The Oncologist, Vol. 2, No. 6, 410–413,
December 1997
© 1997 AlphaMed Press
SPECIAL SECTION
PHYSICIAN EDUCATION |
CD34+ Selection: The Basic Component for Graft Engineering
Stefan Miltenyi
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Correspondence:
Stefan Miltenyi, Miltenyi Biotec GmbH, Friedrich-Ebert Strasse 68, Bergisch Gladbach, 51429, Germany. Telephone: +49-2204-8096; Fax: +49-2204-85197.
The hematopoietic system consists of a network of stem and progenitor cells of varying degrees of maturity interacting with other cells that act in supportive and regulatory capacities (Fig 1
). Many cell types have been identified by their in vivo or ex vivo growth potential, i.e., colony assays, or by physical characteristics like cell-surface markers which can be identified by monoclonal antibodies. One of the most studied antibody markers is the CD34 antigen which is present on the most primitive hematopoietic progenitor cells.
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Figure I. The hematopoietic tree. Hematopoiesis begins with pluripotential stem cells that differentiate into mature cells under the influence of various cytokines and growth factors. Figure is courtesy of Amgen Inc., Thousand Oaks, CA.
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The CD34 antigen was initially identified on a myeloid leukemia cell line [1] and has been found on 0.5% to 29% of cells from normal bone marrow. These hematopoietic progenitor cells are known to circulate in low level(<1%) in the peripheral blood and can be collected by standard apheresis techniques. The use of a growth factor such as Filgrastim (r-metHuG-CSF) greatly increases the number of these progenitor cells in the circulation, a process termed mobilization. The CD34 antigen-positive (CD34+) cell population has been found to contain the complete population of hematopoietic colony-forming cells, but expression of the antigen is lost from the cell surface of blood cells during the normal maturation process.
The CD34+ cell population is heterogeneous, but contains cells required for engraftment and can be used as the starting point for further selection of stem or progenitor cell subtypes. Cells that express the CD34 antigen include both early stem cells as well as more committed progenitors such as CFU-GM (granulocyte-macrophage colony-forming cells). Further classification of the CD34+ population as being either positive or negative for the expression of Lin, Thy-1, CD33 and CD38 is under way to determine the phenotype of the most primitive self-renewing cell. A variety of cell-forting methods have been developed which are based on the binding of cells to macroscopic immunoaffinity surfaces, (i.e., planning, rosetting, or affinity columns), or fluorescence-activated cell sorting. More recent technologies using magnetic cell sorting have been used widely as well. The adoption of these systems in the clinic will depend upon the level of specificity that they provide, the minimized loss of viable CD34+ cells during the processing, and the volume of blood or marrow that can be readily and quickly separated. Figure 2 shows some CD34+ cells that were obtained using magnetic cell sorting.
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Figure 2. Photomicrographs of CD34 cells separated by magnetic-activated cell sorting (MACS). Figure is courtesy of Amgen Inc., Thousand Oaks, CA.
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AUTOLOGOUS BLOOD OR MARROW GRAFT AFTER HIGH-DOSE CHEMOTHERAPY
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The use of CD34+ cell populations from blood (peripheral blood progenitor cells [PBPC]) [2, 3] or bone marrow [4] has been shown to provide durable multilineage hematopoietic reconstitution. The use of positive selection (infusing only the cells selected because of their CD34+ antigen-surface expression) has theoretical advantages for patients. While providing the cells required for engraftment, these techniques simultaneously deplete the graft of CD34- tumor cells without additional manipulations that can result in stem cell loss and delayed engraftment. Reported reductions in tumor contamination with the current systems have varied from 0.5 to 5 logs. No valid comparisons can be made, however, due to differences in starting populations, tumor types, and tumor-cell detection methods. The theoretical residual contaminating cell rate calculated from the purity of the separated population may provide a way to compare various selection systems. Although the elimination or reduction of contaminating tumor cells from the autograft seems intuitively desirable, the contribution of infused tumor cells to relapse is uncertain and a focus of continued investigation.
While certain leukemic cells express CD34, most solid tumors are CD34- (Table 1). Exceptions to this are tumors of vascular endothelial cell origin, which may express the CD34 antigen. As methods of tumor detection improve by use of polymerase chain reaction and other techniques, our ability to determine those rare situations where CD34 separation may enrich for tumor cells will be more precise.
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ALLOGENEIC BLOOD OR MARROW GRAFT AFTER HIGH-DOSE CHEMOTHERAPY
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Allogenic transplantation has often used graft engineering techniques to remove all donor T lymphocytes or subsets of donor T lymphocytes given the patient morbidity and mortality associated with graft-versus-host disease (GVHD) (Table 2). The original use of marrow from a close HLA-matched donor is now gradually being challenged by the use of mobilized PBPC in sibling-matched settings, and randomized trials of this approach are under way by the European Group for Blood and Marrow Transplantation (EBMT) [5].
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Table 2. Comparison of selectively or totally T cell depleted allografts in patients receiving cyclosporine after transplantation [15]
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Because of the greater number of "contaminating" lymphocytes in a PBPC allograft, many investigators are exploring the use of graft-engineering techniques to remove all, or subsets of, donor T lymphocytes. Positive CD34 selection has been reported to provide a two-to-five-log depletion of T cells bearing the CD3, CD4, CD8, and CD56 antigens. MACS-based sorting method provides T-cell depletion at the upper end of this range [6].
Some T-lymphocyte populations and natural killer cells may contribute to or provide a graft-versus-leukemia (GVL) effect [7], as well as the prevention of some infectious complications (including Epstein-Barr virus lymphoproliferative disease). For this reason, there is increasing interest in separating specific T-cell subsets which would be infused later when their biologic effect is required [8, 9]. Innovative and sophisticated methods are being developed to separate the allograft into component parts which may be cryopreserved for future use.
