The clonal composition of human CD4+CD25+Foxp3+ cells determined by a comprehensive DNA-based multiplex PCR for TCRB gene rearrangements

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (TR) have been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cells (PBMC) CD4+ TR and non-TR was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A*0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-γ in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. In the UCB and PBMCs clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3− was observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ TR can share the same clonotypes as CD4+ non-TR in humans

Rapamycin is highly effective in murine models of immune-mediated bone marrow failure

Acquired aplastic anemia, the prototypical bone marrow failure disease, is characterized by pancytopenia and marrow hypoplasia. Most aplastic anemia patients respond to immunosuppressive therapy, usually with anti-thymocyte globulin and cyclosporine, but some relapse on cyclosporine withdrawal or require long-term administration of cyclosporine to maintain blood counts. In this study, we tested efficacy of rapamycin as a new or alternative treatment in mouse models of immune-mediated bone marrow failure. Rapamycin ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of cyclosporine. Rapamycin effectively reduced Th1 inflammatory cytokines interferon-γ and tumor necrosis factor-α, increased the Th2 cytokine interleukin-10, stimulated expansion of functional regulatory T cells, eliminated effector CD8+ T cells (notably T cells specific to target cells bearing minor histocompatibility antigen H60), and preserved hematopoietic stem and progenitor cells. Rapamycin, but not cyclosporine, reduced the proportion of memory and effector T cells and maintained a pool of naïve T cells. Cyclosporine increased cytoplasmic nuclear factor of activated T-cells-1 following T-cell receptor stimulation, whereas rapamycin suppressed phosphorylation of two key signaling molecules in the mammalian target of rapamycin pathway, S6 kinase and protein kinase B. In summary, rapamycin was an effective therapy in mouse models of immune-mediated bone marrow failure, acting through different mechanisms to cyclosporine. Its specific expansion of regulatory T cells and elimination of clonogenic CD8+ effectors support its potential clinical utility in the treatment of aplastic anemia

Decreased TCR ζ-Chain Expression in T Cells from Patients with Aplastic Anemia.

Abstract The ζ-chain is a unique component of the T cell receptor (TCR) with no sequence homology to other CD3 molecules. The ζ-chain acts to stabilize the TCR on the cell surface and to transmit signals to different transduction pathways in the cell. T cells from patients with autoimmune diseases, with chronic inflammation, and suffering malignant tumors are functionally impaired in that they express decreased ζ-chain. Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by T-cell immune mediated destruction of hematopoietic stem cells. Oligoclonal expansion of T cells correlates with disease activity. In this study, we examined TCR ζ-chain protein levels in aplastic anemia patients. Twelve out of sixteen patients (75%) examined displayed decreased ζ-chain protein levels compared to normal controls (p=0.008). As a control, no differences were detected in expression of the TCR ε-chain. Patients and healthy donors displayed comparable amounts of Zap-70, the kinase responsible for full phosphorylation and activation of the ζ-chain. There was no correlation with disease status or medical treatment at the time of sampling. Four patients were re-examined for ζ-chain protein levels at 3 month intervals, but there was no change in their ζ-chain expression pattern despite hematologic improvement. We hypothesized correlation between ζ-chain and IFN-g levels in T cells from patients with AA. When we estimated IFN-g levels by flow cytometry, all 5 patients examined had increased IFN-g levels, of whom 4 had decreased ζ-chain levels: intracellular IFN-g was reciprocally correlated with ζ-chain protein levels. In summary, our data indicate that TCR ζ-chain expression is decreased in the majority of patients with AA, regardless of disease activity or treatment status. ζ-chain-deficient T cells from AA patients also overproduce IFN-g. Is ζ-chain down-regulation the primary event leading to IFN-g over-production, or is IFN-g responsible for the ζ-chain down-regulation? One possible explanation is an intrinsic defect in the TCR leading to alterations in kinases and transcription factors downstream of the ζ-chain-Zap-70 association. Whether ζ-chain expression correlates with expanded Vb clones observed is to be established. The seqeunce of molecular events described in the current work may lead to the Th1 shift observed in aplastic anemia, but further studies are needed to explore the possible role of ζ-chain downregulation to the pathogenesis of AA.</jats:p

