Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.     

SIRPα polymorphisms,but not the prion protein,control phagocytosis of apoptotic cells

Prnp^−/− mice lack the prion protein PrP^C and are resistant to prion infections, but variable phenotypes have been reported in Prnp^−/− mice and the physiological function of PrP^C remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp^−/− mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrP^C is instead controlled by a linked locus encoding the signal regulatory protein α (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrP^C suggests that additional phenotypes reported in Prnp^−/− mice may actually relate to Sirpa or other genetic confounders.The cellular prion protein PrP^C, encoded by the Prnp gene, is tethered to the membrane of most mammalian cells by a glycosylphosphatidylinositol anchor. Conversion and aggregation of PrP^C into a misfolded conformer (termed PrP^Sc) triggers transmissible spongiform encephalopathies, also termed prion diseases (Aguzzi and Calella, 2009). Disparate functions have been ascribed to PrP^C on the basis of phenotypes described in Prnp^−/− mice (Steele et al., 2007; Linden et al., 2008), yet none of these functions has been clarified mechanistically, and their validity was frequently challenged.All currently available Prnp^−/− lines were generated using embryonic stem (ES) cells derived from the 129 strain of Mus musculus. Typically, chimeric founder mice were crossed with WT (Prnp^wt/wt) mice of the C57BL/6 strain (B6; Sparkes et al., 1986). Consequently, congenic Prnp^wt/wt and Prnp^−/− mice may differ at additional polymorphic loci (Smithies and Maeda, 1995; Gerlai, 1996). We hypothesized that co-segregation of linked genes may have confounded the attribution of functions to PrP^C based on phenotypes observed in Prnp^−/− mice (Collinge et al., 1994; Lledo et al., 1996; Walz et al., 1999; Rangel et al., 2007; Laurén et al., 2009; Calella et al., 2010; Gimbel et al., 2010; Ratté et al., 2011; Striebel et al., 2013).

Table 1.

Prnp KO mouse lines analyzed in this study

Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation?

Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4(+) T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies.     

Long-lived intestinal tuft cells serve as colon cancer–initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth–sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT–dependent genetic lineage–tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate–induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer.     

Myeloid cells,BAFF, and IFN-γ establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient mice

