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DEVELOPMENT OF CONDITIONS ALLOWING RESTORATION OF HEMOPOIESIS BY ALLOGENEIC PURIFIED AND IN VITRO MULTIPLIED PLURIPOTENT HEMOPOIETIC STEM CELLS

Objective

The contracts represent a joint effort of four Community laboratories to improve the scientific basis and the practice for the diagnosis and treatment of radiation accident victims. The participating laboratories have a long standing expertise in this area and, for many years, have cooperated closely in research and treatment. They now aim to develop a European network for radiation accident mangement to serve as a centre of expertise for the development of strategies to manage radiation accidents should they ever occur in the Community. Moreover, such a network will have an important role in the training of doctors and nurses in the particular handling of accident victims and, thereby, transmit the unique personnal experience of the participating groups to other institutions.
The purification of rhesus monkey and human bone marrowhaemopoietic stem cells was pursued by a combination of physical methods followed by positive selection of stem cells either on the basis of expression of class II (DR) major histocompatibility complex (MHC) antigens or expression of HPCA-1 (CD34) antigens. It was concluded that selection of haemopoietic stem cells (HSC) by means of the anti-CD34 monoclonal ICH3 conjugated to Protein A beads is a safe, rapid and reliable large scale method to enrich stem cells and to remove undesired cells from a basement membrane (BM) graft.

It has been recognised that proliferation of mouse haemoprietic stem cells in vitro is dependent on interleukin-3 and codependent on other haemoprietic growth factors, such as interleukin-1, interleukin-6 and granulocyte macrophage colony stimulating factor, as well as on more generally acting agents, such as hydrocotisone and adrenergic agonists. In addition, stem cells have histamine receptors. As yet, stimulation of proliferation of stem cells results after a few cell divisions in cell differentiation, leading to the production of pathway restricted progenitor cells and loss of stem cell capacity as tested in transplantation experiments. Thus, although interleukin-3 has been identified as a primary proliferative stimulus for stem cells, stem cell replication is probably dependent on a complex interplay of stimuli.

The research investigated 2 areas:
tests with immunosuppressive monoclonal antibodies as adjuvants to total body irradiation for bone marrow transplantation in mice;
the development of a method to purify and produce the lymphokine that suppresses the action of T-lymphocytes.

Based on the hypothesis that graft rejection has similar effector cells as graft versus host reactions, the immunosuppressive effectiveness of anti-LFA-1 was tested in rhesus monkeys in a graft versus host model (a fully mismatched donor recipient combination) as well as a host versus graft model (8.5 Gy X-rays as conditioning, using a 3-4 log T-lymphocyte depleted, fully mismatched bone marrow graft). Anti-LFA-1 appeared to be incapable of preventing lethal graft versus host disease or graft rejection and it is concluded that its immunosuppressive action as a single agent is weak. Therefore, the efficacy of such a function blocking antibody is probably codependent on other immunosuppressive agents.

By studying the kinetics of high molecular weight soluable suppressor factor (SUF) moiety mediated suppression it became evident that it interferes at an early stage of T-lymphocyte activation. The factor is not species specific. Its further characterisation and production awaits the development of a large scale purification method followed by molecular approaches.

This investigation concerns the development of a simple, rapid, safe and generally applicable method to positively select for and purify hemopoietic stem cells for routine use in autologous as well as allogenic bone marrow transplant (BMT). The method is based on recognition of the stem cells by the anti-human CD34 monoclonal antibody ICH3. ICH3 is a high avidity mouse immunoglobulin (Ig) G2a that neither modulates nor has any effector functions such as cytotoxicity. In pilot experiments, sorted ICH3 positive cells were shown to effectively reconstitute hemopoiesis in autologous lethally irradiated rhesus monkeys. To develop a method suitable for the large scale preparation of bone marrow (BM) stem cells, Protein A was covalently bound to immunomagnetic beads and ICH3 was conjugated to Protein A. Cells bound to the antibodies can then be eluted from the protein A beads by competitive elution using excess soluble lgG.

Experiments were performed to assess quality control and reproducibility of the ICH3: Protein A: immunomagnetic beads method and of allogeneic transplantation in major histocompatibility complex (MHC) matched, sex mismatched rhesus monkey donor/recipient pairs, to assess the potential of these stem cell concentrates, to establish the radiation dose required for acceptance of such highly purified cells and to establish the number of T-lymphocytes that can be allowed in a bone marrow graft without causing unacceptable graft versus host disease (GvHD).
A large number of experiments were done to ascertain reproducbility and quality control of the CD34 positive bone marrow fractions to be used for transplantation purposes. Using an optimal cells to beads ratio, the method appeared to be equally efficient for prefractionated stem cell concentrates obtained by density centrifugation and E-rosette sedimentation to remove residual T-lymphocytes, and for unfractionated bone marrow subjected to Ficoll centrifugation to remove granulocytes and red cells. In bot h cases, about 1% of CD34 positive cells were obtained, which contained on average 60 to 70% progenitor cells as measured by colonly formation in response to granulocyte macrophage colony stimulating factor (GM-CSF). It was established earlier by an assay based on measuring regeneration rate after autologous transplantation, that the number of regenerating stem cells in vivo in fluorescence activated cell sorter (FACS) sorted CD34 positive cell fractions runs closely parallel to the coentent of in vitro colony forming cells. It may be safely assumed that this result is also indicative of the number of regenerating stem cells in the isolated CD34 positive cells. The CD34 positive cell fractions can be easily depleted of a small fractions of monocytes and macrophages by a similar procedure using a CD11b monoclonal. The quality control of the fractions obtained was further done by measuring the forward and perpendicular light scatter, which showed that the majority of the cells had light scatter properties compatible with those of stem cells as identified in the window set in the light scatter plot of unfractionated bone marrow. In addition the content of T-lymphocytes was routinely measured CD4 and CD8 monoclonal antibodies which showed that in all cases the CD34 postitive cell fractions were depleted by 2 to 3 logs of T-lymphocytes. By these experiments a standard was set for CD34 positive fractions suitable for allogeneic bone marrow transplantation, using a method that handle equally well 10{6} as well as 10{10} bone marrow cells.