T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia

In aplastic anemia, immune destruction of hematopoietic cells results in bone marrow failure. Type 1 cytokines, especially IFN-γ, have been implicated in the pathophysiology of T-cell–mediated, Fas-mediated stem cell apoptosis of hematopoietic cells. Here, we show that the transcription factor T-bet (T-box expressed in T cells) is increased in T cells from patients with aplastic anemia. Patients' T-bet bound directly to the proximal site of the IFN-γ promoter without any prior stimulation, in contrast to healthy controls. Increased levels of Itk kinase participated in T-bet up-regulation and active transcription of the IFN-γ gene observed in these patients. Blocking PKC-θ, a kinase that lies downstream of Itk kinase, decreased T-bet protein and IFN-γ intracellular levels. These data suggest that the increased IFN-γ levels observed in aplastic anemia patients are the result of active transcription of the IFN-γ gene by T-bet. Blocking the transcription of the IFN-γ gene with kinase inhibitors might lead to the development of novel therapeutic agents for patients with aplastic anemia and other autoimmune diseases

Enrichment of Hematopoietic Stem Cells from Normal and Trp53 Null Mice Using SLAM Receptor CD150 as a Positive Selection Marker.

Abstract CD150 is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors which can be used as a positive selection marker for hematopoietic stem cells (HSCs) from mouse bone marrow (BM) and fetal liver (Kiel et al., Cell 2005; Yilmaz et al., Blood 2006; Kim et al., Blood 2006). In the current study, we combined CD150 with a set of lineage-specific markers to enrich HSCs, comparing normal C57BL/6 (B6) mice with B6-Trp53-deficient (Trp53 null) mice which were previously shown to have elevated HSC activity. Using an anti-mouse CD150 antibody (Clone TC 15-12F 12.2, BioLegend), we defined a population of Lin−CD41−CD48−CD150+(SLAM) cells that is 0.0078 ± 0.0010% and 0.0135 ± 0.0010% of total BM cells from B6 and Trp53 null mice. The size of the SLAM cell fraction was strongly correlated (R2= 0.7116, P&amp;lt;0.0013) to the population of well defined Lin−Sca1+CD117+ (LSK) cells (at 0.0165 ± 0.0036% and 0.0276 ± 0.0036% respectively) for the same B6 and Trp53 null mice we have tested (results consistent with previous findings indicating that the HSC pool is larger in Trp53 null mice than in B6 mice). To test HSC function in vivo, we sorted SLAM cells and Lin−CD41−CD48−CD150+ (SLAM−) cells from B6 and Trp53 null donors and transplanted them into lethally irradiated (11 Gys) B6 recipients. In a day 12 colony forming unit-spleen (CFU-S) assay, both SLAM and SLAM−cells formed colonies but the SLAM cells contained 12–15 fold higher day 12 CFU-S than did SLAM−cells. In a competitive repopulation assay, when donor SLAM and SLAM−cells were mixed with fresh B6-CD45.1 BM cells and engrafted into lethally-irradiated B6 recipients for six months, the density of repopulating units (RUs) was 22.48 ± 12.05 and 11.06 ± 8.78 per thousand SLAM cells from B6 and Trp53 null donors, dramatically higher than the RU density of 0.43 ± 0.09 and 0.55 ± 0.43 per thousand SLAM−cells from the same B6 and Trp53 null donors. When BM cells from the competitive repopulation recipients were serially-transplanted into secondary recipients, the SLAM donor cell contribution increased while the SLAM−donor cell contribution decreased with time when measured at two, six and fourteen weeks in the secondary recipients, indicating that SLAM cells were the primitive HSCs that sustained hematopoiesis long-term. No recipient that received Try53 null SLAM or SLAM−cells died, significantly different from our previous observation that whole BM cells from Trp53 null donors yielded high donor reconstitution in a competitive repopulation assay but 56% recipient died within five months after reconstitution (TeKippe et al., Exp Hematol 2003). SLAM markers may have enriched HSCs by excluding any Try53 null BM cell responsible for early recipient death. Analysis of intracellular reactive oxygen species (ROS) revealed higher proportions of ROS−cells in the CD150+ than in the CD150− BM cell fraction in both B6 and Trp53 null mice, consistent with the hypothesis that HSCs are metabolically inactive and therefore contain lower level of ROS. While SLAM− cells may still contain some residual HSC activity, results from the present study are in strong support of utilization of SLAM receptor CD150 as a positive selection marker for HSCs. The Lin− CD41−CD48−CD150+SLAM combination provides a simple and effective method for HSC enrichment.</jats:p

