Prevention of graft-versus-host disease in high risk patients by depletion of CD4 and reduction of CD8 lymphocytes in the marrow graft

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1 Bone Marrow Transplantation, (1999) 23, Stockton Press All rights reserved /99 $12. Prevention of graft-versus-host disease in high risk patients by depletion of CD4 and reduction of CD8 lymphocytes in the marrow graft C Herrera, A Torres, JM García-Castellano, J Roman, C Martin, J Serrano, M Falcon, MA Alvarez, P Gomez and F Martinez Department of Hematology, Hospital Reina Sofia, Córdoba, Spain Summary: From March 1994 to September 1997, 3 patients with hematological malignancies (12 ANLL, 1 CML, four ALL and four multiple myeloma) received HLA-identical allogeneic bone marrow transplants with the marrow graft selectively depleted of CD4 lymphocytes and the CD8 cell content adjusted to /kg. Total depletion of CD4 and partial depletion of CD8 lymphocytes was carried out by an immunomagnetical method. All patients were considered as having high risk for developing GVHD by at least one of the following criteria: patient age 35 years; donor age 35 years; donor multiparity or marrow from an unrelated donor. Twenty-four cases received marrow from an identical sibling and six from an unrelated donor. In order to assess the role of methotrexate (MTX) in addition to cyclosporin A (CsA) after transplant, patients were randomly assigned to received either CsA alone (n = 15) or CsA plus a short course of MTX (n = 15). No case of primary graft failure was observed, but two patients developed late graft failure. Six patients presented grade II acute GVHD and no case of severe III IV GVHD was seen. The actuarial probability of developing grade II IV acute GVHD was % for the entire population. Patients receiving post-transplant CsA MTX had significantly less probability of acute GVHD than those receiving CsA exclusively ( % vs %, P =.3) and the schedule of post-transplant immunosuppression was the only factor associated with the incidence of acute GVHD in a multivariate analysis. The actuarial incidence of chronic GVHD for the entire population was , and there was no significant difference between both groups with additional prophylaxis. Four patients with CML and three with ANLL relapsed: the actuarial probability of remaining in complete remission for all patients was %. For patients with acute leukemia, the probability of remaining in complete remission did not differ significantly between those transplanted in first complete remission and those Correspondence: Dr C Herrera, Department of Hematology, Hospital Reina Sofia, Avda, Menendez Pidal s/n, 14 Cordoba, Spain Received 8 May 1998; accepted 28 July 1998 receiving a transplant in more advanced phases of the disease ( % vs %; P =.44). The incidence of mixed chimerism assessed by PCR was 34%. Nineteen patients are alive between 2 and 43 months post-transplant, the probability of overall survival being %. Our data indicate that this method of selective T cell depletion is very effective in preventing acute GVHD in high risk patients, particularly when used in combination with post-transplant CsA MTX. Keywords: GVHD; selective T cell depletion; post-transplant immunosuppression Ex vivo depletion of T lymphocytes from the donor marrow is the most effective method to prevent graft-versus-host disease, but it is uniformly associated with increased risk of graft failure and leukemic relapse. Thus the reduction in morbidity and mortality due to GVHD is not translated into an increase in disease-free survival. 1 4 During the last few years, several methods of selective or partial T cell depletion have been described 5 12 in an attempt to eliminate the potential effectors of GVHD exclusively and to leave some alloreactive T lymphocytes in the donor marrow that are able to reduce graft failure and maintain a graft-versusleukemia reaction. Although the clinical results of these selective T cell depletion protocols generally are better than those of exhaustive T cell depletion, the goal of a marked reduction in GVHD without a concomitant loss of graft enhancing and GVL effects is still to be achieved. Experimental evidence in animal models 13 as well as preliminary clinical results suggest that the presence of low doses of CD8 T lymphocytes in donor bone marrow depleted of CD4 T cells can ensure engraftment. In fact, Gallardo et al 17 have recently reported no graft failure and a low incidence of severe acute GVHD in 12 chronic myeloid leukemia patients with high risk for developing GVHD who received allogeneic bone marrow depleted of CD4 lymphocytes with the content of CD8 cells adjusted to /kg; similarly, Verdonck et al 11 reported a 1% engraftment with a fixed low number of T cells, that obviously contained CD8 lymphocytes, in the marrow graft. In contrast, in four series with CD8 T cell depletion, 5 8 the reported incidence of primary graft failure ranges from 4.5 to 1%, which is not different from that reported with

