Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-gamma secretion

Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4+ and CD8+ T cells responded to allostimulation with interferon (IFN gamma) secretion. Interleukin-I (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFN gamma secretion. Exogenous IL-1 beta, IL-2 and IL-7 increased allostimulated IFN gamma secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFN gamma-neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4+ and CD8+ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFN gamma released during this response can modulate the function of allogeneic AML blasts.     

Differential display analysis of murine collagen-induced arthritis: cloning of the cDNA-encoding murine ATPase inhibitor

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.     

IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity

We have recently demonstrated, in a fully MHC-mismatched murine bone marrow transplantation model, that administration of a short course of high dose IL-2 markedly diminishes graft-vs-host disease (GVHD) without compromising alloengraftment or the graft-vs-leukemia (GVL) effect of allogeneic T cells. We have now evaluated the mechanism of the dissociation of GVL and GVHD observed in this model. We demonstrate that CD4+ T cells were required to produce severe, acute GVHD in the fully MHC-mismatched plus minor histocompatibility Ag-mismatched A/J-->B10 strain combination. The GVHD-producing activity of A/J CD4+ T cells administered without CD8+ T cells was inhibited by IL-2 treatment. In contrast, CD8+ T cells alone mediated the GVL effect observed in the EL4 leukemia/lymphoma model, and CD4+ cells did not contribute to this effect. This CD8-mediated GVL activity was not inhibited by IL-2 treatment. Because naive A/J CD8+ T cells administered without CD4+ T cells did not produce acute GVHD, we were unable to evaluate the effect of IL-2 in this model. However, when A/J donors were presensitized with B10 skin grafts, CD4-depleted A/J spleen cells were capable of causing acute GVHD in B10 recipients. This CD8-mediated GVHD was not inhibited by treatment with IL-2. However, IL-2 did partially inhibit the GVHD produced by nondepleted presensitized A/J spleen cells, probably due to selective inhibition of the function of presensitized A/J CD4+ T cells. The dissociation of GVHD and GVL against the EL4 leukemia/lymphoma in IL-2-treated mice can therefore be explained by selective inhibition by IL-2 of CD4 activity.     

Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects

We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)-promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4-2. BALB/c mice receiving 2.5 x 10(5) 2B-4-2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5-day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.     

Allogenic peripheral blood stem cell (PBSC) transplantation in two patients with graft failure

Allogenic peripheral blood stem cells (PBSC) were used for graft failure after BMT in two patients. These PBSC were mobilized by G-CSF in the same donors, harvested and given without reconditioning to the patients. In one patient, PBSC with a very high T cell number were given unprocessed, in the other patient, CD34+ cells were positively enriched due to a 2-antigen difference. None of the patients had hyperacute GVHD. Trilineage engraftment was seen after 13 days. Acute GVHD grade II to III developed on days +31 in patient 1 and +16 in patient 2, involving predominantly gut and liver, but sparing the skin. Thus, allogeneic PBSCT for graft failure did not cause hyperacute GVHD even with very high T cell numbers in patient 1, and graft failure with CD34 selected PBSC was successfully reversed even with a low number of T cells in patient 2.     

Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells

We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and CD34+ HSCs from the same leukapheresis collection from G-CSF-primed normal donors and cancer patients in sufficient numbers for the purpose of combined PBSCT and immunization strategies.     

Interleukin-10 dose-dependent regulation of CD4+ and CD8+ T cell-mediated graft-versus-host disease

BACKGROUND: Endogenous interleukin (IL)-10 production has been associated with the lack of graft-versus-host disease (GVHD) in human recipients of MHC-disparate donor grafts. Paradoxically, we have shown that the exogenous administration of high doses (30 microg/dose) of IL-10 to murine recipients of MHC-disparate grafts accelerates GVHD lethality. METHODS: The effects of IL-10 on GVHD mediated by either CD4+ or CD8+ T cells was examined in studies involving exogenous IL-10 administration or the infusion of T cells from IL-10-deficient (-/-) donor mice. The role of interferon (IFN)-gamma on IL-10-induced GVHD acceleration was studied using IFN-gamma-deficient (-/-) donor mice or neutralizing monoclonal antibody. RESULTS: IL-10 was found to have a dose-dependent effect on the GVHD lethality mediated by either CD4+ or CD8+ T cells. High doses of exogenous IL-10 accelerated GVHD lethality. IFN-gamma release was not responsible for the IL-10 facilitation of GVHD lethality. Paradoxically, low doses of IL-10 protected mice against GVHD lethality. The GVHD protective effect of the bioavailability of small amounts of IL-10 was confirmed by demonstrating that the infusion of T cells from IL-10 -/- donors accelerated GVHD lethality. CONCLUSIONS: The results suggest that IL-10 has a dose-dependent effect on the GVHD lethality mediated by CD4+ or CD8+ T cells, such that high doses accelerate lethality, while low amounts of bioavailable IL-10 are protective.     

Modulation of in vitro and in vivo T-cell responses by transferrin-gallium and gallium nitrate

Gallium is a group IIIa metal that has efficacy in the therapy of malignant disorders such as lymphoma and urothelial tract tumors. Preclinical studies also indicate a role for gallium in autoimmune disorders, suggesting that gallium is able to modulate T-cell immune reactivity. The purpose of this study was to examine the in vitro and in vivo immunomodulatory action of gallium on T-cell function. Since gallium binds to transferrin in vivo, in vitro studies evaluated the effect of transferrin-gallium (Tf-Ga) on human T cells. Tf-Ga inhibited the mitogen-induced proliferative response of peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. Alloantigen-induced proliferation was also potently suppressed when evaluated in a mixed lymphocyte culture assay. Tf-Ga affected a significant reduction in the density of IL-2 receptors on activated T cells and a slight reduction in the number of CD3+/CD25+ T cells in PHA-stimulated cultures. Neither secretion of interleukin-2 (IL-2) nor the induction of IL-2-stimulated lymphokine-activated killer activity, however, was inhibited by Tf-Ga. Tf-Ga produced significant upregulation of the transferrin receptor (CD71) in T cells as determined by flow cytometric analysis and northern blot assay, but did not affect the percentage of CD3+/ CD71+ T cells after mitogen stimulation. To assess the in vivo effects of gallium on alloreactive T cells, we evaluated the immunosuppressive effect of gallium in a murine model of graft-versus-host disease (GVHD). Administration of gallium significantly prolonged survival in mice undergoing severe GVHD, suggesting that gallium can ameliorate GVH reactivity. Collectively, these data demonstrate that, at clinically achievable concentrations, Tf-Ga potently inhibits T-cell activation and that this immunosuppressive property of gallium may be of adjunctive therapeutic value in the management of disorders characterized by the presence of autoreactive or alloreactive T-cell populations.     

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.     

Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin

The Gal beta(1-3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4+ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4+ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-gamma and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3- Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4- Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars.     

CD34 positive blood cells for allogeneic progenitor and stem cell transplantation

The transplantation of allogeneic peripheral blood progenitor cells (PBPC) provides complete and sustained hematopoietic and lymphopoietic engraftment. In healthy donors, large amounts of PBPC can be mobilized with hematopoietic growth factors. However, the high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic PBPC to an elevated risk of acute and chronic graft-versus-host disease. Thus, the use of appropriate T-cell reduction, but not depletion might reduce this risk. The hazards of graft rejection and a higher relapse rate can be avoided by maintaining a portion of the T-cells in the graft. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1000-fold reduction of T-cells. These purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation and can be used in the following instances: 1. As hematopoietic stem and progenitor cell transplantation instead of bone marrow cells, from HLA-identical family donors; 2. for increasing the stem cell numbers from HLA-mismatched or three HLA-loci different family donors in order to reduce the incidence of rejection but without increasing the T-cell number; 3. boosting of poor marrow graft function with stem cells from the same family donors; 4. transplantation from volunteer matched unrelated donors; 5. split transplantation of CD34+ and T-cells; 6. addition of ex vivo expanded CD34+ cells to blood cell or bone marrow transplantation; 7. generation of antigen specific immune effector cells and antigen presenting cells for cell therapy.     

Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood

We have prospectively evaluated the feasibility and results of the biotin-avidin immunoadsorption method (Ceprate SC system) for a phase I/II study of T-cell depletion of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) for allogeneic transplantation. Twenty consecutive patients, median age, 40 years (21 to 54) and diagnoses of chronic myeloid leukemia in chronic phase (n = 5), acute myeloblastic leukemia (n = 7), acute lymphoblastic leukemia (n = 2), chronic myelomonocytic leukemia (n = 1), refractory anemia with excess of blasts in transformation (n = 3), histiocytosis X (n = 1), and chronic lymphocytic leukemia (n = 1), were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (13 Gy; 4 fractions). HLA identical sibling donors received G-CSF at 10 microg/kg/d subcutaneously (SC); on days 5 and 6 (19 cases) and days 5 to 8 (1 case) donors underwent 10 L leukapheresis. PBPC were purified by positive selection of CD34+ cells using immunoadsorption biotin-avidin method (Ceprate SC) and were infused in the patients as the sole source of progenitor cells. No growth factors were administered posttransplant. The median recovery of CD34+ cells after the procedure was of 65%. The median number of CD34+ cells infused in the patients was 2.9 (range, 1.5 to 8.6) x 10(6)/kg. The median number of CD3+ cells administered was 0.42 x 10(6)/kg (range, 0.1 to 2). All patients engrafted. Neutrophil counts >500 and >1,000/microL were achieved at a median of 14 days (range, 10 to 18) and 15 days (range, 11 to 27), respectively. Likewise, platelet counts >20,000 and >50,000/microL were observed at a median of 10 days (range, 6 to 23) and 17 days (range, 12 to 130), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine plus methylprednisolone. No patient developed either grade II to IV acute or extensive chronic GVHD. After a median follow-up of 7.5 months (range, 2 to 22) three patients have relapsed, and one of them is again in hematologic and cytogenetic remission after infusion of the donor lymphocytes. Two patients died in remission: one on day +109 of pulmonary aspergillosis and the other on day +251 of metastasic relapse of a previous breast cancer. Sixteen of the 20 patients are alive in remission after a median follow-up of 7.5 months (range, 2 to 22). In conclusion, despite the small number of patients and limited follow-up, it appears that this method allows a high CD34+ cell recovery from G-CSF mobilized PBPC and is associated with rapid engraftment without significant GVHD, and with low transplant related mortality.     

Effects of beta2 adrenoceptor agonists on T-cell subpopulations

The aim of the present communication is to determine the effects of beta2 adrenoceptor agonists on growth and cytokine secretion using allergen-specific T cells. Four beta2 adrenoceptor agonists were administered at therapeutically relevant doses (salbutamol 1-2 microM; salmeterol 0.03-0.06 microM; terbutaline 0.56-1.12 microM, and fenoterol 0.7-1.4 microM to: a) Cultures of human peripheral mononuclear cells (PBMC) b) Positively selected CD4+ and CD8+ subsets, c) Allergen-specific T-cell lines (TCL). Drug effects on growth kinetics and the secretion of IL-4, IL-5, INF-gamma and IgE following T-cell stimulation were investigated. Comparing the growth inhibitory effect of the 4 beta2 agonists at 2 different concentrations, using 12 PBMC, 10 CD4+ and CD8+ and 10 TCL cultures, the following patterns were observed: PBMC-, CD4+- and CD8+-cultures: salmeterol, followed by salbutamol and fenoterol, was a more potent inhibitor than terbutaline. In long-term TCL-cultures, salmeterol was the most potent drug, followed by fenoterol. No significant differences were observed between salbutamol and terbutaline. TCL secretion of IL-4 and IL-5 (TH2 cytokines) was also significantly inhibited. In one patient, INF-gamma secretion (TH1/THO cytokine) could be enhanced by drug administration. High IgE secretion, from 1% remaining B cells in one of the patients, following PHA+IL-2 stimulation, could be reduced by the drugs. The results showed that the beta2 agonists could influence T-cell growth and function. The changes regarding cell function were individual and related to T-cell phenotypes secreting TH1/THO or TH2 cytokines. These results suggest that administration of beta2 adrenoceptor agonists could be beneficial, not only for bronchodilation, but also for suppressing the underlying inflammatory process dominated by TH2-like cytokine secretion.     