Autoimmunity is traditionally attributed to altered lymphoid cell selection and/or tolerance, whereas the contribution of innate immune cells is less well understood. Autoimmunity is also associated with increased levels of B cell–activating factor of the TNF family (BAFF; also known as B lymphocyte stimulator), a cytokine that promotes survival of self-reactive B cell clones. We describe an important role for myeloid cells in autoimmune disease progression. Using Lyn-deficient mice, we show that overproduction of BAFF by hyperactive myeloid cells contributes to inflammation and autoimmunity in part by acting directly on T cells to induce the release of IFN-γ. Genetic deletion of IFN-γ or reduction of BAFF activity, achieved by either reducing myeloid cell hyperproduction or by treating with an anti-BAFF monoclonal antibody, reduced disease development in lyn^−/− mice. The increased production of IFN-γ in lyn^−/− mice feeds back on the myeloid cells to further stimulate BAFF release. Expression of BAFF receptor on T cells was required for their full activation and IFN-γ release. Overall, our data suggest that the reciprocal production of BAFF and IFN-γ establishes an inflammatory loop between myeloid cells and T cells that exacerbates autoimmunity in this model. Our findings uncover an important pathological role of BAFF in autoimmune disorders.Systemic lupus erythematosus is a prototypic autoimmune disease with complex and unclear etiology (Rahman and Isenberg, 2008). Most studies of this disease have focused on the defects of B and T cell tolerance as an underlying cause of the disorder. Recently, however, greater attention has been given to the pathological roles of myeloid cells in autoimmunity (Cohen et al., 2002; Hanada et al., 2003; Zhu et al., 2005; Stranges et al., 2007).Mice lacking Lyn, an Src family kinase mainly expressed in B and myeloid cells, are a well-established model of lupus-like autoimmunity (Xu et al., 2005). lyn^−/− mice develop progressive autoimmunity characterized by autoantibody production, lymphocyte activation, immune complex deposition, and nephritis (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997; Yu et al., 2001). The development of autoimmunity in lyn^−/− mice has been mainly attributed to alterations in B cell signaling thresholds, leading to abnormal B cell selection and/or tolerance resulting in production of self-reactive antibodies (Chan et al., 1998; Xu et al., 2005). The Lyn mutation directly affects B cell development, as lyn^−/− mice have an ∼30–50% reduction in mature B cell numbers because of the reduction of specific B cell subtypes such as marginal zone and follicular B cells (Xu et al., 2005; Gross et al., 2009).Lyn is also expressed in innate immune cells, where it regulates cell signaling thresholds to several CSFs, such as G-CSF, GM-CSF, and M-CSF (Harder et al., 2001, 2004; Scapini et al., 2009). lyn^−/− myeloid cells are hyperresponsive to engagement of surface integrins, leading to hyperadhesion, enhanced respiratory burst, and increased secondary granule release (Pereira and Lowell, 2003). Despite this experimental evidence in vitro, the contribution of myeloid cells to the development of autoimmunity in lyn^−/− mice has not been investigated.Autoimmunity is often associated, both in mice and humans, with excess production of B cell–activating factor of the TNF family (BAFF), a member of the TNF superfamily of cytokines also known as B lymphocyte stimulator (Mackay et al., 2007; Stadanlick and Cancro, 2008; Mackay and Schneider, 2009). Both autoimmune-prone mice (such as MRL^lpr/lpr and NZB×W F1) and human patients suffering from autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis have elevated serum levels of BAFF (Kalled, 2005; Mackay and Schneider, 2009). This cytokine is thought to exert its pathogenic role, under conditions of excess production, through its ability to support survival and proliferation of autoreactive B cells, which have a higher BAFF dependence (Lesley et al., 2004). However, in addition to its effect on B cells, recent work has suggested that BAFF can also promote T cell activation (Ye et al., 2004; Sutherland et al., 2005; Mackay and Leung, 2006; Lai Kwan Lam et al., 2008). Despite this evidence, it remains unclear if BAFF exerts a direct pathogenic role on T cells in vivo during autoimmunity. Furthermore, the mechanisms responsible for deregulated BAFF production in autoimmune diseases have been poorly investigated. Studies in mice have shown that there are two distinct pools of BAFF: a constitutive pool produced by stromal cells, which is thought to regulate the size and maturation stage of the peripheral B cell compartment, and an accessory pool, produced mainly by myeloid cells during inflammatory or immune responses (Schneider, 2005). Which of these two pools contributes to autoimmune pathologies is unknown. Different attempts to neutralize BAFF activity in autoimmune disorders have been performed in both mice and humans, but despite a general agreement on the efficacy of the treatments, the mechanisms of this protection are still not fully understood (Ding, 2008; Ramanujam and Davidson, 2008; Moisini and Davidson, 2009).Another important cytokine that has been shown to be involved in lupus pathogenesis is IFN-γ. Several studies in MRL^lpr/lpr and NZB×W F1 autoimmune-prone mice observed significant reduction of histological and serological disease characteristics, and extended survival in these strains after IFN-γ genetic deletion or after anti–IFN-γ mAb treatment (Theofilopoulos et al., 2001).We found that the levels of BAFF were dramatically higher in the sera of lyn^−/− mice compared with WT animals, and that the deregulated production of BAFF by lyn^−/− myeloid cells can contribute to autoimmunity in these animals by affecting not only B cell activation but, more interestingly, by directly promoting T cell activation and IFN-γ production by the latter cells. These findings shed new insight on the pathological mechanisms of interplay between innate and adaptive immunity, as well as the consequences of BAFF and IFN-γ overproduction, in autoimmune disorders.     

Adhesion and cytotoxicity susceptibility of B16 melanoma cells to immune effector cells enhanced after IL-2, IL-4 or IL-6 gene-transfection

The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of tumor cells to effector cells, and as aconsequence, the tumorigenicity decreased. Beforekilling tumor cells, effector cells required first torecognize non-specific surface adhesion molecules on     

In liver fibrosis,dendritic cells govern hepatic inflammation in mice via TNF-α

Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b⁺CD8^– fraction, a lower B220⁺ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-α. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease.     

Polζ ablation in B cells impairs the germinal center reaction,class switch recombination,DNA break repair,and genome stability

