Martha Kirby

Postnatal NG2 proteoglycan–expressing progenitor cells are intrinsically multipotent and generate functional neurons

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan–positive progenitor cells that express the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Regulation of CSF1 Promoter by the SWI/SNF-like BAF Complex

The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.

Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure Syndromes

Aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) are rare hematologic diseases that often appear in the same patient. Patients with aplastic anemia have severe thrombocytopenia, neutropenia, and anemia accompanied by absent hematopoietic precursors in an empty bone marrow (1). In contrast, the classic evidence of PNH is the intermittent appearance of dark urine due to excretion of hemoglobin, the result of intravascular hemolysis (2). The knowledge that this peculiar form of erythrocyte destruction resulted from increased susceptibility of the PNH erythrocyte to complement led to the development of laboratory assays, such as the Ham and sugar hemolysis tests. Modern clinical studies have shown that patients with PNH experience serious morbidity and mortality, mainly from venous thromboses and, especially in younger patients, pancytopenia (3). On the basis of results of the Ham test in several patients, Lewis and Dacie (4) formalized the overlap between the two diseases as the aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome. With improved survival in aplastic anemia, many patients show laboratory and clinical evidence of PNH, often months or years after completion of successful immunosuppressive therapy (5, 6). In aplastic anemia, hematopoietic cells appear to be destroyed by the patients own immune system (7). In contrast, the basis of PNH is a somatically acquired mutation in a hematopoietic stem cell. Almost all patients with PNH have molecular lesions in the PIG-A gene, which is located on the X chromosome (8). The PIG-A gene product is required at an early step in the synthesis of a glycosylphosphatidylinositol (GPI) structure, which serves as an anchor for a group of proteins that are linked to the cell surface by this greasy foot rather than by the more typical transmembrane configuration. As a result, affected cells are globally deficient in GPI-anchored proteins. Deficiency in one of these proteins, CD59, which inhibits late-acting complement component activity on the erythrocyte surface, accounts for the hemolytic component of the disease (9). The pathophysiologic basis of the clinical relation between PNH and aplastic anemia is unknown. Lewis and Dacie (4) postulated the development of an abnormal clone of haemopoietic cells in a regenerating, previously aplastic marrow as a cause, and Rotoli and Luzzatto (10, 11) speculated that an insult leading to a hypocellular environment or some feature of marrow failure might lead to either aplasia or PNH. Observations of patients with lymphoma who received monoclonal antibody treatment directed against a GPI-anchored protein have demonstrated the appearance of PNH-like lymphocytes as an inadvertent result of therapy (12). These results confirmed the in vivo plausibility of a third hypothetical mechanism: that preexisting PNH clones might be selected as a result of their relative insusceptibility to autoimmune attack (6, 10). To test these hypotheses, we used a sensitive flow cytometric method to analyze blood cells from patients in various states of marrow failure for evidence of PNH clones. Methods Patients We developed a rigorous three-color flow cytometric protocol for identification of GPI-anchored protein-deficient granulocyte populations and applied this method over 1.5 years to patients presenting to our clinic at the National Heart, Lung, and Blood Institute with newly diagnosed or previously treated bone marrow failure syndromes. Most of the patients with previously treated bone marrow syndromes had received therapy at the National Institutes of Health up to 11 years before sampling for this study. Samples from 254 participants were analyzed from April 1997 to October 1998. Of these participants, 115 had aplastic anemia, 39 had myelodysplasia, 28 had recently undergone bone marrow transplantation, 20 were controls who had undergone renal transplantation and had received antithymocyte globulin as treatment for graft rejection, 13 had large granular lymphocytosis, 18 had undergone multiple cycles of chemotherapy for cancer, and 21 were healthy controls. All clinical research samples were obtained after informed consent was given under protocols approved by the institutional review board of the National Heart, Lung, and Blood Institute. Other specimens were collected during routine phlebotomy procedures and were studied without unique patient identifiers. Aplastic anemia was defined as bone marrow cellularity of 30% or less and two of the following three laboratory abnormalities: absolute neutrophil count less than 0.5 109 cells/L, platelet count less than 20 109 cells/L, and reticulocyte count less than 60 109 cells/L. Myelodysplasia was defined by the standard morphologic criteria of either