Alloantigen presenting capacity, T cell alloreactivity and NK function of G-CSF-mobilized peripheral blood cells

In this study we addressed whether the proportion and the function of antigen presenting cells (APC), T and NK lymphocytes are modified in the apheresis product of six healthy donors who received a stem cell mobilizing treatment with glycosylated G-CSF at 10 microg/kg/day x 5 days s.c. Flow cytometry analysis showed comparable percentages of HLA-DR+, CD19+, CD86+, CD80+ and CD1a+ cells in preG-CSF-peripheral blood mononuclear cells (preG-PBMC) and after mobilization in G-PBMC, whereas the proportion of CD14+ monocytes significantly increased in G-PBMC (3+/-1% vs 17+/-8%, P = 0.003). Analysis of lymphocyte subsets in preG-PBMC and G-PBMC showed similar proportions of CD3+, CD4+, CD8+ and CD28+ T cells, but a significantly lower percentage of CD16+ (11+/-7% vs 4+/-1%, P=0.01), CD56+ (15+/-6% vs 5+/-2%, P= 0.008), CD57+ (16+/-9% vs 5+/-2%, P=0.04), CD25+ (19+/-2% vs 9+/-6%, p=0.009) and CD122+ (5+/-2% vs 2+/-1%, P = 0.05) cells in G-PBMC. Unfractionated preG-PBMC and G-PBMC were irradiated and tested in primary mixed leukocyte culture (MLC) with two HLA-incompatible responders and induced efficient alloresponses in four of six cases, whereas G-PBMC stimulated poorly in the remaining two cases. Also, in allo-MLC with irradiated G-PBMC we detected lower amounts of IFN-gamma (P = 0.04) and of IL-2 (P = 0.06) than in allo-MLC with preG-PBMC. Furthermore, freshly isolated preG-PBMC and G-PBMC from each donor exerted comparable allogeneic responses to HLA-incompatible irradiated mononuclear cells in all cases. However, G-PBMC showed no NK activity against K562 target cells at any effector:target ratio tested. These data suggest that normal G-PBMC may prevent Thl alloresponses, maintain efficient alloreactivity to HLA mismatched antigens and have impaired NK activity.     

CD14+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells induce secretion of interleukin-6 and G-CSF by marrow stroma

The ability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) to induce secretion of cytokines in primary long-term marrow cultures (LTC) or in the human marrow stromal cell line HS23 was compared with that of marrow mononuclear cells. Equal numbers of G-PBMCs or marrow mononuclear cells were added to stromal cultures, supernatants were harvested at day 4 and levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, G-CSF, and tumor necrosis factor alpha (TNF alpha) were determined. G-PBMCs induced 21.4-fold higher levels of IL-6 and 12.5-fold higher levels of G-CSF in LTC cocultures compared with marrow mononuclear cells and induced 20.6-fold more IL-6 and 6.3-fold more G-CSF when added to HS23 cells. Experiments using sorted populations of CD20+, CD3+, and CD14+ cells showed that CD14+ cells within G-PBMCs were responsible for triggering the production of IL-6 and G-CSF. The effect did not require cell-cell contact and was inhibited when neutralizing antibodies to IL-1 alpha and IL-1 beta were used in combination. In these experiments, the greater stimulating ability of G-PBMCs is most likely attributable to the greater number of CD14+ cells in G-PBMCs (26.1+% +/- 2.3%) compared with marrow (2.5% +/- 0.8%), because equal numbers of CD14+ cells sorted from marrow and G-PBMCs showed comparable ability to induce IL-6 and G-CSF when placed directly on stromal cells.     

Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony-stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.     

Mechanisms associated with defective TH1 cytokine production in HIV infection

Qualitative and quantitative changes in immune functions of different T-cell subsets associated with infection by human immunodeficiency virus type 1 (HIV-1) were analyzed by flow cytometric assessment of intracytoplasmic cytokines. The T(H)1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), were produced by both CD4 and CD8 T-cell subsets. When normal peripheral blood mononuclear cells (PBMC) were activated in culture, both cytokines were produced predominantly by CD4 (CD4) cell and only a minor fraction of normal CD8 cells produced these cytokines. In the cultures of PBMC from HIV-1-infected individuals (HIV+PBMC), more HIV+CD8 cells produced IL-2 and IFN-gamma. Production of IFN-gamma by HIV+CD4 cells was markedly reduced, while IL-2nd tumor necrosis factor-alpha (TNF-alpha) production by HIV+CD4 remained relatively intact until the disease progressed further. Normal CD4 cells which were isolated by using a cell sorter, FACSCalibur was still able to produce IL-2 and TNF-alpha. But for full production of IFN-gamma, normal CD4 required some accessory cells, the identity of which could not yet be established.     

Interleukin-1 alpha-mediated promotion of long-term alloengraftment and short-term neutrophil expansion does not require the presence of either donor or host T cells

In earlier studies, we showed that a 14-day continuous subcutaneous infusion of recombinant human interleukin (IL)-1 accelerated neutrophil recovery and enhanced long-term chimerism in a bone marrow (BM) transplant model in which T-cell-depleted BALB/c donor BM was given to irradiated C57BL/6 fully allogeneic recipients. We have extended these studies to a model entirely devoid of donor and host T cells. In the model, donor BALB/c congenic severe combined immunodeficient (C.B-17-scid/scid) BM cells are T cell depleted. The cells are then transplanted into adult irradiated C57BL/6 hosts that have been thymectomized and treated with anti-CD4 and CD8. When IL-1 alpha was delivered subcutaneously using a mini-osmotic pump, it enhanced short-term neutrophil recovery and longer term alloengraftment despite the absence of T cells in the donors and the hosts. Therefore, T cells were not required for the promotional effects of IL-1 alpha on neutrophil recovery and alloengraftment. Studies also showed that the potency of the IL-1 alpha effects was related to the degree of donor cell engraftment, which was related to the irradiation dose and the presence of T cells. We conclude that IL-1 alpha can augment post-BM transplantation hematopoietic recovery and alloengraftment via a T-cell-independent mechanism by favoring donor allogeneic hematopoietic progenitor cell competition over limited numbers of host progenitor cells.     

Effect of FLT3 ligand and granulocyte colony-stimulating factor on expansion and mobilization of facilitating cells and hematopoietic stem cells in mice: kinetics and repopulating potential

We have previously identified a cellular population in murine bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex (MHC) barriers without causing graft-versus-host disease. Here we investigated the effect of flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of facilitating cells (FC) and HSC into peripheral blood (PB). Mice were injected with FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or both in combination. The number of FC (CD8(+)/alpha betaTCR-/gamma deltaTCR-) and HSC (lineage-/Sca-1(+)/c-kit+) was assessed daily by flow cytometry. Lethally irradiated allogeneic mice were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF showed a highly significant synergy on the mobilization of FC and HSC. The peak efficiency for mobilization of FC (21-fold increase) and HSC (200-fold increase) was reached on day 10. Our data further suggest that the proliferation of FC and HSC induced by FL in addition to the mobilizing effect mediated by G-CSF might be responsible for the observed synergy of both growth factors. Finally, the engraftment potential of PBMC mobilized with FL and G-CSF or FL alone was superior to PBMC obtained from animals treated with G-CSF alone. Experiments comparing the engraftment potential of day 7 and day 10 mobilized PBMC indicate that day 10, during which both FC and HSC reached their maximum, might be the ideal time point for the collection of both populations.