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AUTOLOGOUS OR ALLOGENEIC BLOOD OR MARROW GRAFT AFTER HIGH-DOSE CHEMOTHERAPY FOR SEVERE AUTOIMMUNE DISEASE
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There is no truly curative therapy for severe autoimmune diseases. Treatment has often consisted of chemotherapy to suppress the lymphocyte population, which is the genesis of the autoimmune reaction. Because it has been noted anecdotally that some patients undergoing allotransplant for malignancies have been cured of underlying autoimmune diseases, more clinical work in this area has been planned. The EBMT is developing treatment strategies where both allogeneic and autologous transplants will be administered to carefully selected patients with multiple sclerosis, scleroderma, lupus, and other diseases. The role of T-cell depletion (particularly in the autologous setting) will be evaluated as part of these trials, as certain T-cell populations cause the pathophysiology, and are targeted for ablation. The role of infusion of other favorable T-cell subsets may be explored in the future, as well.
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EX VIVO EXPANSION OF CD34+ PROGENITOR CELLS OR MARROW
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Given the low incidence of progenitor cells in the blood or marrow, it is important to emphasize the role of CD34+ selection for the ex vivo manipulation. To begin the procedure with "clean" populations of progenitor cells is preferable, as the inclusion of other unnecessary or unwanted cell types would result in a high density of cells that would use up nutrients, produce toxic metabolites, and perhaps additional cytokines that may be detrimental to optimal hematopoietic cell growth. Also, plating unselected cell samples at optimal cell densities results in unmanageably large cell cultures. Highly purified CD34+ selected starting populations produce a more predictable expanded cell product.
Reduction of the Duration or Depth of Cytopenia
Despite advances with high CD34+ cell doses and cytokine use after transplant, the period of profound pancytopenia during which patients are at risk of infection and hemorrhage until the infused marrow cells repopulate the peripheral blood with mature progeny remains at least 10 days. Any substantial decrease in this cytopenia would lead to the rapid adoption of outpatient transplant, and make tandem or multiple transplants realistic as a therapeutic modality.
Additional Progenitor Cells
The ability to expand progenitor cells may make transplant possible for some patients with extremely low CD34+ cell numbers in their grafts, i.e., poor mobilizers. For other patients who mobilize PBPC in normal amounts, ex vivo expansion may reduce their apheresis requirements.
Additional Depletion of Unwanted Cells
Many primary cancer cells grow poorly, if at all, in cultures optimized for the growth of myeloid progenitor cells, suggesting that ex vivo expansion allows for the selective expansion of normal hematopoietic cells, with the important extra benefit of providing additional tumor depletion. In addition, a culture optimized for hematopoietic expansion does not support the optimum growth/maturation of mature T lymphocytes.
Use of Small Graft Populations Such as Umbilical Cord Blood
CD34+ cells can be obtained from umbilical cord blood (UCB) [10], and these cells can be expanded greatly [11]. The number of hematopoietic progenitor cells in a UCB aliquot is low, however, compared with the number of marrow or mobilized peripheral blood cells that one would ideally use to transplant an adult patient. The future promise of the use of UCB will depend on its feasibility, and the role that successful expansion of hematopoietic progenitors may bring to the field may be extremely important. This field and its potential have been discussed in depth [12, 13].
Gene Therapy
A selected CD34+ cell population contains the progenitor cells used for transfection in gene therapeutic strategies. The controlled manipulation of cycling of stem cells ex vivo is required to enable effective gene insertion for gene therapy. In addition, a major advantage of starting with only the rare CD34+ cell population is that the cell number, and thus the volume of viral supernatant required for the process, are significantly reduced.
The focus of these strategies is to replace either missing or defective genes, so that normal protein will be produced in the progeny of CD34+ cells to provide a circulating pool. To provide an ample population of genetically modified cells for infusion, ex vivo expansion of these populations would be desirable.
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THE CD34- FRACTION: AUTOLOGOUS OR ALLOGENEIC CD34 CELLS USED FOR ADOPTIVE IMMUNOTHERAPY
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As stated above, positive-selection techniques may minimize the transfer of undesirable cell populations in the graft at the time when hematopoietic reconstitution is of key importance. Later in the process of recovery, however, other factors come into play such as using a lymphocyte subset for immune protection against tumor relapse or infection. The ability to use different components of a graft at different times during the patient’s treatment confers great flexibility in devising treatment strategies to the physician-investigator. The ability of some CD34+ separation systems to provide a viable and usable CD34- fraction which contains these other cell populations may prove instrumental in the generation of new treatment schemas.
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SUMMARY
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The implementation of clinical hematopoietic graft engineering has moved as fast as technologic advances have permitted. That CD34+-selected cells remain viable after cryopreservation, and that they are able to reconstitute hematopoiesis at the same rate as unmanipulated marrow or blood, have led the way into this rapidly expanding field. The clinical acceptance of these therapeutic manipulations will ultimately depend on the value of the clinical benefit that they provide to patients. The future use of elements from the CD34- fraction as "addbacks," and of the ex vivo-manipulated cells will be of great interest.
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REFERENCES
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- Shpall EJ, Jones RB, Bearman SI et al. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral blood progenitors and growth factors on engraftment. J Clin Oncol 1984;12:28–36.[Abstract]
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- Champlin R, Giralt S, Przepiorka D et al. Selective depletion of CD8-positive T-lymphocytes for allogeneic bone marrow transplantation: engraftment, graft-versus-host disease and graft-versus-leukemia. In: Worthington-White DA, Gee AP, Gross S, eds. Advances in Bone Marrow Purging and Processing. New York: Wiley-Liss, Inc., 1992:385-398.
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