M. J. M. L. Gilmore

Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation

Summary. Bone marrow graft rejection following HLA‐matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T‐lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T‐depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T‐depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T‐depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7‐5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T‐depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.

A technique for rapid isolation of bone marrow mononuclear cells using Ficoll-Metrizoate and the IBM 2991 blood cell processor

Marrow from seven normal donors and patients has been layered onto Ficoll‐Metrizoate (FM) under pressure in the IBM 2991 blood cell processor to isolate the mononuclear cell (MNC) population prior to allogeneic transplantation or cryopreservation. This separation method, which takes less than 90 min, is a further development since our previous report detailing the use of the IBM 2991 to produce a concentrated marrow‘buffy coat’for infusion (Gilmore & Prentice, 1981). By adding FM to the system, marrow stem cells are further concentrated in a small volume with removal of unwanted granulocytes and red blood cells. This facilitates the in vitro treatment of marrow with monoclonal antibodies (Granger et al, 1982) or drugs, for either the selective elimination of malignant cells prior to autologous bone marrow transplantation (BMT), or T lymphocytes in an attempt to prevent graft versus host disease (GvHD) in allogeneic BMT.

ABO-INCOMPATIBLE BONE-MARROW TRANSPLANTATION: REMOVAL OF RED BLOOD CELLS FROM DONOR MARROW AVOIDING RECIPIENT ANTIBODY DEPLETION

Eight patients with acute leukaemia undergoing allogeneic bone-marrow transplantation from ABO-incompatible donors received red-cell-depleted donor marrow without any procedure to diminish their anti-ABO antibody titres. Successful marrow red-cell removal (mean 98.8%) was achieved by means of a large-volume separation technique on Ficoll-Metrizoate in the IBM 2991 blood-cell processor. Clinically significant ABO-haemolytic reaction was prevented in all patients, and there was neither failure of engraftment nor rejection. This approach used alone is satisfactory for most ABO-incompatible marrow-transplant recipients, although combining this with some method of recipient antibody depletion, such as plasma exchange, is recommended in the occasional patient with high anti-ABO titres.

A technique for the concentration of nucleated bone marrow cells for in vitro manipulation or cryopreservation using the IBM 2991 blood cell processor.

Abstract. 27 bone marrow aspirates were processed on the IBM 2991 Blood Cell Processor to achieve a reduction in volume (>80%) and in red blood cell contamination (>75%), without loss (<20%) of nucleated cells. The procedure was used to concentrate nucleated bone marrow cells either prior to cryopreservation for subsequent autologous transplantation, or prior to incubation with a murine monoclonal antibody (OKT3) for allogeneic transplantation. We conclude that the procedures used for the concentration, cryopreservation, thawing and infusion do not adversely affect the viability of cells as assessed by in vitro culture (CFU‐GM). Of the marrows processed, 12 have been reinfused and resulted in successful engraftment.

Standardization of the Processing of Human Bone Marrow for Allogeneic Transplantation

Abstract. Allogeneic bone marrow transplantation has been undertaken within this centre on 62 patients with acute or chronic leukaemia. Employing the standard separation protocol described, all bone marrows were processed on the 2991 Blood Cell Processor to isolate the ‘buffy coat’ cells and subsequently the mononuclear cell component using a density separation medium of Ficoll‐metrizoate. Following the mononuclear cell separation, the cells were identified for their T lymphocyte component using a combination of two murine monoclonal antibodies, MBG6 reacting with a pan T cell antigen (CD6) and RFT8 detecting the ‘cytotoxic/supressor’ cell antigen (CD8). The numerical results for nucleated cells, red blood cells, T lymphocytes and colony forming units‐granulocyte macrophage are presented.

Standardization of T-cell depletion in HLA matched bone marrow transplantation

Summary. The IBM 2991 Blood Cell Processor has been used to isolate a mononuclear cell (MNC) fraction from the marrow of 31 allogeneic donors. The MNC fraction was then incubated with a combination of two murine monoclonal antibodies MBG6 (CD6) and RFT8 (CD8) followed by two rounds of treatment with rabbit complement resulting in a marrow inoculum significantly reduced in the number of T‐lymphocytes. We report here new specifications for the use of Ficoll‐Metrizoate, the method used to calculate T‐lymphocyte depletion and the details of our attempts to improve T‐depletion. Following marrow transplantation with this T‐depleted fraction, 29 patients are evaluable for engraftment, one patient failed to engraft and one died too early for evaluation. Twenty‐two had no acute graft‐versus‐host disease (aGvHD), at a minimum of 60 d, six had grade I acute GvHD and one grade III. No correlation was found between the absolute number of MNC infused and time to engraftment, nor any relationship between the number of residual viable T‐lymphocytes in the infused marrow and the incidence of GvHD, but the patient with the most severe aGvHD also had the highest number of T‐lymphocytes infused.

