Emergence of antitumor cytolytic T cells is associated with maintenance of hematologic remission in children with acute myeloid leukemia

Although the graft-versus-leukemia effect of allogeneic bone marrow transplantation (BMT) is of paramount importance in the maintenance of disease remission, the role played by the autologous T-cell response in antitumor immune surveillance is less defined. We evaluated the emergence of antileukemia cytotoxic T-lymphocyte precursors (CTLp's) and the correlation of this phenomenon with maintenance of hematologic remission in 16 children with acute myeloid leukemia (AML), treated with either chemotherapy alone (5 patients) or with autologous BMT (A-BMT, 11 patients). Antileukemia CTLp's were detectable in 8 patients in remission after induction chemotherapy; none of them subsequently had a relapse. Of the 8 patients who did not show detectable CTLp frequency while in remission after induction chemotherapy, 7 subsequently experienced leukemia relapse. In patients undergoing A-BMT, molecular fingerprinting of the TCR-Vbeta repertoire, performed on antileukemia lines, demonstrated that selected antileukemia T-cell clonotypes, detectable in bone marrow before transplantation, survived ex vivo pharmacologic purging and were found in the recipient after A-BMT. These data provide evidence for an active role of autologous T cells in the maintenance of hematologic remission and also suggest that quantification of antileukemia CTLp frequency may be a useful tool to identify patients at high risk for relapse, thus potentially benefiting from an allogeneic antitumor effect.     

One hundred twenty-five adult patients with primary acute leukemia autografted with marrow purged by mafosfamide: a 10-year single institution experience

A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The median follow-up period was 64 months (range, 3 to 126). There were 84 acute myeloblastic leukemias (AMLs) and 41 acute lymphoblastic leukemias (ALLs). At time of autologous BM transplantation (ABMT); 64 AMLs were in first complete remission (CR1), and 20 were in second CR (CR2); 35 ALL were in CR1, and 6 were in CR2. The median age of the patients was 33 years (range, 16 to 55). The median interval between achieving CR and autografting was 5 months (range, 1.3 to 23). The pretransplant regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation. All patients were grafted with autologous BM treated in vitro with mafosfamide used at levels individually adjusted in 95 patients and at a standard dose in 30 patients. The initial richness in granulomacrophagic progenitors (CFU-GM) of the harvested BMs was 5.16 x 10(4) CFU-GM/kg (range, 0.55 to 33). After mafosfamide purging, the residual CFU-GM number was 0.021 x 10(4)/kg (range, 0 to 1.78). The probability of successful engraftment was significantly higher and the time to engraftment was significantly shorter in ALL. Of 33 patients grafted with BM containing no residual CFU-GM, those with AML (n = 22) had platelet recoveries that were significantly longer than those for AML patients receiving BM with residual CFU-GM. At 8 years, patients autografted in CR1 for AML and ALL had a leukemia-free survival (LFS) of 58% and 56%, respectively, with a relapse incidence (RI) of 25% and 37%, respectively. Patients autografted in CR2 for AML had an LFS of 34% and an RI of 48% at 5 years. The incidence of late relapses was significantly higher in ALLs. By multivariate analysis, four factors were found to influence favorably engraftment in addition to a diagnosis of ALL, a younger age, ABMT performed in CR1, the adjusted dose technique of purging, and a shorter interval from CR to ABMT. Two factors were correlated with a better outcome. (1) The LFS was significantly higher and the transplant-related mortality significantly lower in patients who received richer BM. (2) The RI was significantly lower in patients autografted within 150 days from CR. Our results reinforce the view that ABMT is one approach to improve the outcome of adult patients with acute leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Predictive value of colony-forming unit assays for engraftment and leukemia-free survival after transplantation of chemopurged syngeneic bone marrow in rats.

