Towards immunogene therapy of hematological malignancies

030

doi:

Experimental Hematology 34 (2006) 251–263

Towards immunogene therapy of hematological malignancies

Roman Ivanov, Anton Hagenbeek, and Saskia Ebeling

Jordan Laboratory for Haemato-Oncology, Department of Haematology, University Medical Centre Utrecht, Utrecht, The Netherlands

(Received 31 August 2005; revised 10 October 2005; accepted 11 October 2005)

The growing understanding of immunological processesthat follow allogeneic hematopoietic stem cell transplanta-tion and donor lymphocyte infusion provides a rationale foruse of leukemia-specific T cells in treatment of hematolog-ical malignancies. In this review, results of preclinical andclinical studies of T-cell–based immunotherapies are sum-marized. Moreover, those aspects of T-cell biology thatare relevant for development of a successful immunothera-peutic approach are discussed. Furthermore, the novel ap-proach of T-cell receptor transfer is emphasized.Generation of T cells with antileukemic specificity throughgenetic engineering avoids the long-term in vitro expansionthat inevitably leads to loss of T-cell ability to engraft andfunction in vivo.

Immunological effects of allogeneic hematopoieticstem cell transplantation and donor lymphocyteinfusionTransplantation of allogeneic hematopoietic stem cells(allo-HSCT) is a curative approach for a variety of hemato-logic malignancies that respond poorly to conventional che-motherapy or progress despite the initial response tochemotherapy. Its therapeutic potential was first exploitedin allogeneic bone marrow transplantation (allo-BMT),although the success of BMT was initially thought to beassociated with the myeloablative conditioning regimen.However, clinical studies led to recognition of the role ofdonor T cells in the immune eradication of residual tumorcells.

Decisive proof came from excellent clinical results of do-nor lymphocyte infusion (DLI) used to treat chronic myeloidleukemia (CML) patients who had relapsed after allo-BMT.Lymphocyte transfusion from the original bone marrow do-nor induced hematologic and cytogenetic response in 70%to 80% of CML patients in chronic phase [1]. In these

Offprint requests to: Saskia Ebeling, Ph.D., Jordan Laboratory for

Haemato-Oncology, Department of Haematology, University Medical

Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands;

E-mail: [email protected]

1-472X/06 $–see front matter. Copyright � 2006 International Society fo

10.1016/j.exphem.2005.10.004

patients, DLI led to a complete cytogenetic response 1 to4 months after administration of T cells and about 80% ofresponders reached a molecular remission within a meanof 6 months. The lower efficacy of DLI in acute leukemia[2] may be caused by rapid tumor growth that outpacesthe immune response and/or by inferior immunogenicityof leukemic blasts. The same factors may be responsiblefor poorer results of DLI in an accelerated phase of CMLand blast crisis (!33% response).

Introduction of DLI into the clinical practice made mye-loablative conditioning a dispensable component of tumortherapy. Instead, nonmyeloablative regimens of HSCTgained popularity. In nonmyeloablative HSCT, only a mildimmunosuppressive conditioning is administered to allowdonor-cell engraftment; hence the antitumor activity of Tcells is fully responsible for tumor eradication [3]. Thelow toxicity of nonmyeloablative HSCT allows its use inolder patients who are not eligible for BMT.

The excellent clinical results of allo-BMT and DLI inCML are dependent on development of the graft-vs-leuke-mia (GVL) effectdan immune-mediated response that con-serves a state of continued remission of a hematologicalmalignancy following transfer of allogeneic hematopoieticcells [4]. Unfortunately, donor immune cells are alsoresponsible for the major complication of allo-BMT andDLIdalloreactive response against healthy patient tissues,graft-vs-host disease (GVHD). Its occurrence is tightly as-sociated with the dose of T cells [2] Importantly, GVHDdevelopment has a strong temporal association and statisti-cal correlation with induction of a disease response, be-cause a GVL effect is observed in 90% of CML patientsexperiencing GVHD.

Despite recent successes, in the majority of hematologicmalignancies, allo-BMT and DLI efficacy leave plenty ofroom for perfection. Improvement of outcomes of allo-BMT and DLI may be achieved via two complementaryapproachesdsuppression of GVHD and enhancement ofGVL. Importantly, 55% of patients with subclinical GVHDhave a measurable disease response [5]. This demonstratesa partial divergence of the antigenic basis of GVL andGVHD and suggests a possibility for alteration of the

r Experimental Hematology. Published by Elsevier Inc.

mailto:[email protected]

252 R. Ivanov et al./ Experimental Hematology 34 (2006) 251–263

balance between them in favor of GVL. Enhancement ofGVL-mediating immune responses towards leukemia-spe-cific antigens will be discussed in the following sectionsof this review.

Common strategies to control GVHD include use ofcyclosporine A and methotrexate. Experimental methodsemployed anti-interleukin-2 receptor (IL-2R), antitumornecrosis factor-a (TNF-a), induction of anergy by CD28/B7 blockade, and T-helper 2 (Th2)-deviation of the immuneresponse. However, results of those studies are not conclu-sive and clinical trials are required to address the efficacy ofthe proposed approaches.

Decreased GVHD with preserved GVL effect was ob-tained by BMT with T-cell depletion followed by delayedDLI [6]. A low starting dose of donor T cells (107/kg), fol-lowed by gradual escalation of numbers of infused leuko-cytes for nonresponding patients, also led to a reducedoccurrence of acute GVHD [7].

The threatening GVHD may also be controlled throughuse of a suicide gene transferred to T cells prior to admin-istration to a patient. The well-characterized herpes simplexvirus thymidine kinase system may be used to controlGVHD. It has been shown to preserve the GVL effect incase of delayed administration of ganciclovir [8]. However,this system has some drawbacks associated with partial re-sistance to ganciclovir and immunogenicity of the trans-gene. Equipping donor T cells with the CD20 antigenprior to infusion allows elimination of T cells with CD20-specific antibodies in case of necessity [9].

An alternative approach to reduce GVHD is to depletealloreactive donor T cells that become activated soon afteradministration due to an encounter with abundant alloanti-gens. Recent studies demonstrated that use of an immuno-toxin specific to the activation marker CD25 alloweddepletion of alloreactive T cells, while T cells specific toEpstein-Barr virus (EBV), cytomegalovirus (CMV), andleukemia-associated antigens PR1 and HA-1 were pre-served [10].

Despite the positive prospects of selective GVHD sup-pression, this approach may not improve DLI results in rap-idly progressing malignancies due to the low number ofcells in transplant, which mediate the GVL effect. There-fore, methods to selectively enhance GVL through enrich-ment of infused cells for GVL effectors are of great interest.

Role of T cells in GVL and GVH effectsThe clinical success of DLI in patients with CML and thehigh frequency of relapses in T-cell–depleted BMT provideample evidence that T cells mediate an antitumor effect [5].T-cell depletion prior to the human leukocyte antigen(HLA)-identical BMT leads to a fivefold increased risk ofrelapse compared to transplantation of nonmanipulatedbone marrow [11], while nonspecific augmentation of T-cell responses by administration of IL-2 and interferon

(IFN)-a diminishes incidence of relapse [12]. Multiplelaboratory studies demonstrated specific recognition of leu-kemic cells by T cells. T-cell clones specific for leukemia-associated antigens have been isolated from blood samplesobtained after allogeneic HCT and shown to possess an an-tileukemic activity both in vitro and in vivo. In mousemodels, adoptive transfer of antigen-specific CD8D T cellseradicated leukemia [13].

There are conflicting reports concerning the contributionof different T-cell subsets to the GVL effect. Specific Thcells have been shown to be crucial for maintenance ofcytotoxic T lymphocyte (CTL) function [14]. Moreover,the role of CD4D T cells is not limited to amplification ofthe cytolytic immune response mediated by CD8D T cells;antigen-specific CD4D T cells can lyse tumor cells by Fas-independent mechanisms. CD8D-depleted HSCT provideslow GVHD and good survival, which points to the impor-tant role of CD4D T cells in development of GVL and gen-eral antitumor immunity [15].

Adoptive transfer of purified CD4D T cells alone can me-diate tumor regression in most murine models [4]. Moreover,depletion of CD4D T cells decreases the number of antileu-kemia T cells more significantly than CD8D depletion [4].Most of the clinical benefit of CD4D cell infusion is exertedthrough a helper effect by recruiting leukemia-reactiveCD8D T cells. Apparently, cotransfusion of antigen-specificCD4D T cells may be necessary for the survival and optimalfunctioning of adoptively transferred CTL.

The key to dissection of cellular and molecular mecha-nisms of the GVL effect is proper understanding of T-cellfunction. Modulation of T-cell function or a pattern ofT-cell traffic in vivo may influence the antitumor effect ofleukemia-specific T cells (Figure 1).

Effector mechanisms of T cellsNaı̈ve T cells circulate between the periphery and second-ary lymphoid organs in search of their cognate antigen.To become effector cells, they have to encounter a maturedendritic cell (DC). Upon maturation, DCs rapidly upregu-late CD80 and CD86 molecules that amplify T-cell receptor(TCR) signaling by engaging CD28 on the T-cell surface.Mature DCs also express costimulatory 4-1BBL andOX40L molecules. Antigen recognition together with costi-mulatory interactions leads to clonal expansion of T cells[16]. Under the influence of cytokines produced by DCs,T cells acquire certain functional characteristics. For exam-ple, DC-derived IL-12 drives differentiation of naı̈ve Thcells into Th1 subtype of T cells, essential for tumor erad-ication. Subsequently, an activated T cell has to migrate tothe tumor site and gain access to the tumor microenviron-ment to exert its antitumor activity. Direct cytotoxic effectof T lymphocytes on tumor cells is mediated by perforinmechanism or Fas/Fas-ligand interactions.

CD4D T cells are crucial for development of an effectiveantitumor immune response. They provide help to CD8D T

253R. Ivanov et al. / Experimental Hematology 34 (2006) 251–263

Figure 1. Antigenic basis of graft-vs-leukemia (GVL) and graft-vs-host (GVH) immune responses. GVL reactivity is mediated by immune responses specific

to antigens with expression restricted to leukemic cells (leukemia-specific and certain viral antigens) and patient hematopoietic cells (hematopoietic-lineage–

restricted minor histocompatibility antigens, hematopoietic differentiation antigens). Reactivity to leukemia-associated antigens contributes to GVL mostly,

although these antigens are expressed at low levels in normal tissues. Ubiquitously expressed antigens are a substrate of GVH disease.

cells and natural killer cells by production of IL-2 andIL-12 [14] and activate eosinophils and macrophages[17]. Also, CD4D T cells’ help to CTL is mediated throughactivation of DCs via CD402CD40 ligand interaction [16].Moreover, Th2 CD4D T cells can activate productionof tumor-specific antibodies by B lymphocytes.

An important role belongs to T-cell–derived cytokines,which suppress tumor growth or activate other effectorcells. For instance, production of Th1-type cytokines(e.g., IFN-g, TNF-a) inhibits proliferation of leukemic cellsdirectly as well as upregulates expression of Fas and HLAmolecules [18,19]. IFN-g may also mediate tumor rejectionthrough the action on host cells [20]. Potential mechanismsof such antitumor effect include angiostasis, which occursas a response of the tumor stroma to IFN-g [21]. Notably,leukemic precursor cells have an increased susceptibilityto the inhibitory effect of Th1-type cytokines comparedto normal hematopoietic cells, which makes them more vul-nerable to T-cell attack.

T-cell migration and homingT cells follow organ-specific trafficking pathways to exitfrom blood into the extravascular compartment. The mech-anism of T-cell extravasation is similar to that of other leu-kocytes. During the first stage of this process, T cellsestablish a transient interaction (rolling) with the endotheli-al surface, which is mediated by constitutively expressed

selectins, a4 integrins, and their ligands. If the T cell en-counters an activating signal, usually in the form of an in-flammation-associated pattern of expression of chemokinesand their receptors, a firmer binding (sticking) occurs asa result of conformational change of integrins. Lastly,a transmigration of T cells through the endothelial layertakes place. Despite the similarity of extravasation mecha-nisms, migration of different subsets of T lymphocytes,namely, naı̈ve, effector, and memory T cells, follows differ-ent patterns. Their homing is regulated at the level of adhe-sion molecules and chemokine receptors.

Naı̈ve T cells primarily home to secondary lymphoid or-gans upon adoptive transfer. Highly specialized postcapil-lary venules of lymphoid organs (high endothelialvenules) express an array of glycoproteins, collectivelynamed peripheral node addressin, which are recognizedby L-selectin expressed by naı̈ve T cells. An activation sig-nal is provided by the interaction of CCL21 and its receptorCCR7. Engagement of CCR7 leads to a conformationalchange of leukocyte function-associated antigen-1 (LFA-1) enabling its binding to intracellular adhesion molecule(ICAM)-1 or ICAM-2, which makes the T cell stick.

Priming of naı̈ve T cells by DC induces a high-levelexpression of tissue-specific homing receptors. Th1 cells,which are of the utmost importance for the antitumor im-mune response, upregulate P-selectin glycoprotein ligandor E-selectin ligand-1. Interaction of these ligands with


P- and E-selectins that are expressed on the endothelium ofinflamed tissues drives Th1 cells into the site of inflamma-tion. Rolling arrest of effector T cells may be a result of in-teraction between a4b1 integrin and vascular cell adhesionmolecule-1 or LFA-1 and ICAM-1/2.

The altered expression of adhesion molecules on the T-cell surface after in vitro manipulation may lead to distor-tion of the normal pattern of T-cell homing. This issuehas to be addressed properly to provide insights into T-cellbehavior in vivo and maximize the therapeutic effect ofadoptive transfer.

Regulatory T cellsCD4DCD25D regulatory T cells (Tregs) suppress activa-tion of T cells by acting directly on target T cells or on an-tigen-presenting cells (APCs) [22]. The tolerizing effect ofadoptive transfer of these T lymphocytes was shown inmouse models [23]. Notably, only recipient-specific Tregsexerted their inhibitory effect on GVHD when coadminis-tered during allo-SCT [24], suggesting an antigen-specificmechanism of Treg activity. Importantly, Tregs were dem-onstrated to preserve the GVL activity of donor T cellswhile inhibiting GVHD after BMT in mice [25]. Althoughthe exact mechanism of Treg activity is not yet known,adoptive transfer or selective in vivo expansion of this T-cell subset may become a promising approach to separationof GVL and GVHD effects.

GVL target antigensTheoretical feasibility of selective GVL enhancement relieson the specific nature of the T-cell recognition and an as-sumption that certain antigens can induce leukemia-specificimmune responses. Data that support the existence of suchantigens came from clinical as well as from in vitro studies.The observation that the GVL effect may occur in theabsence of significant GVHD is the strongest argument infavor of the distinct antigenic basis of GVL and GVHD[4]. Furthermore, T-cell clones mediating GVL and GVHDhave a distinct repertoire of TCRb variable sequences [26].

Several classes of antigens are able to elicit T-cell re-sponses that preferentially target leukemic cells. These anti-gens are immunogenic for donor T cells either because oftheir absence in the donor or because of their overexpres-sion in leukemic cells.

Leukemia-specific antigensLeukemia-specific antigens arise as a result of oncogenicmutations. These antigens are limited to the malignant tis-sue and represent an attractive target for immunotherapy.One of the best-studied leukemia-specific proteins is theBCR-ABL fusion protein, which is a product of a chimericgene resulting from a translocation t(9;22). This genetic le-sion is typical for CML but may be also found in a numberof ALL and AML patients. The fusion between BCR andABL proteins generates a number of peptides that are

unique for leukemic cells. The most common BCR-ABLtranscript b3a2 contains a fusion between exon b3 of themajor breakpoint region of BCR gene and exon a2 ofABL gene. The fusion results in a sequence encoding lysineresidue absent in either BCR or ABL. Peptides spanningthis region are able to bind to HLA-A2, -A3, -A22, andB8; they are able to elicit CTL that lyse CML cells loadedwith the peptide [27]. However, in most of the cases theseCTL have a low avidity and their cytolytic activity againstnative CML cells is poor. Another prominent leukemia-specific antigendthe chimeric PML/RARA proteindis aproduct of the reciprocal translocation t(15;17), specificfor acute promyelocytic leukemia. Recognition of a PML/RARA peptide by CD4D T cells has been described [28].

The very limited repertoire of antigenic peptides gen-erated by oncogenic mutations makes generation of high-avidity T-cell clones rather problematic. Moreover, theoverwhelming majority of genetic aberrations is not recur-rent and, therefore, not suitable for immunotargeting. Idiot-ypic immunoglobulins and CDR3 variable region sequencesmay be used to elicit immune responses specific to clonalpopulation of leukemic B cells. However, such individualtherapies are unlikely to be applied routinely in the clinic.

Leukemia-associated antigensAntigens that are expressed at low levels in normal cellsand overexpressed in a variety of cancers, represent an at-tractive target for immunotherapy. Frequently, these pro-teins are essential for survival of tumor cells, whichdiminishes the probability of immune escape through lossof antigen expression or mutations.

The Wilm’s tumor antigen-1 (WT-1) is a zinc finger tran-scription factor that is normally expressed in the kidney,testes, ovary, uterus, and lung, and at low levels in normalCD34D hematopoietic cells. Importantly, WT-1 is aberrant-ly expressed in both lymphoid and myeloid acute leukemiaand contains a number of promising antigenic peptides.WT-1 peptides that bind to HLA-A2 and -A24 have beenused to elicit T cells reactive with WT-1 in vitro and in vivo[29]. WT-1–specific T cells were also able to eliminate leu-kemic progenitors in immunodeficient mice engrafted withhuman leukemia [30], but do not recognize normal CD34D

cells.Human telomerase reverse transcriptase (hTERT) is

a component of the ribonucleoprotein complex that is re-sponsible for maintenance of the telomere length and ishighly expressed in the majority of tumors, including leuke-mia. APCs pulsed with HLA-A2– and HLA-A24–restrictedpeptides of hTERT have been used to generate T-cell linesand clones that recognized tumor cells expressing high lev-els of endogenous hTERT [31,32]. Interestingly, thesehTERT-specific T cells do not recognize normal hematopoi-etic cells in vitro [32].

The p53 tumor suppressor protein is mutated and/oraberrantly expressed in approximately 50% of all human


malignancies [33]. Usually, normal p53 function is lostthrough deletion of one allele of the gene and mutation of an-other allele. The mutant protein is devoid of tumor suppressoractivity and has an increased stability, which leads to its higherintracellular concentration. In a mouse model p53-specificCTL were able to eradicate not only p53-overexpressing tu-mors but also tumors where the p53 expression level is closeto a normal, suggesting that an increased p53 turnover maylead to enhanced tumor sensitivity to T-cell attack [34].However, due to the intrathymic expression of p53, T cellsspecific to this antigen are absent in the T-cell repertoire.

The human homologue of the mdm-2 oncoprotein, hdm-2, is overexpressed in a variety of malignancies where it in-activates the p53 tumor suppressor protein. Although thehigh-affinity hdm-2–specific T cells are absent in T-cellrepertoire due to self-tolerance, they can be elicited by im-munizing HLA-A2 transgenic mice with hdm-2 peptides orby stimulating T cells from HLA-A2–negative donors withHLA-A2D cells pulsed with hdm-2 peptide [35]. Thesecells are reactive with hdm-2–positive tumor cells; donorT cells equipped with hdm-2–specific T-cell receptor maybe used in adoptive immunotherapy [35]. The same ap-proach may be potentially applied for generation of p53-specific GVL effector T cells.

Differentiation- and lineage-specific antigensAntigens, which are restricted in their pattern of expressionto a certain tissue or a differentiation stage, are of specialinterest for immunotherapy. For instance, the myeloid-restricted proteins, proteinase 3 and myeloperoxidase, arehighly expressed in 75% of CML patients, 50% of AML,and 30% of myelodysplastic syndromes [36]. PR1, a nona-peptide of proteinase 3, was used to elicit HLA-A2–restrictedCTL responses from normal donors. These PR1-specificCTL preferentially lysed CML cells and were able to inhibitproliferation of leukemic progenitors in vitro [37]. More-over, allogeneic PR1-specific CTL able to lyse CML cellsrather than normal bone marrow cells were also isolatedfrom the peripheral blood of CML patients who had re-ceived BMT [37]. The surface antigen CD45, the myeloidmarker CD33 and the B-lymphoid antigen CD19 may beconsidered potential targets for the immunotherapy becauseof their hematopoietic-restricted pattern of expression[38,39]. Importantly, the polymorphisms that have beenidentified in these genes permit allospecific responses thatleave the donor hematopoietic cells untouched.

Despite frequent expression of tumor-associated andlineage-restricted antigens on leukemic cells, their use forimmunotherapy is problematic. As mentioned previously,the encounter with these antigens often depletes high-affinity T cells or converts T cells into a state of anergy.

Minor histocompatibility antigensMinor histocompatibility antigens (minor H antigens) argu-ably represent the largest group of candidate targets for

immunotherapy of hematologic malignancies. Disparity inminor H antigens occurs when amino acid differences be-tween polymorphic housekeeping genes lie within peptidesthat are presented on patient’s HLA molecules [40]. Be-cause of these polymorphisms, the repertoire of peptidesdisplayed in the peptide-binding groove of HLA moleculesmay differ substantially, even in HLA-identical siblings. Incontrast to tumor-associated antigens, high-avidity T-cellresponses toward minor H antigens can be induced, becausedonor T cells are not tolerant to them. Another cause of an-tigen disparity may be a homozygous gene deletion in a do-nor, as was shown for UGT2B17 antigen [41].Polymorphism can also occur as a result of differentialAg processing [42].

Depending on their pattern of expression, minor H anti-gens may be classified as ubiquitous or tissue-specific.Ubiquitous minor H antigens (HA-3, HA-4, HA-6, HA-8)induce immune responses that target multiple tissues ofa patient and cause indiscriminate GVHD/GVL reactions[40]. For example, recipients who were positive for theubiquitously expressed HA-8 minor H antigen and receiveda transplant from an HA-8–negative donor developed acuteGVHD more frequently than recipients of transplants fromHA-8–compatible donors [43].

Few minor H antigens with the expression restricted tocells of hematopoietic lineage have been identified, suchas HA-1, HA-2, HB-1, BCL2A1 [40]. The HA-1 antigenwas found to be encoded by an autosomal gene KIAA0223;HA-2 is the product of the class II myosin gene. Interesting-ly, HA-1 incompatibility was associated with a lower rateof leukemic relapse in one study [44]. Furthermore, HA-1–and HA-2–specific T-cell clones did not produce signifi-cant response against a skin explant in an in vitro modelof GVHD [45]. The response to DLI was shown to corre-late with the presence of HA-1– and HA-2–specific CTLs[46]. Paradoxically, a mismatch between donors and recip-ients for HA-1 was also found to be associated witha higher risk of GVHD following HLA-identical BMT[47]. Probably, HA-1–specific donor T cells induce de-struction of host blood cells, which leads to release ofubiquitous mHags. Consequently, T cells specific for theseantigens are activated, exacerbating GVHD [48]. The pos-sibility of such ‘‘epitope spreading’’ dictates the necessityof maximal transplant enrichment for T cells specific tohematopoietic lineage-restricted mHags. BCL2A1 belongsto the BCL2 family of antiapoptotic genes and is highlyexpressed in hematological malignancies. It encodesHLA-A*2402- and HLA-B*4403-restricted minor H anti-gens. Disparity in BCL2A1 does not lead to an increase inGVHD incidence [49]. Another minor H antigens, HB-1,was shown to be expressed on leukemic and precursorB cells with expression in normal tissues restricted to verylow levels in testis, thymus, and tonsils with B-cell hyperpla-sia. HB-1–specific CTLs are able to lyse ALL blasts. How-ever, it is restricted by a nonfrequent allele HLA-B44. In


general, identification of additional hematopoietic lineage-restricted minor H antigens remains challenging.

H-Y antigens represent a separate class of minor H anti-gens. These antigens are encoded by X-degenerate geneslocated on the Y chromosome; their X-chromosome allelespossess numerous amino acid differences [50]. H-Y anti-gens can induce both T-cell and antibody immune responseafter sex-mismatched allo-HSCT and during pregnancy[51,52]. Therefore, female patients transplanted with malebone marrow have a higher risk of developing graft rejec-tion than when transplanted with bone marrow of femaleorigin [53]. Interestingly, male recipients of transplantsfrom female BMT donors are at the lowest risk of relapsecompared with other patients, even after controlling forGVHD as a time-dependent covariate [54]. This suggeststhat anti–H-Y responses produce a selective GVL effectdistinct from the one contributed by GVHD. Therefore,H-Y antigens should exist that elicit immune responses se-lectively targeting leukemic cells.

To date, four H-Y genesdSMCY, DBY, UTY, andDFFRYdhave been identified as targets of CTL responses[55–58]. CTL responses against an HLA-A1–restricted epi-tope of DFFRY and an HLA-B60–restricted epitope ofUTY were observed in female patients who rejectedHLA-identical male stem cell graft [57,59]. CD4D T-helpercells specific to a RPS4Y peptide presented on HLA-DRb3*0301 also participated in graft rejection [60].DBY-specific HLA-DQ5–restricted T cells were isolatedfrom a patient with an acute GVHD [58], corroborating da-ta on the ubiquitous pattern of expression of this antigen.Likewise, T cells specific for the HLA-A2–restricted pep-tide of SMCY were shown to mediate GVHD in an in situskin explant model [45]. Moreover, tetramer staining ofT cells from patients undergoing allo-HSCT demonstrateda correlation between expansion of SMCY-reactive T cellsand development of an acute GVHD [61]. In contrast toSMCY-reactive T cells, CTL specific to HLA-B8–restrictedepitope of UTY selectively lysed hematopoietic cells andnot fibroblasts [62]. This differential pattern of recognitioncorrelated with the increased level of UTY mRNA in hema-topoietic cells. Furthermore, these T cells lysed AML blastsin vitro and eliminated leukemic progenitors in nonobesediabetic/severe combined immunodeficient mice [63].

In our studies H-Y epitopes were identified, that are ableto elicit a CTL response differentially targeting male lym-phoblasts [64,65]. These nonapeptides were derived fromthe RPS4Y and UTY proteins. Although the patterns of re-activity of RPS4Y- and UTY-specific T cells were very sim-ilar, the mechanisms that are responsible for the differentialrecognition of these epitopes are different. As for RPS4Y,overexpression of the RPS4 protein in rapidly proliferatingcells, including activated lymphocytes and samples ofleukemic cells, was demonstrated. On contrast, UTYmRNA expression did not correlate with the recognitionpattern of UTY-specific T cells. Nor was expression of

costimulatory molecules by target cells crucial for their sus-ceptibility to lysis. The differential recognition of the UTYpeptide appeared to be caused by a lymphoblast-restrictedantigen-processing mechanism that governed generationof this peptide. The fact that these H-Y epitopes are pre-dominantly presented on lymphoblasts, which are the onlytargets of the CTL specific to these epitopes, makes themattractive targets for immunotherapy of leukemia. Takinginto account the widespread association of the increasedcontent of ribosomal proteins with malignant transfor-mation, RPS4Y, in our opinion, can be regarded as aleukemia-associated minor H antigen [66,67]. As forUTY, the described epitope is the second peptide of thisprotein that is selectively recognized on hematopoietic cells[62]. Therefore, UTY is of a certain interest as a potentialleukemia-associated minor H antigen.

T-cell–based immunotherapy of leukemiaGreater understanding of the antigenic basis of GVL effectcreates new opportunities for antigen-specific therapy ofleukemia. Tumor-specific T-cell responses can be inducedby two different strategiesdactive or passive immuniza-tion. During active immunization (or vaccination), the de-sired immune response is caused by exposure of thepatient’s immune system to antigens expressed on tumorcells. The introduction of an immunogenic substance leadsto activation and expansion of the endogenous immune rep-ertoire. Passive immunization induces immunity by transferof preformed tumor-specific T cells (adoptive transfer).

VaccinationVaccination has been proven to be an effective method forinduction of T-cell responses. DC-based vaccine for malig-nant B cells was shown to induce anti-idiotype T-cell re-sponses that resulted in a prominent clinical response[68]. Additional increase of the vaccine’s immunogenicitycan be achieved by coupling an antigen to a highly immu-nogenic molecule, such as a fragment of the tetanus toxin.This approach was successfully used for induction of im-mune responses by DNA vaccine encoding variable regiongenes of an idiotypic immunoglobulin and Fragment C oftetanus toxin [69]. It led to suppression of tumor growthin mouse models and in patients with follicular lymphoma.

In general, vaccination provides an advantage of contin-uous immune vigilance once the immunity is established.However, while vaccination is successful in induction ofT-cell responses toward foreign antigens in immunocompe-tent individuals, responses against tumor-associated self-antigens are likely to be suboptimal. Thymic selectionusually eliminates the T-cell repertoire of T cells bearingself-specific high-affinity TCRs. In addition, cancer patientsare frequently immunocompromised, which further limitsapplication of therapeutic vaccination strategies. Anotherfactor that is absolutely necessary for an effective antitumor


immune response is an adequate immunogenicity of leuke-mic cells. It is well documented that administration ofIFN-g and granulocyte macrophage colony-stimulating fac-tor (GM-CSF) upregulate major histocompatibility class(MHC) expression and makes tumor cells more susceptibleto cytotoxic T cells [70]. IFN-a and GM-CSF treatment im-proves results of DLI in accelerate phase CML patients byupregulating HLA and costimulatory molecule expressionon tumor cells [71]. Alternatively, ligation of CD40 onB-ALL cells or their stimulation with TNF-a increasesCD80 expression on tumor cells, improving their antigen-presenting capacity [72]. Exposure of malignant CD34D

progenitors to GM-CSF, IL-4, and TNF-a leads to develop-ment of a potent APC that induces a powerful antileukemicresponse [73]. Moreover, leukemic cells can be transfectedex vivo with CD80 [74] or IL-12 [75] in order to upregulateT-cell responses to the native tumor in vivo.

Finally, to improve results of immunotherapy, the tumorburden should be decreased prior to DLI. Chemotherapyhas to be minimally toxic to avoid mucosal damage and re-sulting exacerbation of GVHD. Therefore, combinationtherapy with biologic agents possessing a highly selectiveantitumor activity like STI-571 or rituximab may result ina better clinical outcome.

Irrespective of the immunogenicity of tumor cells, suc-cess of an immunotherapeutic approach depends on its abil-ity to create a large number of tumor-specific T cells ofa high avidity that are in the optimal activation state. Theserequirements can be met by adoptive T-cell transfer.

Adoptive transferThe antitumor effect of adoptive transfer of T cells from animmunocompetent donor has been known since allogeneicbone marrow transplantation was introduced [5]. Transferof allogeneic T cells allows targeting of tumor-associatedself-antigens that are normally ignored by the host immunesystem due to self-tolerance. These self-antigens may berecognized by allogeneic T cells because of their polymor-phism or an HLA mismatch. However, tumor-associatedantigens represent a minority in the antigenic repertoire thatdrives the allogeneic immune response. Therefore, an en-richment of the T-cell transplant for tumor-specific T cellsis of high importance for a successful immunotherapy.

Ex vivo expansion of antigen-specific T cellsEBV lymphoma represents an ideal model for evaluation ofthe feasibility of immunotherapy with expanded antigen-specific T cells. EBV-positive cells have good antigen-presenting capacity and express the whole range of latentcycle EBV antigens, including the immunodominant EBNA3antigen. At the same time, most donors are EBV-seroposi-tive and possess a significant amount of high-avidity EBV-specific T cells. In a clinical trial, polyclonal EBV-specificCTL were used for prophylaxis or therapy of EBV lympho-ma in high-risk HSCT patients; T cells survived in vivo up

to 86 months and a reduction of the viral load was ob-served. EBV-specific CTL were also able to prevent EBVlymphoma progression in these patients because none ofthem developed tumors, compared with 11.5% in controls[76]. Remarkably, in five of six patients with an establishedtumor, complete remission was achieved.

Interestingly, leukemia-specific CTL lines were success-fully used to induce remission in a patient with acceleratedphase CML after allo-BMT [77]. However, the most out-standing studies of the adoptive transfer of expanded T cellswere performed in melanoma patients [78,79]. Notably, thedose of tumor-reactive T cells that was required to achieveclinical responses was about 100 times higher than the doseof antiviral T cells. The remarkable difference in T-cell dos-age and clinical results is explained by the fact that T cellsspecific to viral antigens are well represented in the naturalT-cell repertoire, while tumor-specific T cells are usuallyvery rare. Low numbers of GVL effectors are, however,not the only problem. T-cell activity is frequently sup-pressed in cancer patients by tumor immune escape mech-anisms. Moreover, tumor-associated antigens are frequentlycoexpressed on normal cells on certain developmentalstages. Due to the arising self-tolerance, T cells with a highavidity to such antigens are normally absent in the T-cellrepertoire. Therefore, isolation of tumor-specific T cellsfrom individual cancer patients and expansion to clinicallyapplicable numbers has proven to be an unreliable ap-proach. In addition, the clinically significant numbers ofantigen-specific T cells may be achieved only throughlong-term in vitro culture, which inevitably compromisesfunctional properties of T cells. Long-term expansion ofT cells with use of anti-CD3 and IL-2 is associated witha reduction of T-cell receptor diversity and increased sus-ceptibility to apoptosis [80]. In vivo, these expanded T cellshave limited persistence after adoptive transfer [81].

In conclusion, a method to create a significant number ofGVL effectors in a short period of time is preferred. Trans-fer of genes coding for a TCR specific to a GVL-associatedantigen to large numbers of donor T cells may provide suchan alternative.

TCR-transferIn 1986, Dembic et al. [82] were the first to demonstratethat transfer of a- and b-TCR chains from a donor T-cellclone endows a recipient T cell with donor T-cell reactivity[82]. Nine years later, Cole et al. [83] successfully trans-ferred a human TCR specific to the melanoma differentia-tion antigen MART-1.

However, low expression of exogenous TCR allowedrecognition of peptide-pulsed cells only. Generation of Tcells reactive to tumor cell lines became possible in 1999,with use of the efficient retroviral transduction system[84]. Genes encoding TCR a- and b-chains can be clonedinto a retroviral vector; infection of T cells with such retro-viruses leads to a high surface expression of the exogenous


TCR. Diverse tumor-associated antigens, such as gp100[85] and MAGE-3 [86], p53 [87], and hMDM2 [35] werealso successfully targeted by TCR-transfer.

Despite preservation of the original clone’s specificityafter TCR transfer, some studies demonstrated that avidityof TCR-transgenic cells was inferior to that of the parentalclone and their reactivity was proportional to the level ofexpression of exogenous TCR chains [88,89]. However,with use of efficient retroviral vectors and TCRs of high af-finity, preservation of the parental clones’ avidity wasachieved [85,90]. In fact, reduction of TCR levels by5- to 20-fold had little effect on T-cell sensitivity [91].Therefore, it seems that if the amount of TCR on the T-cellsurface exceeds a certain threshold, the sensitivity of TCR-transgenic cells is defined by the affinity of the individualTCR rather than by the level of expression of the exogenousTCR. Interestingly, by transfer of a TCR from an HLA classI-restricted CD8-independent T-cell clone specific to tyros-inase into a CD8-negative murine T-cell line, Roszkowskiet al. [89] were able to demonstrate that CD8-independenttarget cell recognition is also a property of the TCR and notthe T cell. This has important implications for immunother-apy because TCR transfer into an unselected T-cell popula-tion would result in a significant number of CD4D T cellsexpressing HLA class I-restricted TCR. If this TCR hasa high avidity towards the complex of HLA class I andthe antigenic peptide, the CD8 coengagement is not neces-sary for T-cell activation. The response of CD4D T cells viathe exogenous TCR may significantly broaden the array ofeffector mechanisms contributing to the antitumor effect.

TCR transfer can be exploited to endow CTL with novelspecificities that are absent in the natural TCR repertoire.Immunization of mice transgenic for human HLA mole-cules is an efficient method for generation of T cells withhigh-affinity TCR specific to human self-antigens. TheseTCRs, normally absent in the human T-cell repertoire,can be cloned and used for grafting to human T cells, asshown by Stanislawski et al. [35]. Human peripheral bloodlymphocytes (PBL) transduced with an MDM-2–specificmurine TCR obtained the ability to lyse tumor cell lineswith a peptide sensitivity close to that of mouse-derivedT cells. The similar approach of immunization of anHLA-A*0201 transgenic mice was used by Liu et al. [87]to demonstrate the possibility of selective targeting of dif-ferentially expressed human p53 by murine TCR trans-ferred to Jurkat cells. However, mouse-derived TCRs maybe immunogenic and lead to eradication of TCR-transgenicT cells by the host immune system. An elegant way to cir-cumvent the problem of the immunologic tolerance is tostimulate T cells with APCs presenting the peptide of theself-antigen on an allogeneic HLA molecule [92]. For in-stance, TCRs specific to the HLA-A2–restricted hemato-poietic lineage-specific minor H antigen HA-2 weresuccessfully used for redirection of peripheral T cells to-wards leukemic cells [93]. Another method of generation

of high-affinity self-antigen–specific TCR is through intro-duction of mutations into the variable regions of the origi-nal low-affinity TCR and selection of suitable TCRs byretroviral TCR display [94]. However, artificial high-affin-ity TCR may be prone to cross-reactivity with self-antigens,causing autoimmune responses. Moreover, prolonged inter-action of high-affinity TCR with the HLA-peptide complexmay cause antigen-induced T-cell death. Therefore, biolog-ical properties of TCR with supraphysiological affinityhave to be studied thoroughly prior to their clinicalapplication.

