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Bone Marrow Transplantation, (1997) 19, 989–999 1997 Stockton Press All rights reserved 0268–3369/97 $12.00 Immune escape from a graft-versus-leukemia effect may play a role in the relapse of myeloid leukemias following allogeneic bone marrow transplantation S Dermime, D Mavroudis, Y-Z Jiang, N Hensel, J Molldrem and AJ Barrett BMT Unit, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, USA Summary: We studied patients relapsing with myeloid leukemias following allogeneic bone marrow transplantation (BMT) for evidence of immune escape by clonal evolution of the leukemia. Relapsed cells from four out of five patients had a reduced ability to stimulate proliferation of lymphocytes from an HLA-mismatched responder. There was decreased susceptibility to lysis by CTL in three and reduced susceptibility to NKmediated lysis in one. Relapsed leukemias had marked alterations in expression of critical surface molecules involved in immune responsiveness. Three had decreased expression of MHC class I and II, with no change or increase in CD54 (ICAM-1) or CD80 (B7.1). None of these responded to treatment with donor lymphocytes. Three patients showed no change, or increased expression of MHC with no change or decrease in ICAM-1 or B7.1. Two achieved remission – one in response to donor lymphocytes and one following withdrawal of cyclosporine. In one patient transplanted with myelodysplastic syndrome in transformation, interferon-gamma upregulated expression of MHC molecules in relapsed cells and increased their stimulatory capacity and target susceptibility to unmatched responder lymphocytes. These results suggest that immune escape through clonal evolution of the leukemia is a common occurrence in patients who relapse with myelogenous leukemias after BMT. Keywords: BMT; GVL; relapse; CML; AML; immune escape Although allogeneic BMT from an HLA-identical sibling donor has a high curative potential in the treatment of myeloid leukemias, relapse of the disease occurs in about 20% of acute myeloid leukemias (AML) and 15% of chronic myelogenous leukemias (CML).1,2 The risk of relapse has been linked to the degree to which the allograft can exert a graft-versus-leukemia (GVL) effect against residual leukemia remaining after the myeloablative preparative regimen. Clinical data supports a role for T lymCorrespondence: Dr AJ Barrett, Building 10, Room 7C-103, National Institutes of Health, 9000 Rockville Pike, Bethesda MD 20892-1652, USA Received 21 October 1996; accepted 26 January 1997 phocytes in the GVL response: relapse rates are higher after T cell-depleted BMT3–5 while remissions can be reinduced with donor lymphocyte transfusions in patients who relapse after BMT.6 There is also experimental evidence for the involvement of NK cells in GVL.7,8 It is therefore generally assumed that leukemic relapse after BMT is related to qualitative or quantitative defects in donor immune function.9 One reason for the failure of GVL could be immune escape by clonal evolution in the leukemia which selects malignant cells that can avoid immune recognition. Malignancies that resist the effects of cellular immunotherapy have been demonstrated in murine models10,11 and suggested in man.12 As described in other tumors, the failure to recognize leukemia cells directly may result from lack of expression of MHC-complex molecules,13–16 lack of a suitable tumor antigen,17 defective antigen processing,18,19 production of inhibitors that actively suppress antitumor response,20 lack of the appropriate costimulatory molecules necessary to induce an immune response,21–23 downregulation of CD54 (ICAM-1)24,25 or lack of expression of CD95 (Fas) antigen,26,27 a cell surface molecule involved in apoptosis. In this report, we compared the immunogenicity of myeloid leukemia cells before BMT and at relapse in patients with AML, myelodysplastic syndrome (MDS) and CML. Using mismatched responder lymphocytes we used proliferative and cytotoxic alloimmune responses to detect and quantitate defects in lymphocyte stimulation and target susceptibility of leukemia cells. We also compared the immunophenotype of the leukemia before and after relapse using a direct immunofluorescence assay to measure critical molecules involved in antigen presentation and lymphocyte responses: MHC class I and II (DR and DQ), ICAM-1, Fas and B7.1. We present data supporting the possibility that leukemias relapsing after