Analysis of Rejection in HLA Matched T-Depleted Bone Marrow Transplants

In February 1984 a collaborative trial was begun using the Royal Free (RF) cocktail of MBG6 and RFT8 monoclonal antibodies to T-deplete donor marrow for allogeneic BMT for leukaemia. The trial followed the successful use of the cocktail in 25 patients. A proportion of patients from all centres experienced rejection of the graft. Because of this serious problem, T-depletion was temporarily suspended at the other centres. In the parent institution all but 1 of the subsequent 13 grafts were successful. Rejection occurred in 3 forms: no engraftment at all; partial or complete engraftment followed by early loss of graft, or late loss of graft (at 4 months). Preliminary data from centres using other anti-T cell monoclonal antibodies and from this study strongly implicate the pre-transplant conditioning as being critical to successful, sustained engraftment.

T Depletion Using MBG6 and RFT8 Monoclonal Antibody Combination and Complement Lysis Prevents Significant Acute and Chronic GvHD in HLA Matched Allogeneic Marrow Transplants

Forty-one patients have received marrow from HLA-matched sibling donors with in vitro T-depletion using 2 monoclonal antibodies MBG6 and RFT8 and rabbit complement. No other immunosuppression was given. Engraftment occurred in 39 of 40 evaluable patients. The incidence of acute and chronic GvHD is minimal with a Royal Free acute GvHD score for the group of 0.07. The incidence of viral infections is low with no fatalities. Of the 20 evaluable patients transplanted in 1st CR of AL or 1st CP of CGL, only one patient has had a leukaemic relapse to date (CGL/BT). In poor risk patients who have a high relapse incidence the conditioning has been modified

Allogeneic bone marrow transplantation: the monitoring of granulocyte macrophage colonies following the collection of bone marrow mononuclear cells and after the subsequent in‐vitro cytolysis of OKT3 positive lymphocytes

Summary. Marrow nucleated cells from eight normal allogeneic donors was layered on Ficoll‐Metrizoate to isolate the mononuclear cell fraction. The cells were then washed to remove Ficoll‐Metrizoate and coagulation factors prior to resuspension in a balanced salt solution and the addition of the murine anti‐human T‐lymphocyte monoclonal antibody OKT3 and rabbit complement. The procedures were assessed for their effect on mononuclear cell viability (mean recovery 84·4%); the ability of the cells to proliferate granulocyte‐macrophage colonies (mean recovery 57·4%); the in‐vitro T‐lymphocytolysis (mean 75·7%) and the removal of rabbit complement (> 99%). Following marrow transplantation with this treated mononuclear fraction the mean day to recovery of ≥ 1·0 × 109/1 leucocytes was 20 d, with three patients developing ≥ Grade II acute graft versus host disease (GvHD). Thus, treatment of donor marrow with OKT3 and complement in a large volume system was not detrimental to subsequent engraftment, nor effective in complete T‐lymphocytolysis, nor in prevention of severe GvHD.

Bone Marrow Transplantation in First CR of Acute Leukaemia using T-Depleted Marrow from HLA Identical Sibling Donors

Since May 1983 and following the institution of our present studies of “total” T-lymphocyte depletion for graft versus host disease (GvHD) prevention we have transplanted 22 patients in first complete remission (1st CR) of acute leukaemia using HLA identical sibling donors. The murine monoclonal antibodies (McAbs) RFT8 + MBG6 (or RFT12 in 4) were used in combination with rabbit complement (C′) to achieve a mean 97.7% T-lymphocyte lysis in vitro. We have used a fast dose rate (mean 15.45cGy/min received mid-plane) total body irradiation (mean average mid-plane dose 730cGy) combined with cyclophosphamide 120mg/kg. Five patients had high dose Ara-C 4 of whom received only 90mg cyclophosphamide: the fifth received 120mg/kg. In the absence of GvHD no post-BMT immunosuppressive drugs were given. Engraftment assessed by a granulocyte count of 0.5 × 109/l took a mean 23 days (range 14–72). Seventeen patients survived in CR from 18 to 881 days. Five patients have died, 2 from heart failure (CCF), 1 from cytomegalovirus pneumonitis (CMV I.pn), 1 from idiopathic pneumonitis (ID I.pn) and 1 following delayed graft failure. Five patients have had acute graft versus host disease (aGvHD), grade 1 in four and grade 2 in one. Three had mild chronic graft versus host disease (cGVHD). One patient has cholestatic jaundice of unknown cause. Preliminary analysis of immune reconstitution shows only modest T subset imbalance and preservation of NK cell function. Additionally we have demonstrated effective and useful adoptive transfer of B-cell immunity from donor to recipient. No patient has so far suffered a leukaemic relapse. The actuarial predicted survival at 21/2 years is 74% in this patient group. In conclusion T-depleted allogeneic BMT using single fraction TBI is practical, reduces considerably the risks of GvHD and the need for post-transplant immunosuppressive drugs, is not associated with any increased risk of leukaemic relapse (in this series) and appears to allow regulated regeneration of donor derived immune function.