2 444 complete T cell depletion. Therefore, it seems clear that allogeneic engraftment is dependent on the presence of even very low numbers of donor CD8 cells in the marrow graft. We present here the clinical results of 3 patients with high risk of GVHD that received allogeneic HLA-identical bone marrow depleted of CD4 lymphocytes containing a fixed number of /kg CD8 cells. The aim of the work was to evaluate this method of partial T cell depletion in terms of GVHD incidence, engraftment and relapse rate. In order to assess the impact of additional prophylaxis on GVHD incidence, patients were randomly assigned to receive either cyclosporin A or cyclosporin A plus methotrexate. Materials and methods Patients Between March 1994 and September 1997, 3 patients with hematological malignancies were entered into the study, after giving written informed consent. The clinical characteristics of the patients are shown in Table 1. Twenty-four patients received bone marrow from HLA-identical sibling donors, and the remaining six from a matched unrelated donor (MUD). All patients were considered as high risk for developing GVHD by at least one of the following criteria: (1) patient age 35 years; 18 (2) donor age 35 years; 18 (3) donor multiparity; 21 (4) unrelated donor. 22 Ten patients had one risk factor (always patient age 35 or marrow from MUD), 13 patients had two risk factors, and seven had three risk factors. Conditioning regimens All patients received conventional conditioning regimens that were as follows: 19 patients were conditioned with cyclophosphamide ( mg/kg/day 2 days) and total body Table 1 Characteristics of the patients Age (years) Median 44.5 Range 6 59 Sex (M/F) 18/12 Diagnosis ANLL 12 1st CR 7 2nd or subsequent CR 2 No remission 3 ALL 4 1st CR 1 2nd or subsequent CR 3 CML 1 1st chronic phase 1 MM 4 CR 2 PR 2 ALL = acute lymphoblastic leukemia; ANLL = acute non-lymphoblastic leukemia; CML = chronic myeloid leukemia; MM = multiple myeloma; CR = complete remission; PR = partial remission. irradiation (TBI), 13.2 Gy divided into 8 fractions in 15 patients, and 9 Gy in a single dose in the remaining four; eight patients received busulfan (4 mg/kg/day 4 days) plus cyclophosphamide ( mg/kg/day 2 days); two patients received TBI 13.2 Gy divided into 8 fractions plus melphalan 1 mg/m 2, and one patient received busulfan (4 mg/kg/day 4 days) plus melphalan 1 mg/m 2. Supportive care Patients were treated in either laminar air flow or positivepressure reverse isolation rooms, and received routine antibiotic prophylaxis with ciprofloxacin 5 mg p.o. every 12 h and fluconazole mg p.o. daily, starting on the day of admission. All patients but one received G-CSF (Filgrastim; Amgen Roche, Thousand Oaks, CA, USA) at a dose of 5 g/kg/day, starting on day (four patients) or 7 (25 patients), until they reached an ANC 1/ l for 3 consecutive days. As part of a randomized trial, 14 patients received human intravenous immunoglobulin at a dose of 15 mg/kg every other week until day 9, and monthly thereafter during the first year post transplant. Patients received filtered (leukocyte-poor) red blood transfusions and platelet concentrates. All blood components were irradiated ( Gy) to prevent transfusion-related GVHD. GVHD prophylaxis and treatment In order to assess the role of two regimens of additional pharmacological prophylaxis of the GVHD, patients were randomly assigned to receive either cyclosporin A at an initial dose of 3 mg/kg/day i.v. (n = 15), or cyclosporin A (initial dose 1.5 mg/kg/day) plus methotrexate (15 mg/m 2 day 1, and 1 mg/m 2 days 3, 6 and 11) followed by folinic acid (n = 15). In both groups, as soon as the patient was capable of taking oral nutrition, cyclosporin A was switched to an every 12 h oral administration schedule and maintained for 3 6 months. The six patients receiving marrow from MUD were randomised separately, and so three patients were assigned to each arm. The diagnosis and grading of GVHD was established according to previously defined criteria. 