Because of the availability of suitable MHC-matched, sex-mismatched sibling donors for monkeys selected for allogeneic bone marrow transplantation, pilot experiments were started on the use of these fractions for allogeneic transplantation. For these purposes the regeneration rate of peripheral blood cells was studied in comparison to conventional T-lymphocyte depleted bone marrow grafts, and chimerism wasmonitored as a function of tim e after transplantation. Chimerism was determined in bone marrow by measuring the number of donor-type karyotypes in interleukin (IL)-3 and GM-CSF stimulated bone marrow samples, as well as in T-lymphocytes stimulated with IL-2 and phytohaemagglutinin (PHA). In addition, in 2 monkeys, 500000 (per kg body weight) peripheral blood T-lymphocytes were added to the bone marrow grafts in an attempt to establish an upper limit for the number of T-lymphocytes.
Conventional T-lymphocyte depleted control grafts following a relative low total body irradiation (TBI) dose of 7.4 Gy (orthovolt X-rays) will result in sustained partial bone marrow and peripheral blood T-lymphocyte chimersim without causing GvHD. The 2 monkeys treated in this way were used as standards. The CD34 positive cell engrafted as rapidly as the controls. Karyotyping 3 weeks after transplantation showed that this rapid engraftment was donor derived. However, after more prolonged periods of time, monkeys conditioned with 8.3 Gy TBI showed only 2 to 5% donor type karyotypes in their reconstituted bone marrow, suggesting that either the endogenous residual stem cells had taken over, or that the graft had been partially rejected. Surprisingly, however, donor type peripheral blood T-lymphocytes in these monkeys have been very high during the entire observation period, which is a result comparable to a successful bone marrow transplantation for severe combined immunodeficiency (SCID) in humans. To date, a suitable explanation for this peculiar type of split chimerism is not available. To exclude that this result was attributable to a limited capacity of CD34 positive cells to supply sustained bone marrow progeny, the conditioning TBI dose was rasied to 9 Gy. In 3 of 3 evaluable monkeys transplanted following 9 Gy TBI, bone marrow chimerism remained as high as in the conventional controls. These results demonstrate that CD34-positive cells are very well capable of producing myeloid as well as T-lymphocyte progeny for sustained periods of time, a feature that was hitherto unexplored. In addition, the CD34 positive grafts apparently require a more intensive conditioning regimen that the conventional control grafts.
The same data demonstrated that GvHD did not occur from grafts that contained 100000 T-lymphocytes per kg or less, but did occur, and lethally so, in 1 of 2 recipients of grafts to which 500000 peripheral blood T-lymphocytes were deliberately added. This result indicates that the upper limit of T-lymphocytes allowable in an MHC matched bone marrow graft, a situation in which GvHD is entirely determined by minor histocompatibility disparities, is between 100000 and 500000 per kg body weight.
The management of radiation accidents is typically hampered by the lack of information available on the extent of damage inflicted on the organism and on the amount of tissue spared from radiation which might bring about the recovery of the organism. It should now be possible to develop an expert system based on the experience of past accidents and to use a spectrum of biological indicators to determine the extent of damage, the likelihood of recovery and the risk of permanent damage with emphasis on effects on the haemopoietic and immune systems.

New treatment modalities have recently become feasible thanks to the development of bioengineered haemopoietic growth factors. It can be envisaged that a judicious treatment with these factors could allow an optimal stimulation to promote a more speedy and complete recovery of the haemopoietic and immune system.

The development of new techniques for the isolation of haemopoietic stem cells has enabled bone marrow transplantation to become a more reliable and safer procedure in cases of very severe radiation accidents. Moreover, new methods of supportive care have become available.

Finally, it may now be possible to reduce the risks to persons who have to participate in recovery operations by prophylactic measures. Haemopoietic injury could be mitigated after a planned radiation exposure during a rescue operation by reinfusing autologous stem cells taken previously from the patient. Such cells could be obtained most readily from blood. Factors,such as dextran sulphate, which mobilize stem cells from bone marrow into human blood so that greater numbers of stem cells can be collected, will be investigated. The logistics needed for the establishment of such banks will also be studied. (Ulm. Univ.).

Diagnosis and treatment of radiation accident victims must be based on a sound understanding of the underlying pathophysiological mechanisms of damage and recovery after total and partial body irradiation, including the definition of the conditions which might lead permanent damage.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

RADIOBIOLOGICAL INSTITUTE TNO
Address
Division For Health Research Lange Kleiweg 151
2280 HV Rijswijk
Netherlands