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Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts,several studies have reported relatively unimpaired resistance by recipients who lack perforin,Fas ligand (FasL),and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched,minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin-/-/ gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 ?? 107) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT,and anti-CD8 monoclonal antibody infusion abolished resistance,thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced - consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance,newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast,low or absent colony-forming unit levels were detected in allogeneic recipients,including those that lacked perforin and FasL and that received anti-TWEAK,anti-tumor necrosis factor-related apoptosis-inducing ligand,and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings,these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin,FasL,and the known death ligand receptor pathways. ?? 2005 American Society for Blood and Marrow Transplantation. View Publication

The deregulation of the immune response is a critical component in inflammatory disease. Recent in vitro data show that T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of cytokine signaling. Furthermore,tc-ptp(-/-) mice display immune defects and die within 5 weeks of birth. We report here that tc-ptp(-/-) mice develop progressive systemic inflammatory disease as shown by chronic myocarditis,gastritis,nephritis,and sialadenitis as well as elevated serum interferon-gamma. The widespread mononuclear cellular infiltrates correlate with exaggerated interferon-gamma,tumor necrosis factor-alpha,interleukin-12,and nitric oxide production in vivo. Macrophages grown from tc-ptp(-/-) mice are inherently hypersensitive to lipopolysaccharide,which can also be detected in vivo as an increased susceptibility to endotoxic shock. These results identify T-cell protein tyrosine phosphatase as a key modulator of inflammatory signals and macrophage function. View Publication

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma,an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis,bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2,respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors,and transduced cells were cultured in a semisolid medium permissive for the development of erythroid,myeloid,and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro,in contrast to Tax2-transduced cells,which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected,suggesting that Tax1 inhibited the maturation of relatively early,uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis,lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells,an activity that is not displayed by Tax2,and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo,the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

Memory B cells (MBCs) are long-lived sources of rapid,isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2,independently of isotype,identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely,CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence,the differentiation and regeneration of MBCs are compartmentalized. View Publication

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue,making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1$\$)-hESC lines using chEF1$\$ system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology,karyotype,and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers,OCT-3/4 and SSEA-4,in undifferentiated mHLA-G(EF1$\$)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors,expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1$\$)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives. View Publication

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T,$\$-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate,the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%,whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular,atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified,functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling,drug discovery,and regenerative medicine. View Publication

The p110δ isoform of PI3K is known to play an important role in immunity,yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells,we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation,proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases,our study suggests caution in using these drugs in patients,as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens. View Publication

The youngest peripheral T cells (recent thymic emigrants [RTEs]) are functionally distinct from naive T cells that have completed postthymic maturation. We assessed the RTE memory response and found that RTEs produced less granzyme B than their mature counterparts during infection but proliferated more and,therefore,generated equivalent target killing in vivo. Postinfection,RTE numbers contracted less dramatically than those of mature T cells,but RTEs were delayed in their transition to central memory,displaying impaired expression of CD62L,IL-2,Eomesodermin,and CXCR4,which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge,indicating that the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus,the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity,driving the efficacy of the RTE response to that of mature T cells. View Publication

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation,which is only partially successful in the absence of an HLA genoidentical donor,thus justifying research to find an alternative therapeutic approach. To this end,RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer,negative control region deleted,dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later,T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens,allogeneic cells,and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy,a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether,this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice,constituting a significant step toward clinical application. View Publication

We evaluated the effects of ectopic granulocyte/macrophage colony-stimulating factor (GM-CSF) signals on hematopoietic commitment and differentiation. Lineage-restricted progenitors purified from mice with the ubiquitous transgenic human GM-CSF receptor (hGM-CSFR) were used for the analysis. In cultures with hGM-CSF alone,hGM-CSFR-expressing (hGM-CSFR+) granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs) exclusively gave rise to granulocyte/monocyte (GM) and megakaryocyte/erythroid (MegE) colonies,respectively,providing formal proof that GM-CSF signals support the GM and MegE lineage differentiation without affecting the physiological myeloid fate. hGM-CSFR transgenic mice were crossed with mice deficient in interleukin (IL)-7,an essential cytokine for T and B cell development. Administration of hGM-CSF in these mice could not restore T or B lymphopoiesis,indicating that enforced GM-CSF signals cannot substitute for IL-7 to promote lymphopoiesis. Strikingly,textgreater50% hGM-CSFR+ common lymphoid progenitors (CLPs) and textgreater20% hGM-CSFR+ pro-T cells gave rise to granulocyte,monocyte,and/or myeloid dendritic cells,but not MegE lineage cells in the presence of hGM-CSF. Injection of hGM-CSF into mice transplanted with hGM-CSFR+ CLPs blocked their lymphoid differentiation,but induced development of GM cells in vivo. Thus,hGM-CSF transduces permissive signals for myeloerythroid differentiation,whereas it transmits potent instructive signals for the GM differentiation to CLPs and early T cell progenitors. These data suggest that a majority of CLPs and a fraction of pro-T cells possess plasticity for myelomonocytic differentiation that can be activated by ectopic GM-CSF signals,supporting the hypothesis that the down-regulation of GM-CSFR is a critical event in producing cells with a lymphoid-restricted lineage potential. View Publication