Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplant

Experimental Hematology 27 (1999) 1477–1486

Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplant: Where we now stand Francesco Dazzi, Richard M. Szydlo, and John M. Goldman From the Department of Haematology, Hammersmith Hospital/ICSM, London, United Kingdom (Received 21 June 1999; accepted 28 June 1999)

The infusion of lymphocytes from the original marrow donor (donor lymphocyte infusion [DLI]) reinduces complete remission in a high percentage of patients with chronic myeloid leukemia (CML) who relapse after allogeneic stem cell transplant, and thus, is probably the best initial approach to their management. The major predictive factor for response is the disease stage at time of treatment, because patients in molecular or cytogenetic relapse fare better than those in hematologic relapse. Moreover, patients with a short interval between transplant and DLI have a higher probability of response than those with longer intervals. The durability of DLI-induced remissions has not yet been established, but they appear to be prolonged. The observation that DLI can be highly effective for patients in relapse has encouraged the recent development of new strategies designed to minimize the myeloablative regimen and exploit the immunotherapeutic component of the transplant. The principal complication associated with use of DLI is the occurrence of graft-versus-host disease (GVHD). Several approaches have been tested to reduce the incidence or impact of GVHD, based on the ex vivo depletion of alloreactive donor cells or the use of donor T cells transduced with a suicide gene. The incidence of GVHD can also be reduced by starting with low doses of donor cells and “escalating” subsequent doses as required. However, the identification of selective targets for leukemia-reactive immunity is probably the optimal strategy to resolve the problem of GVHD. Although currently minor histocompatibility antigens appear to be the most likely targets for DLI, several groups are focusing on the generation of leukemia-specific immunity. The results obtained by use of tumor-associated antigens presented by dendritic cells are encouraging and may lay the foundations for the use of adoptive immunotherapy in the autologous setting. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Offprint requests to: Dr. Francesco Dazzi, Department of Haematology, Hammersmith Hospital/ICSM, Du Cane Road, London W12 0NN, UK; E-mail: [email protected]

Keywords: Donor lymphocyte infusion—Chronic myeloid leukemia—Bone marrow transplantation—Graft-vs-leukemia—Graft-vs-host disease

Introduction One of the most important observations in clinical stem cell transplantation over the last decade has been the clear demonstration that chronic myeloid leukemia (CML) can be cured by the administration of allogeneic T lymphocytes. This has prompted investigators to develop novel strategies of adoptive immunotherapy to treat malignant diseases and to attempt to identify the safest and most effective methodology to exploit this new powerful tool. Chronic myeloid leukemia Chronic myeloid leukemia (CML) is a neoplastic disorder originating in a primitive hematopoietic stem cell. The hallmark of the disease is the Philadelphia (Ph) chromosomal translocation t(9;22) that generates a BCR-ABL fusion gene by juxtaposing the ABL proto-oncogene to the BCR gene. The BCR-ABL gene encodes a 210 kD protein (p210BCR-ABL) that probably initiates the neoplastic process [1]. Despite many attempts to isolate p210BCR-ABL-specific antibodies [2], at present leukemia cells can be reliably characterized only at the molecular level. Clinically, CML usually progresses from a chronic phase of variable length to an accelerated phase and then to a blastic phase that terminates with the patient’s death. Blastic transformation is typically accompanied by nonrandom secondary chromosomal changes that may be responsible in some way for the acquisition of the more malignant phenotype [3,4]. Therapeutic options for CML Although single agent chemotherapy (hydroxyurea) induces hematologic remissions in the majority of patients, it rarely results in any degree of Ph-negative hematopoiesis and probably does little or nothing to delay progression to the