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining.In T cell–dependent antibody responses, B cells are triggered to undergo a second round of antibody diversification in germinal centers (GCs) (1). Somatic hypermutation (SHM) introduces mutations into rearranged variable (V) regions of Ig genes allowing antibody affinity maturation (2, 3), whereas class switch recombination (CSR) exchanges the Ig constant (C) region to modify the effector function of the antibody (4). Both SHM and CSR rely on activation-induced deaminase (AID), an enzyme which deaminates cytidine residues in single-stranded DNA (5). The DNA deamination model of SHM suggests the conversion of G-C basepairs into G-U mismatches within the V region by AID (6, 7), which are subsequently processed in one of three ways: direct replication across the G-U mismatches results in G-C to A-T mutations; the removal of the uracil residues by the uracil deglycosylase UNG creates abasic sites, and DNA synthesis by error-prone DNA polymerases generates additional mutations; or the recognition of the G-U mismatches by the mismatch repair enzymes MSH2 and MSH6 leads to subsequent error-prone short-patch DNA synthesis, which introduces mutations outside the initial site of the lesion.In the case of CSR, it is widely believed that upon cytidine deamination by AID, staggered DNA double-strand breaks (DSBs) are generated by the removal of the uracil residues by UNG, followed by the cleavage of the abasic sites by APE1/2 during the G1 phase of the cell cycle (5, 8–13). Alternatively, the mismatch–repair pathway can lead to the generation of staggered DSBs via the recognition of uracil by MSH2 and MSH6 (8, 14, 15). The DSBs are then resolved by a process that includes DNA damage response proteins such as H2AX, MDC1, ATM, 53BP1, and the Nibrin–Mre11–Rad50 complex, the mismatch repair enzymes Pms2 and Mlh1, the exonuclease Exo1, and the classical and alternative nonhomologous end-joining (NHEJ) machinery (16–28).Although the DNA polymerases required for filling in the staggered DNA breaks generated in CSR have not been identified, a possible involvement of several error-prone DNA polymerases in SHM has been tested using both hypermutating cell lines and KO mice deficient of these enzymes. From this work, Polη, Polθ, and perhaps Polι have emerged as important components of the SHM mechanism, whereas Polβ, Polκ, Polλ, and Polμ do not appear to play a significant role (29–35). Polζ is an error-prone DNA polymerase that is characterized by its ability to extend mismatched primer-template termini (36). Polζ, together with Polι, has been suggested as the prime example of the two-step inserter-extender model of translesion synthesis, in which a first DNA polymerase (Polι) synthesizes across the DNA lesion and a second polymerase (Polζ) extends the resulting mismatch (36). These features highlighted Polζ as an additional candidate enzyme of the SHM machinery. Indeed, studies in a hypermutating cell line and a transgenic mouse strain that express antisense RNA against Rev3, the catalytic subunit of Polζ, demonstrated a reduction of the frequency of somatic mutations in rearranged Ig V region genes, suggesting an involvement of Polζ in SHM (37, 38).Attempts to address this issue in vivo by genetically ablating Polζ in mice have been hampered by the embryonic lethality observed upon deletion of the Polζ gene in the mouse germ line (39–41). This embryonic lethality could be a result of the pronounced genomic instability observed upon Polζ ablation in a wide variety of cellular systems (42), in turn suggesting a role of this enzyme in DNA repair and thus, potentially, CSR (16). To assess a possible contribution of Polζ to SHM and CSR in vivo, we generated mice that carry a deletion of Rev3 selectively in mature B cells (subsequently called Polζ^f/Δ/CD21-cre mice). In this paper, we show that Polζ-deficient B cells are impaired in their ability to proliferate and to maintain a stable genome. The mutant cells fail to undergo an efficient GC reaction and exhibit a reduced frequency of SHM and impaired CSR. The CSR defect is associated with an increased frequency of aberrantly or unrepaired DNA breaks within the IgH locus, suggesting that Polζ plays a nonredundant role in DNA DSB repair through NHEJ.     

Wnt/β-catenin signaling may be involved with the maturation,but not the differentiation,of insulin-producing cells

Wnt/β-catenin signaling (WNT) has widespread roles during stem cell differentiation. Whether WNT suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of WNT signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of WNT were dynamically regulated in a 12-day IPC differentiation assay. Specifically, protein levels of Wnt1, β-catenin, and GSK3β steadily increased from day 0 to day 9 and rapidly decreased by day 12 of differentiation. Similarly, endonuclear β-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two WNT pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that WNT signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of WNT signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of WNT signaling. Next, we examined the effect of maintaining active WNT signaling from day 9 to day 12 of IPC differentiation. Differentiating cells were treated with Wnt1 on day 9, when WNT signaling is typically turned off, and subjected to gene expression analysis on day 12. Remarkably, Wnt1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that WNT may not be necessary for IPC differentiation but may be involved in IPC maturation.     

Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8⁺ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34⁺ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that arises from break point cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR/ABL)–transformed hematopoietic stem (HSCs) or early progenitor cells known as leukemia stem cells (LSCs; Kavalerchik et al., 2008). LSCs have been first characterized as the tumor-initiating cells in acute myeloid leukemia (Lapidot et al., 1994) and have also been defined in other hematopoietic neoplasms since then (Cox et al., 2004; Matsui et al., 2004).BCR/ABL-specific tyrosine kinase inhibitors (TKIs) such as Imatinib mesylate (Glivec) have revolutionized the therapy of CML (Druker et al., 2001a,b; Baccarani et al., 2006). Nevertheless, LSCs seem resistant to TKIs and traditional chemotherapy (Weiden et al., 1979; Deininger et al., 2000; Savona and Talpaz, 2008) and CML inevitably progresses to incurable acute leukemia (Faderl et al., 1999). Quiescent, self-renewing LSCs remain in the BM and are responsible for refractoriness and relapse of CML after treatment (Hughes et al., 2003). Therefore, novel cytotoxic agents that selectively target LSCs are under investigation (Jin et al., 2006; Guzman et al., 2007; Neviani et al., 2007; Ito et al., 2008; Bellodi et al., 2009; Majeti et al., 2009; Wang et al., 2010; Schürch et al., 2012).Another promising approach in the treatment of CML is immunotherapy. In fact, currently, the only curative treatment for CML remains allogeneic stem cell transplantation (alloSCT). The graft-versus-leukemia effect of alloSCT is most likely executed by donor CD8⁺ effector CTLs specific for minor histocompatibility antigens (Weiden et al., 1979; Kolb et al., 1990; Gale et al., 1994; Druker et al., 2002). Patients who receive T cell–depleted alloSCT grafts have a higher risk of disease relapse, and donor lymphocyte infusions are able to induce complete remission after relapse (Thomas et al., 1979; Horowitz et al., 1990; Kolb et al., 1995; Sehn et al., 1999). Furthermore, endogenous CTLs directed against leukemia antigens have been detected in the peripheral blood of chronic phase CML patients (Molldrem et al., 2000; Butt et al., 2005). Several proteins may potentially act as potent leukemia-specific antigens for T cells, including BCR/ABL, Wilms’ tumor 1 protein (WT1), and proteinase 3 (Pr3; Van Driessche et al., 2005). Peptides from the junctional region of BCR/ABL are not present in healthy individuals and therefore are leukemia-specific. Yotnda et al. (1998) identified a BCR/ABL junctional nonapeptide that binds to human leukocyte antigen (HLA)-A2.1 and elicits specific CTL responses in vitro and in vivo. Additional studies confirmed and extended the finding of immunogenic BCR/ABL junction peptides (Bocchia et al., 1996; Clark et al., 2001).CTLs have been shown to kill CML target cells in vitro via Fas-receptor triggering (Selleri and Maciejewski, 2000). In a BCR/ABL-induced murine CML model, we have shown that CD8⁺ T cells crucially contribute to disease control in vivo. However, programmed death ligand 1 (PD-L1) expression by the malignant cells induced T cell dysfunction leading to disease progression (Mumprecht et al., 2009b).Despite these advances in the understanding of the immunosurveillance of CML and the development of immunotherapy strategies, the interaction of effector CTLs with the disease-originating LSCs has not been analyzed so far. In the present study, we analyzed the immunogenicity of LSCs in vitro and in vivo using the glycoprotein of lymphocytic choriomeningitis virus (LCMV) as model leukemia antigen. LSCs expressed MHC and co-stimulatory molecules. LSCs isolated from CD8⁺ T cell–depleted CML mice were more immunogenic than LSCs from control CML mice, indicating that CD8⁺ T cells interact with LSCs in vivo and select for low immunogenic variants. To analyze whether LSCs can be recognized and lysed by activated leukemia-specific effector CTLs, we treated CML mice with large numbers of specific T cells. Although these effector CTLs efficiently lysed LSCs in vitro, adoptive immunotherapy in vivo failed to eradicate LSCs but paradoxically increased LSC numbers. LSC proliferation was induced by CTL-secreted IFN-γ. Specific effector CTLs secreted high amounts of IFN-γ when transferred to CML mice in advanced disease stage with high antigen load, but not after transfer to CML mice in early stage of disease. Importantly, our results were confirmed in a humanized CML model using HLA-A2.1 transgenic mice and a BCR/ABL b3a2 junctional peptide. In addition, IFN-γ induced the proliferation of primary CD34⁺ stem/progenitor cells from newly diagnosed CML patients.     

Stem cells: What's happening?

Stem cell enthusiasts are probably aware that we are experiencing an exciting period of new development in the field of progenitor cells. This brief report focuses on selected topics which have been recently reported in two special interest group meetings in the U.K. The potential advantage of stem cells derived from human umbilical cord (HUC) blood over normal bone marrow (NBM) or peripheral blood stem cells (PBSC), as source products for reconstitution of the immune system is briefly mentioned, together with some potential applications of HUC blood in gene therapy.     

Antigen-pulsed, GM-CSF gene-modified bone marrow stromal cells present tumor antigen to T lymphocytes in vitro

It has been demonstrated that tbe critical role ofbone marrow stromal cells (HMSCs ) is to sustain theselfrenewal of pluripotent hematopoietic stem cells andmaintain the homeostasis of bone marrow hematopoiesismicroenvironment. BMSC progenitor can differentiateinto several clements including macrophages, endothelialcells, fibroblasts and some other cells. Almost all