Cytotoxic T cells are not the only type of T cells that canbe generated by TCR transfer. Antigen-specific regulatoryT cells may be also engineered by transduction of CD4D

T cells with both TCR and a CTLA4Ig molecule [95].TCR/CTLA4Ig-transgenic cells specific to the nucleosomalantigen were able to accumulate in spleen of mice affectedwith systemic lupus erythematosus and suppress the patho-genic autoantibody production.

A frequent concern is downregulation of exogenous TCRexpression due to competition with endogenous TCR. How-ever, decreased TCR expression on the surface does notnecessarily lead to diminished peptide sensitivity [64,65].

Another related problem is generation of multispecific Tcells after TCR transfer into mature T cells due to the pres-ence of the endogenous TCR. Its a- and b-chains may formheterodimers with chains of exogenous TCR resulting in theemergence of new specificities, some of which may poten-tially lead to autoimmune responses. One solution to thisproblem may be TCR transduction of hematopoietic pro-genitor cells and their differentiation into mature T cells.Rearrangement of endogenous TCR genes will be preventedby expression of exogenous TCR so that monospecificT cells will be generated [96]. Moreover, TCR-transgenicT cells derived from hematopoietic progenitors are likelyto have greater lifespan and proliferative potential. Anotherway to prevent the appearance of T cells able to induce au-toimmune responses is to limit the repertoire of T cells thatundergo TCR transfer. This may be achieved by transduc-tion of CMV-specific T cells isolated from the peripheralblood of the patient with the help of MHC tetramers [97].Upon infusion into a patient, these dual-specific T cellsare expected to be in the activated state and survive longerbecause of the frequent encounters with APCs presentingCMV antigens.

Prevention of pairing of exogenous and endogenousTCR chains can be achieved with use of single-chainTCR constructs [98]. This also increases the level of ex-pression of the receptor because competition from exoge-nous TCR chains is avoided. TCRa expression frequentlylimits TCR formation; in case of a single-chain TCR con-struct this is no longer the case. Chimeric receptors thatcombine the Fab fragment recognizing the MHC-peptidecomplex and TCR-signaling domains also ensure efficientMHC-restricted recognition of tumor cells [99].


Artificial receptors consisting of single-chain variablefragments of antibodies specific to antigens expressed ontumor cells and intracellular domains of T-cell signalingmolecules, such as CD3z or CD28, are attractive alterna-tives to regular TCRs. Such receptors (chimeric antigen re-ceptors [CARs]) are not HLA-restricted and therefore canbe applied in every patient. Moreover, T cells bearing thesereceptors are able to recognize tumor cells even if HLA ex-pression and antigen processing are downregulated to avoidrecognition by the immune system. Several studies demon-strated tumor clearance in mice after infusion of T cellsequipped with tumor-specific CARs [100,101]. Concomi-tant infusion of Th1 and Tc1 cells specific to the carci-noembryonic antigen (CEA) tumor-associated antigen ledto eradication of CEA-positive tumors in a mouse model[102]. Human T cells transduced with a CAR consistingof single-chain variable fragment of CD19 antibody, CD8hinge, and transmembrane domains and TCRz-chain cyto-plasmic domain were able to efficiently lyse B cells in vitroand eradicate Raji tumors in a mouse model [103]. Thesecells were shown to persist in vivo and travel to the bonemarrow; their ability to engraft was increased significantlyif IL-15 was used during their in vitro expansion. CAR-transduced CD19-specific T cells were able to lyseCD19D target cells irrespective of the level of expressionof accessory molecules on their surface [104]. Lymphomacells were also successfully targeted by CARs recognizingCD20 [105] and CD30 antigens [106]. However, despite theadvantages of CAR-mediated immunotherapy, its applica-bility is still debated. Chimeric receptors unavoidably con-tain fusion sequences that can be recognized by the immunesystem. The high affinity of the antibody-based receptorsmay cause significant prolongation of contact betweentransduced T cells and their targets. This may decreasethe cytotoxic effect and even cause T-cell apoptosis. Intra-cellular domains of CAR do not seem to provide the fullspectrum of T-cell activation signals [107]. Finally, it is un-clear whether CAR ligation leads to formation of the func-tional immunological synapse.

The success of T-cell–based immunotherapy depends onthe susceptibility of tumor cells to the T-cell–mediated cy-totoxicity. Unfortunately, the genetic instability of tumorsleads to a significant phenotypic heterogeneity. In combina-tion with fast proliferation, this may result in outgrowth oftumor cells that escape the immune response of TCR-transgenic T cells. An immune escape can be preventedby simultaneous transduction of T cells with several TCRsand application of immunotherapy at the early stages of tu-mor development when genetic heterogeneity of tumorcells is minimal.

Nonspecific enhancement of T cell activityIn addition to redirection of T-cell specificity via TCR trans-fer, antitumor T-cell response can be enhanced nonspecifi-cally by transduction of T cells with an apoptosis-inducing

ligand [108]. Tumor-specific T cells may also be used for de-livery of stimulatory cytokines into the tumor microenviron-ment. An additional strategy for improving in vivo functionof adoptively transferred T cells is to equip them with mole-cules that can overcome certain tumor escape mechanisms[109]. In addition, prolongation of T-cell lifespan may beachieved through retroviral transduction of T lymphocyteswith the hTERT [110]. Transduction with hTERT allows cellproliferation but does not cause cell-cycle entry or growthderegulation [111]. Accordingly, hTERT-transduced T cellsremain dependent on activating signals [110]. Their abilityto suppress growth of human tumors was shown in vivo[112]. However, an increased genomic instability in hTERT-transduced T cells was reported recently [113]. Therefore,a safety measure, such as a suicide gene, should be includedin the hTERT vector for a clinical application. Survival oftransferred T cells may also be enhanced by introduction ofthe GM-CSF cytokine receptor. Transduced CD8D T cellsproduce GM-CSF upon activation and stimulate themselvesvia this autocrine loop [114]. Genetic modification of T cellsto optimize their migration pattern may enhance clinicalefficacy of adoptive transfer as well. For instance, T cellsmay be redirected to the bone marrow through expressionof the chemokine receptor CXCR4. Its ligand SDF1 is pro-duced by bone marrow stromal cells [115].

In vivo engraftmentand expansion of adoptively transferred T cellsAll aforementioned approaches to T-cell–based immuno-therapy require expansion or genetic modification of T cellsex vivo. Unfortunately, these procedures result in poor sur-vival and reduction of T-cell immune function in vivo[80,116]. This remains a major obstacle for clinical applica-tion of immunotherapy [117]. This problem may be over-come by using both CD3 and CD28 for T-cell activation.Administration of CD3/CD28-activated T cells to patientswith relapsed or refractory non-Hodgkin’s lymphoma afterhigh-dose chemotherapy led to rapid recovery of lympho-cyte counts, though no data on antitumor activity are avail-able [118]. IL-15 was shown to be helpful in prevention ofapoptotic T-cell death and preservation of in vivo antitumoractivity [103]. Interestingly, T-cell engraftment is facilitatedin patients after stem cell transplantation. Most likely, a con-ditioning treatment with fludarabine and cyclophosphamidecreates the proliferative environment that favors engraft-ment [79].

Studies of the behavior of TCR transgenic cells in vivoare of great importance. The pioneering work of Kesselset al. [119] has demonstrated the feasibility of an adoptivetherapy using TCR-transgenic T cells. Mouse splenocytestransduced with TCR specific to a peptide of the influenzaprotein expanded more than 1000-fold upon the antigenicchallenge in vivo. As little as 28 3 103 CD8D T cells car-rying the influenza-specific TCR were able to eradicatetumors expressing influenza antigen. Less-dramatic effects


were found in the work of Tahara et al. [120], where infu-sion of chicken ovalbumin (OVA)-specific TCR-transducedcells was shown to induce eradication of an establishedOVAD tumor in a mouse [121]. However, in the latter work,the effect was complete only when cyclophosphamide con-ditioning was performed prior to adoptive immunotherapy.This indicates that an inflammatory microenvironment inthe tumor was necessary for an effective immune response.

However, all these studies were performed with use ofmouse lymphocytes. All studies where the in vivo efficacyof human T cells was demonstrated were performed using Tcells transduced with CAR. Gyobu et al. [102] found thatthe growth of CEAD tumor was inhibited if T-cell bodiesconsisting of Tc1 and Th1 lymphocytes transduced withCEA-specific CAR were coinjected [102]. Significant re-sponses were achieved by targeting tumor cells expressingthe prostate-specific membrane antigen (PSMA) with hu-man T lymphocytes equipped with a PSMA-specific CAR[122]. Growth of adenocarcinoma of the prostate was alsoinhibited by the intratumor delivery of human T cells trans-duced with a chimeric receptor specific to the ErgB2growth factor receptor [123]. The most elaborate study onin vivo efficacy of immunogenetically modified lympho-cytes demonstrated complete clearance of tumors formedby the Raji Burkitt lymphoma cell line by T cells trans-duced with a CD19-specific CAR [103]. A dependence ofthe antitumor response on an adequate costimulation of Tcells both in vitro and in vivo and the presence of IL-15in the culture medium during expansion was demonstrated.However, the question of T-cell homing and engraftmenthas yet to be addressed properly.

ConclusionsImmunotherapy has the potential to become a decisive weaponin the fight against malignancies, providing a tool for a majorbreakthrough in this field of medicine. However, despite theremarkable clinical success of approaches exploiting GVL,further progress might only be achieved by manipulatingthe graft in a way that will shift the balance between GVLand GVH activity in favor of GVL. Although there is a signif-icant amount of data on the in vitro efficacy of such ap-proaches, only few studies have demonstrated their in vivousefulness. In part, this may be explained by the fact thatlong-lasting in vitro expansion of tumor-specific T cells,which until recently was an essential part of immunotherapyprotocols, inevitably compromised T-cell reactivity. Adventof TCR-transfer offers an elegant way to circumvent thisobstacle. Use of genetically engineered immune cells, forwhich immuno genetherapy would be a proper term, is gain-ing momentum as a promising clinical approach.

AcknowledgmentsThe authors thank Dr. Andy Dale and Dr. Tuna Mutis for their helpin preparation of this article. This work was supported by EC grantQLK3 CT 2001 01265.

References1. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia

effect of donor lymphocyte transfusions in marrow grafted patients.

European Group for Blood and Marrow Transplantation Working

Party Chronic Leukemia. Blood. 1995;86:2041–2050.

2. Collins RH Jr, Shpilberg O, Drobyski WR, et al. Donor leukocyte in-

fusions in 140 patients with relapsed malignancy after allogeneic

bone marrow transplantation. J Clin Oncol. 1997;15:433–444.

3. Tabbara IA, Ingram RM. Nonmyeloablative therapy and allogeneic

haematopoietic stem cell transplantation. Exp Hematol. 2003;31:

559–566.

4. Barrett AJ. Mechanisms of the graft–versus-leukemia reaction. Stem

Cells. 1997;15:248–258.

5. Kolb HJ, Schmid C, Barrett AJ, Schendel DJ. Graft-versus-leukemia

reactions in allogeneic chimeras. Blood. 2004;103:767–776.

6. Johnson BD, Truitt RL. Delayed infusion of immunocompetent do-

nor cells after bone marrow transplantation breaks graft-host toler-

ance allows for persistent antileukemic reactivity without severe

graft-versus-host disease. Blood. 1995;85:3302–3312.

7. Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive im-

munotherapy evaluating escalating doses of donor leukocytes for re-

lapse of chronic myeloid leukemia after bone marrow

transplantation: separation of graft-versus-leukemia responses from

graft-versus-host disease. Blood. 1995;86:1261–1268.

8. Litvinova E, Maury S, Boyer O, et al. Graft-versus-leukemia effect

after suicide-gene-mediated control of graft-versus-host disease.

Blood. 2002;100:2020–2025.

9. Introna M, Barbui AM, Bambacioni F, et al. Genetic modification of

human T cells with CD20: a strategy to purify and lyse transduced

cells with anti-CD20 antibodies. Hum Gene Ther. 2000;11:611–620.

10. Amrolia PJ, Muccioli-Casadei G, Yvon E, et al. Selective depletion

of donor alloreactive T cells without loss of antiviral or antileukemic

responses. Blood. 2003;102:2292–2299.

11. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia re-

actions after bone marrow transplantation. Blood. 1990;75:555–562.

12. Mehta J, Powles R, Singhal S, Tait D, Swansbury J, Treleaven J. Cy-

tokine-mediated immunotherapy with or without donor leukocytes

for poor-risk acute myeloid leukemia relapsing after allogeneic bone

marrow transplantation. Bone Marrow Transplant. 1995;16:133–137.

13. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, Perreault C.

Adoptive transfer of minor histocompatibility antigen-specific T lym-

phocytes eradicates leukemia cells without causing graft-versus-host

disease. Nat Med. 2001;7:789–794.

14. Kalams SA, Walker BD. The critical need for CD4 help in maintain-

ing effective cytotoxic T lymphocyte responses. J Exp Med. 1998;

188:2199–2204.

15. Cohen PA, Peng L, Plautz GE, Kim JA, Weng DE, Shu S. CD4D T

cells in adoptive immunotherapy and the indirect mechanism of

tumor rejection. Crit Rev Immunol. 2000;20:17–56.

16. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by

dendritic cells. Cell. 2001;106:263–266.

17. Blattman JN, Greenberg PD. Cancer immunotherapy: a treatment for

the masses. Science. 2004;305:200–205.

18. Hill GR, Teshima T, Gerbitz A, et al. Differential roles of IL-1 and

TNF-alpha on graft-versus-host disease and graft versus leukemia.

J Clin Invest. 1999;104:459–467.

19. Mumberg D, Monach PA, Wanderling S, et al. CD4(D) T cells elim-

inate MHC class II-negative cancer cells in vivo by indirect effects of

IFN-gamma. Proc Natl Acad Sci U S A. 1999;96:8633–8638.

20. Schuler T, Blankenstein T. Cutting edge: CD8D effector T cells re-

ject tumors by direct antigen recognition but indirect action on host

cells. J Immunol. 2003;170:4427–4431.

21. Ibe S, Qin Z, Schuler T, Preiss S, Blankenstein T. Tumor rejection by

disturbing tumor stroma cell interactions. J Exp Med. 2001;194:

1549–1559.


22. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM. Control of

T-cell activation by CD4D CD25D suppressor T cells. Immunol

Rev. 2001;182:58–67.

23. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Donor-

type CD4(D)CD25(D) regulatory T cells suppress lethal acute graft-

versus-host disease after allogeneic bone marrow transplantation.

J Exp Med. 2002;196:389–399.

24. Trenado A, Charlotte F, Fisson S. Recipient-type specific

CD4DCD25D regulatory T cells favor immune reconstitution and

control graft-versus-host disease while maintaining graft-versus-

leukemia. J Clin Invest. 2003;112:1688–1696.

25. Edinger M, Hoffmann P, Ermann J, et al. CD4DCD25D regulatory

T cells preserve graft-versus-tumor activity while inhibiting graft-

versus-host disease after bone marrow transplantation. Nat Med.

2003;9:1144–1150.

26. Michalek J, Collins RH, Durrani HP, et al. Definitive separation of

graft-versus-leukemia- and graft-versus-host-specific CD4D T cells

by virtue of their receptor beta loci sequences. Proc Natl Acad Sci

U S A. 2003;100:1180–1184.

27. Bocchia M, Korontsvit T, Xu Q, et al. Specific human cellular immu-

nity to bcr-abl oncogene-derived peptides. Blood. 1996;87:3587–

3592.

28. Gambacorti-Passerini C, Grignani F, Arienti F, Pandolfi PP,

Pelicci PG, Parmiani G. Human CD4 lymphocytes specifically recog-

nize a peptide representing the fusion region of the hybrid protein

pml/RAR alpha present in acute promyelocytic leukemia cells.

Blood. 1993;81:1369–1375.

29. Bellantuono I, Gao L, Parry S, et al. Two distinct HLA-A0201-pre-

sented epitopes of the Wilms tumor antigen 1 can function as targets

for leukemia-reactive CTL. Blood. 2002;100:3835–3837.

30. Gao L, Bellantuono I, Elsasser A, et al. Selective elimination of leu-

kemic CD34(D) progenitor cells by cytotoxic T lymphocytes specif-

ic for WT1. Blood. 2000;95:2198–2203.

31. Arai J, Yasukawa M, Ohminami H, Kakimoto M, Hasegawa A,

Fujita S. Identification of human telomerase reverse transcriptase-

derived peptides that induce HLA-A24-restricted antileukemia cyto-

toxic T lymphocytes. Blood. 2001;97:2903–2907.

32. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomer-

ase catalytic subunit is a widely expressed tumor-associated antigen

recognized by cytotoxic T lymphocytes. Immunity. 1999;10:673–

679.

33. Offringa R, Vierboom MP, van der Burg SH, Erdile L, Melief CJ.

p53: a potential target antigen for immunotherapy of cancer. Ann

N Y Acad Sci. 2000;910:223–233.

34. Vierboom MP, Zwaveling S, Bos GMJ, et al. High steady-state levels

of p53 are not a prerequisite for tumor eradication by wild-type p53-

specific cytotoxic T lymphocytes. Cancer Res. 2000;60:5508–5513.

35. Stanislawski T, Voss RH, Lotz C, et al. Circumventing tolerance to

a human MDM2-derived tumor antigen by TCR gene transfer. Nat

Immunol. 2001;2:962–970.

36. Dengler R, Munstermann U, al Batran S, et al. Immunocytochemical

and flow cytometric detection of proteinase 3 (myeloblastin) in nor-

mal and leukaemic myeloid cells. Br J Haematol. 1995;89:250–257.

37. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lym-

phocytes may participate in the elimination of chronic myelogenous

leukemia. Nat Med. 2000;6:1018–1023.

38. Amrolia PJ, Reid SD, Gao L, et al. Allorestricted cytotoxic T cells

specific for human CD45 show potent antileukemic activity. Blood.

2003;101:1007–1014.

39. Raptis A, Clave E, Mavroudis D, Molldrem J, van Rhee F, Barrett AJ.

Polymorphism in CD33 and CD34 genes: a source of minor histo-

compatibility antigens on haemopoietic progenitor cells? Br J Hae-

matol. 1998;102:1354–1358.

40. Bleakley M, Riddell SR. Molecules and mechanisms of the graft-

versus-leukaemia effect. Nat Rev Cancer. 2004;4:371–380.

41. Murata M, Warren EH, Riddell SR. A human minor histocompatibil-

ity antigen resulting from differential expression due to a gene dele-

tion. J Exp Med. 2003;197:1279–1289.

42. Brickner AG, Warren EH, Caldwell JA, et al. The immunogenicity of

a new human minor histocompatibility antigen results from differen-

tial antigen processing. J Exp Med. 2001;193:195–206.

43. Akatsuka Y, Warren EH, Gooley TA, et al. Disparity for a newly

identified minor histocompatibility antigen, HA-8, correlates with

acute graft-versus-host disease after haematopoietic stem cell trans-

plantation from an HLA-identical sibling. Br J Haematol. 2003;

123:671–675.

44. Murata M, Emi N, Hirabayashi N, et al. No significant association

between HA-1 incompatibility and incidence of acute graft-versus-

host disease after HLA-identical sibling bone marrow transplantation

in Japanese patients. Int J Hematol. 2000;72:371–375.

45. Dickinson AM, Wang XN, Sviland L, et al. In situ dissection of the

graft-versus-host activities of cytotoxic T cells specific for minor

histocompatibility antigens. Nat Med. 2002;8:410–414.

46. Marijt WA, Heemskerk MH, Kloosterboer FM, et al. Hematopoiesis-

restricted minor histocompatibility antigens HA-1- or HA-2-specific

T cells can induce complete remissions of relapsed leukemia. Proc

Natl Acad Sci U S A. 2003;100:2742–2747.

47. Goulmy E, Schipper R, Pool J, et al. Mismatches of minor histocom-

patibility antigens between HLA-identical donors and recipients and

the development of graft-versus-host disease after bone marrow

transplantation. N Engl J Med. 1996;334:281–285.

48. Teshima T, Ordemann R, Reddy P, et al. Acute graft-versus-host

disease does not require alloantigen expression on host epithelium.

Nat Med. 2002;8:575–581.

49. Akatsuka Y, Nishida T, Kondo E, et al. Identification of a polymor-

phic gene, BCL2A1, encoding two novel hematopoietic lineage-

specific minor histocompatibility antigens. J Exp Med. 2003;197:

1489–1500.

50. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The male-specific

region of the human Y chromosome is a mosaic of discrete sequence

classes. Nature. 2003;423:825–837.

51. Miklos DB, Kim HT, Zorn E, et al. Antibody response to DBY mi-

nor histocompatibility antigen is induced after allogeneic stem cell

transplantation and in healthy female donors. Blood. 2004;103:

353–359.

52. Verdijk RM, Kloosterman A, Pool J, et al. Pregnancy induces minor

histocompatibility antigen-specific cytotoxic T cells: implications for

stem cell transplantation and immunotherapy. Blood. 2004;103:

1961–1964.

53. Simpson E, Scott D, Chandler P. The male-specific histocompatibil-

ity antigen, H-Y: a history of transplantation, immune response

genes, sex determination and expression cloning. Annu Rev Immu-

nol. 1997;15:39–61.

54. Randolph SS, Gooley TA, Warren EH, Appelbaum FR, Riddell SR.

Female donors contribute to a selective graft-versus-leukemia effect

in male recipients of HLA-matched, related hematopoietic stem cell

transplants. Blood. 2004;103:347–352.

55. Wang W, Meadows LR, den Haan JM, et al. Human H-Y: a male-

specific histocompatibility antigen derived from the SMCY protein.

Science. 1995;269:1588–1590.

56. Pierce RA, Field ED, den Haan JM, et al. Cutting edge: the HLA-

A*0101-restricted HY minor histocompatibility antigen originates

from DFFRY and contains a cysteinylated cysteine residue as identi-

fied by a novel mass spectrometric technique. J Immunol. 1999;163:

6360–6364.

57. Vogt MH, Goulmy E, Kloosterboer FM, et al. UTY gene codes for an

HLA-B60-restricted human male-specific minor histocompatibility

antigen involved in stem cell graft rejection: characterization of the

critical polymorphic amino acid residues for T-cell recognition.

Blood. 2000;96:3126–3132.


58. Vogt MH, van den Muijsenberg JW, Goulmy E, et al. The DBY gene

codes for an HLA-DQ5-restricted human male-specific minor histo-

compatibility antigen involved in graft-versus-host disease. Blood.

2002;99:3027–3032.

59. Vogt MH, de Paus RA, Voogt PJ, Willemze R, Falkenburg JH.

DFFRY codes for a new human male-specific minor transplantation

antigen involved in bone marrow graft rejection. Blood. 2000;95:

1100–1105.

60. Spierings E, Vermeulen CJ, Vogt MH, et al. Identification of HLA

class II-restricted H-Y-specific T-helper epitope evoking CD4D

T-helper cells in H-Y-mismatched transplantation. Lancet. 2003;

362:610–615.

61. Mutis T, Gillespie G, Schrama E, Falkenburg JH, Moss P, Goulmy E.

Tetrameric HLA class I-minor histocompatibility antigen peptide

complexes demonstrate minor histocompatibility antigen-specific

cytotoxic T lymphocytes in patients with graft-versus-host disease.

Nat Med. 1999;5:839–842.

62. Warren EH, Gavin MA, Simpson E, et al. The human UTY gene enc-

odes a novel HLA-B8-restricted H-Y antigen. J Immunol. 2000;164:

2807–2814.

63. Bonnet D, Warren EH, Greenberg PD, Dick JE, Riddell SR. CD8(D)

minor histocompatibility antigen-specific cytotoxic T lymphocyte

clones eliminate human acute myeloid leukemia stem cells. Proc Natl

Acad Sci U S A. 1999;96:8639–8644.

64. Ivanov R, Hol S, Aarts T, Hagenbeek A, Slager EH, Ebeling S. UTY-

specific TCR-transfer generates potential graft-versus-leukaemia

effector T cells. Br J Haematol. 2005;129:392–402.

65. Ivanov R, Aarts T, Hol S, et al. Identification of a 40S ribosomal

protein S4-derived H-Y epitope able to elicit a lymphoblast-specific

cytotoxic T lymphocyte response. Clin Cancer Res. 2005;11:

1694–1703.

66. Zhang L, Zhou W, Velculescu VE, et al. Gene expression profiles in

normal and cancer cells. Science. 1997;276:1268–1272.

67. Tsukasaki K, Tanosaki S, DeVos S, et al. Identifying progression-

associated genes in adult T-cell leukemia/lymphoma by using oligo-

nucleotide microarrays. Int J Cancer. 2004;109:875–881.

68. Timmerman JM, Czerwinski DK, Davis TA, et al. Idiotype-pulsed

dendritic cell vaccination for B-cell lymphoma: clinical and immune

responses in 35 patients. Blood. 2002;99:1517–1526.

69. King CA, Spellerberg MB, Zhu D, et al. DNA vaccines with single-

chain Fv fused to fragment C of tetanus toxin induce protective im-

munity against lymphoma and myeloma. Nat Med. 1998;4:1281–

1286.

70. Dermime S, Mavroudis D, Jiang YZ, Hensel N, Molldrem J,

Barrett AJ. Immune escape from a graft-versus-leukemia effect

may play a role in the relapse of myeloid leukemias following allo-

geneic bone marrow transplantation. Bone Marrow Transplant. 1997;

19:989–999.

71. Chen X, Regn S, Raffegerst S, Kolb HJ, Roskrow M. Interferon alpha

in combination with GM-CSF induces the differentiation of leukae-

mic antigen-presenting cells that have the capacity to stimulate a

specific anti-leukaemic cytotoxic T-cell response from patients with

chronic myeloid leukaemia. Br J Haematol. 2000;111:596–607.

72. Cardoso AA, Seamon MJ, Afonso HM, et al. Ex vivo generation of

human anti-pre-B leukemia-specific autologous cytolytic T cells.

Blood. 1997;90:549–561.

73. Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendrit-

ic cells for the generation of antileukemic cellular cytotoxicity

against Philadelphia chromosome-positive chronic myelogenous leu-

kemia. Blood. 1997;89:1133–1142.

74. Mutis T, Schrama E, Melief CJ, Goulmy E. CD80-Transfected acute

myeloid leukemia cells induce primary allogeneic T-cell responses

directed at patient specific minor histocompatibility antigens and

leukemia-associated antigens. Blood. 1998;92:1677–1684.

75. Hoshino T, Jiang YZ, Dunn D, et al. Transfection of interleukin-12

cDNAs into tumor cells induces cytotoxic immune responses against

native tumor: implications for tumor vaccination. Cancer Gene Ther.

1998;5:150–157.

76. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells

for the prevention and treatment of Epstein-Barr virus-induced lym-

phoma in allogeneic transplant recipients. Blood. 1998;92:1549–

1555.

77. Falkenburg JH, Wafelman AR, Joosten P, et al. Complete remission

of accelerated phase chronic myeloid leukemia by treatment with

leukemia-reactive cytotoxic T lymphocytes. Blood. 1999;94:1201–

1208.

78. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using

antigen-specific CD8D T cell clones for the treatment of patients

with metastatic melanoma: in vivo persistence, migration, and antitu-

mor effect of transferred T cells. Proc Natl Acad Sci U S A. 2002;99:

16168–16173.

79. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and

autoimmunity in patients after clonal repopulation with antitumor

lymphocytes. Science. 2002;298:850–854.

80. Sauce D, Bodinier M, Garin M, et al. Retrovirus-mediated gene

transfer in primary T lymphocytes impairs their anti-Epstein-Barr

virus potential through both culture-dependent and selection pro-

cess-dependent mechanisms. Blood. 2002;99:1165–1173.

81. Rosenberg SA, Aebersold P, Cornetta K, et al. Gene transfer into

humans–immunotherapy of patients with advanced melanoma, using

tumor-infiltrating lymphocytes modified by retroviral gene transduc-

tion. N Engl J Med. 1990;323:570–578.

82. Dembic Z, Haas W, Weiss S, et al. Transfer of specificity by murine

alpha and beta T-cell receptor genes. Nature. 1986;320:232–238.

83. Cole DJ, Weil DP, Shilyansky J, et al. Characterization of the func-

tional specificity of a cloned T-cell receptor heterodimer recognizing

the MART-1 melanoma antigen. Cancer Res. 1995;55:748–752.

84. Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI.

Efficient transfer of a tumor antigen-reactive TCR to human periph-

eral blood lymphocytes confers anti-tumor reactivity. J Immunol.

1999;163:507–513.

85. Schaft N, Willemsen RA, de Vries J, et al. Peptide fine specificity of

anti-glycoprotein 100 CTL is preserved following transfer of engi-

neered TCR alpha beta genes into primary human T lymphocytes.

J Immunol. 2003;170:2186–2194.

86. Calogero A, Hospers GA, Kruse KM, et al. Retargeting of a T cell

line by anti MAGE-3/HLA-A2 alpha beta TCR gene transfer. Anti-

cancer Res. 2000;20:1793–1799.

87. Liu X, Peralta EA, Ellenhorn JD, Diamond DJ. Targeting of human

p53-overexpressing tumor cells by an HLA A*0201-restricted

murine T-cell receptor expressed in Jurkat T cells. Cancer Res.

2000;60:693–701.

88. Cooper LJ, Kalos M, Lewinsohn DA, Riddell SR, Greenberg PD.

Transfer of specificity for human immunodeficiency virus type 1 into

primary human T lymphocytes by introduction of T-cell receptor

genes. J Virol. 2000;74:8207–8212.

89. Roszkowski JJ, Yu DC, Rubinstein MP, McKee MD, Cole DJ,

Nishimura MI. CD8-independent tumor cell recognition is a property

of the T cell receptor and not the T cell. J Immunol. 2003;170:2582–

2589.

90. Rubinstein MP, Kadima AN, Salem ML, et al. Transfer of TCR genes

into mature T cells is accompanied by the maintenance of parental

T cell avidity. J Immunol. 2003;170:1209–1217.

91. Labrecque N, Whitfield LS, Obst R, Waltzinger C, Benoist C,

Mathis D. How much TCR does a T cell need? Immunity. 2001;

15:71–82.

92. Sadovnikova E, Stauss HJ. Peptide-specific cytotoxic T lymphocytes

restricted by nonself major histocompatibility complex class I


molecules: reagents for tumor immunotherapy. Proc Natl Acad Sci U

S A. 1996;93:13114–13118.

93. Heemskerk MH, Hoogeboom M, de Paus RA, et al. Redirection of

antileukemic reactivity of peripheral T lymphocytes using gene trans-

fer of minor histocompatibility antigen HA-2-specific T-cell receptor

complexes expressing a conserved alpha joining region. Blood. 2003;

102:3530–3540.

94. Kessels HW, van DB, Spits H, Hooijberg E, Schumacher TN. Chang-

ing T cell specificity by retroviral T cell receptor display. Proc Natl

Acad Sci U S A. 2000;97:14578–14583.

95. Fujio K, Okamoto A, Tahara H, et al. Nucleosome-specific regulatory

T cells engineered by triple gene transfer suppress a systemic auto-

immune disease. J Immunol. 2004;173:2118–2125.

96. Yang L, Qin XF, Baltimore D, Van Parijs L. Generation of functional

antigen-specific T cells in defined genetic backgrounds by retrovirus-

mediated expression of TCR cDNAs in hematopoietic precursor

cells. Proc Natl Acad Sci U S A. 2002;99:6204–6209.

97. Heemskerk MH, Hoogeboom M, Hagedoorn R, Kester MG,

Willemze R, Falkenburg JH. Reprogramming of virus-specific T cells

into leukemia-reactive T cells using T cell receptor gene transfer.

J Exp Med. 2004;199:885–894.

98. Willemsen RA, Weijtens ME, Ronteltap C, et al. Grafting primary

human T lymphocytes with cancer-specific chimeric single chain

and two chain TCR. Gene Ther. 2000;7:1369–1377.

99. Willemsen RA, Debets R, Hart E, Hoogenboom HR, Bolhuis RL,

Chames P. A phage display selected fab fragment with MHC class I-

restricted specificity for MAGE-A1 allows for retargeting of primary

human T lymphocytes. Gene Ther. 2001;8:1601–1608.

100. Haynes NM, Snook MB, Trapani JA, et al. Redirecting mouse CTL

against colon carcinoma: superior signaling efficacy of single-chain

variable domain chimeras containing TCR-zeta vs Fc epsilon RI-

gamma. J Immunol. 2001;166:182–187.

101. Kershaw MH, Westwood JA, Hwu P. Dual-specific T cells combine

proliferation and antitumor activity. Nat Biotechnol. 2002;20:1221–

1227.

102. Gyobu H, Tsuji T, Suzuki Y, et al. Generation and targeting of human

tumor-specific Tc1 and Th1 cells transduced with a lentivirus con-

taining a chimeric immunoglobulin T-cell receptor. Cancer Res.

2004;64:1490–1495.

103. Brentjens RJ, Latouche JB, Santos E, et al. Eradication of systemic

B-cell tumors by genetically targeted human T lymphocytes co-stim-

ulated by CD80 and interleukin-15. Nat Med. 2003;9:279–286.

104. Cooper LJ, Topp MS, Serrano LM, et al. T-cell clones can be ren-

dered specific for CD19: toward the selective augmentation of the

graft-versus-B-lineage leukemia effect. Blood. 2003;101:1637–1644.

105. Jensen MC, Cooper LJ, Wu AM, Forman SJ, Raubitschek A. Engi-

neered CD20-specific primary human cytotoxic T lymphocytes for

targeting B-cell malignancy. Cytotherapy. 2003;5:131–138.

106. Hombach A, Heuser C, Sircar R, et al. An anti-CD30 chimeric recep-

tor that mediates CD3-zeta-independent T-cell activation against

Hodgkin’s lymphoma cells in the presence of soluble CD30. Cancer

Res. 1998;58:1116–1119.

107. Brocker T. Chimeric Fv-zeta or Fv-epsilon receptors are not suffi-

cient to induce activation or cytokine production in peripheral T

cells. Blood. 2000;96:1999–2001.

108. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor

necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med.

1999;5:157–163.

109. Bollard CM, Rossig C, Calonge MJ, et al. Adapting a transforming

growth factor beta-related tumor protection strategy to enhance anti-

tumor immunity. Blood. 2002;99:3179–3187.

110. Luiten RM, Pene J, Yssel H, Spits H. Ectopic hTERT expression ex-

tends the life span of human CD4D helper and regulatory T-cell

clones and confers resistance to oxidative stress-induced apoptosis.

Blood. 2003;101:4512–4519.

111. Harley CB. Telomerase is not an oncogene. Oncogene. 2002;21:494–

502.

112. Verra NC, Jorritsma A, Weijer K, et al. Human telomerase reverse

transcriptase-transduced human cytotoxic T cells suppress the growth

of human melanoma in immunodeficient mice. Cancer Res. 2004;64:

2153–2161.

113. Schreurs MW, Hermsen MA, Geltink RI, et al. Genomic stability and

functional activity may be lost in telomerase-transduced human

CD8D T lymphocytes. Blood. 2005;106:2663–2670.

114. Cheng LE, Ohlen C, Nelson BH, Greenberg PD. Enhanced signaling

through the IL-2 receptor in CD8D T cells regulated by antigen rec-

ognition results in preferential proliferation and expansion of re-

sponding CD8D T cells rather than promotion of cell death. Proc

Natl Acad Sci U S A. 2002;99:3001–3006.

115. Mohle R, Bautz F, Rafii S, Moore MA, Brugger W, Kanz L. The che-

mokine receptor CXCR-4 is expressed on CD34D hematopoietic

progenitors and leukemic cells and mediates transendothelial migra-

tion induced by stromal cell-derived factor-1. Blood. 1998;91:4523–

4530.

116. Duarte RF, Chen FE, Lowdell MW, et al. Functional impairment of

human T-lymphocytes following PHA-induced expansion and retro-

viral transduction: implications for gene therapy. Gene Ther. 2002;

9:1359–1368.

117. Sadelain M, Riviere I. Sturm und drang over suicidal lymphocytes.

Mol Ther. 2002;5:655–657.

118. Laport GG, Levine BL, Stadtmauer EA, et al. Adoptive transfer of

costimulated T cells induces lymphocytosis in patients with relapsed/

refractory non-Hodgkin lymphoma following CD34D-selected he-

matopoietic cell transplantation. Blood. 2003;102:2004–2013.

119. Kessels HW, Wolkers MC, van DB, van der Valk MA,

Schumacher TN. Immunotherapy through TCR gene transfer. Nat

Immunol. 2001;2:957–961.

120. Tahara H, Fujio K, Araki Y, et al. Reconstitution of CD8D T cells by

retroviral transfer of the TCR alpha beta-chain genes isolated from

a clonally expanded P815-infiltrating lymphocyte. J Immunol.

2003;171:2154–2160.

121. Chamoto K, Tsuji T, Funamoto H, et al. Potentiation of tumor erad-

ication by adoptive immunotherapy with T-cell receptor gene-trans-

duced T-helper type 1 cells. Cancer Res. 2004;64:386–390.

122. Ma Q, Safar M, Holmes E, Wang Y, Boynton AL, Junghans RP. Anti-

prostate specific membrane antigen designer T cells for prostate can-

cer therapy. Prostate. 2004;61:12–25.

123. Pinthus JH, Waks T, Kaufman-Francis K, et al. Immuno-gene therapy

of established prostate tumors using chimeric receptor-redirected

human lymphocytes. Cancer Res. 2003;63:2470–2476.

Towards immunogene therapy of hematological malignancies

Documents

Transcript of Towards immunogene therapy of hematological malignancies