interactions between tumor cells, FDC and Tfh. We observed that FL tumor cells spontaneously produced low levels of LTA but its production was significantly enhanced when tumor cells were cultured with either recombinant BAFF or CD40L. Dose response curves from FL tumor cells treated with both BAFF and CD40L recombinant proteins showed synergistic increase in LTA secretion. Recombinant BAFF and CD40L also synergistically enhanced the survival and proliferation of FL tumor cells. HK cells spontaneously produced low levels of BAFF and CXCL13 but their production was significantly enhanced by recombinant LTA in a dose-dependent manner. Consistent with the above studies, co-culturing FL tumor cells with HK cells significantly enhanced LTA secretion, possibly due to the effects of BAFF secreted by HK cells. In addition, in this co-culture, we also observed enhanced production of BAFF and CXCL13 possibly due to the effects of LTA secreted by the tumor cells. The production of BAFF and CXCL13 in this co-culture could be inhibited by an LTA neutralizing antibody in a dose-dependent manner suggesting the role of LTA in this mechanism. Co-culturing FL tumor cells with HK cells also significantly enhanced the survival and proliferation of FL tumor cells greater than two-fold compared with FL tumor cells alone. This effect also could be inhibited by LTA neutralizing antibody but not isotype control antibody. The survival benefit for tumor cells was further enhanced upon co-culture with autologous Tfh cells FACSorted from the same tumor sample. We also studied the downstream NFkB signaling mediated by BAFF on tumor B cells and by LTA on HK cells in the co-culture. We found that both tumor B cells and HK cells had significantly higher NFkB activity upon co-culture compared to cells alone. Conclusions: Our results suggest a model where LTA secreted by tumor B cells forms the afferent axis and induces production of BAFF and CXCL13 by FDC. CXCL13 preferentially recruits CXCR5-expressing Tfh into the tumor microenvironment and CD40L expressed by Tfh and BAFF secreted by FDC form the efferent axis and promote further production of LTA by tumor cells. In addition, CD40L and BAFF synergistically enhance the survival and proliferation of FL tumor cells via activation of NFkB. Our results suggest that because of their redundant functions, blocking BAFF-BAFF-R pathway alone or CD40-CD40L pathway alone with monoclonal antibodies as is being done in ongoing clinical trials may not be optimal. In contrast, blocking both pathways simultaneously is likely to significantly enhance the clinical responses. Our results also suggest that neutralizing LTA or blocking its receptor may be another novel therapeutic strategy for management of FL. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Impact of Human Exome Sequencing on Clinical Research

Recent advances in human exome sequencing and the associated advantages have made it a technology of choice in various domains. The savings in time, cost and data storage compared with whole genome sequencing make this technology a potential game changer in clinical research settings. Recent advances in NGS have made it feasible to use exome sequencing in clinical research for identifying novel and rare variants that can lead to change in protein structure and function which may finally culminate into a totally different phenotype. If whole exome is not desired the same technology can be used for studying target exonic regions to investigate causative genes for a specific phenotype associated with disease. Exome sequencing has emerged as an effective and efficient tool for the translational and clinical research. There is a demand for systematically storing variant information in large databanks. Meaningful information from the exome-seq data can be combined with other data. This can be correlated with clinical findings within a clinical trial setting for a better study outcome.</jats:p

Abstract 1908: TH2 cells interact with tumor cells to facilitate recruitment of regulatory T cells into the tumor microenvironment in human B-cell lymphomas

Abstract Immunosuppressive Foxp3+ regulatory T cells (Tregs) were shown to be markedly increased in the tumor microenvironment in patients with B-cell non-Hodgkin lymphomas and may impair the efficacy of immunotherapeutic strategies. While the induction of Tregs from conventional T cells by tumor B-cells may be one reason for the increased number, we hypothesized that Tregs may also be actively recruited into the lymphoma tumor microenvironment. Here, we used a combination of genomic and immunologic approaches to elucidate the mechanism of recruitment of Tregs into the tumor microenvironment in various human B-cell lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and splenic marginal zone lymphoma. To determine whether human lymphoma tumor cells hyperexpressed specific chemokines, we analyzed cDNA microarray data on enriched tumor B-cells and compared them with those on enriched B-cells from normal tonsils. We observed that lymphoma tumor cells hyperexpressed CCL17 and CCL22 but not other chemokines compared with tonsillar B-cells, a finding confirmed by real-time PCR. CCL17 and CCL22 are known chemotactic factors for Tregs but are not known to be constitutively expressed by B cells. Since production of CCL17 and CCL22 was previously shown to be induced by IL-4 and/or IL-13 in various tissues, we reasoned that the expression of these chemokines by lymphoma tumor cells is induced by IL-4 and/or IL-13 produced in the tumor microenvironment. Using immunohistochemical studies, we observed that IL-4 is hyperexpressed in the B-cell lymphoma tumor tissues compared with normal tonsillar tissues. Using cytokine induction assay and intracellular cytokine assay, we showed that IL-4 and/or IL-13 are produced by intratumoral TH2 cells but not by TH1 or tumor B-cells. IL-4 and/or IL-13 induced secretion of CCL17 and CCL22 by primary human B-cell lymphoma tumor cells. The expression of CCL17 and CCL22 by tumor B-cells was dependent on STAT6 since knock-down of STAT6 using siRNA inhibited the production of the chemokines. Finally, we observed that culture supernatants from IL-4-treated lymphoma tumor cells, significantly enhanced the migration of Tregs compared with non-Tregs in a transwell chemotaxis assay. The chemotaxis of Tregs in response to culture supernatants was abrogated in the presence of neutralizing antibodies against CCL17 and/or CCL22 in a dose-dependent manner. Tregs migration was also inhibited by anti-CCR4 blocking antibody suggesting that the chemotaxis of Tregs is mediated via CCR4, the receptor for CCL17 and CCL22. Taken together, these results suggest that the TH2 cells produce IL-4 and IL-13 which in turn exert a paracrine effect on tumor B-cells and induce production of CCL17 and CCL22 to facilitate recruitment of Tregs into the lymphoma tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1908.</jats:p

Host DNA Repair Proteins in Response to Pseudomonas aeruginosa in Lung Epithelial Cells and in Mice ▿

Although DNA repair proteins in bacteria are critical for pathogens' genome stability and for subverting the host defense, the role of host DNA repair proteins in response to bacterial infection is poorly defined. Here, we demonstrate, for the first time, that infection with the Gram-negative bacterium Pseudomonas aeruginosa significantly altered the expression and enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1) in lung epithelial cells. Downregulation of OGG1 by a small interfering RNA strategy resulted in severe DNA damage and cell death. In addition, acetylation of OGG1 is required for host responses to bacterial genotoxicity, as mutations of OGG1 acetylation sites increased Cockayne syndrome group B (CSB) protein expression. These results also indicate that CSB may be involved in DNA repair activity during infection. Furthermore, OGG1 knockout mice exhibited increased lung injury after infection with P. aeruginosa, as demonstrated by higher myeloperoxidase activity and lipid peroxidation. Together, our studies indicate that P. aeruginosa infection induces significant DNA damage in host cells and that DNA repair proteins play a critical role in the host response to P. aeruginosa infection, serving as promising targets for the treatment of this condition and perhaps more broadly Gram-negative bacterial infections