ALBERT

Description: As recent case reports have suggested that proto-oncogene activation by replication-deficient retroviral vectors may trigger leukemia in mice and men, there is an urgent need for models allowing predictive investigations into the pathomechanisms of this complication. Previously, we have shown that dose-escalated replication-defective retrovirus vectors (such as HaMDR1 or SF91dsRedpre) may trigger combinatorial leukemia-initiating events in single cells, with a probability that allows prospective studies in simple mouse models (C57Bl/6). Meanwhile, we have performed molecular analyses for 5 different leukemias, harboring 6 to 12 vector insertions according to Southern blot. 34 insertions recovered from these clones by LM-PCR and LAM-PCR showed that 4 leukemias had combinatorial hits in at least one established proto-oncogene (class 1 genes: Hivep1, Mllt3, Fli1, HoxA7, Brd2, Evi1, Csfr3, Bcl11a, Ski) with at least one hit in other signaling genes (class 2 genes). One leukemia for which 2 insertions remain to be identified showed at least 3 class 2 hits, so far without a class 1 event. All leukemias presented with hits in genes involved in cytoplasmic signal cascades along with genes contributing to nuclear regulatory networks. Some of these leukemias had a normal karyotype. A comparison with 62 insertion sites from healthy control animals (determined in peripheral blood DNA harvested 7 months after transplantation) revealed a significant enrichment of class 1 hits in leukemias (14-fold over expected and 3-fold over control). Healthy animals still showed 6 hits (~10%) in class 1 genes, obviously not being sufficient for leukemia induction. Three of these occurred upstream of Evi1, one upstream of Nfic, one upstream of Pdclg2, and one in Bcl11a. Interestingly, upstream vector insertion was preferred in class 1 gene hits. Overall, insertions recovered from both normal and leukemic samples revealed an overrepresentation of hits within (or upstream of) known or predicted genes (89 and 90%, respectively). In line with the data of a related murine marking study (Kustikova, Fehse et al., ASH 2004), our study strongly suggests that single hits in class 1 or class 2 genes are not sufficient to induce leukemia in mice. However, certain combinations of such events in single cells may well be leukemogenic with a given latency. Our findings establish a powerful approach for both oncogene discovery and safety validation in gene therapy.

Description: The integration of a retroviral vector as used in hematopoietic gene therapy trials produces a transition sequence from the vector DNA into the genomic DNA and may thus serve as a stable molecular marker unique for each cell clone. High sensitive linear amplification mediated PCR (LAM-PCR) allows the detection of specific retroviral integration sites. Thus it is possible to determine the clonal composition of the hematopoietic system in vivo (1). We could show that the hematopoietic repopulation in human SCID-X1 patients was derived from various, long-lived progenitor cell clones indicating retroviral transduction into pluripotent cells (manuscript submitted). In two cases of lymphoproliferative disorder after successful SCID-X1 gene therapy integration of the retroviral vector into the LMO-2 oncogene was probable the main reason for malignancy (2). The distribution of integration sites over the whole genome, the potential preference for integration at certain loci and which cells receive genetic correction and engraftment are therefore of considerable interest. Recently, another gene therapy trial has successfully corrected 4 infants suffering from SCID-X1. A GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene was used for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. We analyzed lymphoid and myeloid DNA from all patients. The transduction efficiency of T lymphocytes and myeloid cells was up to 100 and 1 %, respectively. In vivo clonality analysis of CD3+ cells showed a polyclonal composition 1 to 2 years after transplantation. The myeloid repopulation also consisted of various different clones. These data may indicate transduction of pluripotent and long term active stem or progenitor cells. We here report on more than 300 sequenced integration sites of the patients, whereas 250 sequences could be assigned unequivocally to a unique locus. So far, no vector integration in the LMO-2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to specific regions of genes. We found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strongly support the presumption that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells and provide an important insight into the integration manner of GALV-pseudotyped MLV-based vectors.