23 Acute GVHD grade I or more was treated with methylprednisolone 1 2 mg/kg /day. Patients were considered at risk of developing chronic GVHD when they were alive at day 15 without evidence of relapse. Chronic GVHD was treated with prednisone.5 1 mg/kg every other day and cyclosporin A. Bone marrow processing Donor bone marrow was obtained by multiple aspirations from the iliac crests, either at our hospital or at various collection centers. Mononuclear cells were isolated by gradient density centrifugation on Ficoll Hypaque using a COBE 2991 (Cobe, Lakewood, IL, USA). The recovered mononuclear cells were washed three times and resuspended in saline containing 1% human serum albumin at a concentration of cells/ml. A sample was taken

3 to determine the percentages of CD2, CD34, CD4 and CD8 by flow cytometric analysis as described later. Depletion of CD4 and CD8 T cells was carried out by immunomagnetic separation using Dynabeads M-45 CD4 and M-45 CD8 (Dynal, Oslo, Norway) in two consecutive steps. First, marrow MNC were incubated with Dynabeads M-45 CD4 (ratio beads:target cell 5:1) for 3 min at 4 C on a rotator. After incubation, magnetic separation was accomplished on the MaxSep Magnetic Cell Separator (Baxter, Deerfield, IL, USA). A sample of the depleted product was obtained to determine the efficacy of depletion and the percentage of CD8 cells. In a second step, an aliquot of the cellular suspension that contained CD8 cells/kg patient body weight was reserved without further manipulation, and the rest of the marrow underwent a second procedure of depletion by the same method using Dynabeads M-45 CD8. Once the efficacy of the CD8 depletion was assessed by flow cytometry, the two cellular fractions were mixed and immediately infused to the patient through a central venous access. Flow cytometry CD2, CD4, CD8 and CD34 surface markers were determined by direct immunofluorescence using the following FITC-labeled antibodies: anti-leu 5b (CD2), anti-leu 3a (CD4) anti-leu 2a (CD8) and HPCA-2 (CD34) purchased from Becton Dickinson (San Jose, CA, USA). After staining according to the manufacturer s instructions, the cells were analyzed using a EPICS-Profile Cytometer (Coulter, Hialeah, FL, USA). The percentages of positive cells were calculated by subtracting the respective number of isotypelabeled negative control cells. The sensitivity of flow cytometric analysis was.1% according to previous experiments. Progenitor cell assay The CFU-GM progenitor cell assay was performed in semisolid medium consisting of Iscove s modified Dulbecco s medium (IMDM),.9% methylcellulose, % fetal calf serum and 12% human placental conditioned medium. Cultures were plated in duplicated in 35 mm Petri dishes, incubated at 37 C in5%co 2 in air, and scored on day 7. Analysis of chimerism High molecular weight DNA was extracted from peripheral blood mononuclear cells obtained from the donor and recipient before transplant using a salting out procedure. 24 After transplant, DNA was isolated from the peripheral blood of the patients to allow determination of chimerism by a polymerase chain reaction amplification method (PCR). For PCR amplification, we used specific primers designed to flank the repeat units of the following human minisatellite regions: D1S, 33.6, 33.1, YNZ-22, APO-B, g3, DXS52 and 3 HVR. The sequence of the primers and conditions for each reaction have been described elsewhere All products of amplification were electropheresed in tris-borate buffer on 1 3% agarose gels which were stained with ethidium bromide. The gels were photographed by ultraviolet transilumination. We defined a minisatellite locus as being informative if analysis of recipient and donor samples prior to BMT showed a unique band for the recipient and a unique band for the donor, or showed a unique band for the recipient only. To assess the sensitivity of this method, DNA from several donors and recipients were mixed in the following proportions of host DNA: 25, 1, 5, 3.5, 2, 1.5, 1,.5 and.1%. Each DNA mixture was subjected to PCR analysis. Sensitivity was considered as the lowest proportion of host DNA in the mixtures for which host DNA could be detected. The sensitivity of the different minisatellites ranged between.5 and 1% (data not shown). Patients who exhibited complete donor hematopoiesis with all markers tested at all times were defined as donor or complete chimeras. Patients who exhibited mixed populations of donor and host cells on more than one occasion with at least two different markers after day 45 or when the recipient was transfusion independent were considered to exhibit mixed chimeras. Statistical analysis The median duration of follow-up for the patient population was 21 months (range 2 43 months). Cumulative actuarial probabilities of engraftment, GVHD, remaining in complete remission and overall survival were calculated using the Kaplan Meier method. 