We evaluated the efficacy of in vitro clonogenic assays for acute myeloid leukemia (AML) (CFU-Leuk) and granulocyte-macrophage progenitor cells derived from normal bone marrow (BM) (CFU-GM) to predict hematopoietic engraftment, median survival time (MST) and leukemia-free survival (LFS) in LBN rats that received injections of untreated or drug-treated AML and/or normal BM cells. Injection of untreated AML cells resulted in a log-linear relationship between AML cell dose and time of death from leukemia; LBN rats given 10(6) cells died with AML (MST, 24 days; range, 19-28) after injection. A minimum of 0.5-1.0 X 10(6) untreated normal BM cells was needed to insure satisfactory hematopoietic reconstitution in at least 50% of lethally irradiated LBN rats. After ex vivo incubation with graded concentrations of 4-hydroperoxycyclophosphamide (4HC) or bleomycin (BLEO), LBN AML or normal BM cells were cultured for CFU-Leuk or CFU-GM and injected into untreated or lethally irradiated syngeneic recipients. Over a variety of drug concentrations (4HC, 3-30 micrograms/ml; BLEO, 100-10,000 mU/ml) and cell doses (10(6)-10(7)/animal) examined, the log-kill estimates derived from in vitro CFU-Leuk assays correlated with the observed MST or LFS. Recovery of greater than 1% CFU-GM from 4HC- or BLEO-treated suspensions of normal BM was associated with satisfactory engraftment in lethally irradiated LBN rats. Clonogenic assays also predicted for engraftment and LFS in animals that received mixtures of AML and normal BM cells (1:10) treated with 4HC and/or BLEO. We conclude that CFU-Leuk and CFU-GM assays are useful screening techniques to develop and evaluate strategies for ex vivo purging with chemotherapeutic agents in this preclinical model of autologous marrow transplantation for AML.     

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.     

Lymphoid subsets in acute myeloid leukemias: Increased number of cells with NK phenotype and normal T-cell distribution

Summary Natural killer (NK) and T subsets were analyzed with appropriate dual labeling by flow cytometry in peripheral blood (PB) (66 cases) and bone marrow (BM) (55 cases) from patients with de novo AML in order to determine: (a) their distribution at diagnosis, (b) the correlation between PB and BM in NK subpopulations, (c) their relationship with the clinical and hematological disease characteristics, and (d) the changes occurring upon achieving complete remission (CR). NK cells defined by the expression of CD56 in the absence of CD3 were significantly increased at diagnosis and their levels in PB correlated with those of BM. By contrast, NK subsets defined by CD16 expression (CD16+ CD2+ and CD16+ CD2– NK-cell subsets) as well as T lymphocytes with NK activity (CD56+ CD3+), although increased in PB, displayed normal levels in BM. An additional observation of interest was the expansion of an immature NK population lacking CD16 Ag expression (CD56+CD16–). AML cases were divided into two groups according to the absolute number of NK cells in PB; patients with the highest levels showed an increased proportion of blast cells in PB (p=0.01), monocytic subtypes (p=0.03), and expression of CD11b, CD14, and CD4 antigens (p=0.05). Infections at diagnosis were not related to the level of NK cells. In 19 patients who achieved complete remission the number of CD56+CD3– cells tended to be reduced to within the normal range. Other T-cell populations, including the CD4 naive and memory cells, were also explored, their distribution being normal in the PB of AML patients. By contrast, the cytotoxic subset CD8+/CD57+was significantly increased (p< 0.001). These data point to the existence of marked alterations of NK cells in AML patients, possibly reflecting a host-tumor immunological interaction.     

The use of recombinant cytokines for enhancing immunohematopoietic reconstitution following bone marrow transplantation. II. The influence of lymphokines on CFU-GM colonies from human untreated, ASTA-Z or Campath-1M treated bone marrow

Patients undergoing bone marrow transplantation (BMT) are subjected to the risk of pancytopenia in the immediate post-BMT period. Recipients of bone marrow (BM) autografts purged in vitro by chemical agents such as mafosfamide (ASTA-Z) are even more likely to develop a delayed engraftment. In a previous study in mice, we showed earlier immunohematopoietic reconstitution after syngeneic marrow grafting with BM cells precultured with single or combined cytokines. In this work we determined optimal culture conditions for the use of single and various cytokine combinations in order to activate progenitor cells following in vitro cultures of untreated, ASTA-Z purged or T cell-depleted human BM preparations prior to BMT. The best single cytokine for enhancing CFU-GM was found to be GM-CSF (0.1 mg/ml) which produced an increase of up to 8-fold over controls after 3 days' incubation. Addition of recombinant human interleukin 3 (rhIL3) (0.1 mg/ml) to rhGM-CSF had an additive effect. The same enhancing effect of in vitro CFU-GM by both cytokines was observed following ASTA-Z purging of BM cells obtained from patients undergoing autologous BMT. Similarly, depletion of lymphocytes from BM using the monoclonal rat anti-human lymphocyte antibody Campath-1M and human complement did not diminish the beneficial influence of rhIL3 and rhGM-CSF. When combined with rhGM-CSF and rhIL3, rhIL1 and rhIL2 had no appreciable enhancing effect on CFU-GM and occasionally even reduced it. We suggest that enhancement of differentiation of hematopoietic progenitor cells may be accomplished by in vitro incubation of BM cells without continuous administration of cytokines in vivo following BMT.     

Comparison of three techniques for the ex vivo elimination of T cells from human bone marrow

The high incidence of graft-vs-host disease (GVHD) in allogeneic bone marrow transplantation (BMT) is a significant cause of morbidity and mortality, despite pharmacological prophylactic regimes. Laboratory technologies have been developed to eliminate the immunocompetent T-lymphocyte, the proposed effector cell in the GVHD reaction. In this study, three techniques for the ex vivo purging of T cells from human bone marrow (BM) were compared. BM treatment groups consisted of T-cell depletion by the monoclonal antibodies OKT3 and OKT11A plus complement (MoAb + C), soybean agglutination followed by sheep erythrocyte rosette depletion, or triple rosetting with neuraminidase-treated sheep erythrocytes. Mean final cell yields were 37.2 +/- 4.0%, 2.8 +/- 0.8%, and 2.5 +/- 1.3%, respectively, while final yields of BM progenitor cells, assayed in the double-layer soft-agar CFU-c assay, were 28.5 +/- 6.5%, 3.9 +/- 2.1%, and 10.5 +/- 3.8%, respectively. The three techniques were comparably efficient in elimination of mitogenic responses to irradiated allogeneic lymphoblastoid cells and cytotoxic lymphocyte responses of the BM. Immunofluorescence after T-cell depletion showed greater than 97.5% of all OKT3-positive cells to be eliminated by each technique. Despite the fact that all three techniques were effective in T-cell depletion, treatment with anti-T-cell MoAbs + C proved less labor-intensive and resulted in higher cell yields.     

Recovery of the ability to induce immune resistance against L1210 lymphatic leukemia in semisyngeneic CD2F1 mice after lethal irradiation and reconstitution with bone marrow purged of leukemia with mafosfamide (ASTA Z 7654)

Balb/c x DBA/2 F1 (CD2F1) mice were lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells (SBMT) untreated or treated with mafosfamide (ASTA Z 7654) for ex vivo purging of semisyngeneic L1210 leukemia (TBI + SBMT or TBI + SBMT-Maf mice, respectively). At various times after irradiation and reconstitution mice were injected intraperitoneally four times at weekly intervals with 10(6) immunogenic L1210-Maf cells (L1210 cells treated in vitro with mafosfamide for inhibition of their growth in vivo). As positive controls we immunized normal (non-irradiated) CD2F1 mice. Full resistance against L1210 leukemia (as compared to normal immunized mice) could be obtained in TBI + SBMT and TBI + SBMT-Maf mice when the immunization procedure was started from day +28 or day +56 after transplantation, respectively. Earlier immunization of TBI + SBMT mice (from day +14) or TBI + SBMT-Maf mice (from day +14 or +28) caused only partial resistance against the leukemia.     

Newborn blood can engraft adult mice without inducing graft-versus-host disease across non H-2 antigens

Cells derived from human cord blood were recently used instead of bone marrow (BM) for transplantation. However, several questions concerning the potential of these cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce a graft-versus-host disease (GVHD) remain unanswered. Here we used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non H-2 antigens. Few mature T cells were found in NBB as in adult BM and both contain around 10% to 20% SCA-1+ pluripotent progenitor cells. Yet numerous immature CD4+CD8+ TCR alpha/beta low Thy-1high T cells were present in NBB. In contrast, adult peripheral blood (PB) exhibits a mature T-cell phenotype and contains few progenitor cells. The injection of blood pooled from one to three newborn mice resulted in the engraftment of 71% to 86% of F1 recipients, which survived more than 100 days without clinical signs of GVHD. The injection of BM leads to 100% survival whereas the injection of adult PB resulted in rapid mortality, both without signs of GVHD. In NBB-engrafted F1 surviving mice, T-cell reconstitution preceded B-cell reconstitution, and the degree of donor cell chimerism increased progressively with time. Additionally, 2 to 4 months after transplantation, T cells from these mice were tolerant to host non H-2 antigens but kept reactivity against third-party Ags. Host specific tolerance in NBB-engrafted F1 mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells were able to reconstitute the lymphohematopoietic system of lethally irradiated adult mice without inducing GVHD against incompatible non H-2 antigens. Thus, this experimental model in vivo may be relevant to the human cord blood transplantation.     