dysplastic myeloid or megakaryocytic (but not solely erythroid) bone marrow elements in patients who required 2 or more units of red blood cell transfusions per month for 2 or more months, with or without thrombocytopenia or neutropenia. Monoclonal Antibodies We used CD55-PE (clone 143-30, mouse IgG1 [Research Diagnosis, Inc., Flanders, New Jersey]) and CD59-PE (clone MEM 43, mouse IgG2a [Research Diagnosis, Inc.]) for analysis of GPI-anchored protein expression on erythrocytes. Glycophorin-A-FITC (clone D2.10, mouse IgG1 [Immunotech, Westbrook, Maine]) was used as a nonanchored marker to positively identify erythrocytes. The isotypic control for erythrocyte phenotyping consisted of mouse IgG1-PE (clone X40 [Becton Dickinson, San Diego, California]) and mouse IgG2a-PE (clone X39 [Becton Dickinson]). For analysis of granulocyte GPI-anchored protein expression, we used CD66b-FITC (clone 80H3, mouse IgG1 [Immunotech]) and CD16-PECy5 (clone 3G8, mouse IgG1 [Caltag]), with CD15-PE (clone 80H5, mouse IgM [Immunotech]) as a non-GPI-anchored marker to positively identify granulocytes. The isotypic controls for granulocyte staining consisted of mouse IgG1-FITC (clone X40 [Becton Dickinson]) and mouse IgG1-PECy5 (clone MOPC-21 [Caltag]). Nonspecific Fc receptor-mediated binding of conjugated antibodies to granulocytes or erythrocytes was blocked by pre-incubating 1 mL of blood with 30 L of mouse IgG (Caltag). Antibody Staining and Flow Cytometry Analysis Blood was drawn by venipuncture into tubes containing EDTA. Samples were stained within 48 hours of collection; staining was usually done within 8 hours. (Pilot studies revealed no significant change in cytofluorometric results in samples stored for up to 48 hours.) Erythrocyte GPI-anchored protein expression was evaluated by incubating for 30 minutes at room temperature 50 L of a 1:20 dilution of whole blood with 20 L of Gly-A-FITC and either 10 L each of CD55-PE plus CD59-PE or the appropriate isotype controls. Samples were washed and resuspended in 1 mL of phosphate-buffered saline before flow cytometry. Granulocyte GPI-anchored protein expression was evaluated by incubating for 60 minutes at room temperature 100 L of whole blood with 10 L of CD15-PE and either 20 L of CD66b-FITC plus 5 L of CD16-PECy5 or the appropriate isotype controls. Erythrocytes in these samples were lysed by using a Q-Prep apparatus (Coulter, Fullerton, California) and were then fixed with paraformaldehyde. All of the samples were analyzed by using a Coulter XL flow cytometry machine equipped with a 488-nm argon laser and XL or XLII software. Strict criteria to distinguish cells lacking GPI-anchored proteins were applied for two reasons. First, like many cell surface transmembrane proteins, GPI-anchored protein can vary in expression according to the stage of cellular differentiationfor example, as a result of myeloid or erythroid maturation. In addition, in some hematologic diseases, differentiation may be abnormal, as reflected in abnormal structure and aberrant expression of specific proteins. Therefore, polymorphonuclear cells were identified on the basis of light-scatter properties that correlate with cell size and granularity and by staining with a specific antigranulocyte antibody (anti-CD15, conjugated to the fluorescent dye phycoerythrin). Two antibodies directed against distinct GPI-anchored protein (anti-CD16, conjugated to the fluorochrome PE-Cy5, and anti-CD66b, conjugated to fluorescein isothiocyanate) were used to determine the PNH phenotype. Erythrocytes were similarly analyzed by using appropriate antibody combinations (anti-glycophorin for identification of erythrocytes and anti-CD59 and anti-CD55 as labels for GPI-anchored protein). Interpretation of Flow Cytometry Data The gates used to define CD16 /CD66b granulocyte or CD55 /CD59 erythrocyte populations were set on the basis of the isotypic control analyses performed on the same day. The mean SD of double negative cells for 21 healthy participants was 0.129% 0.101% for granulocytes and 0.195% 0.116% for erythrocytes. Because many patients required red blood cell transfusion at the time of initial sampling, the classification of patients was based on granulocyte analysis; a population of GPI-anchored protein-negative granulocytes of 1% or more that was clearly separate from the wild-type CD16+/CD66b+ cells on a two-dimensional histogram (Figure 1) was required for categorization of a patient as harboring PNH cells. Figure 1. Protocol for identification of paroxysmal nocturnal hemoglobinuria ( PNH) phenotype granulocytes. A. B. C. D. E. Response Criteria Response to treatment in aplastic anemia was defined as failure to fulfill criteria for severity for at least 3 months (see above); this was previously shown to correlate with transfusion independence and absence of infections. Criteria for response to therapy in myelodysplasia were no need for transfusion for 8 or more weeks, hemoglobin values of 80 g/L or more, platelet counts greater than 20 109 cells/L, and absolute neutrophil counts of 0.5 109 cells/L or more. Statistical Analysis A Fisher exact test was used to determine two-tailed P values in 2 2 contingency tables. The chi-square test was used