29 Comparisons of time to event distributions were made using the log rank test. The Cox regression model 3 was used to investigate factors associated with clinically significant grades of GVHD. Results Ex vivo bone marrow treatment The mean initial percentages of CD4 and CD8 cells were % and %, respectively. After depletion procedures these percentages fell to.3.1% and %, respectively. In 29 cases after CD4 depletion and in 26 after CD8 depletion no residual positive cells were detected by flow cytometry after subtraction of the isotype control. In these cases, a percentage of.9% residual positive cells was assumed for the purpose of calculation. The calculated purging efficiency for CD4 cells is log and for CD8 lymphocytes. Non-specific loss of hematopoietic progenitors was % for CFU-GM, and % for CD34 cells. The cellular composition of the final infused graft is shown in Table 2. Engraftment Two patients were not evaluable for engraftment due to leukemia post transplant in one case and to early death in the other. No case of primary graft failure was observed, 445

4 446 Table 2 Cells/kg Composition of the infused marrow graft Mean s.d. MNC ( 1 7 ) CD4 ( 1 4 ) CD8 ( 1 6 ) 1 CD34 ( 1 6 ) CFU-GM ( 1 4 ) and all 28 evaluable patients engrafted promptly. The median days to reach an ANC /l and /l were 15 (11 24) and 16 (12 25), respectively, for patients receiving CsA; patients receiving CsA MTX reached an ANC /l and /l on days 17 (13 23) and 18 (15 23), respectively. Four patients did not reach platelets/l, but all of them maintained platelet counts between 25 and /l without the need for transfusion support. For the remaining 26 patients, the median time for platelet recovery to /l was 27 days ( 425) for patients receiving CsA and 26 (18 177) for those receiving CsA MTX. The cumulative probabilities of myeloid and platelet engraftment are shown in Figure 1. As can be seen, there 1 1 a b was no significant difference in the probability of engraftment between patients receiving CsA or CsA MTX. We have not found statistically significant differences in the probability of primary engraftment between patients receiving or not receiving TBI in the conditioning, nor between those receiving or not receiving hyperfractionated TBI (data not shown). Two patients among the 28 evaluable presented late graft failure. The first was a female diagnosed with ANLL-M 7 with extensive myelofibrosis, who received marrow transplant in partial remission, and after initial engraftment developed pancytopenia on day 165 and died from Candida krusei sepsis. The second was a male with CML who received marrow from an unrelated donor and developed graft failure on day 94; he received unmanipulated BMT from the original donor, and after initial engraftment with complete donor chimera, on day 2 he showed autologous hematopoiesis and relapse of his CML. Graft-versus-host disease Fourteen patients (5% of 28 evaluable) had no signs of acute GVHD, eight (28%) had grade I and six (21%) had grade II acute GVHD. Patients with grade I or II acute GVHD were treated with steroids for a median time of 5 days (range 13 64) for those receiving CsA alone, and 25 days (range 12 ) for those receiving CsA MTX. No patient developed grades III IV GVHD, and all six patients with grade II responded completely to steroids. The cumulative probability of developing clinically significant agvhd was % (Figure 2). We did not find any significant difference in the incidence of acute GVHD between patients receiving or not receiving TBI, or receiving or not receiving hyperfractionated TBI in the conditioning (data not shown). Among the 15 patients who had received additional prophylaxis with CsA five (33%) developed grade II agvhd, whereas only one patient among those receiving CsA MTX (6%) had this complication. The actuarial incidence of grade II agvhd was % in the CsA group vs % in the CsA MTX group ± 9.6 Figure 1 Actuarial probabilities of (a) myeloid (ANC /l) and (b) platelet (platelets /l) engraftment for patients receiving CsA ( ) or CsA MTX ( ). Log rank test, P =.27 and P =.29, respectively Figure 2 Actuarial probability of developing grade II IV acute GVHD for the entire patient population.

