ALBERT

Description: We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion 〉 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( 〉 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( 〈 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells.

Description: One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin−CD34+ CD38− cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin− CD34+ CD38− cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.

Description: Human neutrophils have been shown recently to express both the beta and the gamma chains of the interleukin-2 receptor (IL-2R). IL-15, a cytokine that has recently been cloned and characterized, was found to share many of the biological functions of IL-2 and is known to mediate signals through IL-2R beta and IL-2R gamma. In recent studies, we observed that IL-2 exerts few effects on various neutrophil functions, but information on IL-15-neutrophil interactions is lacking. In this study, we observed that IL-15, in contrast to IL-2, induces important morphological cell shape changes that are typical of activated neutrophils. Furthermore, phagocytosis of opsonized sheep red blood cells was significantly increased by IL-15 but not by IL-2. However, similar to IL-2, IL-15 did not modulate the oxidative burst response. Furthermore, we observed that de novo RNA synthesis is increased in neutrophils by IL-15 along with de novo protein synthesis, whereas no significant effect of IL-2 was noted. Among the different proteins that were found to be upregulated by IL-15, one was identified by microsequencing as the cytoskeletal protein actin. Finally, we found that IL-15 delays apoptosis of neutrophils more efficiently than IL-2 when evaluated by both microscopic observations and flow cytometry procedures. Furthermore, this phenomenon was dose-dependent (10 to 500 ng/mL), and, at 500 ng/mL, IL-15 delayed apoptosis as strongly as granulocyte-macrophage colony-stimulating factor. This study is the first to show that IL-15 is a significant neutrophil agonist. Moreover, in view of the differential effects of IL-15 and IL-2 on this cell type, our results support the existence of a specific IL-15R component(s) on human neutrophils.

Description: We have studied the reconstitution of the T-cell compartment after bone marrow transplantation (BMT) in five patients who received a graft-versus-host disease (GVHD) prophylaxis consisting of methotrexate, cyclosporin, and 10 daily injections (day −4 to day +5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/μL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool was repopulated exclusively through expansion of residual T cells with all CD4+ T cells expressing the CD45RO-memory marker. In two patients, the expansion was extensive and within 2 months, the total number of T cells (CD8〉〉CD4) exceeded 1,000/μL. In the other three patients, T cells remained low (87 ± 64 T cells/μL at 6 months) and remained below normal values during the 2 years of the study. In all patients, the first CD4+CD45RA+RO− T cells appeared after 6 months and accumulated thereafter. In the youngest patient (age 13), the increase was relatively fast and naive CD4+ T cells reached normal levels (600 T cells/μL) 1 year later. In the four adult patients (age 25 ± 5), the levels reached at that time-point were significantly lower (71 ± 50 T cells/μL). In all patients, the T-cell repertoire that had been very limited, diversified with the advent of the CD4+CD45RA+RO− T cells. Cell sorting experiments showed that this could be attributed to the complexity of the T-cell repertoire of the CD4+CD45RA+RO− T cells that was comparable to that of a normal individual and that, therefore, it is likely that these cells are thymic emigrants. We conclude that after BMT, the thymus is essential for the restoration of the T-cell repertoire. Because the thymic activity is restored with a lag time of approximately 6 months, this might explain why, in particular in recipients of a T-cell–depleted graft, immune recovery is delayed.

Description: We have studied the reconstitution of the T-cell compartment after bone marrow transplantation (BMT) in five patients who received a graft-versus-host disease (GVHD) prophylaxis consisting of methotrexate, cyclosporin, and 10 daily injections (day −4 to day +5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/μL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool was repopulated exclusively through expansion of residual T cells with all CD4+ T cells expressing the CD45RO-memory marker. In two patients, the expansion was extensive and within 2 months, the total number of T cells (CD8〉〉CD4) exceeded 1,000/μL. In the other three patients, T cells remained low (87 ± 64 T cells/μL at 6 months) and remained below normal values during the 2 years of the study. In all patients, the first CD4+CD45RA+RO− T cells appeared after 6 months and accumulated thereafter. In the youngest patient (age 13), the increase was relatively fast and naive CD4+ T cells reached normal levels (600 T cells/μL) 1 year later. In the four adult patients (age 25 ± 5), the levels reached at that time-point were significantly lower (71 ± 50 T cells/μL). In all patients, the T-cell repertoire that had been very limited, diversified with the advent of the CD4+CD45RA+RO− T cells. Cell sorting experiments showed that this could be attributed to the complexity of the T-cell repertoire of the CD4+CD45RA+RO− T cells that was comparable to that of a normal individual and that, therefore, it is likely that these cells are thymic emigrants. We conclude that after BMT, the thymus is essential for the restoration of the T-cell repertoire. Because the thymic activity is restored with a lag time of approximately 6 months, this might explain why, in particular in recipients of a T-cell–depleted graft, immune recovery is delayed.

Description: In several models of lymphocyte apoptosis, two alterations of mitochondrial function precede advanced DNA fragmentation: (1) a reduction of mitochondrial transmembrane potential (delta psi m) and (2) an increase in mitochondrial generation of superoxide anion. Here we show that two fluorochromes allow for the identification of analogous mitochondrial perturbations in circulating T lymphocytes from human immunodeficiency virus (HIV)-1+ donors. The first among these fluorochromes, the cationic lipophilic dye DiOC6(3), measures delta psi m; the second marker, hydroethidine (HE), is nonfluorescent, unless it is oxidized by superoxide anions to the product ethidium (Eth). CD4+ or CD8+ cells from clinically asymptomatic HIV-1 carriers contain a significantly elevated percentage of cells endowed with enhanced HE --〉 Eth conversion and/or reduced DiOC6(3) uptake as compared with normal controls. Phenotypic characterization of (HE --〉 Eth)high cells from HIV+ donors shows that these cells possess a low delta psi m, thus demonstrating a functional alteration of mitochondria. In addition, (HE --〉 Eth)high cells display a reduced incorporation of the cardiolipin-specific dye nonyl-acridine orange (NAO), showing a structural defect of the cardiolipin-containing inner mitochondrial membrane. Control experiments involving rotenone, an inhibitor of the respiratory chain complex I, indicate that the reactive oxygen species responsible for HE --〉 Eth conversion is generated during mitochondrial electron transport. In synthesis, it appears that mitochondrial alterations occur in a significant percentage of circulating T lymphocytes from HIV-1 carriers. The extent of delta psi m reduction, as determined ex vivo, correlates with the frequency of cells undergoing DNA fragmentation after overnight in vitro culture. These observations may be important for the understanding and for the direct ex vivo quantitation of HIV-triggered lymphocyte destruction.

Description: In several models of lymphocyte apoptosis, two alterations of mitochondrial function precede advanced DNA fragmentation: (1) a reduction of mitochondrial transmembrane potential (delta psi m) and (2) an increase in mitochondrial generation of superoxide anion. Here we show that two fluorochromes allow for the identification of analogous mitochondrial perturbations in circulating T lymphocytes from human immunodeficiency virus (HIV)-1+ donors. The first among these fluorochromes, the cationic lipophilic dye DiOC6(3), measures delta psi m; the second marker, hydroethidine (HE), is nonfluorescent, unless it is oxidized by superoxide anions to the product ethidium (Eth). CD4+ or CD8+ cells from clinically asymptomatic HIV-1 carriers contain a significantly elevated percentage of cells endowed with enhanced HE --〉 Eth conversion and/or reduced DiOC6(3) uptake as compared with normal controls. Phenotypic characterization of (HE --〉 Eth)high cells from HIV+ donors shows that these cells possess a low delta psi m, thus demonstrating a functional alteration of mitochondria. In addition, (HE --〉 Eth)high cells display a reduced incorporation of the cardiolipin-specific dye nonyl-acridine orange (NAO), showing a structural defect of the cardiolipin-containing inner mitochondrial membrane. Control experiments involving rotenone, an inhibitor of the respiratory chain complex I, indicate that the reactive oxygen species responsible for HE --〉 Eth conversion is generated during mitochondrial electron transport. In synthesis, it appears that mitochondrial alterations occur in a significant percentage of circulating T lymphocytes from HIV-1 carriers. The extent of delta psi m reduction, as determined ex vivo, correlates with the frequency of cells undergoing DNA fragmentation after overnight in vitro culture. These observations may be important for the understanding and for the direct ex vivo quantitation of HIV-triggered lymphocyte destruction.

Description: Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA- 1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA- 1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T- lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks posttransplant, animals that received cells transduced by the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) count than control animals that received cells transduced with a virus containing a neor gene. Erythropoiesis was stimulated in GATA-1 and control animals by phlebotomy. GATA-1 animals required more extensive phlebotomy to reach a hematocrit less than 25 and their hematocrit returned to normal levels sooner than control animals. The effect of twice-daily injections of 10 U recombinant erythropoietin (epo) was also examined. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data suggest that dysregulated expression of GATA-1 in primitive hematopoietic cells enlarges the pool of epo-responsive erythroid progenitor cells.