|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Oncogenic Fusion Genes and Proteins Unit,
Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy;
INSERM U.487, Institut Gustave Roussy, Villejuif, France; and
Unité d'Immunité Cellulaire Antivirale, Department
SIDA-Retrovirus, Institut Pasteur, Paris, France.
Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK
and associated variants) are expressed in about 60% of anaplastic
large cell lymphomas (ALCLs) but are absent in normal tissues. In this
study, we investigated whether ALK, which is expressed at high
levels in lymphoma cells, could be a target for antigen-specific
cell-mediated immunotherapy. A panel of ALK-derived peptides was tested
for their binding affinity to HLA-A*0201 molecules. Binding peptides
were assessed for their capacity to elicit a specific immune response
mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201
transgenic mice, and in vitro in the peripheral blood lymphocytes
(PBLs) from healthy donors. Two HLA-A*0201-restricted CTL epitopes,
p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK
kinase domain were identified. The p280-89- and
p375-86-induced peptide-specific CTL lines were able to specifically release interferon- The discovery of tumor-associated antigens (TAAs)
demonstrated that tumor cells can be specifically recognized by the
immune system, raising the hypothesis that most if not all tumors
express antigens that cytotoxic T lymphocytes (CTLs) can potentially
attack.1 The identification of immunogenic epitopes led to
their use as targets to mediate the specific clearance of neoplastic
cells by TAA targeting strategies such as
vaccination.2-4
A major issue in the development of efficient vaccination protocols is
the identification of the appropriate and effective TAAs, able to
induce an immune response that will affect tumor growth. The ideal
target for immune destruction is a tumor-specific protein that is not
expressed in normal cells and is essential for the malignant
phenotype.5
In an effort to identify new and specific tumor antigens, we examined
whether anaplastic lymphoma kinase (ALK) could induce a CTL-mediated
antitumor immune response, and therefore be considered as a TAA.
More than half the patients with anaplastic large cell lymphoma
(ALCL) possess a t(2;5) chromosomal translocation that leads to the
expression of the NPM/ALK fusion protein. This fusion protein is
composed of the N-terminal portion of the nuclear phosphoprotein nucleophosmin (NPM) linked to the entire intracellular portion of ALK,
which contains its kinase catalytic domain.6,7 It has been
extensively demonstrated that constitutive activated NPM/ALK is a
potent oncogene having transforming and tumorigenic properties.8-11
Although NPM is a "housekeeping" gene that is
ubiquitously expressed in normal cells, ALK expression is restricted in
embryonic and adult mouse to the central nervous system
(CNS).12,13 In humans, ALK was detected at low levels in
some pericytes and scattered glial cells in specific areas of the CNS.
ALK is not normally expressed in lymphohematopoietic
tissues.14 However, as a result of the t(2;5) chromosomal
translocation, ALK is ectopically expressed at high levels in lymphoid
cells and its constitutive tyrosine kinase activity leads to
deregulated lymphocyte growth.9,15
It has been demonstrated that partners other than NPM can promote the
oncogenic expression of the ALK intracytoplasmic domain. ALK variant
fusion proteins so far identified in patients with ALCL are
TPM3/ALK,16,17 TFG/ALK,18,19
ATIC/ALK,20,21 CLTCL/ALK,22 and
MSN/ALK.23
The high level of expression of NPM/ALK and its variants in lymphoma
cells and their direct role in lymphomagenesis, combined with the fact
that normal ALK is expressed at low levels in the immune-privileged CNS, suggests that ALK could potentially be an ideal
target for immunotherapy. Indeed, circulating antibodies recognizing
NPM/ALK were recently detected in ALCL patient sera.24
Here we studied a panel of ALK-derived peptides with binding motif for
the HLA-A*0201 molecules. We evaluated their ability to provoke an in
vivo and in vitro peptide-specific CTL response in HLA-A*0201
transgenic mice and in peripheral blood lymphocytes (PBLs) of
HLA-matched healthy donors. We demonstrated that the anti-ALK CTLs
generated from PBLs of healthy donors elicited an antigen-specific,
HLA-A2.1-restricted response, able to effectively kill tumor targets
endogenously expressing ALK.