BMT are clonally selected to evade donor-derived immune control. Patients and methods Patients and donors Between September 1993 and January 1996, 28 patients with acute or chronic myeloid leukemia or myelodysplastic syndrome received a bone marrow transplant from an HLAmatched sibling. Six developed hematological relapse of leukemia and form the basis of this study (Table 1). After Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 990 informed consent, cells from patients with leukemia and from healthy donors were obtained from the peripheral blood or bone marrow. All patients received a BMT from serologically HLA-A, B and DR-matched donors. The BMT dose of T lymphocytes was adjusted to 2 × 105/kg CD3+ cells by elutriation, but subsequently all patients received a donor lymphocyte add-back of 2 × 106 and 5 × 107 CD3+ cells/kg on days 30 and 45 post-BMT. All patients received cyclosporin GVHD prophylaxis from day −4 to day +100 post-BMT. The cells were separated using Ficoll–Hypaque gradient-density (Organon Teknika, Durham, NC, USA) and subsequently frozen in RPMI-1640 complete medium (CM, 25 mm HEPES buffer, 2 mm l-glutamine, 20% gentamicin; GIBCO BRL, Gaithersburg, MD, USA) supplemented with 20% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, USA), or 20% heat-inactivated pooled human AB serum (HS; PelFreez, Brown Deer, WI, USA) and 10% DMSO according to standard protocols. Before use, the cells were thawed, washed and suspended in CM + 10% HS. Cell lines Lymphoblastoid cell lines (LCL) were obtained by incubating 107 peripheral blood mononuclear cells (PBMC) for 1 h at 37°C with 1 ml of supernatant from the Epstein–Barr virus (EBV)-producing cell line B95-8 (ATCC, Rockville, MD, USA) and then culturing in CM + 10% FBS. Cyclosporin A (2 mg/ml) was added to the cultures during the first 4 weeks. LCL were used after 8 weeks of culture. The K562 NK-sensitive leukemia cell line (ATCC) was maintained in CM + 10% FBS. Differential cell count of patients’ leukemia samples The percentage of leukemia cells from Ficoll–Hypaque separated samples were determined by examination of cytospins of pre-BMT and relapsed bone marrow cells stained with Wright’s Giemsa. Two hundred cells per sample were counted. Proliferative assay The proliferative assay was performed as previously described. 28 In brief, 105 responder mismatched normal individual (D1) PBMCs were cultured with 105 5000 cGY irradiated pre-BMT or relapsed leukemic cells as stimulators. Patient and donor HLA typing data are shown in Table 2. Before the test, the stimulators were thawed, washed and cultured for 24 h in the presence or absence of 103 U/ml recombinant interferon-g (IFN-g; Biosource, Camarillo, CA, USA). The test was set up in six replicates in 96-well U-bottom plates in a total volume of 200 ml CM + 10% HS in each well. Controls were responder cells with 10 000 cGY irradiated K562 cells, responder cells alone and stimulators alone. Cultures were incubated at 37°C in 5% CO2 for 5 days and pulsed for the last 18 h with 1 mCi/well of 3H-labeled methyl thymidine (3HTdR; Amersham International, Amersham, UK). Cells were harvested and 3HTdR incorporation was measured in a beta-counter (Wallac, Gaithersburg, MD, USA). The results are expressed as mean counts per minute (c.p.m.) ± s.d. Cell-mediated cytotoxicity (CMC) Cytotoxic T lymphocyte (CTL) activity: A total of 107 normal PBMCs from donor D1 (used in the proliferative assay) were cultured with 107 5000 cGY irradiated stimulating pre-BMT cells in 25-cm2 flasks containing 10 ml CM + 10% HS. Recombinant interleukin-2 (IL-2; Biosource) was added to the cultures on day 3 and day 7 at 60 IU/ml. On day 10, lymphocytes were harvested, resuspended, and serially diluted to be used in the CTL assay as effectors. Natural killer (NK) cell activity: Fresh PBMCs isolated from the heparinized peripheral blood of a normal individual donor D2 were separated by gradient-density using Ficoll–Hypaque and used immediately as effectors in the cytotoxicity assay against pre-BMT and relapsed leukemia cells. Cytotoxicity assay: A semi-automated mini-cytotoxic assay was used.29 Effector cells (T lymphocytes or NK cells) were prepared in doubling dilutions from 6 × 103 to 24 × 103 or 25 × 103 to 105 cell/well and plated in 40 ml, 60-well Terasaki trays (Robbins Scientific, Mountain View, CA, USA) with six replicates per dilution. Target cells (preBMT or post-BMT leukemia cells, K562 cells) at a concentration of 2 × 106 cell/ml were stained with 10 mg/ml of Calcein-AM (CAM; Molecular Probes, Eugene, OR, USA) for 30 min in a 37°C water bath. After washing three times in CM + 10% HS, target cells were resuspended at 105 cell/ml for K562 and 2 × 105 cells/ml for leukemia cells (103 or 2 × 103 target cells in 10 ml medium were added to each well containing effector cells). Wells with target cells alone and medium alone were used for maximum (Mx) and minimum (Mn) fluorescence emission, respectively. After 4 h incubation at 37°C in 5% CO2, 5 ml FluoroQuench (EB Stain-Quench Reagent; One Lambda, Canoga Park, CA, USA) was added to each well and the trays were centrifuged for 1 min at 60 g before measurement of fluorescence using an automated Lambda FluoroScan (One Lambda). A decrease in the fluorescence emission is proportional to the degree of lysis of target cells, once the released dye is quenched by hemoglobin in the FluoroQuench reagent. The percentage of lysis was calculated as follows: % lysis = (1 − [(mean experimental emission − mean Mn) / mean Mx − mean Mn]) × 100. Antibodies and flow cytometry Phenotypes of pre-BMT and post-BMT leukemia cells were determined by flow cytometry. Cells (106) were incubated with labeled monoclonal antibodies on ice, in the dark for 30 min. After washing three times in phosphate-buffered saline (PBS), cells were analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA). The fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies used were: HLA class I (CALTAC Laboratories, San Francisco, CA, USA), HLA class II DQ (Becton Dickinson) Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al Table 1 Patient characteristics Patient (UPN) Sex/ Diagnosis Age (years) 991 Status pre-BMT Status at relapse Karyotype pre-BMT Karyotype at relapse Relapse on/off CsA Acute GVHD grade Treatment of relapse t(12;14), del8, t(4;21) complex abnormalities 5q−, add1, inv4 inv X, del19 t(9;22), t(1;12), t(2;20), t(3;8) trisomy 20, monosomy 22 t(9;22), t(4;17), t(7;10), t(2;5) del16q t(9;22) on II skin DLT + IL-2 NR, died day 76 off I skin DLT + IL-2 off 0 DLT + IFN-a DLT + IL-2 second BMT NR, died day 510 CR, alive day 330 off 0 DLT + IFN-a CR, alive day 450 on 0 DLT + IL-2 NR, died day 110 on II gut Stop CsA CR, alive day 320 P1 (029) M/25 AML M0 4th rel M4 NA P2 (005) F/51 AML CMML/tMDS M5 5q− P3 (025) M/23 CML CP AP t(9;22) P4 (019) F/34 CML CP CP t(9;22) P5 (017) M/18 CML BC (myeloid) P6 (026) F/29 CML MF BC (myeloid) MF t(9;22), trisomies, +8, +21 t(9;22) t(9;22) Outcome AML = acute myeloid leukemia; CML = chronic myeloid leukemia; CMML = chronic myelomonocytic leukemia; tMDS = myelodysplastic syndrome in transformation; CP = chronic phase; AP = accelerated phase; BC = blast crisis; MF = myelofibrosis; CsA = cyclosporin A; DLT = donor lymphocyte transfusion; NA = not available; IL-2 = interleukin-2; IFN-a = interferon-alpha; NR = no response; CR = cytogenetic remission. Table 2 HLA typing of patients and third-party donors (D1) used in the mixed lymphocyte cultures and T cell-mediated cytotoxicity and D2 as a source of NK cells Patients (UPN) P1 P2 P3 P4 P5 P6 (029) (005) (025) (019) (017) (026) Donors D1 (0331) D2 HLA typing HLA-A HLA-B 1, 3 24, 29 1, 2 1, 2 2, 26 2, 26 7, 39, 35, 63, 13, 38, 2, — 2, 23 13, 27 58, 62 8 44 62 — 47 50 HLA-DR HLA-DQ 8, 11 7, 15 10, 14 3, 8 7, — 13, 7 4, 7 2, 6 5, 52 6, 4 2, 9 6, 9 1, 7 4, 7 2, 5 2, 8 Numbers in bold represent the HLA shared between each patient and the third-party donor D1. and HLA class II DR, CD54, CD80 and CD95 (all from ImmunoTech, Marseille, France). Phycoerythrin (PE)-conjugated anti-CD33 (ImmunoTech) was used to select for CD33+ cells. Controls were CD33+ bone marrow cells, LCLs from normal donors, and K562 cells. Isotypematched irrelevant monoclonal antibodies were used as controls. Results Patient details and relapse data are shown in Table 1. HLA typing of patients and responders are shown in Table 2. Patient 029 (P1) was in a fourth relapse at BMT with an AML M0. At relapse however, the leukemia had M4 morphology and t(12;14), t(4;21) and del(8) karyotype. Patient 005 (P2) was transplanted with tMDS (.65% blast cells) and relapsed with 100% blast cells showing M5 morphology and 5q−, invX, inv4, add1 and del19 karyotype. She had not received additional chemotherapy prior to BMT or at the time the sample was taken at relapse. Patient 025 (P3) with CML chronic phase t(9;22) relapsed into accelerated phase with additional chromosomal rearrangements (t9;22, t1;12, t2;20, t3;8, trisomy 20, monosomy 22). At the time of collection of the pre-BMT and relapsed samples he was not receiving chemotherapy. Patient 019 (P4) was transplanted in chronic phase CML and relapsed into chronic phase. The karyotype at relapse showed clonal progression from a t9;22 karyotype to include t4;17, t7;10, t2;5 and del16q. Patient 017 (P5) was transplanted in myeloid blast crisis CML and appeared morphologically unchanged at relapse. Patient 026 (P6) with myelofibrosis from Ph+ CML was transplanted and appeared morphologically and karyotypically unchanged at relapse. In order to determine whether the composition of pre- and post-BMT leukemic stimulator/target cells was comparable, we performed differential counts of stained cytospins. No important differences in the cellular composition of the leukemic stimulator/target cells were found (Table 3). Stimulation of third party lymphocytes by leukemia cells before and after BMT Paired samples of Ficoll-purified leukemia cells obtained before BMT and at relapse after BMT were tested for their ability to stimulate proliferation of lymphocytes from a third party responder (D1 in Table 2). Proliferation against K562 cells (not expressing HLA class I and II) was used as a negative control to calculate the stimulation index (SI = c.p.m. of responder vs leukemia cells/c.p.m. of responder to K562 cells). Results are shown in Figure 1. K562 cells induced background level proliferation Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al Table 3 Differential counts of leukemic stimulators/targets Patients (UPN) status P1(029) P2(005) P3(025) P4(019) P5(017) Pre Post Pre Post Pre Post Pre Post Pre Post 77 79 86 96 28 17 38 24 91a 92a % Monocytes — — 1 — 51 57 23 38 — — % Lymphocytes — 3 5 — 11 9 18 19 5 3 % Polymorphs (NEB) 18 14 8 4 10 17 18 18 4 5 Cell type % Blasts/myelocytes a Monocytoid blasts and abnormal monocytes. 50 3HTdR incorporation (c.p.m. × 103) 992 45 Pre-BMT Relapse 14 12 10 8 6 4 2 0 40 35 30 P2 relapse P2 relapse + IFN- γ 25 20 15 10 5 0 P1 P2 P3 P4 P5 K562 Stimulators Figure 1 Proliferative responses of pre-BMT and relapsed leukemia cells to third-party T lymphocytes D1. Responder cells (10 5/well) cultured for 5 days with 5000 cGY irradiated leukemia cells (105 /well), incubated for additional 18 h with 3HTdR, and harvested. Relapsed leukemia cells from P2 were incubated with IFN-g (103 U/ml) for 24 h before the test (inset). Controls were responder cells with 10 000 cGY irradiated K562 cells. Counts per minute (c.p.m.) values represent the mean of six replicates ± s.d. (c.p.m. , 100 c.p.m.). Leukemias pre-transplant differed in their ability to stimulate a third party with absolute c.p.m. varying from 6000 ± 1000 to 40 000 ± 2000. Post-transplant stimulation was generally lower from , 100 to 27 5000 ± 2000 c.p.m. Leukemia cells at relapse post-BMT induced lower proliferative responses than the pre-transplant leukemia in four patients with a ratio of pre:post stimulation indices of 1:0.04 (P1), 1:0.59 (P2), 1:0.7 (P3) and 1:0.07 (P4). One patient P5 who was transplanted in blast crisis of CML and relapsed into blast crisis induced comparable low proliferation before and after transplant (6000 c.p.m.). Pre-transplant and post-transplant leukemia samples were incubated with IFN-g (103 U/ml) for 24 h to test for induction of stimulatory ability to the third party lymphocytes. IFN-g treatment of P2 relapsed cells induced a sevenfold increase in stimulatory capacity (Figure 1 inset). There was no increase in the stimulatory capacity of the other relapsed leukemias. Susceptibility to lysis of leukemia cells by CTLs before and after BMT Irradiated pre-transplant leukemia samples were used to stimulate the lymphocytes from the HLA-mismatched responder D1. After 10 days of culture with IL-2 addition and re-feeding, the responder cells were tested for their ability to lyse paired pre- and post-transplant leukemia samples. Results are shown in Figure 2. At an E:T ratio of 24:1 there was a 27 to 44% lysis of pre-transplant targets. Lysis of post-transplant leukemias was markedly reduced in three patients P1, P2 and P5 (4.6, 6.6 and 3.6% respectively). There was no change in the target susceptibility in patients P3 and P4. In patient P2, incubation of the post-transplant but not the pre-transplant leukemia targets with IFN-g induced an increase in the lysis of the posttransplant leukemia to almost pre-BMT levels (E:T at 24:1 rose from 6.6 to 30%). There was no increase in the lytic activity against other pre-BMT and relapsed leukemias. Susceptibility to NK cell-mediated lysis of leukemia cells before and after BMT Fresh normal PBMC was used as a source of NK cells. Standard NK assays were carried out at three E:T ratios, against paired pre- and post-transplant leukemia cells using K562 cells as a positive control. The results for four patients tested are shown in Figure 3. In three both preand post-BMT leukemia samples gave comparable results and were lysed more readily than K562 cells. In one (P4) the relapsed sample was lysed less effectively than K562 cells and considerably less than the pre-transplant leukemia. Phenotype of leukemia cells before and after BMT The alterations in stimulatory ability and target susceptibility of leukemia at relapse after BMT prompted us to examine the possibility that escape from immune surveillance had occurred by downregulation in surface molecules involved in immune responses. We therefore compared expression of MHC (class I and class II), CD45 (ICAM1), CD80 (B7.1) and CD95 (Fas) in CD33+ gated cell populations of pre-transplant and post-transplant relapsed leukemias. An additional patient (P6) was also studied. Results of flow cytometry were expressed as % positive cells and Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 993 50 P2 P2 (IFN) Pre-BMT Pre-BMT+ IFN-γ Relapse Relapse+IFN-γ K562 cells 40 30 20 10 0 0 6 12 18 12 18 24 50 P3 % Lysis 6 0 24 50 P4 40 40 30 30 20 20 10 10 0 0 0 6 12 18 0 24 6 12 18 24 6 12 18 24 50 50 P5 P1 40 40 30 30 20 20 10 10 0 0 0 6 12 18 24 0 E:T ratio Pre-BMT Relapse K562 cells Figure 2 Susceptibility to cell-mediated cytotoxicity of pre-BMT and relapsed leukemia cells by normal third-party lymphocytes from D1. Effector cells at dilutions of 6 × 103 –48 × 103 cell/well/20 ml were plated with 2 × 103 pre-BMT or relapsed leukemia cells as targets in a 4-h cytotoxic assay. Six replicates were used for each dilution. Leukemia cells from P2 were incubated with IFN-g (103 U/ml) for 24 h before the test (inset). K562 target cells were used as a control. fluorescent intensity (FI) and calibrated against EBV LCL (strong positivity) and K562 (weak to zero positivity). HLA class I expression: With the exception of K562 cells all samples tested contained more than 5% MHC class I positive cells and in most cases the percentage of positive cells was over 90% (Figure 4a). The fluorescence intensity (FI) of MHC class I was in general much lower than that of EBV-LCL cells. In P1, P2 and P3, FI was in the region of 30% of EBV-LCL pre-transplant, and fell to barely detectable levels after BMT. In P3, the post-BMT sample also showed a major reduction in the percentage of cells expressing MHC class I. P4 and P5 showed no difference in FI before and after BMT. An increase in MHC class I expression from 60 to 100% post-transplant with a corresponding increase in FI occurred in P6. Incubation with IFN-g modestly increased FI in most samples. HLA class II DR expression: HLA class II DR was generally less well expressed on leukemia cells, both in terms of % positive cells and their FI, compared with EBV-LCL (Figure 4b). Only patient P5 expressed high levels of DR. Comparing pre- and post-relapse samples, there were three discernable alterations in HLA DR expression: (1) lower percentage of DR expressing cells at relapse after BMT with almost undetectable FI at relapse (P1, P2 and P3); (2) slightly higher percentage of cells expressing HLA DR after BMT but with extremely low FI (P4 and P6); and (3) P5 with high DR expression and high FI pre- and postBMT. IFN-g had no effect on the percentage of cells expressing HLA DR but increased FI in P5 (pre- and postBMT) and P1 post-BMT. HLA class II DQ: Compared with EBV-LCL the % and the FI of leukemia cells expressing HLA DQ was generally Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 994 100 100 % Lysis P1 P2 80 80 60 60 40 40 20 20 0 0 0 100 25 50 75 100 0 100 P4 80 80 60 60 40 40 20 20 0 25 50 75 100 25 50 75 100 P5 0 0 25 50 75 100 0 E:T ratio Pre-BMT; Relapse; K562 cells Figure 3 Lytic susceptibility of pre-BMT and relapsed leukemia cells to NK cells from D2. Effector cells at dilutions to achieve 25 × 103–200 × 103 cell/well/20 ml were plated with 2 × 103 pre-BMT or relapsed leukemia cells as targets in a 4-h cytotoxic assay. Six replicates were used for each dilution. K562 target cells were used as a control. very low (Figure 4c). Only P3 and P5 had more than 10% cells expressing DQ, with FI above the background for K562 cells. Comparing pre- and post-BMT samples, there were two changes: (1) low to undetectable % expression and FI (P1, P2, P4 and P6); and (2) moderate % expression and FI (P3 and P5). DQ expression in P3 was lower at relapse post-BMT, while in P5 expression and FI increased at relapse. IFN-g did not generally affect DQ expression or FI. The exception was P2 who showed an increase in % cells expressing DQ after but not before BMT. CD54 (ICAM-1) expression: Expression of ICAM-1 both in percentage of marked cells and FI was variable (Figure 4d). Some leukemias marked as strongly as EBV-LCL, while in others expression was less than on K562 cells. Comparing pre- and post-BMT samples, three variations were observed: (1) low % expression pre-BMT, with increase at relapse (P1, P2 and P3); (2) lower % expression post-BMT (P4 and P6) and (3) high expression with no change (P5). There were no clear differences in FI before and after BMT. In general, IFN-g did not alter ICAM-1 expression in leukemia cells. In P1 post-BMT and P5 preBMT however IFN-g increased FI. CD80 (B7.1) expression: B7.1 was strongly expressed on EBV LCL but was less than 10% in K562 cells. Expression was variable in the leukemias (Figure 4e). Comparing pre- and post-relapse samples P1, P4, P5 and P6 had lower % expression and usually lower FI after BMT, whereas P2 and P3 showed increased % expression after BMT but decreased FI. IFN-g modestly increased B7.1 expression and FI in some samples. CD95 (Fas) expression: Fas was variably detectable on leukemia cells. Notably P5 had extremely strong Fas expression (Figure 4f). Comparing pre- and post-BMT samples P1, P3, and P5 had moderate to high expression (20–100%) and FI above 20 which did not change at relapse. P2 and P6 showed an increased % of cells expressing Fas at relapse with no change in FI. P4 Fas expression fell from around 80 to 35% post-BMT with no change in FI. IFN-g increased Fas expression in P1 post-relapse cells. Correlation of changes in phenotype with immune function and outcome of immune manipulation Table 4 summarizes the alterations in immune characteristics of the six patients tested and correlates results with changes in immune phenotype and clinical outcome. Behavior at relapse can be categorized into two broad groups. Loss in MHC class I and II expression (P1, P2 and P3): Reduction in stimulatory capacity of relapsed cells Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 995 a LCL 100 Pre-BMT Relapse 80 –IFN-γ 40 % Positive cells Fluorescence intensity (Fl) 60 20 0 K562 P1 P2 P3 P6 P4 P5 LCL 100 80 60 40 +IFN-γ 20 0 K562 100 LCL 1800 1600 1400 1200 1000 800 600 400 200 0 LCL K562 P1 P2 P3 P6 P4 P5 1800 1600 1400 1200 1000 800 600 400 200 0 LCL K562 b 2400 2200 Pre-BMT 2000 Relapse 1800 1600 80 –IFN-γ % Positive cells 40 20 K562 0 P1 P2 P3 P6 P4 P5 LCL 100 80 60 40 Fluorescence intensity (FI) 60 +IFN-γ 20 K562 0 LCL 1400 1200 1000 800 600 400 200 0 K562 P1 P2 P3 P6 P4 P5 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 K562 320 LCL LCL c LCL 100 Pre-BMT 280 Relapse 240 80 –IFN-γ % Positive cells 40 20 K562 0 P1 P2 P3 P6 P4 P5 100 LCL 80 60 40 +IFN-γ 20 0 K562 Fluorescence intensity (FI) 60 200 160 120 80 40 0 K562 P1 P2 P3 P6 P4 P5 320 280 240 200 160 120 80 40 0 LCL K562 Figure 4 HLA class I (a), class II DR (b), class II DQ (c), ICAM-1 (d), B7.1 (e) and Fas (f) expression by pre-BMT and relapsed leukemia cells analyzed by flow cytometry. Percentage positive cells (%) and fluorescence intensity (FI, median channel of fluorescence (log)) were measured in untreated (−IFNg) and IFN-g treated (+IFNg) cells. Mean ± s.d. of percentage marked cells and FI from three × LCL lines and K562 were used as high and low controls (horizontal grey bars). Differences between pre- and post-BMT and + or − IFNg samples were considered significant if they exceeded 2 s.d. of LCL controls. and loss of susceptibility to CTL, but not NK cytotoxicity in the two patients tested, was associated with no significant change, or an increase in B7.1, ICAM-1 and Fas. None of these three patients responded to treatment with donor lymphocytes. No change (P4) or an increase in MHC class I and II expression (P5 and P6): Loss of susceptibility to CTL or NK cells, with low or reduced stimulatory capacity, was associated with loss of B7.1, no change or loss or ICAM1 and variable changes in Fas. Two out of three patients Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 996 d 100 LCL 60 % Positive cells 40 20 0 P1 P2 P3 P6 P4 P5 100 LCL 80 60 40 +IFN-γ 20 Fluorescence intensity (FI) 120 Pre-BMT 100 Relapse 80 60 –IFN-γ 40 20 K562 0 80 K562 0 LCL K562 P1 P2 P3 P6 P4 P5 120 100 80 60 40 20 0 LCL K562 e 100 100 LCL 60 –IFN-γ % Positive cells 40 20 0 K562 P1 P2 P3 P6 P4 P5 100 LCL 80 60 40 Fluorescence intensity (FI) Pre-BMT 80 Relapse 80 +IFN-γ 20 LCL 40 K562 20 0 P1 P2 P3 P6 P4 P5 100 80 60 LCL 40 K562 20 K562 0 60 0 f 100 100 LCL 80 60 –IFN-γ % Positive cells 40 20 0 100 P1 P2 P3 P6 P4 P5 K562 LCL 80 60 +IFN-γ 40 Figure 4 60 40 K562 LCL K562 20 0 P1 P2 P3 P6 P4 P5 100 80 60 40 20 20 0 Fluorescence intensity (FI) Pre-BMT 80 Relapse LCL K562 0 (Continued). responded to immune manipulation (one to DLT (P4)) and one to withdrawal of cyclosporine immunosuppression. Discussion It has long been considered that malignant cells may undergo clonal selection to escape from immune regulation and there is circumstantial evidence supporting this hypothesis.10–12 The demonstration of a GVL effect following allo- geneic BMT and the effect of immune manipulation to induce remissions in patients relapsing after BMT (T celldepletion, DLT, stopping CsA, IFN-a) strongly supports a role of the donor immune system in regulating residual leukemia after BMT. Leukemic relapse following allogeneic BMT is usually attributed to a failure of the preparative regimen or the donor immune system to control residual disease. An alternative possibility however is that the leukemia may have evolved to escape from immune control under the strong negative selection pressure of an Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al Table 4 Correlation of changes in phenotype with immunogenicity of leukemia and outcome of immune manipulation with donor lymphocytes or withdrawal of cyclosporine Diagnosis at relapse Phenotype Functional tests Response to treatment P1 (029) AML M4 P2 (005) MDSt P3 (025) CML AP P4 (019) CML CP P5 (017) CML BC P6 (026) CML MF MHC class I HLA DR HLA DQ ↓ ↓ L ↓ ↓ L ↓ ↓ ↓ 0 0 0 0 0 0 ↑ ↑ 0 ICAM-1 (CD54) B7.1 (CD80) fas (CD95) ↑ L 0 ↑ ↑ ↑ ↑ ↑ 0 0 ↓ 0 ↓ ↓ L ↓ ↓ ↑ stimulation CTL lysis NK lysis ↓ ↓ 0 ↓ ↓ 0 ↓ 0 NT L ↓ 0 ↓ ↑ ↓ NT NT NT No No No No Yes Donor lymphocytes Yes Cyclosporine withdrawal At relapse: ↑ = increased; ↓ = decreased; 0 = no change; L = no change but low; NT = not tested. alloreactive donor immune system. Only one report has associated leukemic relapse after allogeneic BMT with immune escape of the leukemia.12 In this study, relapse occurred after an HLA-haploidentical BMT in a patient with acute lymphocytic leukemia. The weak mixed lymphocyte culture and undetectable primary cell-mediated cytotoxicity responses of the donor to the post-BMT relapse cells suggested that the relapsed leukemia may have been selected in vivo for resistance to the donor immunity. We argued that immune escape leading to relapse after BMT could occur either because the leukemia becomes nonstimulatory to donor T cells or ceases to be susceptible to destruction from effector T cells or NK cells. We therefore examined six patients with various myeloid leukemias who relapsed with leukemia after HLA-matched sibling BMT, comparing pre-transplant leukemia with the relapsed leukemia, for its ability to stimulate an immune response and to be a target for T cell and NK cell attack. We showed that in four of five leukemias, the ability to stimulate a third party lymphocyte proliferative responses was significantly reduced. In the fifth patient the stimulatory capacity of the leukemia was low pre-transplant and remained unchanged. By using the normal donor as an HLA-matched control for the third party responder, we showed that the response was significantly lower than that of a normal stimulator cell. Using a similar approach we used pre-transplant leukemia cells to stimulate CTL from an HLA-mismatched normal responder. (We chose pre-transplant cells to induce CTL, since they were shown to be capable of stimulating a third party proliferative response.) After 10 days, CTL generated in this way had no LAK activity as demonstrated by their inability to lyse K562 cells. However they showed specific cytotoxic activity to the stimulating leukemia. Using a second normal responder, the susceptibility to NKmediated lysis was also tested. Leukemia cells from all patients before the BMT were susceptible to CTL lysis and more susceptible to NK lysis than K562 cells. In three of five patients tested, there was a reduction in the ability of CTL to lyse the post-transplant target, with conserved susceptibility to NK lysis. One patient at relapse showed no change in target susceptibility and one showed an increased susceptibility to CTL lysis, but a reduced sensitivity to NK lysis with cells that were more resistant than K562 cells. Since there were no important differences in the composition of the stimulator/targets before and after relapse, the observed differences in immunogenicity between pre-BMT and relapsed leukemias could not be attributed to the stimulator/target cell composition (Table 3). In order further to define the changes in susceptibility to immune attack, we quantitated major surface molecules involved in the immune response on pre- and post-BMT leukemia samples. CD33+ cell fractions from CML and AML and MDS pre-transplant, showed some variation in the percentage of marked cells and fluorescence intensity. Mostly the expression of MHC molecules, costimulatory molecules and Fas, were in an intermediate range between the highly expressing EBV LCL controls and the low or absent expression found in K562 cells. Leukemias relapsing after transplantation showed two broad patterns of alteration in surface molecule expression: (1) downregulation of MHC class I and II with upregulation of ICAM-1 and upregulation or high expression of B7.1; and (2) increase or no change in MHC expression but decrease or no change in ICAM-1 and B7.1. Expression of Fas antigen was variably altered at relapse (in four it was unchanged or fell but remained highly expressed; in two it was increased). Both loss of MHC expression or loss of costimulation from ICAM-1 or B7.1 could explain the decreased ability of relapsed leukemia to stimulate T cell responses. These findings did not however readily explain the reduced susceptibility of the leukemias to CTL or NK mediated lysis. The decreased ability of the relapsed leukemia to stimulate an immune response and the increased ability to avoid cytotoxic damage from CTL or NK cells were consistent with the hypothesis that immune escape had occurred by clonal evolution leading to relapse. In support of clonal evolution in the leukemia, was the observation of new chromosomal abnormalities in P2, P3 and P4 and a change in morphology in P1 and P2. The alteration in the leukemia cell type could be the result of selection of pre-existing leukemia sub-clones remaining after the preparative regimen or to mutations occurring after BMT. It is of course 997 Mechanisms of escape from GVL by relapsed leukemia cells S Dermime et al 998 possible that many other factors involved in target susceptibility to lysis could also have altered to allow the leukemia to relapse, or that relapse occurred for non-immunological reasons. The existing data are insufficient to link the in vitro changes that we found with responsiveness of the leukemia to treatment with donor lymphocytes, withdrawal of immunosuppressive therapy or use of cytokines to alter the immune response. It is of interest however that none of the three patients who showed downregulation of MHC molecules and upregulation of ICAM-1 and B7.1 responded to donor lymphocyte infusions with or without the addition of IFN-a, while two of the patients who showed MHC upregulation and downregulation of ICAM-1 and B7.1 achieved a further remission (one following cessation of CsA and one following DLT and IFN-a). Finally we investigated the potential of immune modulation to repair defects of stimulatory capacity or target susceptibility. Since IFN-g upregulates MHC molecules30 we tested its effect on upregulating surface molecule expression of relapse leukemia cells and altering immunostimulation and susceptibility to lysis. IFN-g upregulated MHC class I and II expression in some relapsed leukemias tested and variably upregulated ICAM-1 and B7.1 expression. One patient P2 showed a significant response with a marked increase in stimulatory ability of the relapsed leukemia and return of the target susceptibility to pre-transplant levels. These changes were accompanied by small increases in ICAM-1 and MHC expression. In conclusion these studies suggest that escape from immune regulation may be a common accompaniment of clonal evolution leading to relapse after allogeneic BMT. 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