5 ± ± ± 6.4 Figure 3 Actuarial probability of developing grade II IV acute GVHD for patients receiving post-transplant CsA ( ) or CsA MTX ( ). Log rank test, P =.3. (Figure 3); this difference was statistically significant (log rank = 4.5, P =.3). The multivariate analysis of factors potentially associated with agvhd (including dose of infused T cells, number of high risk criteria, sibling donor or MUD, diagnosis and disease s status, and additional GVHD prophylaxis) revealed that the unique factor associated with lower incidence of agvhd was the combination of CsA and MTX as additional prophylaxis (P =.3). Eighteen patients were evaluable for chronic GVHD. Limited cgvhd developed in two patients and extensive forms of the disease in three patients. All but one responded completely to immunosuppressive therapy, and there were no deaths attributable to either cgvhd or its treatment. The actuarial probability of developing cgvhd in this series is %, and that of presenting an extensive form is % (Figure 4). Among nine evaluable patients receiving CsA the actuarial incidence of cgvhd is %, whereas among the nine receiving CsA MTX it is only %; this difference, however, did not reach statistical signification (P =.7), probably because of the small number of patients. None of the factors analyzed had a significant association with cgvhd in the multivariate analysis Months post-bmt Figure 5 Actuarial probability of remaining in complete remission for the entire patient population. Relapse Seven patients have relapsed. Two patients with ANLL who were not in complete remission at the time of BMT relapsed on days and 7, respectively, and one patient with ANLL-M 4 transplanted in first CR relapsed also on day 7. Four patients with CML relapsed in months 5, 8, 12 and 3 post transplant; the remaining six CML patients are in complete molecular remission with bcr/abl persistently negative. The 3.5 year actuarial probability of remaining in complete remission for the entire patient population was %, with a mean remission duration of months (Figure 5). For patients with acute leukemia receiving a transplant in first complete remission the 3 year probability of being in remission was %, and for those transplanted in advanced phases of their disease (second or subsequent complete remission, or nonremission) was % (Figure 6); there was no statistically significant difference between these two groups of patients (P =.44). Survival Nineteen patients are alive between 2 and 43 months post transplant; the overall 3.5 year actuarial survival is % (Figure 7) ± ± ± ± Figure 4 Actuarial probability of developing any form of chronic GVHD ( ) and an extensive form of the disease ( ) Months post-bmt Figure 6 Actuarial probability of remaining in complete remission for patients with acute leukemia transplanted in first complete remission ( ) or in more advanced phases of the disease ( ). Log rank test, P =.44.

6 Months post-bmt 57.8 ± 1.4 Figure 7 Actuarial probability of overall survival for all the patients included in the study. Eleven patients died, four due to leukemic relapse (leukemic mortality rate 13.3%) and seven due to transplant-related complications: bacterial infection three cases, fungal infection one case, hepatic veno-occlusive disease two cases and interstitial pneumonitis one case (transplantrelated mortality rate 23.3%). No deaths attributable to GVHD were observed. Chimerism Data concerning hematopoietic chimerism are available in 23 patients. Fifteen of them are complete hematopoietic chimeras, and eight (34%) developed mixed chimerism after being complete chimeras for periods ranging from 1 to months. No patient among the 15 that are complete chimeras have relapsed; by contrast, five out of eight patients with mixed chimerism relapsed. There is a strong association between mixed chimerism and relapse in patients with CML (four relapses among five mixed chimeras, P =.2 by Fischer s exact test), but not in patients with acute leukemia (one relapse among three mixed chimeras, P =.27). Discussion In this study we analyzed the impact of a model of partial T cell depletion and post-transplant immunosuppression on GVHD incidence, engraftment and leukemic relapse in 3 patients with high risk of developing GVHD. Our results demonstrate that depletion of CD4 lymphocytes plus reduction of the CD8 T cell content in the bone marrow graft, in combination with cyclosporin A or cyclosporin A plus methotrexate, is extremely effective in preventing GVHD in high risk patients. It is well known that complete depletion of T cells in the graft is the most effective method for GVHD prophylaxis, with a reported incidence of this complication of only 1% without the need for additional inmunosuppression. 