Animals
Cell lines
Human cell lines used in the experiments were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, and antibiotics. The following
HLA-A*0201 tumor cell lines were used: SU-DHL1 and SUP-M2
(NPM/ALK+ lymphomas), FM3 (melanoma), HCT 116 (colon
carcinoma), MCF-7 (breast carcinoma), IMR-32 (ALK+
neuroblastoma), NB5 (ALK Peptides The NPM/ALK sequence was reviewed for peptides that could potentially bind to HLA-A*0201, the most common HLA class I allele among whites, using a peptide-motif scoring system (http://bismas.dcrt.nih.gov/molbio/hla_bind/index.html). Twenty-two among the highest scored predicted peptides present in the ALK sequence were considered. Peptides were also chosen to cover the entire ALK sequence: 11 peptides were within the kinase domain (p197-205: GMPNDPSPL; p217-25: SEQDELDFL; p246-54: SLQSLPRFI; p246-55: SLQSLPRFIL; p249-58: SLPRFILLEL; p280-89: SLAMLDLLHV; p281-89: LAMLDLLHV; p282-90: AMLDLLHVA; p376-85: GVLLWEIFSL; p377-85: VLLWEIFSL; p394-402: SNQEVLEFV), 2 were upstream (p139-47: KLSKLRTST; p148-57: IMTDYNPNYC), and 2 were downstream (p438-46: IILERIEYC; p456-65: ALPIEYGPLV) of the kinase domain and 7 in the C-terminal portion of the protein (p592-600: NLGLEGSCTV; p593-601: LGLEGSCTV; p609-17: RLPGASLLL; p614-22: SLLLEPSSL; p615-23: LLLEPSSLT; p615-24: LLLEPSSLTA; p621-29: SLTANMKEV). FLU matrix58-66 (GILGFVFTL) peptide was used as an irrelevant peptide in human CTL assays.Peptides purified by high-performance liquid chromatography were synthesized and purchased from American Peptide Company (APC) (Sunnyvale, CA) at a minimum purity of 90%. HLA-A*0201 binding assays To determine the binding ability of predicted peptides to HLA-A*0201 molecules, an in vitro cellular binding assay was performed using the TAP-deficient cell line T2. T2 cells were incubated with 100 µM peptide in serum-free RPMI 1640 supplemented with 5 µg/mL human 2m (Fluka, Buchs, Germany) for 18 hours at 37°C. HLA-A*0201 expression was then measured by flow cytometry using the
anti-HLA-A2.1 monoclonal antibody (mAb) BB7.2 followed by incubation
with fluorescein isothiocyanate (FITC)-conjugated F(ab')2 goat antimouse Ig (Biosource, Camarillo, CA). The results are expressed
as fluorescence index (FI) defined as a ratio (median channel of
fluorescence) between the sample and a control containing an irrelevant
peptide (PML/RAR : VASGAGEAA). An increase over the control of at
least 65% (FI > 2) was arbitrarily chosen as the cutoff point.
Measurement of HLA-A*0201/peptide complex stability To assess the HLA-A*0201/peptide complex stability, T2 cells (106/mL) were incubated overnight with 100 µM of each peptide in serum-free RPMI 1640 supplemented with 100 ng/mL human2m at 37°C. Cells were then washed 4 times to remove
free peptides, incubated for 1 hour with 10 µg/mL Brefeldin A
(Sigma-Aldrich, Lyon, France) to block cell surface expression
of newly synthesized HLA-A*0201 molecules, washed, and incubated at
37°C for 0, 2, 4, 6, or 8 hours. Subsequently, cells were stained
with anti-HLA-A2.1 mAb BB7.2. For each time point, peptide-induced
HLA-A*0201 expression was calculated as mean fluorescence value of
peptide incubated T2 cells/mean fluorescence value T2 cells in the
absence of the peptide. Dissociation complex50
(DC50) was defined as the time required for the loss of
50% of the HLA-A*0201/peptide complexes stabilized at t = 0.
Generation of CTLs in HHD mice HHD mice were injected subcutaneously at the base of the tail with 100 µg peptide emulsified in incomplete Freund adjuvant (IFA) in the presence of 140 µg IAb-restricted hepatitis B virus (HBV) core antigen-derived T-helper epitope (p128-40: TPPAYRPPNAPIL). After 11 days, spleen cells from primed mice (5 × 107 cells in 10 mL) were restimulated in vitro with 10 µM peptide. On day 6 of culture, cells were tested for specific cytotoxicity in a 4-hour 51Cr release assay using as targets RMA-S/HHD cells pulsed with 1 µM peptide or alone.Generation of CTLs in healthy donors Blood was collected from HLA-A*0201 healthy volunteer donors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Dendritic cells (DCs) were generated according to the protocol of Sallusto and Lanzavecchia.26 Adherent monocyte-enriched PBMCs were cultured in RPMI 1640 medium supplemented with 10% FCS, 20 ng/mL recombinant human interleukin-4 (IL-4) (R &D Systems, Minneapolis, MN) and 800 U/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; Sandoz, Basel, Switzerland). On day 5 of culture, 10 ng/mL recombinant human tumor necrosis factor (TNF)- (Knoll, Ludwigshafen, Germany) was
added to the medium. After 40 hours, the surface phenotype of
monocyte-derived DCs was determined by single-color flow cytometry using the following mAbs: fluorescein-conjugated anti-CD1a (Valter Occhiena, Torino, Italy), anti-CD86 (Caltag, Burlingame, CA), anti-CD83
(Caltag), anti-CD14 (Becton Dickinson, Franklin Lakes, NJ), and
rhodamine-conjugated anti-CD80 (Becton Dickinson). DCs were used to
prime autologous PBLs as follows. DCs were pulsed with 10 µM peptide
for 2 hours at 37°C, irradiated at 30 Gy, and added to autologous
PBLs at a ratio variable from 3:1 to 6:1 (PBL/DC). PBLs
(36 × 106 total) were cultured at
1.5 × 106 cells/2 mL culture medium (CM: 50% RPMI
1640/50% X-VIVO) supplemented with 10% heat-inactivated autologous
plasma and 10 ng/mL IL-7 (R & D Systems) in the presence of autologous
peptide-loaded DCs. At day 10 to 12 of culture, lymphocytes were
restimulated with irradiated (30 Gy) autologous peptide-loaded
monocytes in CM supplemented with 10% heat-inactivated autologous
plasma. After 24 to 48 hours 10 IU/mL IL-2 (Chiron, Emeryville, CA) and
10 ng/mL IL-7 were added to the CM. Lymphocytes were then restimulated
weekly in the same way. Starting from the forth round of stimulation,
the specificity of the resulting T-cell lines was evaluated weekly in a
cytolytic assay. When any specific activity was detected, CD8+ T cells were isolated from the bulk culture by
CD4+ cells negative depletion using mAb-coated beads (Dynal
Dynabeads, Oxoid, Oslo, Norway) and the CTL lines were maintained in
culture in RPMI 1640 containing 10% human AB serum supplemented with
50 U/mL IL-2 in the presence of irradiated (70 Gy) PBMCs as feeder.
CTL assays Cytolytic activity was measured in a conventional 4-hour 51Cr release assay. A 10-fold excess of unlabeled K562 cells was added to offset natural killer (NK) activity. Target cells and effector-to-target (E/T) ratio are indicated in each figure. Specific lysis was determined according to the following formula: % specific lysis = cpm (sample spontaneous)/cpm (totalspontaneous) × 100.
For cytokine release assay, 105 target cells (Epstein-Barr
virus [EBV]-transformed B cells and TAP HLA-A*0201 blocking of T-cell activity (lysis or cytokine release or both) was performed by preincubating target cells with the anti-HLA-A2 mAb CR11.351.
Identification of ALK-derived peptides binding to HLA-A*0201 molecules Using a computer-assisted analysis, the ALK amino acid sequence present in the NPM/ALK fusion protein was screened for the presence of HLA-A*0201 binding motifs. We selected 14 nonamer and 8 decamer candidate peptides with the highest predicted half-time of dissociation from HLA-A*0201 molecules.The ability of the 22 selected ALK-derived peptides to bind to HLA-A*0201 molecules and to form stable major histocompatibility complex (MHC)/peptide complexes was assessed by measuring the binding affinity, expressed as FI, and stability, expressed as DC50. Nine of the 22 predicted peptides were shown to be high-affinity
peptides, with strong binging to HLA-A*0201 (FI > 2.3) and efficient stabilizers (DC50 ranging from 2 to 6 hours;
Table 1). Such HLA binding and
stabilizing capacities correlate well with potential immunogenicity.
In vivo immunogenicity of ALK-derived peptides in HHD mice To evaluate the in vivo immunogenic potential of ALK-derived high-affinity binding peptides, we immunized H-2Db /,
2m/, HLA-A*0201 monochain transgenic
(HHD) mice. In these mice, the peripheral CD8+ T-cell
repertoire is educated, both at the thymic and peripheral levels, with
transgenic HLA class I human molecules. Therefore, the HHD mouse is a
useful animal model to assess the ability of individual peptides to
induce HLA-A*0201-restricted CTL responses in
vivo.27,28
Seven of the 9 HLA-binding peptides were able to induce peptide-specific CTL responses. Taking into account the binding affinity, the number of responding mice and the level of specific lysis, ALK-derived peptides can be classified as strong (p280-89, p282-90), intermediate (p376-85, p377-85, p621-29), weak (p281-89, p456-465), or inefficient (p609-17, p615-24) CTL inducers (Table 1). The ALK-derived peptide CTL induction was comparable to that associated with well-known dominant tumor epitopes, such as gp100p154 and HER-2/neup369,27,29 in HHD mice. As previously described for different HLA-A2.1-associated peptides,29 ALK-derived peptides that failed to generate significant peptide-specific CTLs are also those demonstrating weaker stabilization capacity (DC50, 2 hours), irrespective of their binding affinity. This observation is in accord with the need of MHC affinity and MHC/peptide stability in determining peptide immunogenicity. Stimulation of ALK-specific CTLs derived from healthy donor PBLs To determine the capacity of ALK peptides to mobilize a human repertoire, anti-ALK-specific CTLs were generated by in vitro sensitization of HLA-A*0201 healthy donor PBLs with ALK peptides previously screened in HHD mice. For each peptide PBL samples from 3 donors were tested.To prime ALK-specific lymphocytes, peptide-pulsed autologous DCs
derived from PBMCs were used after their in vitro maturation confirmed
by flow cytometric analysis of DC surface markers: CD1a, CD83, CD80,
CD86, CD14 (data not shown). After 3 cycles of weekly restimulation,
the specificity of T lymphocytes bulk cultures for ALK peptides was
evaluated as antigen-restricted lysis of peptide-loaded T2 cells and
NPM/ALK+ ALCL cell lines. At least 1 of 3 tested donors
were responders to stimulation with peptides p280-89, p376-85,
p377-85, and p456-65 (donors [d]: 01, 02, 03, 04, 05, Figure
1). No specific reactivity was detected
in the cell lines generated from PBLs stimulated with peptides p281-89,
p282-90, and p621-29 (data not shown).
Attempts at generating anti-ALK CTLs from responding donors led to the
establishment of 2 cell lines specific for peptide p280-89 (d01 and
d02) and 1 line specific for peptide p376-85 (d03). Flow cytometric
analysis demonstrated that more than 99% of the cells were
CD3+, CD8+, CD4 Antigen specificity of anti-ALK-specific CTLs was investigated.
Autologous EBV-transformed B cells (LCLs) or T2 cells were prepulsed
with the indicated peptide and used as targets under different
conditions (Figure 2).
CTLs from the 2 donors responding to ALK peptide p280-89 released
IFN- Similar quantities of IFN- A titration assay was performed to further assess the sensitivity and
competence of CTLs in recognizing the antigen. T2 cells were pulsed
with p280-89, p376-85, or FLU peptides at different concentrations, and
then used as stimulators in a cytokine release assay. For
p280-89-specific CTLs, maximal production of IFN-
Taken together these results indicate that ALK peptides, p280-89 and p376-85, previously tested for their ability to be immunogenic in HHD mice, were also able to mobilize a specific CTL repertoire in PBLs from healthy donors. Moreover, dose-response data indicated a high and a moderate affinity of recognition for p280-89- and p376-85-generated CTLs, respectively, providing further evidence that ALK kinase is immunogenic and not subject to tolerance mechanisms. Lysis of tumor cell lines To determine if ALK-derived peptides, demonstrated to be immunogenic in vivo and in vitro, were also naturally processed on human tumor cells, we examined the ability of p280-89- and p376-85-specific CTLs to lyse HLA-A2.1+ ALCL lymphoma cell lines, SU-DHL1 and SUP-M2, expressing the chimeric protein NPM/ALK. ALK-specific CTLs killed these tumor lines and their lytic activity was inhibited by the presence of anti-HLA-A2 mAb CR11.351. No lysis above background level was observed in HLA-A2.1+/ALK FM3
(melanoma), HCT-116 (colon carcinoma), and MCF-7 (breast carcinoma)
human tumor lines expressing irrelevant antigens (Figure 4).
Thus, it can be concluded that ALK peptide-induced CTLs efficiently lyse lymphoma targets, in a HLA-A2-restricted fashion, by relevant antigen-specific recognition. Interestingly, native ALK tyrosine kinase expression has also been
demonstrated in cell lines and tumors of neural origins such as
neuroblastoma.30 Because p280-89 and p376-85 peptides lie
within the kinase domain, which is present in both native and
translocated ALK, we used neuroblastoma cell lines as targets in
cytotoxic experiments to further verify the natural processing of these
2 epitopes. Due to the limited expression of class I MHC molecules
characterizing neuroblastomas that could interfere with the antigen
recognition by CTLs, we treated tumor cells with INF-
These results indicate that both p280-89 and p376-85 epitopes are efficiently processed and presented for CTL recognition at the cell surface of tumor cells and may therefore provide targets for antigen-specific immunotherapy.
In this study, using a reverse immunology approach, we identified 2 new tumor-associated CTL epitopes (p280-89 and p376-85) derived from the ALK tyrosine kinase domain, which (1) bind to HLA-A2.1 molecules, (2) are immunogenic in vivo in the HLA-A*0201 transgenic mice, (3) stimulate in vitro-specific CTL responses using PBLs from healthy donors, and (4) are spontaneously processed and presented at the surface of tumor cells. ALK is a receptor tyrosine kinase identified for the first time by Morris et al6 as part of the fusion protein NPM/ALK in ALCL-derived cell lines. NPM/ALK contains the NPM oligomerization domain and the intracytoplasmic region of ALK. NPM/ALK homodimerization determines the constitutive activation of the ALK catalytic domain in the transformed cell. It has been demonstrated that constitutive activation of ALK plays a key role in lymphomagenesis, by aberrant phosphorylation of intracellular substrates that likely contributes to the molecular pathogenesis of ALCL.8,9,15,31 We postulate that ALK could represent an ideal tumor antigen mostly because it is not expressed by normal cells, except at low levels in some CNS cells.14 As a consequence, we expected that anti-ALK-specific CTL precursors were not physiologically eliminated by clonal deletion in the thymus and were not restricted by self-tolerance in the periphery. Following this hypothesis, ALK-derived peptides that experimentally showed strong HLA-A2.1 binding features and immunogenicity in HHD mice were tested for in vitro anti-ALK repertoire mobilization capacity from human circulating blood. Our results confirm the existence of an efficient anti-ALK precursor population present within the T-cell repertoire of healthy donors. Moreover, considering the relatively low starting number of PBLs (36 × 106) used to start each single sensitization experiment, such precursors may be present at high frequency. Given the known difficulty in generating immune responses in vitro, it is remarkable that the screening of only 3 healthy donors for each peptide induced a specific immune response in at least one donor, suggesting the strength of ALK in being immunogenic. The anti-ALK-specific CTL repertoire is unlikely to react against normal CNS cells, because the CNS is an immunologically privileged site where, under physiologic conditions, antigens are not exposed to the immune system. More importantly, the scarce level of expression in normal CNS is most likely too low to ensure CTL recognition as shown with human MAGE-specific32 and murine p53-specific33 CTLs. In contrast, the high level of expression of NPM/ALK, driven by the strong NPM promoter, should result in a substantial amount of NPM/ALK processing by the proteosome, thereby leading to a significant expression of ALK-derived epitopes at the cell surface. This should make the immune response against ALK selective for malignant cells. The down-regulation of tumor antigen expression represents a problem for the development of immunotherapies because of the possible immunoselection of nonexpressing tumor clones. Because ALK is required for neoplastic transformation of lymphoid cells, it should be unlikely to observe tumor escape phenomena by ALK antigen loss variants. Translocated ALK is expressed in approximately 83% and 31% of children and adults with ALCL, respectively,34 indicating that ALK is a stable lymphoma-specific hallmark. Recently, ALK+ lymphoma or "Alkoma" has been recognized as a distinct tumor entity.35 ALK broad expression not restricted to one or few patients, as in the case of point-mutated oncogenes, should make its hypothetical use in vaccination protocols more easily and widely applicable. Finally, we demonstrated that ALK could be targeted for killing of ALK+ neuroblastoma tumor lines. Despite a lower level of ALK expression compared with ALCL and an unclear involvement in the transformed phenotype,30 our results demonstrate that neuroblastoma cell lines can still be efficiently lysed by ALK-specific CTLs. The killing of neuroblastoma cells by a CTL response specific for ALK epitopes reveals the potential of ALK as a shared tumor antigen for multiple malignancies. In conclusion, ALK kinase is a novel tumor-specific antigen
able to induce an antigen-specific CTL response in humans. Two novel
ALK-derived epitopes (p280-89 and p376-85) were identified. We propose
that these CTL epitopes could serve as good candidates for the design
of future immunotherapy strategies of ALK-expressing tumors such as
ALCL and neuroblastoma. ALK immunogenicity could be linked to the more
favorable prognosis, with a better 5-year survival rate observed in
ALK+ ALCL patients compared with ALK
The contributions of Drs G. Parmiani, R. H. Gunby, P. Dalerba (Istituto Nazionale Tumori, Milan, Italy) and Dr S. Morris (St. Jude Children's Hospital, Memphis, TN) in critically reading the manuscript are gratefully acknowledged. We thank Edoardo Marchesi (Istituto Nazionale Tumori, Milan, Italy) for the excellent technical contribution, S. Ragsdale (St. Jude Children's Hospital, Memphis, TN) for providing the NB neuroblastoma cell lines, Dr F. Arienti (Istituto Nazionale Tumori, Milan, Italy) for supplying us with several buffy coats, and Dr C. Russo (Merck Research Laboratories, West Point, PA) for the anti-HLA-A2 mAb CR11.351.
Submitted July 6, 2001; accepted October 25, 2001.
Supported by grants from Italian Association for Cancer Research (AIRC, Milan), European Community (EUCAPS program), INSERM (PROGRES program), and "Fondation pour la Recherche Medicale."
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Lorena Passoni, Oncogenic Fusion Genes and Proteins Unit, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy; e-mail: passoni{at}istitutotumori.mi.it.
1. Renkvist N, Castelli C, Robbins PF, Parmiani G. A listing of human tumor antigens recognized by T cells. Cancer Immunol Immunother. 2001;50:3-15[CrossRef][Medline] [Order article via Infotrieve]. 2. Bremers AJ, Kuppen PJ, Parmiani G. Tumour immunotherapy: the adjuvant treatment of the 21st century? Eur J Surg Oncol. 2000;26:418-424[CrossRef][Medline] [Order article via Infotrieve]. 3. Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med. 2000;6:332-336[CrossRef][Medline] [Order article via Infotrieve]. 4. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328-332[CrossRef][Medline] [Order article via Infotrieve]. 5. Gilboa E. The makings of a tumor rejection antigen. Immunity. 1999;11:263-270[CrossRef][Medline] [Order article via Infotrieve].
6.
Morris SW, Kirstein MN, Valentine MB, et al.
Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.
Science.
1994;263:1281-1284
7.
Falini B, Pileri S, Zinzani PL, et al.
ALK+ lymphoma: clinico-pathological findings and outcome.
Blood.
1999;93:2697-2706
8.
Fujimoto J, Shiota M, Iwahara T, et al.
Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5),
Proc Natl Acad Sci U S A.
1996;93:4181-4186 9. Bischof D, Pulford K, Mason DY, Morris SW. Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis. Mol Cell Biol. 1997;17:2312-2325[Abstract].
10.
Kuefer MU, Look AT, Pulford K, et al.
Retrovirus-mediated gene transfer of NPM-ALK causes lymphoid malignancy in mice.
Blood.
1997;90:2901-2910 11. Wellmann A, Doseeva V, Butscher W, et al. The activated anaplastic lymphoma kinase increases cellular proliferation and oncogene up-regulation in rat 1a fibroblasts. FASEB J. 1997;11:965-972[Abstract]. 12. Iwahara T, Fujimoto J, Wen D, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene. 1997;14:439-449[CrossRef][Medline] [Order article via Infotrieve]. 13. Morris S, Naeve C, Mathew P, et al. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene. 1997;14:2175-2188[CrossRef][Medline] [Order article via Infotrieve].