Purging of G-CSF-mobilized peripheral autografts in acute leukemia with mafosfamide and amifostine to protect normal progenitor cells

In the present study the in vitro growth of CFU-GM from PBPC of patients with AML (n = 11), purged with mafosfamide alone or a combination of mafosfamide and amifostine, was compared to historical controls of mafosfamide-purged bone marrow (AML CR1, n = 16). Two patients were transplanted with mafosfamide and mafosfamide/amifostine pretreated PBPC autografts. The in vitro experiments demonstrated a significantly higher resistance of peripheral blood derived CFU-GM to mafosfamide (median ID95 190 microg mafosfamide/ml) compared with bone marrow derived CFU-GM (median ID95130 microg/ml). Preincubation with amifostine significantly further increased the median ID95 to 245 microg/ml. The clinical results showed short recovery times for neutrophils >500/microl (9 and 13 days) and platelets >20 000/microl (12 and 21 days) and stable long-term engraftment with one relapse at day +118 and one patient in CR at day 760 after transplantation. The in vitro results show a significant advantage of PBPC over bone marrow-derived progenitors for purging with mafosfamide. Furthermore, a protective effect from mafosfamide of amifostine on normal progenitors could be demonstrated. The clinical results demonstrate the clinical feasibility of using mafosfamide-purged autologous PBPCT without impairing the short-term and long-term repopulating capacities of the autografts.     

The effect of a combined purging with mafosfamide and hyperthermia on murine haemopoietic stem cells and leukaemogenic cells.

The in vitro purging effect of mafosfamide combined with hyperthermia was studied in a murine model. The survival of normal clonogenic progenitors (d-9 CFU-S and CFU-GM) and WEHI 3-B leukaemic clonogenic cells (CFU-L) were compared. At 37 degrees C, CFU-L proved to be significantly more sensitive to mafosfamide than either of the normal progenitors. When mafosfamide was combined with 42.5 degrees C hyperthermia for 1 h, an additive effect was observed: at a dose of 5 micrograms/ml mafosfamide, the survival of CFU-L was nearly two logs lower than that observed at 37 degrees C, while 37.7% of CFU-S survived the purging. The repopulating capacity of surviving bone marrow CFU-S was not altered: a similar 60 d survival of supralethally irradiated recipients transplanted with comparable graft sizes from purged or non-purged bone marrow was observed. When bone marrow suspensions containing WEHI 3-B cells were purged with 5 micrograms/ml mafosfamide at 42.5 degrees C and the minimal amount of bone marrow cells needed to protect supralethally irradiated mice were injected, leukaemia incidence was reduced to less than 10% as opposed to 100% of those injected with untreated bone marrow. Our results suggest that ex vivo hyperthermia may enhance the purging efficiency of mafosfamide.     

T-cell reconstitution and expansion after hematopoietic stem cell transplantation: 'T' it up!

Adoptive immunotherapy is the isolation and infusion of antigen-specific or nonspecific lymphocytes. Adoptive therapy with T cells may have a role in replacing, repairing, or enhancing immune function damaged by cytotoxic therapies, and rapid lymphocyte recovery may improve outcome after autologous and allogeneic stem cell transplantation (SCT). Recently, a plethora of information on the basic mechanisms of T-cell biology and regulation of cellular immune responses has emerged, permitting the development of new forms of adoptive cell therapy. Efficient ex vivo culture method for T-cell subsets affords the possibility of adoptive transfer of T cells engineered with enhanced capacity for central memory, effector cytotoxicity, Th1, Th2, veto cell, and T regulatory functions. Studies show that homeostatic T-cell proliferation is important for effective adoptive immunotherapy and pretreatment with chemotherapy may enhance the effects of infused T cells. Replicative senescence, in part due to telomere erosion, likely limits successful adoptive immunotherapy, though it may be possible to maintain T-cell pools by enforced expression of telomerase. Clinical trials now demonstrate that it is possible to enhance immune reconstitution after SCT with cytokines or infusions of ex vivo costimulated expanded T cells. These data all support the premise that adoptive therapy can accelerate reconstitution of cellular immunity with enhanced antitumor effects following SCT.     

Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-versus-leukemia reactivity, and tolerance after bone marrow transplantation

Graft rejection, mixed chimerism, graft-versus-host disease (GVHD), leukemia relapse, and tolerance are interrelated manifestations of immunologic reactivity between donor and host cells that significantly affect survival after allogeneic bone marrow transplantation (BMT). In this report, a mouse model of BMT, in which the donor and host were compatible at the major histocompatibility complex (MHC), was used (1) to examine the interrelationship of pretransplant conditioning and T-cell content of donor BM with regard to lymphoid chimerism and GVHD and (2) to determine how these factors affected graft-versus-leukemia (GVL) reactivity and donor-host-tolerance. AKR (H-2k) host mice were administered optimal or suboptimal total body irradiation (TBI) as pretransplant conditioning followed by administration of BM cells from B10.BR (H-2k) donor mice with or without added spleen cells as a source of T lymphocytes. Transplanted mice were injected with a supralethal dose of AKR leukemia cells 20 and 45 days post-BMT to assess GVL reactivity in vivo. The pretransplant conditioning of the host and T-cell content of the donor marrow affected the extent of donor T-cell chimerism and the severity of GVH disease. GVL reactivity was dependent on transplantation of mature donor T cells and occurred only in complete chimeras. Transplantation of T-cell-deficient BM resulted in the persistence of host T cells, ie, incomplete donor T-cell chimerism, even when lethal TBI was used. Mixed chimerism was associated with a lack of GVL reactivity, despite the fact that similar numbers of donor T cells were present in the spleens of mixed and complete chimeras. In this model, moderate numbers of donor T cells facilitated complete donor T-cell engraftment, caused only mild GVHD, and provided a significant GVL effect without preventing the subsequent development of tolerance after conditioning with suboptimal TBI. In contrast, severe, often lethal, GVHD developed when the dose of TBI was increased, whereas tolerance and no GVH/GVL reactivity developed when the T-cell content of the marrow was decreased.     

Interleukin 2 production in bone marrow of normal individuals and patients associated with B-cell chronic lymphocytic leukaemia

T-cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) have abnormal T4/T8 ratios and functions. Previously, we demonstrated that peripheral blood (PB) mononuclear cells from B-CLL patients secrete significant amounts of interleukin 2 (IL2) with an apparent dysregulation of accessory cells controlling this production. In this study, IL2 production was investigated in PB and in bone marrow (BM) from patients with previously untreated B-CLL, mostly in early stages of the disease, and compared to normal donors. A significant secretion was observed in both PB and BM from B-CLL patients after stimulation by phytohaemagglutinin (PHA), with lower amounts in patients with nodular involvement of BM, as compared to interstitial or diffuse involvements. To explore the role of accessory cells in controlling IL2 production, we added phorbol ester or indomethacin to the culture system or irradiated the cells before culture. Phorbol ester significantly increased the IL2 secretion in both the PB and the BM of B-CLL patients. The irradiation or the addition of indomethacin did not enhance the IL2 production in PB from B-CLL patients. However, IL2 secretion increased in the BM cells from B-CLL patients after addition of indomethacin or prior irradiation, in a similar way to that observed in PB and BM of normal controls, suggesting an apparent normal control of the IL2 production in BM from B-CLL patients. In normal controls, we demonstrated that IL2 secretion per T-cell from BM was 5.4-fold greater than that from normal PB, suggesting a very efficient role of accessory cells controlling IL2 production in normal BM.     

A comparative study of bone marrow and peripheral blood CD34+ myeloblasts in acute myeloid leukaemia

To examine the differences between primitive bone marrow (BM) and peripheral blood (PB) myeloblasts in acute myeloid leukaemia (AML), we compared CD34⁺ myeloblasts of paired BM and PB samples from 14 AML patients in terms of surface phenotype, homing and engraftment in a xenogeneic transplantation model, and gene expression, based on microarray studies and quantitative polymerase chain reaction. While there was no significant difference in surface phenotypes between these two populations, in vivo assay showed significantly better homing potential of PB CD34⁺ cells than BM CD34⁺ cells. Significant correlation between homing and engraftment in AML samples was also noted. In addition, gene expression profiling of CD34⁺ cells from five paired BM and PB leukaemic samples showed that genes involved in G-protein and prostaglandin signalling, chemotaxis and stress response, cell proliferation and apoptosis were down-regulated in PB CD34⁺ myeloblasts. These data suggested that circulating primitive myeloblasts in AML are functionally different from those residing in the marrow compartment, and such differences may be partly regulated by the BM microenvironment.     

Analysis at the clonal level of T-cell phenotype and functions in severe aplastic anemia patients.

The aim of this study was to analyze at the clonal level the phenotype and functions of T cells from patients with severe aplastic anemia (SAA). For this purpose we studied 175 T-cell clones obtained from peripheral blood (PB) and bone marrow (BM) of four SAA patients and 97 clones from two healthy controls. The percentage of CD8+ T-cell clones obtained from the patients' PB and BM was higher, but not significantly (P = .07 and P = .14, respectively), than that obtained in controls. A higher proportion of T-cell clones from SAA patients exhibited lectin-dependent cytolytic activity and especially natural killer-like activity when compared with controls (PB: P less than .01, P less than .05; BM: P less than .05, P less than .01, respectively). Lymphokine release was tested before and after mitogen stimulation. A number of patients' clones were able to release interferons (IFNs) spontaneously (PB: 28.6% v 0%, P less than .05; BM: 28.6% v 0%, P less than .10). After mitogen stimulation, patients' BM T-cell clones produced IFNs in greater proportions (90.9% v 46.7%, P less than .01) and in greater quantities (PB: 25.5 arbitrary units [AU]/mL v 5.7 AU/mL, P less than .03; BM: 26 AU/mL v 9.1 AU/mL, P = .011) as compared with controls. Tumor necrosis factor (TNF) activity was not found in supernatants of unprimed T-cell clones. After mitogen stimulation, PB T-cell microcultures produced TNF alpha in greater proportions (97.9% v 72.2%, P less than .01) and, also in this case, in greater quantities (PB: 7.2 AU/mL v 1.5 AU/mL, P = .007; BM: 9.9 AU/mL v 1.5 AU/mL, P = .003) than controls. In conclusion, T-cell clones from SAA patients exhibit predominantly a CD8+ phenotype, a greater cytotoxic activity, and can be shown to produce greater quantities of suppressor lymphokines when compared with controls.     

Quantitative lymphocyte subset reconstitution after allogeneic hematopoietic transplantation from matched related donors with CD34+ selected PBPC grafts unselected PBPC grafts or BM grafts.

CD34+ cell selection of PBPC after harvest from G-CSF-treated allogeneic donors results in a more than 200-fold depletion of T lymphocytes in the graft and has been used to reduce the incidence of acute GVHD post transplant. Since transplantation with T cell-depleted BM grafts is associated with a delay in immune reconstitution and an increase of opportunistic infections, we evaluated the immunological reconstitution of patients with hematologic malignancies after therapy followed by CD34+-selected PBPC34 transplantation from matched related donors. Lymphocyte subset reconstitution over the first 12 months post transplant and the incidence of infections were evaluated in 12 patients receiving PBPC34 grafts and compared to that of patients after transplantation with PBPC without CD34+ enrichment (n = 20) or unmanipulated bone marrow grafts (BM; n = 15). PBPC34 grafts contained 264-fold fewer T lymphocytes (median 0.53 x 10(6) kg/body weight) than PBPC grafts and 36-fold fewer than BM grafts (140 x 10(6)/kg and 19 x 10(6)/kg, respectively). Despite a two log depletion of T cells in the PBPC34 grafts, T lymphocyte reconstitution appeared comparable among the three transplant groups over the first 12 months. A positive patient CMV serostatus pretransplant was correlated with a faster T cell reconstitution in all transplant groups. GVHD prophylaxis with methylprednisolone delayed B lymphocyte reconstitution. The incidence of infections post transplant did not appear to be increased in the PBPC34 group compared with the PBPC and BMT groups. It remains to be shown in larger prospective trials, whether these promising preliminary data of lymphocyte reconstitution and the clinical course after transplantation with PBPC34 can be confirmed.     

Bacterial Contamination of Autologous Bone Marrow: Reinfusion of Culture-Positive Grafts Does Not Result in Clinical Sequelae during the Posttransplantation Course

Objectives : Microbiological cultures and posttransplantation course were analyzed in order to investigate the incidence and clinical significance of bacterial contamination of autologous bone marrow (BM) grafts. Methods : Cultures were obtained from BM after collection, BM concentrate after processing, contaminated/cryopreserved BM at thawing, and from peripheral blood (PB) following autologous BM transplantation (ABMT). The posttransplantation course of patients grafted with culture-positive BM was recorded and compared with patients who underwent ABMT with noncontaminated BM grafts. Results : In 239 BM grafts processed, the incidence of microbiological contamination was 26.4% (n = 63). Fifty marrow grafts were contaminated by bacteria from the skin flora: coagulase-negative Staphylococcus (CNSC), Propionibacterium, and Coryne-bacterium species (79%). Thirty-eight patients underwent ABMT (day 0) with cryopreserved culture-positive BM, and 32 patients were evaluable for microbiological cultures at thawing: in 10 of 32 BM grafts CNSC was found prior to reinfusion. Following ABMT, PB cultures revealed CNSC in 5 of 38 patients between days +4 and +12. However, the late occurrence of positive PB cultures after BM reinfusion made a relationship between BM CNSC and PB CNSC unlikely. In 33 of 38 patients, no graft-contaminating bacteria were detected in PB. Comparison of the posttransplantation course of patients who received contaminated BM with that of patients grafted with noncontaminated BM showed no significant differences concerning time to engraftment, febrile days, and days on antibiotics. Conclusion : (1) Collection and/or ex vivo processing can result in microbiological contamination of BM grafts predominantly with bacteria from the skin flora, and (2) only CNSC can be cultured at thawing from previously contaminated/cryopreserved BM. Since patients undergoing ABMT usually receive oral antibiotics from beginning of the conditioning regimen which are active against CNSC, no further administration of antibiotics is recommended for the reinfusion of bacterially contaminated BM grafts.     

Variable cytotoxicity of diphtheria toxin 388-granulocyte-macrophage colony-stimulating factor fusion protein for acute myelogenous leukemia stem cells

In this study, the utility of DT388-granulocyte-macrophage colony-stimulating factor (GM-CSF) for the ex vivo purging and direct administration to patients with acute myeloid leukemia (AML) is tested using clonogenic assays, long-term cultures (LTC), and NOD/SCID mice as assays for leukemic progenitors.We compare the ability of 24-hour exposure to 0.3 microg/mL (4 nM) DT388-GM-CSF to kill AML colony forming cells (CFC) and the more primitive AML progenitors detected after 6 weeks in stromal cocultures (AML LTC-initiating cells or AML LTC-IC) and after 8 weeks in NOD/SCID mice.AML samples (n = 10), expressing a mean of 35 to 1466 GM-CSF receptors/blast, showed mean (range) percent kills of AML CFC and LTC-IC of 61 (17-98) and 46 (0-94) respectively with a direct correlation (r = 0.69) between the % kills detected in the in vitro assays. Among 5 evaluable samples the percent reduction in AML cell engraftment in NOD/SCID marrow following ex vivo DT388-GM-CSF treatment varied from 38% to 100%. 40% to 56% of normal bone marrow CFC and 31% to 48% of normal LTC-IC survived the same ex vivo treatment (n = 3). In subsequent experiments, NOD/SCID mice received AML blast cell injections intravenously followed in 24 hours by 1.5 microg DT388-GM-CSF daily intraperitoneally for 5 days. A reduction of marrow blast cells was seen with 7 of 9 samples tested 4 to 12 weeks post one course of toxin. Repeating the 5-day course of toxin 2 or 3 times at 4-week intervals did not improve the response, while delaying administration until 4 to 8 weeks post AML cell injection reduced the toxin's effectiveness (n = 5).This fusion toxin may prove useful for in vitro purging of stem cell harvests from selected AML patients and for direct administration to such patients.