技术资料

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular,T cell regeneration is generally delayed,resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled,feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect,indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore,the addition of AA to feeder cocultures resulted in development of DP and SP T cells,whereas without AA,a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy. View Publication

TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides,the most common type of cutaneous T-cell lymphoma (CTCL),as compared to normal skin or benign inflammatory dermatoses. However,its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study,we demonstrate that TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome,a leukemic variant of CTCL,and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression,leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore,TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors,CDKN1B and CDKN1C. Lastly,blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively,these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL. View Publication

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here,we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions,resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions,which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. View Publication

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold,recruiting downstream signaling proteins,as well as by proteolytic cleavage of multiple substrates. However,the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation,we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells,B1 B cells,IL-10-producing B cells,regulatory T cells,and mature T and B cells. In general,immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro,inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation,impaired IL-2 and TNF-α production,as well as defective Th17 differentiation. Consequently,Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly,Malt1(PD/PD) animals developed a multiorgan inflammatory pathology,characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels,which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

Canonically,immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE),IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs),a type of cell of the immune system linked to viral defense,which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE,followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses. View Publication

In early life,a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c(+) DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore,we demonstrated that neonatal DC can efficiently take up,process,and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults,this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo. View Publication

Through the regulation of human leukocyte antigen (HLA)-DM (DM) in B cells,HLA-DO (DO) modulates positively or negatively the presentation of specific peptides. Transduction of DO into human blood monocyte-derived dendritic cells (MoDC) has been proposed as a mean of modifying the peptide repertoire of major histocompatibility complex class II molecules. However,maturation of DC induced by inflammatory stimuli or possibly the adenoviral vector itself triggers acidification of vesicles and shuts down transcription of the class II transactivator gene as well as de novo biosynthesis of class II-related molecules and DM activity. In these conditions,it is unclear that transduced DO could alter the peptide repertoire. Our Western blot and reverse transcriptase-polymerase chain reaction analyses revealed that human DC derived from blood monocytes express small amounts of DOalpha. Transduction of DObeta alone resulted in the accumulation of a small pool of DO in DM(+) CD63(+) vesicles and at the plasma membrane of mature DC. The cell-surface increase in class II-associated invariant chain peptide (CLIP)/class II complexes is in line with an inhibitory role of DO on DM. Cotransduction of DOalpha and DObeta only slightly increased CLIP and DO levels at the cell surface. Together with the fact that a large fraction of transduced DO remains in the endoplasmic reticulum,this suggests that DM is limiting in these conditions. DO expression did not affect a mixed lymphocyte reaction but reduced presentation of the exogenous gp100 antigen to a specific T cell clone. These results show that transduced DO modulates antigen presentation in human mature MoDC,evoking the possible use of this chaperone for immunotherapy. View Publication

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation,which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time,attesting to a selective advantage of transduced progenitors. Notably,a high transgene copy number was detected in all lymphoid organs,and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether,these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases. View Publication

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However,the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell,marked with a LacZ transgene,can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer,the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing,properties that define them as MaSCs. View Publication

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines,complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(ptextless0.0001). In t(12;21) samples,efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels,suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H,blocking the CD55 and CD59 complement inhibitors,and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL,alone or in combination with cytotoxic drugs. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication