Immunotherapy in allogeneic hematopoietic stem cell ... - Nature

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Immunotherapy in allogeneic hematopoietic stem cell transplantation – not just a case for effector cells A Troeger1,3, R Meisel1,3, T Moritz2 and D Dilloo1 1 Clinic for Pediatric Oncology, Hematology and Immunology, University Clinic Du¨sseldorf, Germany; and 2Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Duisburg-Essen, Germany

Summary: The concept that in allogeneic hematopoietic stem cell transplantation (alloHSCT) the immune system plays a prominent role in the control of leukemic disease is supported by the clinical observation that immunological effector mechanisms contribute to the elimination of leukemic blasts. The failure to induce prolonged remission after alloHSCT has led to resurgent interest in complementing concepts of immune modulation to improve the antileukemic reponse. While the general focus has been placed on manipulation of cytotoxic effector cell populations, we will explore the dual role of leukemia cells as both antigen-presenting and target cells and describe various vaccination strategies to facilitate a protective antileukemic immune response in this setting. In addition, we will introduce mesenchymal stem cells (MSC) as another cell population recently recognized for their immunomodulatory properties. The potential benefits and hazards of MSC-cotransplantation in alloHSCT with regard to the graft versus leukemia (GvL) and the graft versus host (GvH) response will be discussed. Bone Marrow Transplantation (2005) 35, S59–S64. doi:10.1038/sj.bmt.1704849 Keywords: allogeneic hematopoietic stem cell transplantation; leukemia; vaccination; dendritic cells; mesenchymal stem cells

In patients with acute leukemia, particularly with high risk or advanced disease, allogeneic hematopoietic stem cell transplantation (alloHSCT) has considerably improved prognosis.1,2 Part of the therapeutic efficacy is unequivocally attributed to the graft versus leukemia (GvL) response mediated by donor T lymphocytes and natural killer (NK) cells. Unfortunately, the benefit of the GvL response is often associated with graft versus host disease

Correspondence: Dr D Dilloo, Klinik fu¨r Kinder-Ha¨matologie, -Onkologie und -Immunologie, Heinrich-Heine Universita¨t Du¨sseldorf, Moorenstr. 5, D-40225 Du¨sseldorf, Germany; E-mail: [email protected] 3 These authors contributed equally to this work.

(GvHD) with a significantly reduced risk of post transplant relapse in patients suffering from GvHD.3 While there has been considerable improvement in reducing the toxicity of conditioning regimens in alloHSCT, dissecting the GvL from the GvHD response has remained a challenge. In clinical practice, the general emphasis has been placed on (1) donor selection improving the degree of HLA-match between donor and recipient4 and on (2) graft manipulation either removing allogeneic effector cells with the aim to reduce the risk of GvHD or by post transplant donor lymphocyte infusion (DLI) to enhance the GvL response.5 In this respect, a number of different approaches albeit with variable clinical efficacy have been taken to eliminate effector cells with predominant GvHD reactivity and isolate those in which antileukemic reactivity supervenes.6 Yet, optimizing the GvL response is not a matter of effector cells alone and an ideal HLA match between donor and recipient may not always prove beneficial. Two advances in the field have recently shifted the focus from effector cell to the leukemia cell as both stimulator and target cell of the desired antileukemic response. The recognition that leukemia cells express killer cell immunoglobulin-like receptors (KIR) as well as the identification of minor histocompatibility antigens (mHags) restricted to hematopoietic cells suggests that a certain degree of mismatching for KIR/KIR ligand pairs and tissue-specific mHags may indeed improve antileukemic efficacy in alloHSCT.7,8 At the same time, these observations document that target specificity as well as target susceptibility to immunological cytotoxic mechanisms may be just as critical for a protective antileukemic response as identification and selection of appropriate effector cell populations. When looking at the cell populations involved in modulating the immune response post alloHSCT, the focus has recently been extended to include mesenchymal stem cells (MSC)9,10 in addition to antigen-presenting cells (APC), leukemia and effector cells. It is the profound T-cell inhibitory role of MSC allowing for transplantation across HLA-barriers that has recently elicited considerable interest in the transplantation community in particular with regard to amelioration of GvHD. This review will explore the potential of leukemia cells in their dual role as both antigen-presenting and target cells. In addition, the capacity of MSC as critical players in GvH/GvL modulation is examined.

Immunotherapy in alloHSCT A Troeger et al

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Expanding antileukemic effector cell population by vaccination approaches Modification of leukemia cells for vaccination There is no doubt that in patients with chronic myelogenous leukemia (CML) suffering from relapse after alloHSCT, immunomodulatory intervention by DLI has been highly successful.11 Yet in acute leukemia, treatment of overt morphological relapse with DLI is of little benefit, which has been attributed to the high proliferative potential of leukemic blasts.12 Also due to GvHD as the doselimiting toxicity, numbers of leukemia-specific cytotoxic effector cells applied may not be sufficient to counteract large leukemic burden.11 While both lymphoid and myeloid blasts express MHC molecules on their surface and an array of leukemia-specific target antigens have been identified,13 acute leukemia cells still escape immune surveillance. Thus in addition to the dynamics of the disease, lack of DLI efficacy in acute leukemia may be explained by the reduced T-cell stimulatory capacity of leukemic blasts attributed primarily to low expression of costimulatory molecules.14 In principal, several strategies are available to improve antigen-presentation by leukemic blasts themselves. Among these are transgenic expression of costimulatory signals or receptor–ligand-induced upregulation of costimulatory surface molecules. Murine studies employing genetically modified neoplastic cells for vaccination have been instrumental in identifying cytokines, chemokines and costimulatory surface molecules as the decisive costimulatory signals. These studies also document that in acute leukemia, combinations of various immunomodulators acting on different levels of the immune response are required for a protective antineoplastic response.15–19 With regard to costimulatory surface molecules, transgenic expression of CD80 has been shown to enhance the immunostimulatory quality of acute myeloid and lymphoblastic leukemia cells.15 Of note, CD40L-transduced fibroblasts admixed with nontransduced B lymphoblastic leukemia cells can also stimulate a protective antileukemic immune response, thus documenting that efficacy even in the presence of residual disease may be effectively provided via bystander cells.17 As in the murine model, in primary human chronic lymphocytic leukemia, transgenic expression of CD40 ligand (CD40L) results in upregulation of costimulatory molecules of the B7 family such as CD80 and CD86 delineating the mechanism by which CD40L is thought to afford antileukemic protection.20 In addition to the B7 family, the CD27/CD70 receptor/ ligand pair exhibits well-defined immunomodulatory qualities in B-cell/T-cell interaction. Physiologically, the costimulatory signal CD70 mediates antigen-dependent Tcell activation and increases the memory cell pool.21 CD70mediated costimulation during primary antigen-challenge further allows for a strong secondary response such that antigen-specific T-cell anergy as observed in the absence of adequate costimulation does not take effect.22 In vivo vaccination models have shown surface bound or soluble CD70 to promote expansion of antigen-specific T cells and enhance their cytotoxic capacity facilitating tumor rejecBone Marrow Transplantation

tion.23 Human B-cell malignancies such as CLL and lymphoma also express CD70 albeit at low levels, which contributes to their reduced immunogenicity.24 In acute lymphoblastic leukemia, CD70 expression on B-cell precursors is barely detectable but, as described for CD80 and CD86, may be upregulated by a maturation stimulus via the CD40 receptor.25,26 In analogy to professional APC, upregulation of costimulatory molecules on leukemic blasts may also be achieved via receptor/ligand interaction. In B cells, monocytes and dendritic cells (DC), crosslinking of the CD40 receptor has long been recognized as a decisive step in APC maturation as well as in APC/T-cell interaction.27 More than 80% of B-lineage leukemias express CD4028 and ligation of the receptor has been successfully employed to improve the antigen-presenting capacity in primary ALL and CLL cells.25,26,29 A more recent study in CD40activated primary B-cell precursor ALL describes release of IL-10 but lack of bioactive IL-12 production with the result that activation of naı¨ ve T cells is only temporary and T cells become anergic over time.30

DC-based leukemia vaccines In contrast to blast-derived vaccine cells, professional antigen-presenting cells such as DC are reliable in activating unprimed T cells in an antigen-specific manner as they express high levels of MHC-, costimulatory and adhesion molecules.31 Thus in relapsed acute leukemia postalloHSCT, a vaccine consisting of DC pulsed with leukemia antigens may serve to activate and expand GvL effectors. In the last years, protocols have been developed that allow for large scale ex vivo generation of clinical grade DC derived from either CD14 þ monocytes or CD34 þ hematopoietic progenitor cells. While to date there are no clinical studies that formally compare antineoplastic efficacy of DC derived from CD14 þ monocytes vs CD34 þ hematopoietic cells, tumor-specific immune responses have been observed in clinical studies with each type of DC.32–35 Recent reports suggest however that CD34 þ -derived DC might be more efficient in priming of CD8 þ T cells possibly due to the Langerhans cell component contained in the CD34 þ cell-derived DC pool.31 A decisive advantage of CD34 þ -derived DC is the initial expansion step prior to DC differentiation allowing for large numbers of mature DC to be generated from a relatively small cell culture inoculum.31,36 This is in contrast to the comparatively low DC yield obtained from monocytes.37 Several approaches can be pursued to load DC with target antigens. The use of lysates generated from neoplastic target cells has the advantage that a broad range of antigens is presented to the immune system. Immunological efficacy of lysate-pulsed DC has been demonstrated predominately for solid tumors with clinical responses observed in individual patients.34,38 In the allogeneic setting, the potential risk of lysate-pulsed DC as well as the use of DC matured from malignant cells lies in the induction of GvHD via inadvertent presentation of self-antigens.39 These approaches have therefore been introduced into the clinic mainly in the autologous setting.


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While the use of leukemia-specific antigens may diminish the incidence of GvHD, employing defined antigen-derived peptides to stimulate an antileukemic response carries the risk that leukemic escape mutants are not targeted. This disadvantage may be overcome by the combination of different antigens. Also, the peptide-based vaccine approach is limited to patients carrying the respective HLArestriction elements. On the other hand, employing peptides for DC loading offers the definitive advantage that selected peptides can be synthesized under good manufacturing practice conditions to high quantities and once synthesized are readily available for clinical grade vaccine preparation. In patients with solid tumors, peptide-pulsed DC vaccines have been employed in a number of clinical phase I/II studies documenting immunological and clinical efficacy of this approach in patients with melanoma and prostate cancer.34,35,40,41 Peptide-based DC vaccines have also been assessed in immunotherapy for hematological malignancies initially as a complementary approach to chemotherapy.41,42 More recently, the efficacy of peptide-based vaccination strategies with or without DC for enhanced antigen-presentation has been examined in the context of HSCT. Post autologous HSCT, a clinical feasibility study on idiotype vaccination in multiple myeloma patients demonstrated complete remission in individual patients.42 Of interest, in one patient with relapsed myeloma after alloHSCT from a matched sibling donor, idiotype vaccination was performed and resulted in sustained remission without development of GvHD.43 Similarly, in a peptidebased vaccine approach in CML targeting the breakpoint region of the bcr-abl fusion product post alloHSCT, bcr-abl-specific T-cell responses were observed without severe side effects even when the vaccine was applied in combination with DLI.44 Besides leukemia-specific antigens, so called ‘shared’ antigens that are expressed in different malignancies but not in normal tissue represent potential targets for a leukemia vaccine.45,46 One of them is the Wilms tumor antigen 1 (WT1), a zinc-finger transcription factor involved in cell differentiation and proliferation, which is expressed at high levels in solid tumors and also in acute myeloid and lymphoblastic leukemia.45 In a phase I clinical study, vaccination with WT1-derived peptides for acute myeloid leukemia led to a decrease of WT1 levels as a marker of residual disease in 7/10 evaluable patients.47 Another ‘shared’ antigens is survivin, a member of the inhibitor of apoptosis protein family.48,49 Survivin is an inhibitor of the effector caspases 3 and 7 resulting in blockade of the mutual downstream events of both the death-receptormediated and mitochondrial apoptosis pathways.50 Association with early events in leukemogenesis is documented in patients with myelodysplastic syndrome (MDS) by an increase in survivin expression prior to development of overt leukemia.51 Clinically, the prognostic relevance of survivin accumulation is further documented by the observation that in different hematological malignancies high level of survivin correlates with reduced disease-free survival.52 As in adult leukemia,53 we have shown that in 70% of childhood ALL a more than two-fold increase in survivin protein expression can be observed.54 Also, in murine in vivo models, vaccination with survivin-trans-

duced DC has been shown to afford long-term protection in a highly malignant lymphoblastic lymphoma.55 Thus based on their critical role in regulating apoptosis/ proliferation, WT1 and survivin are attractive targets in DC-based vaccine approaches aiming to expand effector T cells directed towards antigens favoring leukemia cell survival. In Germany two clinical phase I/II trial are about to be opened, our own study employing these antigens in a vaccine approach in combination with DLI for relapsed ALL post-alloHSCT and another study assessing the efficacy of survivin-derived peptides in CLL.49 In addition to careful choice of target-antigen and mode of antigen-presentation, optimal timing of both DLI and vaccine application needs to be considered. Here, murine models of alloHSCT suggest that vaccines application during T-cell regeneration favors the induction GvL over GvHD responses.56 In shifting the balance towards the GvL response post-alloHSCT, tolerization towards alloantigens may be critically influenced by another player in the concert of antigen-presenting, target and effector cells: the MSC.

Immunomodulatory properties of MSC Human bone marrow-derived stromal cells (MSC), also referred to as mesenchymal stem cells, have initially been identified as the marrow stroma compartment constituting the microenvironment for hematopoietic cell engraftment and proliferation. More recently, MSC have gained increasing attention in tissue engineering, cell and gene therapy based on their potential to differentiate into multiple mesenchymal tissues,9,10 weak immunogenicity and the capacity to inhibit allogeneic and antigen-specific T-cell responses.57–60 Owing to their immunomodulatory and engraftment facilitating properties, the concept of MSC cotransplantation has also attracted increasing attention in alloHSCT.61 In this context, a recent case report demonstrates successful treatment of refractory GvHD by MSC administration.62 In the following, the clinical promises and also the potential hazards of MSC application in the setting of alloHSCT are discussed. Several reports have shown that MSC inhibit Tlymphocyte proliferation in response to allogeneic and antigen-specific stimulation in a cell dose-dependent manner.57–60 In mixed lymphocyte cultures (MLR) proliferation of CD4 þ and CD8 þ T lymphocytes is equally inhibited by MSC.58 Likewise, MSC are able to inhibit primary as well as memory type T-cell responses.59 Regarding the impact of MSC on the effector function of cytotoxic T cells, two recent studies have yielded conflicting results.63,64 Yet, in a large animal model, the relevance of MSC-mediated T-cell inhibition has been verified showing prolonged survival of allogeneic skin grafts following infusion of donor type or third party MSC, indicating that cotransfusion of MSC might also hold promise for GvHD control.57 The molecular mechanisms mediating the immunosuppressive properties of MSC have long remained unclear and seem to involve cell contact-dependent mechanisms as well as soluble mediators.58–60,65 One report indicates that MSC Bone Marrow Transplantation


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might selectively induce CD8 þ suppressor T cells.65 As soluble mediators, transforming growth factor-b and hepatocyte growth factor have been implicated in MSCmediated T-cell suppression, which was not confirmed in a series of subsequent studies.58–60,65 We have recently identified the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as an immunosuppressive effector pathway in human MSC.66 Expression of IDO, which is induced by interferon-g (IFN-g) and other proinflammatory cytokines, catalyses conversion from tryptophan to kynurenine and blocks T-cell proliferation by depleting tryptophan from the cellular microenvironment.67 In addition, recent data indicate that kynurenine breakdown products might also be involved in IDO-mediated T-cell inhibition.68 IDO has initially been identified as a major immunosuppressive effector pathway in professional antigen-presenting cells that inhibits T-cell responses to autoantigens and fetal alloantigens in vivo.67,69 In MSC, IDO is not constitutively expressed but requires cell activation by inflammatory cytokines as released in allogeneic T-cell responses. As a result of IDO enzyme activity there is profound depletion of tryptophan from the cellular microenvironment significantly inhibiting allogeneic T-cell proliferation.66 The identification of IDO as an activationdependent T cell inhibitory effector pathway in human MSC might in future allow for modulation of this effector mechanism in different therapeutic applications. In alloHSCT, MSC might be employed for prevention and treatment of GvHD.62 The Acute Leukemia Working Party of the EBMT has established clinical trials evaluating MSC cotransplantation in HLA-matched related and unrelated alloHSCT recipients. However, caution needs to be exercised as based on the current knowledge on the immunosuppressive properties, there are no indications that MSC will selectively impair GvHD but not GvL with the potential risk of an increase in relapse rate. Also since MSC-mediated T-cell inhibition appears not to be antigenspecific, highly desirable immune effector functions including antiviral and antifungal T-cell responses might be inadvertently blunted by MSC cotransfusion. Two recent observations from murine models support a cautious use of MSC in the setting of alloHSCT for the treatment of malignancies: In melanoma, simultaneous administration of MSC and allogeneic tumor cells results in accelerated tumor growth even when MSC are delivered to a distant site, suggesting that MSC might facilitate local as well as systemic immunosuppression.65 In addition, IDO expression by tumor cells themselves has been demonstrated to abrogate T-cell-mediated tumor control.70 Our finding that MSC impair allogeneic T-cell responses via IDO-mediated tryptophan degradation integrates the former two experimental results and strongly suggests that MSC might inadvertently subvert antineoplastic immune responses when coadministered with an allogeneic hematopoietic stem cell graft. Thus, further evaluation of potential beneficial and undesirable effects of MSC transplantation on the fine balance between GvH and GvL reactions is warranted. In conclusion, manipulation of leukemic target cells harnessing their innate capacity for antigen-presentation in a vaccination approach or employing professional antigenBone Marrow Transplantation

presenting cells to this effect in addition to exploration of the potential role of MSC in both GvH and GvL responses may serve to complement immunomodulatory effector cellbased approaches in alloHSCT.

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