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blastic phase. Interferon-a (IFNa) is superior to chemotherapy and up to 30% of patients achieve major or complete cytogenetic responses [5]. The mechanism of action of IFNa is unknown. It has been suggested that IFNa alters some of the malignant properties of CML cells [6,7] and/or enhances sensitivity of leukemic cells to immune recognition [8]. There is currently interest in using high-dose chemotherapy to mobilize and collect Ph-negative stem cells for subsequent use in an autologous transplant [9], but there is as yet no evidence that autografting with these cells prolongs survival. Allogeneic hematopoietic stem cell transplantation (alloSCT) is probably the only treatment that can cure patients with CML. The disease-free survival at 5 years may reach 70% in allografted patients [10,11]. Cure is probably mediated by the combined effects of the high dose chemotherapy and a graft-vs-leukemia (GVL) effect mediated by donorderived lymphoid cells. Thus, the high doses of chemoradiotherapy used in the pretransplant regimen are undoubtedly effective in “de-bulking” the leukemia, but alone are usually insufficient to eradicate the disease. The evidence for this conclusion is based on two principal lines of evidence: (1) patients who receive T-cell depleted donor marrow have significantly higher risk of relapse than those receiving unmanipulated transplants [12]; and (2) a higher incidence of relapse has also been reported using identical twins as compared with HLA-identical sibling donors [13]. Thus, the GVL effect must play a crucial role in eradicating leukemia after allografting. Unfortunately, CML patients who have achieved complete remission after allografting may still relapse. Until the beginning of this decade the therapeutic options for these patients were limited to a second transplant, which was associated with significant mortality [14], or to the use of IFNa, which can restore remission in only a minority of cases [15]. The observation that T lymphocytes present in donor marrow cells can exert an antileukemic effect constituted an important advance and suggested novel strategies for managing CML and other malignant hematologic disorders. Two main conclusions can now be drawn: (1) donor T lymphocytes play a major role in the control or eradication of leukemia by mediating the GVL effect; and (2) such a GVL effect can be mainly achieved with allogeneic T cells. The proof of these principles came with the demonstration that infusions of lymphocytes from the original stem cell donor are sufficient to restore remission in patients with CML who relapse after allografting [16]. Donor lymphocyte infusion (DLI) have since been widely used to treat relapses of all types of leukemia after allogeneic bone marrow transplant (BMT), to reverse defective bone marrow engraftment due to graft rejection, to reconstitute immune deficiency, and to cure viral diseases that may complicate transplant. Use of DLI in CML The now extensive use of adoptive immunotherapy after allografting has shown clearly that CML cells are exquisitely

sensitive to immune recognition and that DLI should be considered as initial treatment for patients who relapse. The results with other hematologic malignancies including acute leukemia, multiple myeloma and lymphomas are less convincing and require further study. Evidence supporting a graft-vs-tumor effect in solid tumors is circumstantial. Therefore, the experience acquired in CML has shed light on the mechanisms underlying the effect of DLI and provided important information to design effective and safe procedures to administer donor cells. Timing of treatment. Our understanding of the molecular biology of CML has led to development of laboratory assays that are now essential for monitoring disease and for diagnosing relapse after transplant. The BCR-ABL fusion gene can be detected and quantitated by RT-PCR such that a progressive rise in the numbers of BCR-ABL mRNA transcripts in the blood indicates relapse of CML at the molecular level before the appearance of any Ph-positive metaphases [17]. Subsequently, relapse can be recognized at the cytogenetic level and thereafter the leukemia, if untreated, will eventually progress to hematologic relapse. Molecular and cytogenetic relapses are more responsive to DLI than hematologic relapses [18]. The interval between BMT and DLI also correlates with the probability of response; when the interval is less than 2 years, response to DLI is significantly more likely [19]. These data suggest that DLI should be used as soon as evidence of relapse is documented. In practice there is still debate as to whether it is necessary to treat molecular relapse because in rare cases it may be transient, but the quantitation of BCR-ABL transcripts by competitive PCR has shown that increasing transcript levels are highly predictive of cytogenetic relapse [17]. Response rate and factors for response. Table 1 illustrates the response rates reported by the most representative studies [18–27]. DLI results in complete remission in a high percentage of patients although the actual values do vary. These differences might be explained by the heterogeneity of the patients treated. Certain features correlate with response to DLI. The most important predictive factor is the disease stage at time of DLI. Patients with a relapse detected only at the molecular or cytogenetic level fare better than those with hematologic evidence of disease [18,19,22,23]. Among hematologic relapses, patients in chronic phase usually respond more favorably than those with advanced disease [18,19,22,23], yet some groups have observed excellent results in patients with accelerated or blastic phase disease [20,26]. The stage of disease at the time of transplantation appears almost as important as the stage of disease at time of DLI to predict response [22], but it is not known which of the two factors is more relevant. The interval between BMT to DLI also predicts response. Thus, response rates are higher if the interval is less than 2 years [19]. This finding does help to identify the mechanisms that underlie susceptibility of leukemic cells to