CD44 Signaling Via PI3K/AKT and MAPK/ERK Pathways Protects CLL Cells from Spontaneous and Drug Induced Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-cells. CLL cell proliferation and survival depends on host factors that are present in the tissue microenvironment. In–vitro these cells rapidly undergo apoptosis but can be supported by culture on stroma cells. The extracellular matrix is an important component of the microenvironment. Interactions between tumor cells and the extracellular matrix are, in part, mediated by CD44, whose principle ligand is hyaluronic acid. To explore a possible role of this adhesion receptor in CLL, we evaluated the effect of CD44 engagement on the survival of CLL cells and the induction of drug resistance. Dimerization of CD44 activated the PI3K/AKT and MAPK/ERK pathways and resulted in increased levels of MCL-1. There was no increase in MCL mRNA in CD44 stimulated cells, consistent with an effect on protein stabilization. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous apoptosis: CD44 stimulated CLL cells had a 46% increase (range 7% – 181%) in viability over the corresponding unstimulated control cells (n=20, p&amp;lt;0.0001). Furthermore, CD44 activation also protected from fludarabine-induced apoptosis: cell viability for isotype treated cells 76 ±6%, for isotype and fludarabine treated cells 47 ±18% and for CD44 stimulated and fludarabine treated cells 69 ±16% (p=0.005). CLL cells of the IgVH unmutated subtype expressed CD44 more highly than IgVH mutated CLL cells (MFI ratio 224 ±43 to 122 ±44, respectively, p&amp;lt;0.0001) and derived a more pronounced survival advantage from CD44 activation: % live cells (CD44 stimulated - control) 21 ±9% vs. 13 ±6%, respectively (p=0.04), which could contribute to the more progressive nature of this subtype. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44 activation. Furthermore, obatoclax was able to sensitize CLL cells to fludarabine. In conclusion, CD44 engagement protects CLL cells from spontaneous and fludarabine induced apoptosis. This prosurvival effect is mediated through PI3K/Akt and MAPK/ERK pathways and increased MCL-1 levels. Targeting either signaling pathway or MCL-1 with specific inhibitors could overcome the protective effect of the microenvironment in CLL and provides a rationale for combination therapy.</jats:p

Utlilizing Mutant Mice To Dissect Immune Bone Marrow Failure: Markedly Reduced Cytotoxicity of Lymphocytes from Fas- and Fas Ligand-Deficient Mice in Lymph Node Cell-Mediated Aplastic Anemia.