Description: SAP is a small protein, consisting of a single SH2 domain which is mutant in humans with X-linked lymphoproliferative disease. Patients with XLP are affected by fatal EBV infections and malignant B cell lymphomas. The increased risk for B cell lymphomas is suggested to result from impaired immunosurveillance of B cell proliferation by T cells. Here, we investigated the role of SLAM/SAP for activation of effector cells with cytotoxic activity (CIK cells), which are generated by unspecific stimulation of the T cell receptor and addition of exogenous IL-2 as described previously. The TCR activation on day +1 resulted not only in a short peak of activated cells, but activation continued and increased in combination with IL-2. We observed a striking peak of SLAM (Signaling Lymphocyte Activation Molecule) on day +6 in form of extracellular detectable CD150 as well as at the level of proteins and mRNA. Interestingly, the cytotoxic activity and the amount of SHP-2 protein showed a similar pattern as the parameters mentioned above but were shifted one day. Comparing these data for correlation, we observed a significant correlation between cytotoxic activity and CD150 expression pattern (P

Description: To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.

Description: Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-γ (IFN-γ) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-γ– or GM-CSF–primed human monocytes can be completely suppressed by preincubation with LPS (fromEscherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E2 (PGE2), tumor necrosis factor- (TNF-), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-γ–induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.

Description: Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncatedPseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti–Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti–Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.

Description: In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

Description: Constitutive expression of a rat CD44 variant isoform, rCD44v4-v7, on murine T cells accelerates immune responsiveness. Because prolonged immunodeficiency can be a major drawback in allogeneic bone marrow transplantation, we considered it of special interest to see whether repopulation of lethally irradiated syngeneic and allogeneic mice may be influenced by constitutive expression of the rCD44v4-v7 transgene. When lethally irradiated syngeneic and allogeneic mice were reconstituted with bone marrow cells (BMC) from rCD44v4-v7 transgenic (TG) or nontransgenic (NTG) mice, the former had a clear repopulation advantage: thymocytes expanded earlier after reconstitution and, as a consequence, higher numbers of lymphocytes were recovered from spleen and lymph nodes. Lymphocytes also displayed functional activity in advance to those from mice reconstituted with BMC from NTG mice. Most importantly, after the transfer of BMC from TG mice into an allogeneic host, the frequency of host-reactive T cells decreased rapidly. Apparently, this was due to accelerated induction of tolerance. Because these effects were counterregulated by an rCD44v6-specific antibody, it is likely that they could be attributed to the rCD44v4-v7 TG product. Thus, expression of a CD44 variant isoform at high levels facilitated reconstitution with allogeneic BMC by accelerated establishment of tolerance and the regaining of immunocompetence.

Description: Constitutive expression of a rat CD44 variant isoform, rCD44v4-v7, on murine T cells accelerates immune responsiveness. Because prolonged immunodeficiency can be a major drawback in allogeneic bone marrow transplantation, we considered it of special interest to see whether repopulation of lethally irradiated syngeneic and allogeneic mice may be influenced by constitutive expression of the rCD44v4-v7 transgene. When lethally irradiated syngeneic and allogeneic mice were reconstituted with bone marrow cells (BMC) from rCD44v4-v7 transgenic (TG) or nontransgenic (NTG) mice, the former had a clear repopulation advantage: thymocytes expanded earlier after reconstitution and, as a consequence, higher numbers of lymphocytes were recovered from spleen and lymph nodes. Lymphocytes also displayed functional activity in advance to those from mice reconstituted with BMC from NTG mice. Most importantly, after the transfer of BMC from TG mice into an allogeneic host, the frequency of host-reactive T cells decreased rapidly. Apparently, this was due to accelerated induction of tolerance. Because these effects were counterregulated by an rCD44v6-specific antibody, it is likely that they could be attributed to the rCD44v4-v7 TG product. Thus, expression of a CD44 variant isoform at high levels facilitated reconstitution with allogeneic BMC by accelerated establishment of tolerance and the regaining of immunocompetence.