F. Dazzi et al./Experimental Hematology 27 (1999) 1477–1486 Table 1. Response rates of chronic myeloid leukemia to donor lymphocyte infusions in major studies Responders/total number of patients (%) at the time of donor lymphocyte infusion Authors

Mrel/Cyrel

CP

van Rhee et al. [18] Collins et al. [19] Drobyski et al. [20] Porter et al. [21] Kolb et al. [22] Mackinnon et al. [23] Bacigalupo et al. [24]* Alyea et al. [25] Verdonck et al. [26] Sehn et al. [27]

11/11 (100) 8/14 (57) 3/3 (100) 25/34 (73) — — — 6/8 (75) 14/17 (82) 39/53 (73) 8/8 (100) 9/10 (90) 6 12 15/19 (79) — 9/9 (100) N/S N/S

AP

Overall

1/5 (20) 5/18 (27) 6/8 (75) 0/3 (0) 1/14 (7) 2/4 (50) N/S 0/5 (0) 4/5 (80) N/S

20/30 (66) 33/42 (78) — 6/11 (54) 54/84 (64) 19/22 (86) 10/18 (55) 15/24 (62) 13/14 (93) 19/23 (82)

AP indicates advanced phase (accelerated and blastic); CP, chronic phase; Cyrel, cytogenetic relapse; Mrel indicates molecular relapse; N/S, not specified. *The number of patients at the time of treatment, not the number of responders per disease stage, was reported in this study.

DLI, since it is likely that this interval, as an indirect measurement of the time from BMT to relapse, reflects the activity of the disease and/or the tumor burden. There is no correlation between response to DLI and the donor type. A recent report showed that the overall probability of obtaining cytogenetic remission was not significantly different in patients receiving DLI from HLAmatched volunteer donors (VUD) and in those transfused with leukocytes from HLA-identical siblings (SIB) [28]. Depletion of T cells from the original BM graft and absence of GVHD after transplant seems to be associated with a higher proportion of responders to DLI [22]. This observation appears to be in conflict with the finding of similar response rates in recipients of VUD and SIB transplants, because in the above-mentioned study the VUD recipients and the SIB recipients received T-cell depleted and T-replete transplants, respectively. The effect of T-cell depletion on DLI response rates has not been confirmed by others [19]. Many DLI recipients also receive IFNa. This agent is thought to enhance the immune response [9]. However, multivariate analyses of large groups of CML patients have failed to identify any difference in response rate [19,22] as initially suggested by others [29]. In conclusion, disease activity remains the major factor influencing response to DLI. However, it is still unclear whether advanced disease is refractory because of an unfavorable ratio between donor T cells and recipient leukemic cells or because leukemic cells become intrinsically resistant to effector T cells. Graft-vs-host disease. The major complication of DLI is graft-vs-host disease (GVHD). Acute and chronic GVHD produce significant morbidity and sometimes mortality. The

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two largest multicenter studies have reported an overall incidence of 41% [22] and 60% [19] for both acute and chronic GVHD in patients receiving DLI. Importantly, GVHD is closely correlated with disease response and in one study only 3 of the 23 patients who did not develop GVHD responded to DLI [19]. However, complete remissions can undoubtedly be attained in the absence of GVHD, suggesting that a GVH/GVL effect can be achieved without producing the specific symptoms of GVHD [22,23]. It has been reported that the incidence of acute GVHD is higher in patients receiving cells from VUD as compared with those receiving cells from their SIB but no difference in the GVL effect has been demonstrated [28]. This observation further corroborates the concept that clinical GVHD is partially distinct from the biologic GVH/GVL. Another major complication of DLI directly related to the GVH effect is bone marrow failure. DLI can lead to a pancytopenia of variable intensity, which in some cases is prolonged and requires the infusion of donor stem cells. Bone marrow failure occurs only in patients treated in hematologic relapse. Kiel et al. [30] reported a correlation between bone marrow aplasia and lack of residual donor hematopoiesis. They also found that the assessment of BCRABL-positive hematopoiesis alone is insufficient for predicting aplasia. Because other studies have failed to detect any correlation between pancytopenia and GVHD, these findings strongly suggest that GVL is closely related to GVH but not to GVHD.