Bias toward use of a specific T cell receptor beta-chain variable region in a subgroup of individuals with sarcoidosis.

To evaluate the concept that biases in the usage of T cell antigen receptor beta variable (V) regions may be manifested in T lymphocytes that accumulate in nonmalignant, T cell-mediated human disorders, a V beta 8-specific antibody (anti-Ti3A, 5REX9H5) was used to evaluate lung and blood T cells in pulmonary sarcoidosis, a chronic granulomatous disorder of unknown etiology. Whereas normal patients had less than 5% Ti3A+ lung (n = 7) and/or blood (n = 9) lymphocytes, strikingly, a subgroup (8 of 21) with active pulmonary sarcoidosis had greater than 7% Ti3A+ lung and/or blood T cells and a higher proportion of Ti3A+ lymphocytes in the lung compared with blood. Dual-color flow cytometry demonstrated compartmentalization of Ti3A+ CD4+ lymphocytes to lung and Ti3A+ CD8+ lymphocytes to blood. Analysis with a 32P-labeled V beta 8 probe revealed that sarcoid lung T lymphocytes contained higher amounts of V beta 8+ mRNA than autologous blood T cells. However, Southern analysis of sarcoid lung and blood T cell DNA demonstrated no evidence of clonal rearrangements of V beta 8 genes. These observations demonstrate a clear bias toward the use of at least one V beta region in sarcoidosis, and suggests T cells accumulate secondary to external selective pressure, rather than in a random polyclonal fashion or by clonal expansion of one or few T cell clones.

Differential Expression of Interleukin-17A and -17F Is Coupled to T Cell Receptor Signaling via Inducible T Cell Kinase

T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.

SAP regulates T cell–mediated help for humoral immunity by a mechanism distinct from cytokine regulation

X-linked lymphoproliferative disease is caused by mutations affecting SH2D1A/SAP, an adaptor that recruits Fyn to signal lymphocyte activation molecule (SLAM)-related receptors. After infection, SLAM-associated protein (SAP)−/− mice show increased T cell activation and impaired humoral responses. Although SAP−/− mice can respond to T-independent immunization, we find impaired primary and secondary T-dependent responses, with defective B cell proliferation, germinal center formation, and antibody production. Nonetheless, transfer of wild-type but not SAP-deficient CD4 cells rescued humoral responses in reconstituted recombination activating gene 2−/− and SAP−/− mice. To investigate these T cell defects, we examined CD4 cell function in vitro and in vivo. Although SAP-deficient CD4 cells have impaired T cell receptor–mediated T helper (Th)2 cytokine production in vitro, we demonstrate that the humoral defects can be uncoupled from cytokine expression defects in vivo. Instead, SAP-deficient T cells exhibit decreased and delayed inducible costimulator (ICOS) induction and heightened CD40L expression. Notably, in contrast to Th2 cytokine defects, humoral responses, ICOS expression, and CD40L down-regulation were rescued by retroviral reconstitution with SAP-R78A, a SAP mutant that impairs Fyn binding. We further demonstrate a role for SLAM/SAP signaling in the regulation of early surface CD40L expression. Thus, SAP affects expression of key molecules required for T–B cell collaboration by mechanisms that are distinct from its role in cytokine regulation.