2,31,32 However, with the new strategies of T cell depletion consisting of the selective elimination of determined T cell subsets, the incidence of clinically significant agvhd is higher, ranging from 18 to 28% for recipients of HLA-identical marrow without special risk factors, 5 11 and from 33 to 63% for recipients of marrow from an unrelated donor. 8,9,12 The low incidence of 21% grade II agvhd without any case of grade III IV in our series is only comparable with that reported by Verdonk et al 11 (28% grade II exclusively) in standard risk patients receiving marrow grafts with T cells/kg. With this sole exception, all the published experiences with partial or selective models of T cell depletion have reported some incidence of severe grade III IV acute GVHD, that varies between 5 and % after CD8 depletion, 5 8 9% using a CD5 or CD6 depletion in standard risk patients, 9,1 and reaches 37% in recipients of CD8-depleted marrow from unrelated donors. 8 It is possible that the use of steroids in our patients as soon as the first sign of acute GVHD appeared, could also have abrogated the progression of the disease to a more severe grade. Although, in fact, many factors can influence both the incidence and the severity of GVHD and can therefore result in marked differences between groups of patients, our results as well as those of the above mentioned series suggest a pivotal role for CD4 T lymphocytes in the pathogenesis of clinical acute GVHD as has been previously demonstrated at the experimental level After exhaustive T cell depletion, there is probably no need for additional immunosuppression as prophylaxis for GVHD. However, data from the IBMTR demonstrated that post-transplant methotrexate plus cyclosporin A reduced the likelihood of acute GVHD after T cell-depleted transplantation whereas this effect is not detected with methotrexate or cyclosporin A alone. 1 Our data are in agreement with these results, and it may be of important clinical relevance for two reasons: first, until now, there were no convincing data in the literature regarding the role of posttransplant immunosuppression in recipients of marrow partial or selectively depleted of T cells; and second, the low incidence and severity of acute GVHD, particularly in the group receiving CsA MTX with only 5% of patients having grade II GVHD, supports the evaluation of this schedule in patients with even higher risk of GVHD, such as recipients of marrow from HLA-mismatched donors. No patient in this series developed primary graft failure, and the rate of engraftment was comparable to that seen after unmodified allogeneic marrow transplants. Gallardo et al 17 have also recently reported a 1% rate of primary engraftment using a method of T cell depletion which is identical to that reported here, except for a lower dose of CD8 lymphocytes in the graft corresponding to /kg. In sharp contrast are the results of three series reporting their results with a model of CD8 selective depletion 5 7 in which the incidence of primary graft failure, ranging from 5 to 1% is not different from that reported with complete T cell depletion. All these findings, considered as a whole, strongly suggest an important role for CD8 donor lymphocytes in the engraftment of allogeneic marrow, as had been previously proven in experimental murine models. 13 It is difficult to evaluate the actual impact of this method of selective T cell depletion on leukemic relapse due to the heterogeneity of the group. However, it seems that, at least for the 16 patients with acute leukemia, the probability of remaining in complete remission is comparable with that

7 reported after conventional unmodified allogeneic transplant, even for those receiving BMT in advanced phases of their disease. Data from the literature regarding the relapse rate after T cell-depleted transplants for acute leukemia are controversial: whereas several trials of both complete and selectively T cell-depleted transplants have reported increased rates of leukemia relapse, 1,1,36 others have found the risk of disease recurrence to be similar to that after conventional BMT. 37,38 It is possible that many factors, such as different intensity of pretransplant chemotherapy and conditioning regimens as well as differences in the proportion of high risk patients in each series, may have contributed to these apparently contradictory results. Following standard preparative regimens, the incidence of relapse in our series is 12% for patients with acute leukemia in first CR and 27% for those in advanced phase of their disease, including three patients transplanted whilst not in remission. In contrast to the results observed in recipients of T celldepleted marrow for acute leukemia, most such studies of CML patients have revealed a dramatically increased risk of relapse, 1,39, and this finding is consistent with a particularly important role for donor lymphocytes in maintaining remission in CML. Among the 1 patients with CML in chronic phase in this series, four relapsed between 5 and 3 months post transplant, and although the number of patients is too small to draw any conclusions, it seems that the post-transplant relapse rate for patients with CML is increased with our method of selective T cell depletion, as it is after complete T cell depletion. Some experimental data have indicated that the GVL effect against CML is mainly exerted by donor CD4 lymphocytes, 41,42 and the elimination of this cellular subset with our method of selective depletion as well as after exhaustive T cell depletion, may be responsible for the loss of the GVL effect that leads to the high relapse rate observed. Consistent with this concept, are the results of four series of selective depletion of CD8 lymphocytes, 5 8 in which no patient with CML has relapsed. In contrast, Gallardo et al 17 have recently reported a % incidence of relapse in 12 patients with CML transplanted with marrow depleted of T cells by a method identical to that reported in our series, except for a lower dose of infused CD8 cells. We have no convincing explanation for the difference in the relapse rates between our CML patients and those reported by Gallardo, but the small number of patients as well as differences in risk factors between both series may account for this. In conclusion, the method of selective T cell depletion that we present here, in combination with post-transplant immunosuppression, reduces dramatically the incidence and severity of acute GVHD, particularly in patients receiving the combination of CsA plus MTX, without adversely influencing either the ability of primary engraftment or the relapse rate in acute leukemia patients, but not in CML. These results support the continued application of the method in patients with acute leukemia at high risk of GVHD and provides rationale for its use in BMT from nonfully-matched donors. Acknowledgements We are indebted to M García and M del Castillo for technical assistance. References 1 Marmont A, Horowitz M, Gale RP et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78: Mitsuyasu RT, Champlin RE, Gale RP et al. Treatment of donor bone marrow with monoclonal anti-t-cell antibody and complement for the prevention of graft-versus-host disease: a prospective, randomized, double-blind trial. Ann Intern Med 1986; 15: Hale G, Cobbold S, Waldmann H. T-cell depletion with Campath-1 in allogeneic bone marrow transplantation. Transplantation 1988; 45: Kernan N, Bordignon C, Heller G et al. Graft failure following T-cell depleted HLA-identical marrow transplants for leukemia: analysis of risk factors and results of secondary transplants. Blood 1989; 74: Champlin R, Ho W, Gajewsky J et al. Selective depletion of CD8 T lymphocytes for prevention of graft-versus-host disease after allogeneic bone marrow transplantation. Blood 199; 76: Champlin R, Jansen J, Ho W et al. Retention of graft-versusleukemia using selective depletion of CD8-positive T lymphocytes for prevention of graft-versus-host disease following bone marrow transplantation for chronic myelogenous leukemia. Transplant Proc 1991; 23: Nimer S, Giorgi J, Gajewsky J et al. Selective depletion of CD8 cells for prevention of graft-versus-host disease after bone marrow transplantation. Transplantation 1994; 57: Jansen J, Hanks S, Akard L et al. Selective T-cell depletion with CD8-conjugated magnetic beads in the prevention of graft-versus-host disease after allogeneic bone marrow transplantation. Leukemia 1995; 9: Antin JH, Bierer BE, Smith BR et al. Selective depletion of bone marrow T lymphocytes with anti-cd5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies. Blood 1991; 78: Soiffer RJ, Murray C, Mauch P et al. Prevention of graftversus-host disease by selective depletion of CD6-positive T lymphocytes from donor bone marrow. J Clin Oncol 1992; 1: Verdonck L, Dekker A, de Gast G et al. Allogeneic bone marrow transplantation with a fixed low number of T cells in the marrow graft. Blood 1994; 83: Drobyski WR, Ash RC, Casper JT et al. Effect of T-cell depletion as graft-versus-host disease prophylaxis on engraftment, relapse, and disease-free survival in unrelated marrow transplantation for chronic myelogenous leukemia. Blood 1994; 83: Martin PJ. Donor CD8 cells prevent allogeneic marrow graft rejection in mice. Potential implications for marrow transplantation in humans. J Exp Med 1993; 178: Grañena A, Canals C, Picón M et al. GVHD prophylaxis in CML: complete depletion of CD4 cells and controlled numbers of CD8 lymphocytes in the donor marrow. Bone Marrow Transplant 1994; 14 (Suppl. 3): 85 (Abstr.). 15 Gallardo D, García-López J, Sureda A et al. Total depletion of CD4 cells and partial depletion of CD8 cells as a GVHD 449

8 45 prophylaxis prevents graft failure in CML patients undergoing allogeneic BMT. Bone Marrow Transplant 1997; 19 (Suppl. 1): 27 (Abstr.). 16 Herrera C, Martin C, García-Castellano JM et al. Prevention of GVHD by selective CD4 T cell depletion plus adjustement of the CD8 cell content in donor bone marrow. Blood 1995; 86 (Suppl. 1): 571 (Abstr.). 17 Gallardo D, García-Lopez J, Sureda A et al. Low dose donor CD8 cells in the CD4-depleted graft prevent allogeneic marrow graft rejection and severe graft-versus-host disease for chronic myeloid leukemia patients in first chronic phase. Bone Marrow Transplant 1997; : Weisdorf D, Hakke R, Blazar B et al. Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation. Transplantation 1991; 51: Bross DS, Tutschka PJ, Farmer ER et al. Predictive factors for acute graft-versus-host disease in patients transplanted with HLA-identical bone marrow. Blood 1984; 63: Gale RP, Bortin MM, van Bekkum DW et al. Risk factors for acute graft-versus-host disease. Br J Haematol 1987; 67: Flowers MED, Pepe MS, Longton G et al. Previous donor pregnancy as a risk factor for acute graft-versus-host disease in patients with aplastic anemia treated by allogeneic marrow transplantation. Br J Haematol 199; 74: Beatty PG, Hansen JA, Longton GM et al. Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies. Transplantation 1991; 51: Przepiorka D, Weisdorf D, Martin P et al. Consensus conference on acute GVHD grading. Bone Marrow Transplant 1995; 15: Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: Boerwinkle E, Xiong W, Fourest E, Chan L. Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3 hypervariable region. Proc Natl Acad Sci USA 1989; 86: Budowle B, Chakraborty R, Giusti AM et al. Analysis of the VNTR locus DIS by PCR followed by high-resolution PAGE. Am J Hum Genet 1991; 48: Mackinnon S, Barnett L, Bourhis JH et al. Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: evaluation of conditioning regimens using polymerase chain reaction to amplify human minisatellite regions of genomic DNA. Blood 1992; : Richards B, Heilig R, Oberle I et al. Rapid PCR analysis of the St14 (DXS52) VNTR. Nucleic Acids Res 1991; 19: Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: Cox DR. Regression models and life-tables. JR Stat Soc 1972; 34: Prentice HG, Janossy G, Price-Jones L et al. Depletion of T lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukemic marrow transplant recipients. Lancet 1984; 1: Waldmann HG, Hale G, Cividalli G et al. Elimination of graftversus-host disease by in vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (Campath-1). Lancet 1984; 2: Korngold R, Sprent J. Variable capacity of L3T4 T cells to cause lethal graft-versus-host disease across minor histocompatibility barriers in mice. J Exp Med 1987; 165: OKunewick JP, Kochiban DL, Buffo MJ. Comparative effects of various T cell subtypes on GVHD in a murine model for MHC-matched unrelated donor transplant. Bone Marrow Transplant 199; 5: Hamilton BL. L3T4-positive T-cells participate in the induction of graft-vs-host disease in response to minor histocompatibility antigens. J Immunol 1987; 139: Pollard CM, Powles RL, Millar JL et al. Leukaemic relapse following Campath-1 treated bone marrow transplantation for leukaemia. Lancet 1986; 2: Prentice HG, Brenner MK. Donor marrow T cell depletion for prevention of GvHD without loss of the GvL effect. Current results in acute myeloblastic leukemia and future directions. In: Gale RP, Champlin RE (eds). Bone Marrow Transplantation: Current Controversies. Alan R Liss: New York, 1989, pp Young JW, Papadopoulos EB, Cunningham I et al. T-celldepleted allogeneic bone marrow transplantation in adults with acute nonlymphocytic leukemia in first remission. Blood 1992; 79: Apperley JF, Jones L, Hale G et al. Bone marrow transplantation for patient with chronic myeloid leukemia: T-cell depletion with Campath-1 reduces the incidence of graft-versus-host disease but may increase the risk of leukaemic relapse. Bone Marrow Transplant 1986; 1: Goldman JM, Gale RP, Horowitz MM et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk for relapse associated with T-cell depletion. Ann Intern Med 1988; 18: Jiang YZ, Mavroudis D, Dermime S et al. Alloreactive CD4 T lymphocytes can exert cytotoxicity to chronic myeloid leukemia cells processing and presenting exogenous antigen. Br J Haematol 1996; 93: Jiang YZ, Barret AJ. Cellular and cytokine-mediated effect of CD4 lymphocyte lines generated in vitro against chronic myelogenous leukemia. Exp Hematol 1995; 23: