Immunotherapy For Hematologic Malignancies Research Articles

Targeting leukemia by CD1c-restricted T cells specific for a novel lipid antigen

A subset of CD1c-restricted T lymphocytes exhibits strong reactivity against leukemia cells. These T cells recognize methyl-lysophosphatidic acid (mLPA), a novel lipid antigen produced by acute leukemia cells. Considering that CD1c-restricted T cells display efficacious anti-leukemia activities in a mouse model, this lipid antigen thus represents a novel target in the immunotherapy of hematological malignancies.

Guest editorial: immunotherapy for hematological malignancies: the quest to overcome tolerogenic drive

How easily a healthy body can defend itself against ordinary pathogens! If only it was possible to do the same against self-derived malignancies. Unfortunately, however, the physiology of the immune system discourages such a persistent fight by establishing the overwhelming hurdle of ‘‘tolerance’’. For this reason, tumor immunotherapy stands as an ultimate challenge to the fundamental basis of immunology. Will we ever win this battle? Recent remarkable advances in related fields are finally beginning to make tumor immunologists confident enough to answer in the affirmative. In fact, it has been hematologists who have led the field of tumor immunotherapy through their work in allogeneic hematopoietic stem cell transplantation, which exploits the power of graft-versus-tumor effects. However, this is inevitably accompanied by graft-versus-host disease, which precludes elderly patients, who represent a major population suffering from hematological malignancies, from receiving allogeneic transplantation. Clearly, we must develop novel therapies for such patients. In contrast to the tremendous power of allogeneic reaction, autologous antitumor responses are usually feeble. Heightening them to a clinically meaningful level is a daunting task in the face of the driving force of tolerance. However, rational strategies for overcoming each step in the series of tolerogenic mechanisms have gradually brought immunotherapy into the arena of cancer therapy. Positive and negative components that, respectively, enhance and suppress immune responses are present in the immune system, as in other physiological systems, and are responsible for maintaining homeostasis. The primordial positive component that triggers virtually any type of immune response is innate immunity. The main reason why the immune system efficiently combats pathogens is that innate immune cells express an abundant array of receptors that recognize various molecules found in microbes. Such recognition provokes intense inflammatory responses and the activation of dendritic cells (DCs), the most potent antigen-presenting cells for T cells. Inflammation and activated DCs subsequently initiate effective antigen-specific immune responses, that is, adaptive immunity. This ‘‘innate immunity-DC-adaptive immunity’’ axis is the essence of the positive immune component. To prevent immune responses to autologous or innocuous antigens, and to avoid excessive responses to noxious antigens, the immune system has several layers of negative (suppressive) components. First of these is the induction of central tolerance in the thymus, through which high-affinity self-reactive T cells are eliminated. To assure tolerance to innocuous antigens in the periphery, peripheral tolerance is maintained by several mechanisms, among which regulatory T cells play a key role. Furthermore, negative signals transmitted to T cells through inhibitory receptors, such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1), represent crucial molecular mechanisms for the timely termination of T cell responses [1]. In addition, tumor tissues co-opt certain immunosuppressive components, including regulatory T cells, PD-1 ligands, and myeloid-derived suppressor cells, thus creating a microenvironment hostile to tumorreactive T cells. These positive and negative components provide targets for provoking antitumor immune responses. In this issue, Dr. Bocchia reviews peptide vaccines derived from antigens that are preferentially expressed by tumor cells, thus N. Kadowaki (&) Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan e-mail: kadowaki@kuhp.kyoto-u.ac.jp

Advances in immunotherapy of hematological malignancies by using chimeric antigen receptor-modified lymphocytes

Chimeric antigen receptors (CAR) are fusion proteins between single-chain variable fragments (scFv) from monoclonal antibodies and signaling domains of T-cells, which allow T-cells recognize specific cell-surface targets in an MHC-unrestricted fashion. The structure of CAR has changed over time, from first generation CAR (scFv + signaling moiety) to 2 and 3 generation CAR (combined with one or multiple costimulatory endodomains, such as CD28, 4-1BB and OX40), which enhance persistence, expansion and cytotoxicity of CAR. Many clinical trials treating hematological malignancies using the CAR-modified T-cells targeting CD19 and CD20 are under evaluation or even finished. These clinical trials indicated that CAR-based immunotherapy prolonged the survival of patients with relapsed/refractory B-cell malignancies. Furthermore, CAR have being studied to translate to other fields like adoptive therapy after hematopoietic stem cell transplantation. As to the treatment toxicity, CAR modified T-cell infusion is tolerant and safe in most patients. However, insertional mutagenesis, off-target effect and inflammatory response are safety issues surrounding CAR-modified T-cell therapy. In this review, the use of CAR technique in treatment of hematologic malignancies and evaluation of CAR safety are summarized.

PASD1: A Promising Target for the Immunotherapy of Haematological Malignancies

In general, there is a lack of good immunotherapy targets within the spectrum of haematological malignancies. However haematopoietic stem cell transplants and continuing antigen discovery have allowed further insight into how further improvements in outcomes for patients might be achieved. Most patients with haematological malignancies can be treated with conventional therapies such as radio- and chemotherapy and will attain first remission. However the removal of residual diseased cells is essential to prevent relapse and its associated high mortality. PASD1 is one of the most tissue restricted cancer-testis (CT) antigens with expression limited to primary spermatagonia in healthy tissue. However, characterisation of PASD1 expression in cancers has been predominantly focussed on haematological malignancies where the inappropriate expression of PASD1 was first identified. PASD1 has one of the highest frequencies of expression of all CT antigens in acute myeloid leukaemia, with some suggestion of its role as a biomarker in diffuse large B-cell lymphoma. Here we describe the characterisation of the function and expression patterns of PASD1 in cell lines and primary tissues. Development of DNA vaccines targeting PASD1 epitopes demonstrate effective ex vivo T-cell responses in terms of IFNγ secretion and tumour cell killing. Of particular note these vaccines have led to the destruction of cells which process and present endogenous PASD1 indicating that effectively primed CTLs could kill PASD1-positive tumour cells.

Precursor B Cell Acute Lymphoblastic Leukemia Induces Pro-Leukemic Changes in the Bone Marrow Microenvironment That Contribute to Therapeutic Resistance

AbstractAbstract 1466Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures:No relevant conflicts of interest to declare.

A Pediatric Case of Systemic Lupus Erythematosus Developed 10 Years after Cord Blood Transplantation for Juvenile Myelomonocytic Leukemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a most powerful immunotherapy for hematological malignancies. However, the impact of immunological disturbances as a result of allo-HSCT is not understood well. We experienced an 11-year-old boy who presented with systemic lupus erythemathosus (SLE) 10 years after unrelated cord blood transplantation of male origin for juvenile myelomonocytic leukemia (JMML) with monosomy 7. Bone marrow examination showed complete remission without monosomy 7. Genetic analysis of peripheral blood revealed mixed chimera with recipient cells consisting of <5% of T cells, 50–60% of B cells, 60–75% of NK cells, 70–80% of macrophages, and 50–60% of granulocytes. Significance of persistent mixed chimera as a cause of SLE is discussed.

Genetic engineering of T cells for the immunotherapy of haematological malignancies

Accumulating experimental and clinical evidence has been obtained over recent years in support of the notion that the immune system has the potential to cure cancer. The most convincing example is the graft versus leukaemia effect observed after allogeneic haematopoietic stem cell transplantation. In the autologous setting, however, the isolation and expansion of naturally occurring tumour-specific T cells is a challenging task. Cancer antigens are often self-antigens and cancer-specific T cells are thus subject to selective mechanisms of central and peripheral tolerance. The significant advances in gene-transfer technologies developed over the last decade have offered new tools to overcome these limitations. Natural T cells can be genetically modified to generate high numbers of 'supernatural' tumour-reactive T cells from virtually every cancer patient. Supernatural T cells may express clonal receptors providing new specificities, factors increasing T-cell performance or safety factors enabling their elimination in case of toxicity. Technological improvements applied to novel concepts of T-cell biology and oncogenesis will allow to simultaneously equip T cells with different transgenes and expand a real 'army' of lymphocytes trained to selectively eradicate cancer cells.

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

Vγ9Vδ2 T cell-based immunotherapy in hematological malignancies: from bench to bedside.

Many hematological malignancies consist of tumor cells that are spontaneously recognized and killed by Vγ9Vδ2 T cells. These tumor cells generate high amounts of intracellular phosphorylated metabolites mimicking the natural ligands and display a wide range of stress-induced self-ligands that are recognized by Vγ9Vδ2 T cells via TCR-dependent and TCR-independent mechanisms. The intrinsic features of Vγ9Vδ2 T cells and that of tumor cells of hematological origin constitute an ideal combination from which to develop Vγ9Vδ2 T cell-based immune interventions. In this review, we will discuss the rationale, preclinical and clinical data in favor of this therapeutic strategy and the future perspectives of its development.

Prevention of GVHD without losing GVL effect: windows of opportunity

Allogeneic hematopoietic cell transplantation has developed into a most successful form of immunotherapy for hematologic malignancies in the past 50years. However, its effectiveness and wider applications have been greatly limited by the development of graft-versus-host disease (GVHD), a potentially lethal side effect associated with this procedure. Since the main effectors for both graft-versus-leukemia (GVL) effect and GVHD are T lymphocytes and these two processes share many similar pathways, it has not been easy to separate GVL from GVHD. Because the clinically used pan immunosuppressive therapy for GVHD prevention also results in decreased GVL effect, the success of allogeneic hematopoietic cell transplantation relies on a small and unpredictable therapeutic window at the present time. This review discusses how we may widen this therapeutic window so that we can reliably prevent GVHD without losing GVL effect.

Vγ9Vδ2 T cells as a promising innovative tool for immunotherapy of hematologic malignancies

The potent anti-tumor activities of γδ T cells, their ability to produce pro-inflammatory cytokines, and their strong cytolytic activity have prompted the development of protocols in which γδ agonists or ex vivo-expanded γδ cells are administered to tumor patients. γδ T cells can be selectively activated by either synthetic phosphoantigens or by drugs that enhance their accumulation into stressed cells as aminobisphosphonates, thus offering new avenues for the development of γδ T cell-based immunotherapies. The recent development of small drugs selectively activating Vγ9Vδ2 T lymphocytes, which upregulate the endogenous phosphoantigens, has enabled the investigators to design the experimental approaches of cancer immunotherapies; several ongoing phase I and II clinical trials are focused on the role of the direct bioactivity of drugs and of adoptive cell therapies involving phosphoantigen- or aminobisphosphonate-activated Vγ9Vδ2 T lymphocytes in humans. In this review, we focus on the recent advances in the activation/expansion of γδ T cells in vitro and in vivo that may represent a promising target for the design of novel and highly innovative immunotherapy in patients with hematologic malignancies.

Immunomodulation in the treatment of haematological malignancies

Despite the continuous advances in immunology and cancer biology, haematological malignancies are often incurable. Conventional chemotherapy and radiation are efficacious for some lymphoma and leukaemia, however relapse and progressive disease often occurs. The evidence that the immune system can play an essential role in controlling cancer progression provide a basis for the development of active therapies, such as immunization, aimed to evoke or amplify a tumour-specific immune response. However, the inability of the patient's own immune system to mount effective responses against tumour antigens is a major limit of vaccination approaches. The adoptive transfer of effectors of the adaptive immune system is an attractive strategy to circumvent the limitations of autologous immune responses. Donor lymphocyte infusion and the transfer of monoclonal antibodies (MoAbs) have been the first forms of adoptive therapy approved for clinical use and are still fundamental components of immunotherapy of haematological malignancies. Due to the continuous characterization of tumour-specific antigen, the development of tumour-tailored therapies that exploit the specificity of antibodies and T cell receptors (TCRs) is progressing rapidly. This review highlights the current advances in the field of adoptive immunotherapy of haematological malignancies, starting by elucidating the ongoing progress in passive transfer of MoAbs. We will also discuss recent advances in the adoptive transfer with tumour-specific high avidity T cells, which can be generated ex vivo by the transfer of gene constructs encoding single chain antibodies or TCRs, thus redirecting T cell specificity to selected tumour antigens. The ability to produce gene-modified T cells of desired specificity and defined functional activity may improve in the future T cell based immunotherapy of cancer.

Successful cell‐mediated cytokine‐activated immunotherapy for relapsed acute myeloid leukemia after hematopoietic stem cell transplantation

Acute myeloid leukemia (AML) is an extremely aggressive disease with a high relapse rate even after allogeneic hematopoietic stem cell transplantation (HSCT). We report the successful outcome of cell-mediated cytokine-activated immunotherapy in a high-risk pediatric AML patient who relapsed shortly after allogeneic HSCT. Donor lymphocyte infusion along with interferon induced a graft-versus-leukemia effect, presenting as a reversible episode of graft-versus-host disease, which led to stable complete donor chimerism and total eradication of AML for over 24 months, at the time of this report. The curative potential of immunotherapy in hematological malignancies is discussed.

CD200: a New Target for Immunotherapy in Hematologic Malignancies.

CD200 is a transmembrane glycoprotein expressed on several tissues in rats and humans. It plays an immunoregulatory function by switching cytokine production from a TH1 to TH2 pattern, thus reducing cytotoxic response while indirectly enabling tumor escape and growth. Interestingly, CD200 is also a target for a novel humanized monoclonal antibody (Anti-CD200 MoAb, Alexion Pharmaceuticals, Cheshire, CT, USA). Anti-CD200 inhibits CD200 binding to its receptor, so modifying cytokine production and improving T-cell mediated cytotoxic response. Expression of CD200 has been already described in chronic lymphocytic leukemia/small lymphocyte lymphoma (CLL/SLL), multiple myeloma (MM) and acute myeloid leukemia (AML). Moreover, CD200 expression is considered an unfavorable prognostic factor in MM and AML. The goal of this work was to study, using flow cytometry, CD200 expression on a large number of onco-hematological samples, with the aim to exactly describe conditions in which anti-CD200 MoAb could be a therapeutic option. Analysis was conduced using a six-color FACSCanto II cytometer (Becton Dickinson, BD, San Jose, CA, USA) equipped with the FACSDiva software (BD). CD200 expression was evaluated by using CD200-PE-conjugated antibody (BD/Pharmingen). During the last year we analyzed CD200 expression in 184 samples: 131 bone marrow aspirates (BM), 33 peripheral blood specimens (PB) and 20 fine needle aspiration cytology samples (FNAC). One hundred and four were lymphoproliferative disorders, 12 MM, 16 myelodysplastic syndromes (MDS) and 52 acute leukemias. CD200 positivity was assigned to every single case when CD200 mean fluorescence intensity (MFI) was higher then 256 arbitrary units, a channel close to the cut-off point between positive and negative cells, in our experience. All results concerning our analysis are showed in the table. Three classes of hematologic neoplasms displayed a constant positivity for CD200 with a high level of MFI: CLL/SLL, hairy cell leukemia (HCL) and B-cell acute lymphoblastic leukemia (B-ALL). Lymphoplasmacytic lymphoma (LPL) was positive in 100% of cases but with lower MFI as compared to CLL/SLL, HCL and B-ALL (p<0.001). MM/MGUS plasma cells showed CD200 positivity in 80% of cases. CD200 was also expressed in cases of marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), T-non Hodgkin lymphoma (T-NHL), AML, acute hybrid leukemia (AHL) and T-cell acute lymphoblastic leukemia (T-ALL). Follicular Lymphoma (FL) samples were always negative but one, which expressed the antigen with a very low MFI. In AML we also compared CD200 expression with that of a large number of antigens (n=40), finding a statistically significant inverse correlation with myeloperoxidase (MPO-7) (Spearman's r= −0.54; p<0.001). Difference in MFI between AML and B-ALL was statistically significant (p=0.003). No case of acute promyelocytic leukemia (APL) was positive for CD200. In conclusion, CD200 is candidate as a new specific target for immunotherapy with anti-CD200 in all cases of CLL/SLL, B-ALL and HCL as well as in selected cases of MZL, DLBCL, MCL, LPL, MM, T-NHL, AML, AHL and T-ALL.DiseaseNPositive N (%)PPC (mean)PPC (25°–75° percentile)MFI (median)MFI (25°–75° percentile)AML3824 (63)32.98–57294138–695APL20 (0)2.5-33-MDS1612 (75)34.215–57516263–1122AHL54 (80)60.227–87420238–1194ALL76 (86)63.657–851099288–1444CLL/SLL4747 (100)98.498–9934102300–4733LPL55 (100)54.841–65492377–715MCL118 (72)433–7166520–835FL101 (10)4.72–84630–83DLCL126 (50)29.83–7120466–754MZL96 (66)39.24–8535247–864HCL66 (100)8772–9838411271–7527MM1211 (92)52.718–792956979–5937T-NHL42 (50)23.52–6228723–2477PPC= percent positive cells MFI= mean fluorescence intensity

Cyclin-dependent Kinaselike 5 is a Novel Target of Immunotherapy in Adult T-cell Leukemia

In the present study, we attempted a comprehensive analysis of human leukocyte antigen (HLA) class I-bound peptides presented on adult T-cell leukemia (ATL) cells by the latest technology of mass spectrometry combined with reversed phase liquid chromatography (LC/MS) to identify novel tumor-associated antigens. We screened the sequenced peptides for those compatible with the motives of the respective HLA class I alleles. Then, we narrowed down the candidate peptides according to the differential expression of their source proteins between ATL cells and normal CD4 T cells. Among these candidates, we focused on cyclin-dependent kinaselike 5 (CDKL5) because it was highly expressed in several ATL cell lines and some ATL clinical samples but not in normal CD4 T cells. To examine its immunogenicity, we stimulated CD8 T cells from an HLA-B62 healthy donor several times with autologous monocyte-derived dendritic cells loaded with HLA-B*62-restricted CDKL5 peptide1012-1021 QVNQAALLTY that we identified. CDKL5-stimulated bulk CD8 T cells exerted higher cytotoxicity against CDKL5 peptide-loaded autologous Epstein Barr virus-transformed B cell line (LCL) than against unloaded LCL. Furthermore these T cells had strong cytotoxic activity against HLA-B*62-positive CDKL5-positive but not HLA-B*62-negative CDKL5-positive ATL cells. These results demonstrate that CDKL5 is a novel tumor (leukemia) antigen in ATL and that the HLA-B*62-restricted CDKL5 peptide can be used for cytotoxic T-lymphocyte-mediated immunotherapy. Identification of tumor-associated antigens by LC/MS is an eligible and efficient method suitable for future taylor-made immunotherapy of hematologic malignancies.

Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies

Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy.

Upregulation of antigen-processing machinery components at mRNA level in acute lymphoblastic leukemia cells after CD40 stimulation

The development of immunotherapy in hematologic malignancies has been observed in the last few years. One of the approaches is the use of cancer vaccines based on leukemia-derived dendritic cells (DC). Recent studies from our laboratory and other laboratories have shown that CD40 stimulation improves leukemia cells immunogenicity and generates an antitumor immune response. The design of future cancer vaccines requires the knowledge concerning the function of dendritic cells including antigen processing. The aim of our present study was the assessment of antigen-processing machinery (APM) components in acute lymphoblastic leukemia (ALL) cells before and after CD40 stimulation at messenger RNA (mRNA) level. Twenty-five children with ALL were enrolled into the study. Leukemic cells were stimulated (or not) with CD40L and IL-4. Elements of the antigen-processing machinery (MB1, LMP2, LMP7, LMP10, TAP1, TAP2, calnexin, calreticulin, tapasin, ERp57, zeta, delta) were determined by real-time PCR technique. The expression of important costimulatory and adhesion molecules considered as DC markers (CD40, CD54, CD80, CD83, CD86) were determined at the mRNA (PCR) and protein (flow cytometry) levels. The following are the results of our study: (1) We noted an upregulation of all costimulatory and adhesion molecules at the mRNA and protein levels in ALL cells after the culture; (2) the significant rise in expression of nearly all APM components after CD40 stimulation was observed. This confirms specific stimulation of the antigen-processing system in ALL cells by CD40L. Future work should focus on the clinical significance of these findings for immunotherapy in leukemias.

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

Identification of a broad array of leukaemia-associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) alpha and beta chains of leukaemia-specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)-specific TCRs was performed and the feasibility of the use of TCR-transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR-transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine-production by TCR-transgenic T cells permits the detection of either small amounts of antigen-positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR-transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR-transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR-transfer generates a large amount of functional antigen-specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.

284. CD137L Gene Transfer Generates Effective and Long-Lasting Immunity in a Murine Plasmocytoma Model

Background: CD137L, a member of the tumour necrosis factor receptor (TNFR) family, is constitutively expressed on activated antigen-presenting cells. In a wide range of solid tumours, over-expression of CD137L has been shown to produce tumour immunity. This was at least partly due to the stimulation of CD8+ CTL. Costimulation by other known interactions was not unconditionally necessary. Anti-tumour effects were even increased when immunotherapy with Interleukin-12 was additionally employed, either systemically or locally. Yet, there is rare data on the effect of CD137L immunotherapy in hematological malignancies. Here, we present data obtained in a murine plasmocytoma model to evaluate the effect of CD137L and IL-12, either as mono- or combination therapy.

Immunotherapy of hematological malignancies with dendritic cells.

Immunotherapy of hematological malignancies with dendritic cells.