CLB, Sanquin Blood Supply Foundation, Department of Transfusion Technology, Stem Cell Laboratory, Laboratory for Experimental and Clinical Immunology, Academic Medical Centre, University of Amsterdam,
1 Department of Internal Medicine, Academic Medical Centre, Amsterdam,
2 Department of Haematology, University Hospital Utrecht, Utrecht,
3 Department of Paediatric Immunology, University Hospital for Children, `het Wilhelmina Kinderziekenhuis', Utrecht and
4 Department of Paediatrics, University Hospital Leiden, Leiden,The Netherlands
Correspondence to:
I. C. M. Slaper-Cortenbach, CLB Sanquin Blood Supply Foundation, Department of Transfusion Technology, Stem Cell Laboratory, PO Box 9190, 1006 AD Amsterdam, The Netherlands.
Abstract
Objective. In our laboratory, we have developed an immunorosette technique for the depletion of T cells from bone marrow transplants. Tetrameric complexes of monoclonal antibodies are able to form very stable immunorosettes, which are efficiently depleted with the aid of a blood cell separator. Major improvements over the original sheep red blood cell depletion are the use of human (patient or donor derived) erythrocytes instead of sheep-derived cells, and the possibility of using a closed system for separation in a cell separator. In contrast to bone marrow, mobilized haematopoietic stem cell transplants obtained after leucocytapheresis contain higher numbers of T cells. Therefore, a different approach is necessary.
Method. We have used two CD34 selection systems (IsolexTM 300SA and the ClinimacsTM) to perform T-cell depletions from peripheral blood stem cell (PBSC) transplants.
Results. Immunorosette T-cell depletion, with CD2/CD3 tetrameric complexes, of bone marrow transplants resulted in a mean 2.5 log depletion of T cells with a yield of 50% of the CD34+ cell population. Stem cell selection of PBSC transplants using one of the CD34 selection procedures resulted in a 4.5 log depletion of T cells for both systems, but with different results for the recovery of CD34+ cells. An increased yield of CD34+ cells was obtained with the ClinimacsTM procedure (57.9±9.0%) in comparison to the IsolexTM procedure (40.1±12.5%).
Conclusion. Our own immunorosette depletion technique and the two tested CD34 selection methods for stem cell transplants both resulted in a very efficient T-cell depletion with the recovery of 4060% of the CD34+ haematopoietic stem cells present in the transplant.
KEY WORDS: T cells, Depletion, Haematopoietic stem cell transplants.
In the past, several different T-cell depletion techniques have been developed to avoid graft-vs-host disease in an allogeneic stem cell transplantation setting. Kernan et al. [1] have applied an agglutination technique using soy bean agglutinin (SBA) followed by T-cell depletion using sheep red blood cells (SRBC). This method was the first and one of the most widely applied T-cell depletion techniques for the removal of T cells from bone marrow. Others have performed methods, for instance, based on the difference in cell size between T cells and haematopoietic stem cells, known as the counterflow elutriation technique [2], or antibody-mediated techniques, like complement-mediated cell lysis using Campath antibody [3].
In our institute, Lansdorp et al. [4] developed a technique using bivalent antibody complexes, known as tetrameric complexes. We have modified the application of this method and introduced an immunorosette procedure to couple the patient's own erythrocytes to target cells for efficient depletion. Originally, this technique was set up to deplete malignant cells from patients with B-cell malignancies [5], but we have also produced tetrameric complexes for the removal of T cells. This technique can now be used to deplete T cells from allogeneic or autologous bone marrow stem cell transplants.
Since peripheral blood stem cells (PBSC) are rapidly replacing bone marrow as a source of haematopoietic stem cells, other techniques have been developed for the removal of T cells. PBSC transplants from normal donors contain higher percentages of T cells, and often >10 times the total number of T cells. This necessitates another approach. CD34 cell selection is an efficient method to achieve T-cell removal. Several different methods for clinical application are commercially available with variable results. Cellpro was the first on the market with a Food and Drug Administration-approved column system (CeprateR SC) using biotin-labelled CD34 monoclonal antibody (MAb) in combination with avidin-coated Sepharose beads. This selection procedure results in a 4050% yield of CD34 stem cells with a purity of ~80%. Then, Baxter introduced the IsolexTM system for positive selection, using DynabeadsR to isolate the stem cells and a CD34-releasing peptide (PR 34+TM ) to dissolve the bond between the stem cells and the beads. This method also results in a 4050% yield of CD34+ cells, but with a much higher purity of >90% CD34+ cells. Recently, Miltenyi Biotec has introduced an immunomagnetic system, ClinimacsTM, using very small beads (colloidal super paramagnetic MicroBeads), which can efficiently enrich the stem cell population (>90% pure) like the IsolexTM system, but with a higher CD34 yield of ±65% [6]. Using this system, the beads, which are very small, are not removed from the surface of the CD34+ stem cells.
Autologous stem cell transplants have recently been introduced for the treatment of patients with autoimmune diseases [7]. Most of these diseases are believed to be T-cell mediated, so a depletion of autologous T cells seems indicated. However, an exact T-cell dose is not yet known, but a more extensive reduction than 1x105 T cells/kg body weight (BW) might not be necessary (van Bekkum [8]).
In this article, we present an outline of our results obtained with immunorosettes for the depletion of T cells from bone marrow, and CD34 selection for the depletion of T cells from PBSC.
Materials and methods
Allogeneic stem cell transplants
Patients suffering from several different haematological malignancies, who have an allogeneic donor available, were treated with high-dose chemotherapy and total body irradiation. On the day of transplant, bone marrow cells were taken from either HLA-identical siblings or matched unrelated donors, and sent to the cell-processing laboratories of the CLB in Amsterdam, or of the Academic Hospital in Utrecht. More recently, the use of PBSC transplants for rapid haematopoietic reconstitution in an HLA-identical setting has become more beneficial for the patients than using bone marrow as a source of stem cells. Donors were given 5 µg granulocyte-colony stimulating factor (G-CSF) for mobilization of PBSC. The leucocytapheresis procedures were performed at the Academic Medical Centre. The PBSC transplants were transported to the CLB and either CD34 selected directly or the next day. After selection, the cells were taken to the hospital and directly infused.
Autologous stem cell transplants
Autologous bone marrow transplants were harvested from children with juvenile chronic arthritis, according to a protocol described in this issue by Wulffraat and Kuis [9].
Techniques for T-cell depletion
Immunorosette depletion of bone marrow.
Tetrameric complexes are formed by the addition of cross-linking RaMIgG1 MAb to a mixture of MAbs, one directed against glycophorin A in the membrane of human erythrocytes and another T-cell-specific MAb (CD2 or CD3). These complexes are then bound to erythrocytes and the coated erythrocytes are washed. The bone marrow harvest is centrifuged, prior to depletion, and a buffy coat suspension is prepared to get rid of the excess erythrocytes. After addition of the coated erythrocytes to the bone marrow buffy coat cells, immunorosettes are formed. These immunorosettes are depleted using a Ficoll density separation (d=1.077 g/cm3 ) in an IBM 2991 cell processor and the light density cells are washed and cryopreserved.
CD34 selection of PBSC.
In our laboratory, we have used two different CD34 selection systems for the removal of T cells from PBSC transplants: IsolexTM 300SA (Baxter Biotech group, USA) and the ClinimacsTM (Miltenyi Biotec, Germany). Procedures were performed according to the manufacturers' instructions.
Quality control
The quality of the transplant was measured using the myeloid progenitor cell assay (CFU-GM, colony forming unit for granulocytes and monocytes) with human placental conditioned medium as a source of growth factors, and by monitoring the CD34 content. The efficacy of the T-cell depletion technique was measured either by means of the immunorosette method or by immunofluorescence using the FACscan flow cytometer (Becton Dickinson, Mountain View, CA, USA).
Results
Immunorosette depletion
The immunorosette technique was successfully applied to allogeneic bone marrow transplants from HLA-identical siblings (Table 1).
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In all CD2/CD3-depleted stem cell transplants, a supplement of unseparated bone marrow cells was necessary to reach 1x105 T cells/kg BW. This enabled transplantation of exact numbers of T cells in these patients. In four patients, the recovery of the CD34 haematopoietic stem cell population was measured and the CD34 yield was 49.1±18.8%.
Furthermore, we have used the same technique to reduce the number of T cells in autologous bone marrow transplants from children with juvenile chronic arthritis (Fig. 1). Here, the aim was to reach an even lower level of T cells: 1x104 T cells/kg BW. By performing a second round of T-cell depletion, this target number was reached in two out of six autologous transplants, with an average of 5.1±4.9x104 T cells/kg BW for all six patients.
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In Utrecht, the same CD2 and CD3 immunorosette procedure was performed on 34 allogeneic bone marrow transplants, resulting in a 2.3 log depletion of T cells (Fig. 2).
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Discussion
The main advantage of using the immunorosette depletion technique instead of the SBA/SRBC procedure is the fact that it can be performed in a closed system. Very stable immunorosettes are formed, which can easily be separated in the IBM cell processor. Furthermore, binding of the patient's own or donor-derived erythrocytes to the T cells circumvents the use of SRBC, which cannot be produced according to good manufacturing practice regulations. In our procedures, MAbs were used that were screened according to the CLB Biosafety Testing protocols, in the absence of bacterial and viral contamination. Moreover, combining the removal of immunorosettes within one round of density separation reduced the processing time from 9 to 6 h, thereby saving ~50% of the CD34+ cells. Using immunorosettes for the depletion of T cells from bone marrow, a significant removal of T cells occurred, averaging a 2.5 log T-cell depletion.
So far, we have only limited experience with the isolation of CD34+ cells from bone marrow (n=2), with varying results: 6% yield with the IsolexTM system and 50.2% with the Clinimacs. Apart from the prolonged selection procedure (density separation followed by selection), the CD34 selection methods are all very expensive. This method is not only costly because of the separation device, but also because the disposables, media and CD34 kits are expensive.
For PBSC transplants, however, we have so far tested both immunomagnetic systems of Baxter and Miltenyi. In a recently published study [11], the CD34 selection systems of Cellpro and Baxter were compared, resulting in a 3.4 median log T-cell depletion for the IsolexTM 300I system and a 2.9 log T-cell depletion for the CeprateR system.
Our own results indicate that similar T-cell depletion efficacies are being reached using the two immunomagnetic selection systems IsolexTM 300SA (Baxter) and the ClinimacsTM (Miltenyi).
In our laboratory, we are currently performing experiments to develop new methods using the tetrameric complexes for depletion of larger numbers of T cells in combination with a nylon wool filtration technique as described in part by Kwekkeboom et al. [12] so that we can perform a much cheaper technique than the CD34 selection for PBSC.
Moreover, we think that CD34 selection is not the optimal method for T-cell depletion, since all other cell types are also excluded from the transplant, including cells which, in an allogeneic setting, might play a role in the engraftment of the haematopoietic stem cells. Further studies will indicate whether we can successfully develop a T-cell depletion technique for PBSC transplants.
References