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Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

Although structurally unrelated,the human killer cell Ig-like (KIR) genes and the rodent lectin-like Ly49 genes serve similar functional roles in NK cells. Moreover,both gene families display variegated,monoallelic expression patterns established at the transcriptional level. DNA methylation has been shown to play an important role in maintenance of expression patterns of KIR genes,which have CpG island promoters. The potential role of DNA methylation in expression of Ly49 genes,which have CpG-poor promoters,is unknown. In this study,we show that hypomethylation of the region encompassing the Pro-2 promoter of Ly49a and Ly49c in primary C57BL/6 NK cells correlates with expression of the gene. Using C57BL/6 x BALB/c F1 hybrid mice,we demonstrate that the expressed allele of Ly49a is hypomethylated while the nonexpressed allele is heavily methylated,indicating a role for epigenetics in maintaining monoallelic Ly49 gene expression. Furthermore,the Ly49a Pro-2 region is heavily methylated in fetal NK cells but variably methylated in nonlymphoid tissues. Finally,in apparent contrast to the KIR genes,we show that DNA methylation and the histone acetylation state of the Pro-2 region are strictly linked with Ly49a expression status. View Publication

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study,we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs),evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect,confirming that VEGF and IL-6,at least in part,mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo,immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs,and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6),DCs only (SI = 9.3),or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM. View Publication

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs,but does not abrogate,V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia,significant serum levels of immunoglobulin (Ig) G,IgM,IgA,and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired,even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire,which is associated with defects in central and peripheral checkpoints of B cell tolerance,is an important,previously unrecognized,aspect of immunodeficiencies associated with hypomorphic RAG mutations. View Publication

OBJECTIVE: The T-cell receptor zeta (TCR zeta)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCR zeta-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCR zeta(dim) T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCR zeta(dim) T cells in coronary artery disease. METHODS AND RESULTS: We examined the expression of TCR zeta-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4(+) TCR zeta(dim) T cells were found in patients with acute coronary syndromes (ACS,n=66; median 5.3%,interquartile 2.6 to 9.1% of total CD4(+) T cells; Ptextless0.0001) compared to chronic stable angina (CSA,n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein,and it persisted after the acute event. Moreover,TCR zeta(dim) cells were also more represented within CD8(+) T cell,NK,and CD4(+)CD28(null) T cell subsets in ACS compared to CSA and controls. Finally,CD4(+) and CD8(+) TCR zeta(dim) T cells isolated from ACS displayed an enhanced transendothelial migratory capacity. CONCLUSIONS: TCR zeta(dim) T cells,an effector T-cell subset with transendothelial migratory ability,are increased in ACS,and may be implicated in coronary instability. View Publication

HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear,but it may involve chronic B-cell activation,inflammation,and/or impaired immunity,possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly,because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region,thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model,and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined,only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells,and were CD19(+)IgM(-)IgD(-)CD93(+)CD43(+)CD21(-)CD23(-)VpreB(+)CXCR4(+) Consistent with the pro-B-cell stage of B-cell development,microarray analysis revealed enrichment of transcripts,including Rag1,Rag2,CD93,Vpreb1,Vpreb3,and Igll1 We confirmed RAG1 expression in Tg26 tumors,and hypothesized that HIV-1 matrix protein p17 may directly induce RAG1 in B cells. Stimulation of human activated B cells with p17 enhanced RAG1 expression in three of seven donors,suggesting that intracellular signaling by p17 may lead to genomic instability and transformation. View Publication

During an immune response,activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this,we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function,independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells,DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation,respectively,suggesting that these two pathways cooperate for optimal DC conditioning. View Publication

Natural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However,immature thymocytes also retain NK potential. Currently,the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here,we show that TCRgamma genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells,and similar levels are detected in NK cells from TCRbeta,delta double-knockout mice,excluding the possibility of T-cell contamination. NK-cell TCRgamma gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRgamma genes. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory,cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression,we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies,we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry,reverse transcriptase-polymerase chain reaction,and immunoblot analysis. Furthermore,CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion,thus confirming the presence of a functional CD33 on these leukemic cells. Moreover,we found that circulating pDCs in healthy individuals also weakly express CD33. Overall,our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies. View Publication

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus,developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein,we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time,we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally,CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets,supporting the role of FA metabolism for Treg function. Furthermore,we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively,our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor,thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication