Robert W. Grady, Johannes Gerdes and Stefano Rivella de Sousa, Eliezer A. Rachmilewitz, John D. Hood, M. Domenica Cappellini, Patricia J. Giardina, Scholzen, Amy Chadburn, YiFang Liu, Margrit Kernbach, Bettina Baron-Lühr, Matteo Porotto, Maria Ilaria V. Libani, Ella C. Guy, Luca Melchiori, Raffaella Schiro, Pedro Ramos, Laura Breda, Thomas -thalassemia β erythropoiesis in Decreased differentiation of erythroid cells exacerbates ineffective

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-gamma (IFN-gamma), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.

Inducible costimulator (ICOS) has been recognized as a costimulatory molecule essential in T cell activation. Studies with ICOS inhibition or ICOS−/− recipients have demonstrated prolonged allograft survival after heart or liver transplantation in animal models. We hypothesized that inhibition of ICOS on alloreactive T cells would ameliorate the development of graft-versus-host-disease (GVHD) in recipients of an allogeneic bone marrow transplantation (allo-BMT). To analyze the expression of ICOS on donor T cells, we transferred CFSE-labeled donor T cells into irradiated allogeneic recipients and observed an increased expression of ICOS on alloreactive T cells compared to non-alloreactive T cells. We then performed GVHD experiments in two models with full MHC class I and II disparity and observed significantly less GVHD morbidity and mortality in recipients of ICOS−/− donor T cells. Interestingly, in GVHD/graft-versus-tumor (GVT) experiments, ICOS−/− donor T cells displayed intact GVT activity, while their GVH activity was diminished. We are currently performing experiments to assess the alloreactivity of ICOS−/− T cells in vivo, the infiltration of ICOS−/− donor T cells into GVHD target organs, and the degree of GVHD damage in target organs by histopathological analysis. In conclusion, we have found that ICOS−/− donor T cells cause less GVHD morbidity and mortality in comparison to wild-type donor T cells while preserving GVT activity. This data suggests that strategies to inhibit ICOS could be useful for the prevention and/or treatment of GVHD in recipients of an allo-BMT.

Inducible costimulator (ICOS) is a member of the B7 family that is expressed on activated and memory T cells and is involved in the regulation of TH1 and TH2 effector cytokine production after CD3/TCR stimulation. Studies with ICOS inhibition or ICOS−/− recipients have demonstrated prolonged allograft survival after heart or liver transplantation in animal models. To study the role of ICOS expression on alloreactive T cells in graft-versus-host disease (GVHD), we used allogeneic MHC class I and II disparate hematopoietic stem cell transplantation (allo-HSCT) models. We first analyzed the expression of ICOS by transferring CFSE-labeled donor T cells into irradiated allogeneic recipients and observed an increased expression of ICOS on alloreactive T cells compared to non-alloreactive T cells. We then studied B6-ICOS−/− alloreactive T cells and found intact proliferation in vivo (as determined by adoptive transfer of CFSE labeled T cells and donor T cell numbers in the spleen of allo-HSCT recipients), intact cytotoxicity, intact up regulation of activation markers, but decreased IFN-γ production in vitro. We then performed GVHD experiments in two models with full MHC class I and II disparity and observed significantly less GVHD morbidity and mortality in recipients of ICOS−/− donor T cells. Furthermore, histopathological analysis demonstrated less GVHD in all target organs (skin, liver, small bowel and large bowel) of recipients of ICOS−/− splenic T cells compared to recipients of wild type T cells. We harvested target organs (spleen, thymus, liver and gut) on days 7, 14, and 21 to examine donor T cell content (naïve and activated T cells) and found no significant difference in the total T cell numbers and subpopulations. Interestingly, in GVHD/graft-versus-tumor (GVT) experiments, ICOS−/− donor T cells displayed intact GVT activity, while their GVH activity was diminished. We then tested the levels of IFN-γ in the sera of mice undergoing GVHD and observed decreased serum levels in recipients of B6-ICOS−/− T cells. In conclusion, alloreactive ICOS−/− donor T cells display less GVHD morbidity and mortality due to decreased IFN-γ production, while proliferation, infiltration and GVT activity remain intact. These data suggests that strategies to inhibit ICOS could be useful for the prevention and/or treatment of GVHD in recipients of an allo-HSCT.

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintained and differentiate in stroma-dependent, long-term bone marrow cultures (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Chang, and T. D. Allen. 1990. Molecular Control of Haemopoiesis). However, 70-80% of primitive progenitors capable of reinitiating secondary stromal cultures (LTBMC-initiating cells [IC]) are lost over a period of 5 wk in such cultures. We have recently described a novel "stroma-noncontact" culture system, in which hematopoietic progenitors are separated from the stromal layer by a 0.4-/~m microporous filter membrane. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater extent than in "Dexter" cultures. However, still only 50% of the originally seeded LTBMC-IC are recovered at week 5. Since maintenance of primitive progenitors may depend not only on growth-promoting factors but also on factors that inhibit differentiation and/or proliferation, we evaluated the effect of macrophage inflammatory protein 1 ol (MIP-lo 0 or "stem cell inhibitor'" in combination with the growth-inducing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stroma-noncontact cultures. We demonstrate that addition of MIP-lot alone to stroma-noncontact cultures does not change the number of LTBMC-IC present after 8 wk, indicating that this factor may not directly inhibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL-3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their terminal differentiation. However, LTBMC-IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP-lc~ plus IL-3. This effect depends on soluble (ill-defined) stromal factors, and results from a direct interaction of these cytokines with the progenitor population or its progeny, but not the stroma.

