技术资料

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

Acute myeloid leukemia (AML) cells are characterized by a constitutive and abnormal activation of the nuclear factor-kappaB (NF-kappaB) transcription factor. This study,conducted in vitro on 18 patients,shows that targeting the IKB kinase 2 (IKK2) kinase with the specific pharmacologic inhibitor AS602868 to block NF-kappaB activation led to apoptosis of human primary AML cells. Moreover,AS602868 potentiated the apoptotic response induced by the current chemotherapeutic drugs doxorubicin,cytarabine,or etoposide (VP16). AS602868-induced cell death was associated with rupture of the mitochondrial transmembrane potential and activation of cellular caspases. NF-kappaB inhibition did not affect normal CD34+ hematopoietic precursors,suggesting that it could represent a new adjuvant strategy for AML treatment. 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

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern,and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy,preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration,without reducing accumulation. In contrast,recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect,which was negated using a specific neutrophil elastase inhibitor. Furthermore,T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase,which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha,which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine,ELC (CCL19),and was not reversed by replenishment of SDF-1alpha,indicating endothelial retention of the inactivated chemokine. In summary,transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors,and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities. View Publication

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection,but do not correlate with control of viremia in chronic infection,suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here,we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection,but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies,and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection,but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions. View Publication

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread,requires CD4 and coreceptor expression in target cells,and may also lead to syncytium formation and cell death. Here,we show that in addition to this classical coreceptor-mediated transmission,the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped,captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions,without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion,we describe a novel mechanism of HIV transmission,activated by the contact of infected and uninfected primary CD4 T cells,by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir. View Publication

NK cells play a critical role in the rejection of xenografts. In this study,we report on an investigation of the effect of complement regulatory protein,a decay accelerating factor (DAF: CD55),in particular,on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested,and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses,delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However,delta-SCR4-DAF showed a clear complement regulatory effect,but had no effect on NK cells. Conversely,the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function,but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins,such as the cell membrane-bound form factor H,fH-PI,and C1-inactivator,C1-INH-PI,and CD59 were also assessed,but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest,for DAF to function on NK cells,SCR2-4 is required but no relation to its complement regulatory function exists. View Publication

NK cells protect hosts against viral pathogens and transformed cells,and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma,despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast,IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition,blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally,presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming. View Publication

Mixed chimaerism and graft rejection are higher after reduced-intensity allogeneic stem cell transplantation (RIST) with T-cell depleted (TCD) allografts. As host immune status before RIST affects engraftment,we hypothesized that targeted depletion of host lymphocytes prior to RIST would abrogate graft rejection and promote donor chimaerism. Lymphocyte-depleting chemotherapy was administered at conventional doses to subjects prior to RIST with the intent of decreasing CD4(+) counts to textless0.05 x 10(9)cells/l. Subjects (n = 18) then received reduced-intensity conditioning followed by ex vivo TCD human leucocyte antigen-matched sibling allografts. All evaluable patients (n = 17) were engrafted; there were no late graft failures. At day +28 post-RIST,12 patients showed complete donor chimaerism. Mixed chimaerism in the remaining five patients was associated with higher numbers of circulating host CD3(+) cells (P = 0.0032) after lymphocyte-depleting chemotherapy and was preferentially observed in T lymphoid rather than myeloid cells. Full donor chimaerism was achieved in all patients after planned donor lymphocyte infusions. These data reflect the importance of host immune status prior to RIST and suggest that targeted host lymphocyte depletion facilitates the engraftment of TCD allografts. Targeted lymphocyte depletion may permit an individualized approach to conditioning based on host immune status prior to RIST. View Publication

PURPOSE: Allogeneic T lymphocytes can induce regression of metastatic breast cancer through an immune-mediated graft-versus-tumor (GVT) effect in murine models. To determine if a clinical GVT effect exists against metastatic breast cancer,allogeneic lymphocytes were used as adoptive cellular therapy after a reduced-intensity chemotherapy conditioning regimen and allogeneic hematopoietic stem-cell transplantation (HSCT) from human leukocyte antigen-matched siblings. PATIENTS AND METHODS: Sixteen patients with metastatic breast cancer that had progressed after treatment with anthracyclines,taxanes,hormonal agents,and trastuzumab,received allogeneic HSCT. The reduced-intensity transplant conditioning regimen consisted of cyclophosphamide and fludarabine. To distinguish an immunological GVT effect from any antitumor effect of cytotoxic chemotherapy in the transplant-conditioning regimen,allogeneic T lymphocytes were removed from the stem-cell graft and were subsequently administered late postallogeneic HSCT. Allogeneic lymphocytes containing 1 x 10(6),5 x 10(6),and 10 x 10(6) CD3(+) cells/kg were infused on days +42,+70,and +98 post-allogeneic HSCT,respectively. RESULTS: Objective tumor regressions occurred after day +28 post-allogeneic HSCT in six patients and were attributed to allogeneic lymphocyte infusions. Two of these responding patients had disease progression post-allogeneic HSCT before subsequent tumor regression. Tumor regressions occurred concomitantly with the establishment of complete donor T-lymphoid engraftment,were associated with the development of graft-versus-host disease (GVHD),and were abrogated by subsequent systemic immunosuppression for GVHD. CONCLUSION: Allogeneic lymphocytes can induce regression of advanced metastatic breast cancer. These results indicate that an immunological GVT effect from allogeneic lymphocytes exists against metastatic breast cancer and provide rationale for further development of allogeneic cellular therapy for this largely incurable disease. View Publication