Increased cytotoxic T cells with effector phenotype in aplastic anemia and myelodysplasia

OBJECTIVE We hypothesized that an active autoimmune process in aplastic anemia (AA) corresponds to the expansion of cytotoxic lymphocytes (CTLs) displaying mature effector phenotype. We determined whether the numbers of effector CTLs in blood of patients with bone marrow failure syndromes are elevated and correlate with the disease activity and responsiveness to immunosuppression. PATIENTS AND METHODS We analyzed samples from patients with AA, myelodysplastic syndrome (MDS), polytransfused patients with nonimmune-mediated hematologic disease, and normal controls for the presence of effector T lymphocytes using four-color flow cytometry. Expression of CD57 and loss of CD28 on CD8+CD3+ CTL were used as markers for the terminal effector phenotype. In addition, intracellular staining for perforin and granzyme B was preformed. The numbers of effector CTL did not differ between healthy individuals and hematologic controls and the two groups were pooled. RESULTS The percentages of CD8+CD28- and CD8+CD28-CD57+ cells were significantly higher in AA and MDS patients than in controls. There was a trend toward a gradual decrease in the effector CTLs from the high values observed in untreated new patients and patients who did not respond to immunosuppression, intermediate levels for partial responders and complete responders, to the lowest levels seen in controls. However, severity of pancytopenia did not correlate with the size of the effector cell population. In contrast to CD57+ CTLs, expression of perforin or granzyme B in the cytotoxic effector cells did not differ in AA patients from those of controls. CONCLUSIONS Our results indicate that phenotypically defined effector CTLs are increased in AA and MDS and the effector phenotype may be useful to isolate and characterize antigen-specific T cells in AA in order to delineate the possible inciting or driving agents in AA.

Increased numbers of T lymphocytes with gamma delta-positive antigen receptors in a subgroup of individuals with pulmonary sarcoidosis.

Individuals with sarcoidosis were evaluated for preferential usage of T cells with the gamma delta-positive (+) type of T cell antigen receptor. Compared with normal subjects (n = 19), the group with sarcoidosis had increased numbers of CD3+ alpha beta-negative (-) T cells in the blood (normal, 58 +/- 12 cells/microliters; sarcoid, 192 +/- 45 cells/microliters, P less than 0.05) and in the epithelial lining fluid of the lung (normal, 78 14 cells/microliters; sarcoid, 240 +/- 60 cells/microliters, P less than 0.04) and a concomitant elevated number of blood and lung CD3+ gamma delta+ T cells, owing to a striking increase in the number of CD3+ gamma delta+ T cells in a subgroup (7 of 20) of sarcoid individuals. The elevated numbers of sarcoid blood gamma delta+ T lymphocytes were mostly Ti gamma A+ and delta TCS1-, a pattern also seen in normal individuals, consistent with the majority of gamma delta+ T cells expressing one gamma-chain variable region, V gamma 9. The observation of an increase in the total gamma delta+ T cell numbers in a sarcoid subgroup suggests that various specific stimuli may trigger the expansion of different T cell subpopulations within different groups of individuals with sarcoidosis.

Second-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes somatic mosaicism in two WAS siblings

Revertant mosaicism due to true back mutations or second-site mutations has been identified in several inherited disorders. The occurrence of revertants is considered rare, and the underlying genetic mechanisms remain mostly unknown. Here we describe somatic mosaicism in two brothers affected with Wiskott-Aldrich syndrome (WAS). The original mutation causing disease in this family is a single base insertion (1305insG) in the WAS protein (WASP) gene, which results in frameshift and abrogates protein expression. Both patients, however, showed expression of WASP in a fraction of their T cells that were demonstrated to carry a second-site mutation causing the deletion of 19 nucleotides from nucleotide 1299 to 1316. This deletion abrogated the effects of the original mutation and restored the WASP reading frame. In vitro expression studies indicated that mutant protein encoded by the second-site mutation was expressed and functional, since it was able to bind to cellular partners and mediate T cell receptor/CD3 downregulation. These observations were consistent with evidence of in vivo selective advantage of WASP-expressing lymphocytes. Molecular analysis revealed that the sequence surrounding the deletion contained two 4-bp direct repeats and that a hairpin structure could be formed by five GC pairs within the deleted fragment. These findings strongly suggest that slipped mispairing was the cause of this second-site mutation and that selective accumulation of WASP-expressing T lymphocytes led to revertant mosaicism in these patients.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells

Loss of the Tec family kinase Itk results in a bias to FoxP3+ Treg cell differentiation and reduced TCR-induced phosphorylation of mTOR targets.