BACKGROUND:Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution.METHODS:To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration.RESULTS:IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality.CONCLUSION:IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.

Correction to: Bone Marrow Transplant (2002)29, 453–456. doi:10.1038/sj.bmt.1703372 In the above paper reference 22 is incorrect. The correct reference appears below.

Autologous transplantation is a treatment option for relapsed childhood acute lymphoblastic leukemia (ALL) in second complete remission (CR2) when a suitable donor is not available. In an attempt to prevent relapses originating from graft leukemic contamination, the experimental protocol of in vitro purification of leukapheretic products with monoclonal antibodies (MoAbs), previously reported for adults, was adopted in 11 of 12 consecutive patients (median age, 9 years) with B cell precursor ALL in CR2 after late relapse (median, 37; range, 31–51 months after the onset) enrolled between July 1997 and July 1999 at a single pediatric center. At a median of 12 days after the mobilizing chemotherapy followed by G-CSF, a median of 13.9 (range, 5.9–18.7) × 10 6 CD34 + cells/kg were collected from each patient and a median of 7.5 (range, 4.1–12.6) × 10 6 CD34 + cells/kg underwent the purification procedure. The first step of immunorosetting allowed a one-log reduction of the total cell count, by eliminating more than 90% of the CD11b + cells; the second step, performed after incubation with anti-CD19 MoAbs, allowed the depletion of 99% (range, 93–100) of the CD19 + cells, kept within the magnetic field of the immunodepletion column, with a median recovery of 73% (range, 55–87) of the collected CD34 + cells. Molecular analysis assessed the in vitro eradication of detectable leukemic cells. A median reinfusion of 5.2 (range, 3.2–9.1) × 10 6 CD34 + cells/kg for each patient (median viability, 90%), after conditioning with the ‘TBI-VP16-CY’ regimen, allowed prompt engraftment and immunological reconstitution; no patients experienced severe transplant-related toxicity or major infections. One patient relapsed 7 months after transplantation, while 10 patients are alive in clinical and molecular remission, at a median follow-up of 29 months (range, 15–40) (2-year EFS, 89%, s.e. 9). In conclusion, the procedure proved to be reproducible for pediatric purified autografting, highly efficient concerning stem cell recovery and depletion of leukemia-lineage specific cells, and promising in terms of final outcome.

One hundred and 43 consecutive pediatric patients (June 1985-December 1996) with at least 18 months of follow-up, were considered: most of the patients (111/143, 77.6%) underwent allogeneic BMT. The median follow-up was 5.7 years. Overall survival and 5 years EFS were 48.6% and 46.9%, respectively. For patients who underwent allogeneic BMT from HLA-identical siblings, the 5 years EFS for ALL was 75% in 1st CR, 60.4% in 2nd CR, 22.3% in > 2nd CR and 86.7% for AML in 1st CR. The EFS for Allo-BMT in "good" and "poor" prognosis patients was 68.6% and 21.8%, respectively (p value = 0.001). Early mortality in Allo-BMT patients was 17.7% between 1985-1990 and 10.3% between 1991-1996. Early treatment-related organ complications occurred mostly in patients who underwent BMT from an unrelated or a mismatched family donor. Late toxicity was evaluated in 57 patients (median follow-up of 82 months): none of the patients complained of significant late cardiac or respiratory dysfunction. With regards to growth, 18/57 patients (31.6%) lost more than two height centile channels. Three cases of thyroid neoplasms were observed. Evaluation of psychosocial functioning, studied in 39 patients who had at least 2 years of follow-up in CR, did not reveal any evident quality of life impairment. The possibility of curing childhood hematological malignancies is based on a global pediatric and multidisciplinary approach. A continuous need to improve results in terms of EFS and quality of life suggests that further multicenter prospective studies should be carried out.

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP(+) cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP(+) cells, all present within the CD3(+) population. with CD4(+) and CD8(+) cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34(+) cells were also susceptible, Varying from 6% to 10% GFP(+) cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP(+) T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored, In all cases the difference in percentage of GFP(+) cells did not correlate with the percentage of S/G(2)/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cell lines in vitro, we tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. We have cloned a full- length human ALDH1 cDNA and used retroviral vectors to transduce it into human (U937) and murine (L1210) hematopoietic cell lines that were then tested for resistance to maphosphamide, an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced L1210 and U937 cells (50% inhibition concentration [IC50], approximately 13 mumol/L). The resistant phenotype was specifically caused by ALDH1 overexpression as shown by its reversion by disulfiram, a specific ALDH1 inhibitor. ALDH1 transduction into peripheral blood human hematopoietic progenitor cells also led to significant increases (4- to 10-fold; IC50, approximately 3 to 4 mumol/L) in cyclophosphamide resistance in an in vitro colony-forming assay. These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.