Regimen of administration. The conventional approach to administration of DLI has been to infuse single relatively large doses containing variable numbers of CD31 T cells (bulk dose regimen [BDR]). Although this regimen is undoubtedly effective, it is associated with a high incidence of acute and chronic GVHD [1,22]. Based on the observation that a proportion of responders do not experience GVHD, the Sloan-Kettering Transplant Group transfused donor lymphocytes in multiple aliquots starting at a low cell number and escalating subsequent doses until remission was achieved (escalating dose regimen [EDR]) [23]. By avoiding administration of unnecessarily high cell doses, this approach can limit the incidence and severity of GVHD without apparently jeopardizing the GVL effect. A preliminary study conducted by the Hammersmith Transplant Group on a larger cohort of patients showed that the GVL effect is similar with the two regimens but that the incidence and severity of GVHD are reduced using EDR. Intriguingly, they found that it is the fact that the lymphoid cells are administered on an escalating dose schedule over a considerable number of months and not the fact that the final dose is lower that is responsible for the lower incidence of GVHD in recipients of DLI administered by the EDR [31]. These findings suggest the initial dose(s) of donor cells that is inadequate to exert a useful GVL effect may be “anergized”

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by contact with recipient tissues and thereby reduce the capacity of the subsequent higher dose or doses to produce GVHD. This speculation is in accord with the notion of ‘transferable anergy’ whereby anergic T cells can inhibit an allospecific T-cell response [32]. The existence of a suppressor cell population responsible for the resistance to reinduction of GVHD has been postulated also on the basis of data from BMT in experimental systems [33]. Another interesting observation relates to the interval between DLI infusions. From four studies adopting the EDR protocol with different intervals between infusions [23,24, 26,31], it appears that regardless of the cell dose administered, the lowest incidence of GVHD was observed in the study where DLI were given at the longest intervals [30]. Notably, delayed administration of donor splenocytes in a murine model of allogeneic bone marrow transplantation greatly reduces the incidence and severity of GVHD [34]. Effective cell dose. There is no information about the cell dose required to achieve remission (effective cell dose [ECD]). The identification of the minimal ECD for different phases of CML would be desirable. Furthermore, it may help to determine whether the ECD is related to the leukemic cell number or to a differential sensitivity of leukemic cells characteristic of the different disease phases of CML. In vivo studies conducted in murine models of allogeneic BMT have shown that the outcome of the antileukemic response is determined, at least in part, by the balance between recipient leukemic cells and donor effector cells [35,36]. The Hammersmith Transplant Group [37] has recently reported the results of a preliminary study on 30 CML patients treated according to an EDR treatment schedule. They found that although DLI is subject to a dose-response effect, the ECD does not correlate with disease stage and, thus, to the leukemic burden. The ECD is lower for VUD than for SIB recipients although no difference in overall response rate was observed between the two groups. They suggested that the ECD is influenced only partially by the tumor burden and that non-MHC mismatches (donor type) may play a major role in determining the efficacy of donor lymphocytes [37]. Durability of response. Although DLI is clearly effective at least in the short term, information about the durability of remission is still preliminary, since the follow-up is short. Furthermore, the optimal end-point for treatment remains unclear. Important unanswered queries include: 1. What level of remission is required for a response to be durable? 2. Does molecular remission guarantee complete eradication of the leukemic clone? 3. For how long can donor T cells control residual leukemic cells?

In a preliminary study of 37 CML patients achieving cytogenetic remission 4 to 70 months after DLI, 22 patients (59%) remained in complete remission [38]. More recently, Dazzi et al. [39] have shown that of 44 CML patients achieving at least two consecutive negative PCR studies after DLI, only 4 have become PCR-positive with a follow-up of 4 to 70 months. These preliminary results suggest that attainment of molecular remission is indeed highly predictive of durable remission. It is not known whether these remissions are durable due to the leukemia being eradicated or because donor T cells are able to produce active immune surveillance over the leukemia. Studies of adoptive immunotherapy using genemarked donor T cell lines to treat post-transplant viral diseases have shown that donor T cells can be detected at least 18 months after infusion and can be recruited again in case of viral reactivation [40]. Similarly, the life span of memory T cells is relatively long [41]. On the other hand, in the DLI setting tumor escape mechanisms and the fact that donor T-cell ontogeny might be affected by the allogeneic environment, could reduce stimulation and life span of donor T cells, respectively. There is evidence that some patients may respond only transiently to DLI, which suggests that in some instances donor lymphocytes can be effective only for a limited period of time [42]. Although transient responses might simply be a matter of insufficient donor cell number [37], the hypothesis that donor T lymphocytes can be anergised by the tumor cells [43] is also plausible, which might also account for DLI failures in advanced phase CML. Innovative strategies to prevent GVHD Because GVHD is the major complication of DLI, various approaches have been proposed to circumvent it based on manipulation in vitro of donor lymphocytes prior to infusion into patients (Table 2). Because data obtained from mice [44] and humans [45] suggested CD81 cytotoxic/suppressor T lymphocytes contain most of the GVH effectors, Giralt et al. [46] selectively depleted CD81 cells from the donor lymphocyte collection. The hypothesis that CD8depleted DLI can reduce the risk of GVHD without affecting the GVL activity has since been confirmed in a more recent study, although a significant proportion (50%) of responders still experienced GVHD [25]. An alternative method to deplete potentially harmful cells has been developed and pursued independently by sev-

Table 2. Approaches to reduce DLI-induced GVHD Approaches Depletion of alloreactive T cells Depletion of CD81 cells Transduction of a suicide gene into T cells Use of analogue peptides Administration of escalating doses of cells

References [47–50] [29, 44–46] [53–55] [56, 57] [23, 24, 26, 31]

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eral groups [47–49]. Donor alloreactive T cells can be specifically activated in vitro by recipient cells and selectively removed using an antibody specific for the a-chain of the IL-2 receptor (CD25) that is expressed by activated T cells. CD25-positive cells can be removed either by use of a toxin bound to the antibody [47,49] or by immunomagnetic separation [48]. This approach appears to efficiently delete alloreactive cells, while sparing antileukemic and antiviral cytotoxic responses [49]. These conclusions are, however, based on in vitro studies and need to be tested in the clinic before conclusions can be drawn. Guinan et al. [50] have recently adopted a similar approach in a clinical stem cell transplantation setting. Knowing that T cells can be rendered anergic in vitro by blocking the interaction between the B7 molecule and CD28 [51], they cocultured donor bone marrow cells with mononuclear cells from the recipient in the presence of CTLA-4-Ig, an inhibitor of B7-CD28-mediated costimulation. This ex vivo treatment was performed on the donor bone marrow preparation prior to infusion in 12 transplant patients mismatched for one HLA haplotype. All 11 evaluable patients engrafted and only 3 had GVHD. A further strategy to avoid GVHD following DLI involves the use of suicide genes. Suicide genes encode for enzymes that render cells sensitive to otherwise non-toxic compounds. The thymidine kinase encoded by the herpes simplex virus type 1 (HSV-tk) converts ganciclovir (GCV) to an active metabolite that inhibits DNA extension [52] and leads to death of the cell. The artificial transfer of the HSVtk gene into T lymphocytes, therefore, can provide a means to kill dividing T cells on demand. This approach has proved effective for reversing GVHD induced by DLI [53,54]. However, there are two major problems apparently related to this approach: (1) the induction of a strong immune response against transduced lymphocytes, and (2) the lack of efficacy in avoiding chronic GVHD [54]. Furthermore, because the technique does not spare the lymphocytes that mediate the antileukemic effect, the therapeutic effect of DLI may be jeopardized. This fascinating approach warrants a larger clinical study to assess its true efficacy. An elegant approach has also been proposed based on blockade of MHC molecules with analogue peptides. Although conducted exclusively in murine models, these studies have shown the effectiveness of synthetic peptides in blocking MHC on antigen presenting cells and preventing T-cell activation and GVHD [56,57]. Potential use of DLI in CML The case for nonmyeloablative conditioning. The therapeutic potential of donor lymphocytes has provoked a fundamental reassessment of transplantation strategies. In the last year several attempts to generate an allogeneic GVL in conjunction with minimal myeloablative conditioning therapy have been reported. Low toxicity nonmyeloablative transplant regimens can be designed exclusively to establish do-

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nor hematopoiesis, thus producing host-vs-graft tolerance and the condition for subsequent use of DLI [58,59]. Extending this strategy to HLA-mismatched transplants [60] might allow novel immunotherapeutic procedures to be pursued and so increase the number of CML patients who may be cured. Prophylactic DLI. The techniques used to reduce the incidence of GVHD after allogeneic BMT are associated with a significantly higher relapse rate. Similarly, the new nonmyeloablative transplantation regimens are associated with a relatively high risk of relapse. Because DLI is most effective when the tumor burden is low, the administration of DLI should be considered in those cases before detection of relapse. Furthermore, there is evidence that the incidence of GVHD is lower if T cells are infused 45 days after BMT [61]. In vitro activated DLI. Patients with advanced CML are often refractory to DLI treatment, and some chronic phase patients respond only partially and/or transiently [42]. In these cases, IL-2 may be a suitable adjuvant to minimize DLI failures [62,63]. IL-2 can recruit and expand donor T cells that are infused with DLI and primed by the allogeneic leukemic cells. IL-2 can also potentiate natural cell immunity amongst donor lymphocytes and increase the growth and survival of T cell in vivo even without prior immunosuppression [64]. The molecular basis of DLI The immune system does not generally appear to be defective in CML patients [65], but the invariably fatal outcome of untreated patients suggests that it fails to control disease progression. Moreover, increasing the ratio between the number of lymphocytes and the number of leukemic cells does not restore autologous antitumor immune reactivity to any great extent. In fact, the use of high-dose chemotherapy followed by reconstitution with autologous hematopoietic cells does not significantly alter the outcome of CML patients. Despite the selective unresponsiveness of autologous T cells against leukemic cells, the lymphocytes infused with allogeneic BMT or DLI do exert a powerful anti-leukemic reaction against CD341 progenitor cells [66]. The GVL effect of allogeneic lymphocytes can be explained by the fact that they are derived from a supposedly healthy individual where T cells have not been inactivated by the tumor escape mechanisms that presumably facilitates the spread of tumor in the patient [67]. This explanation would favor the argument that it is possible, in the appropriate setting, to generate tumor-specific immunity. For example, tumor infiltrating lymphocytes isolated from melanoma patients exert a powerful and specific cytotoxic activity on autologous tumor cells in vitro [68]. Alternatively, the efficacy of allogeneic donor lymphocytes could be based on the differences between donors and recipients at the level of

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polymorphic molecules. In such a case, these polymorphisms—the target for the GVH effect—would also be the major target for GVL. However, a tumor-specific immune response could still be postulated in this context, although it would represent a minor component within the effector cell population. We will review the evidence in favor of the two hypotheses.

Table 3. Generation of antileukemia CTL

The GVL effect is merely a GVH effect. The striking association between GVHD and GVL has encouraged research aimed at analyzing the role of transplantation antigens in the DLI effect. Because human transplantation donors and recipients are matched for the MHC, GVH and graft rejection might be caused by disparities at the level of histocompatibility antigens that are not encoded by the MHC, namely the minor histocompatibility (H) antigens. Minor H antigens are polymorphic (generally di-allelic) cell-derived self-peptides inherited independently of MHC. They are presented on the cell surface by MHC molecules [69] and recognized by alloreactive T cells. A correlation between mismatches for minor H antigens between donor and recipient and GVHD has been defined, suggesting that these molecules may play a major role in allogeneic BMT [70]. A few minor H antigens have been identified in humans and in some cases the immunodominant peptide has also been characterized [71,72]. The evidence that allogeneic molecules appear to be the major target for GVL argues against the possibility that GVH can be separated from GVL. However, an interesting feature of minor H antigens is that some exhibit a restricted tissue distribution. Minor H antigen-specific cytotoxic T lymphocytes (CTL) have been identified, which recognize hematopoietic cells but not other tissues [73,74], thus providing an opportunity to selectively drive an immune response against tumor cells of hematopoietic origin [75]. Because the minor H antigens HA-1 and HA-2 are expressed exclusively on hematopoietic cells, HA-1 and HA-2-specific CTL have recently been generated using synthetic peptides. These CTL efficiently lyse leukemic cells but not skin fibroblasts and might, therefore, be used to treat relapse of leukemia after allogeneic BMT with a low risk of GVHD [76].

CTL = cytotoxic T lymphocytes.

Evidence for leukemia-specific immune response. Numerous attempts have been made to identify leukemia-specific antigens. Although no evidence for such specificity has been provided, the majority of studies have led investigators to focus on a restricted number of candidates (Table 3). The CML-specific chromosomal translocation encodes a fusion peptide that is unique to the leukemic cells. Some synthetic peptides spanning the BCR-ABL fusion region are capable of binding to HLA-A3, -A11, and -B8 molecules [77,78] and can induce a specific T-cell response [79–81]. A recent multicenter case-control study comparing patients with CML with unaffected individuals tends to support the view that presentation of BCR-ABL breakpoint peptides in association with HLA molecules can induce a protective

Allogeneic setting

Autologous setting

Minor histocompatibility antigens [73–76] “Allo-restricted” CTL [89, 90]

Proteinase 3 [88] BCR-ABL peptides [77–81] Leukemic dendritic cells [86]

“Leukemia-reactive” CTL [66]

immune response [82]. The results indicate that HLA-B8, especially when coexpressed with HLA-A3, is associated with a reduced incidence of CML. However, others have shown that BCR-ABL-specific CD41 CTL clones do not inhibit but actually augment CML cell growth [83]. Another interesting strategy for generating leukemiaspecific T cells is the use of dendritic cells. Dendritic cells are extremely potent antigen presenting cells for initiation of primary immune responses and have been using as adjuvants for cancer vaccines [84]. Because of their ability to overcome tolerance [85], they can be regarded as potential tools for tumor immunotherapy. In CML, dendritic cells belong to the leukemic clone and are thus capable of presenting endogenously derived tumor peptides. In fact, dendritic cells generated from CML peripheral blood cells can induce autologous T cells to mount a leukemia-specific immune response. These T cells exhibit vigorous proliferative and cytotoxic activity against CML cells but low reactivity to allogeneic normal cells [86]. Although their specificity has not been identified, this approach appears promising. The identification of normal differentiation antigens as tumor-specific antigens has been pioneered by Boon and colleagues [87]. A similar approach has been exploited to raise CML-specific CTL against a peptide derived from the primary granule enzyme proteinase 3 [88]. These CTL showed HLA-restricted inhibition of myeloid colony formation by CML cells, which overexpress proteinase 3, but were ineffective on normal hematopoietic cells. An alternative strategy to overcoming tumor-specific unresponsiveness is to bypass self MHC restriction by generating T cells specific for tumor antigens and restricted by an allogeneic MHC molecule. Allo-restricted CTL clones have been generated against a mouse mdm-2 peptide that specifically lyse tumor cells and delay tumor growth in vivo [89]. The same results have been reproduced in a human system using cyclin-D1 [90]. These findings indicate that it is possible to generate in vitro allo-restricted CTL clones specific for peptides presented at high levels in leukemic cells, and suggest their therapeutic exploitation in HLA-mismatched allogeneic combinations without GVH effect. This approach is however subject to the development of minimally toxic and efficient transplantation regimens for haplotypemismatched combinations.

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Conclusions Greater understanding of the mechanisms responsible for the efficacy of allogeneic BMT has led to the conclusion that the immune system plays a key role in eradicating leukemic cells. Such a powerful tool is not, however, free of complications and its exploitation still requires complex therapeutic maneuvers for conditioning of the patient. It is likely that, in the near future, increasing knowledge of the molecular events underlying transplantation immunology and developments in the identification of tumor specific antigens will give rise to effective and safe immunotherapeutic strategies for the treatment of patients with CML.

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