Abstract Key cellular events in the development of aplastic anemia are activation and expansion of T cells that are destructive of hematopoietic tissue. Human immune-mediated bone marrow failure has been modeled in mice by infusion of lymph node (LN) cells into major- or minor-histocompatibility antigen mismatched recipients; oligoclonal T cell expansion causes indiscriminant hematopoietic cell destruction, marked marrow hypoplasia, and fatal pancytopenia. We have utilized this simple model in order to dissect the molecular events responsible for T cell mediated, selective organ destruction. In mice with spontaneous mutations: lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld), animals have an abnormal increase in lymphocytes and develop clinical manifestations similar to those seen in patients with systemic lupus erythematosus (SLE). In contrast to human SLE, the lpr and gld bone marrow appear unaffected by this typically self-destructive process. We examined C57BL/6 (B6) mice that carry the lpr and gld mutations along with normal B6 mice to define cellular and functional differences as they relate to BM damage. In our initial analyses of peripheral blood (PB), we found a reduced CD4 T cell proportion in lpr and gld mice (5.5±1.5%, 4.3±0.6%, 9.9±0.9%) and a reduced CD8 T cell proportion in gld mice when compared to B6 controls (13.4±1.9%, 7.9±0.9%, 12.7±1.8%). In the BM, both CD4 (1.5±0.3%, 2.3±0.3%, 1.0±0.1%) and CD8 (2.9±0.3%, 3.1±0.7%, 2.6±0.2%) T cell proportions were slightly increased in lpr and gld mice, while total number of BM cells was not significantly different among the three genotypes (438±48, 324±18, 350±27, millions). These findings indicated no PB lymphocyte accumulation and no BM destruction in lpr or gld mice. We then tested the hypothesis that lpr and gld BM cells were more resistant to immune attack by infusing 5 x 106 lymph node (LN) cells from minor-histocompatibility antigen-mismatched C.B10 mice into sublethally-irradiated (5 Gys) lpr, gld, and B6 recipients. At two weeks, all recipients infused with CB10 LN cells had reduced blood neutrophils, and gld and B6 recipients also had reduced RBCs, as compared to mice that did not receive LN cell infusions. This result suggests that lpr and gld hematopoietic cells, like those from B6 mice, are susceptible to immune-mediated destruction. We then examined lymphocyte function by co-incubating lpr, gld and B6 LN cells (effectors) with C.B10 BM cells (targets) in a cytotoxilux assay in vitro. B6 effectors produced 51–78% more apoptotic targets than did gld and lpr effectors. This difference was confirmed by the infusion of lpr, gld (15 x 106 cells/recipient) and B6 (5 x 106 cells/recipient) LN cells into sub-lethally irradiated CB10 mice in vivo. At three weeks, enumeration of blood neutrophils (0.69±0.60, 1.27±0.83, 0.08±0.03), RBCs (3.58±0.42, 4.80±1.20, 4.76±0.36), platelets (274± 94, 400±156, 298±91) and total BM cells (87± 23, 195±78, 82±6, millions) demonstrated that, despite massive lymphoproliferation, lymphocytes from lpr and gld mice were less effective in mediating BM cell destruction. Our results provide evidence supporting the role of Fas and Fas-ligand mediated programmed cell death in immune-mediated BM destruction. The ready availability of genetically modified mice with well characterized functional defects should allow a full description of the molecular pathways in immune-mediated marrow failure in this model.</jats:p

A Longitudinal Analysis of the T-Cell Receptor Vβ Repertoire in Aplastic Anemia Patients at Initial Presentation, Remission, and Relapse.

Abstract Aplastic anemia is a bone-marrow-failure syndrome characterized by low blood-cell counts and a fatty bone marrow. In most cases, no obvious etiological factor can be identified, but clinical responses to immunosuppressive treatment (IST) strongly suggest an immune pathophysiology. Our previous study of T-cell receptor (TCR) Vβ (variable region of β-chain) repertoire usage by flow cytometry suggested that aplastic anemia results from antigen-specific lymphocyte attack on hematopoietic progenitors (Risitano et al. Lancet2004; 364:355). In the current work, 7 patients were investigated for Vβ pattern expression before first immunosuppresive treatment, at the remission, and again on relapse. The TCR Vβ repertoire was analyzed for CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 22 different Vβ chains. Most patients had very different patterns of Vβ usage from healthy individuals, and all but one showed expansion of at least one Vβ family before immunosuppressive treatment (Vβ family expansions were defined as 2 standard deviations (SD) from the means in controls). The median number of expanded Vβ families was 4 per patient among CD8CD28dim effector cells. At remission, almost all the initially expanded Vβ subfamilies decreased to less than 2SD of controls. At relapse, most of the expanded Vβ subsets were increased again. However, 5/7 patients showed new expanded Vβ subsets at recurrence of cytopenias, suggesting antigenic spread of new epitopes recognized by immune systems. Although no common pattern of specific expanded Vβ subsets could be identified among different patients, some Vβ subfamilies appeared to be more frequently involved (Vβ 5.1 and Vβ 5.2 were expanded in 4 of 7 patients both at initial presentation and relapse ). These data suggest that monitoring Vβ subsets in aplastic anemia, and potentially in other immune-mediated human diseases of a similar pathophysiology could be used to guide individual therapeutic decisions and in the development of new treatments.</jats:p