14.
Pulford K, Lamant L, Morris SW.
Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1.
Blood.
1997;89:1394-1404
15.
Bai RY, Dieter P, Peschel C, Morris SW, Duyster J.
Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-gamma to mediate its mitogenicity.
Mol Cell Biol.
1998;18:6951-6961
16.
Siebert R, Gesk S, Harder L, et al.
Complex variant translocation t(1;2) with TPM3-ALK fusion due to cryptic ALK gene rearrangement in anaplastic large-cell lymphoma.
Blood.
1999;94:3614-3617
17.
Lamant L, Dastugue N, Pulford K, Delsol G, Mariame B.
A new fusion gene TPM3-ALK in anaplastic large cell lymphoma created by a (1;2)(q25;p23) translocation.
Blood.
1999;93:3088-3095
18.
Hernandez L, Pinyol M, Hernandez S, et al.
TRK-fused gene (TFG) is a new partner of ALK in anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.
Blood.
1999;94:3265-3268
19.
Rosenwald A, Ott G, Pulford K, et al.
t(1;2)(q21;p23) and t(2;3)(p23;q21): two novel variant translocations of the t(2;5)(p23;q35) in anaplastic large cell lymphoma.
Blood.
1999;94:362-364
20.
Trinei M, Lanfrancone L, Campo E, et al.
A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive anaplastic large cell lymphoma.
Cancer Res.
2000;60:793-798
21.
Ma Z, Cools J, Marynen P, et al.
Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis.
Blood.
2000;95:2144-2149
22.
Touriol C, Greenland C, Lamant L, et al.
Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like).
Blood.
2000;95:3204-3207 23. Tort F, Pinyol M, Pulford K, et al. Molecular characterization of a new ALK translocation involving moesin (MSN-ALK) in anaplastic large cell lymphoma. Lab Invest. 2001;81:419-426[Medline] [Order article via Infotrieve].
24.
Pulford K, Falini B, Banham AH, et al.
Immune response to the ALK oncogenic tyrosine kinase in patients with anaplastic large-cell lymphoma.
Blood.
2000;96:1605-1607
25.
Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau B.
HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.
J Exp Med.
1997;185:2043-2051
26.
Sallusto F, Lanzavecchia A.
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.
J Exp Med.
1994;179:1109-1118 27. Firat H, Garcia-Pons F, Tourdot S, et al. H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol. 1999;29:3112-3121[CrossRef][Medline] [Order article via Infotrieve].
28.
Minev B, Hipp J, Firat H, Schmidt JD, Langlade-Demoyen P, Zanetti M.
Cytotoxic T cell immunity against telomerase reverse transcriptase in humans.
Proc Natl Acad Sci U S A.
2000;97:4796-4801 29. Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000;30:3411-3421[CrossRef][Medline] [Order article via Infotrieve].
30.
Lamant L, Pulford K, Bischof D, et al.
Expression of the ALK tyrosine kinase gene in neuroblastoma.
Am J Pathol.
2000;156:1711-1721
31.
Bai RY, Ouyang T, Miething C, Morris SW, Peschel C, Duyster J.
Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway.
Blood.
2000;96:4319-4327 32. Lethe B, van der Bruggen P, Brasseur F, Boon T. MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte. Melanoma Res. 1997;7(suppl 2):S83-S88.
33.
Vierboom MP, Nijman HW, Offringa R, et al.
Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes.
J Exp Med.
1997;186:695-704 34. Drexler HG, Gignac SM, von Wasielewski R, Werner M, Dirks WG. Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas. Leukemia. 2000;14:1533-1559[CrossRef][Medline] [Order article via Infotrieve].
35.
Benharroch D, Megueria-Bedoyan Z, Lamant L, et al.
ALK-positive lymphoma: a single disease with a broad spectrum of morphology.
Blood.
1998;91:2076-2084
36.
Shiota M, Nakamura S, Ichinohasama R, et al.
Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.
Blood.
1995;86:1954-1960
37.
Gascoyne RD, Aoun P, Wu D, et al.
Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma.
Blood.
1999;93:3913-3921
38.
Suzuki R, Kagami Y, Takeuchi K, et al.
Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype.
Blood.
2000;96:2993-3000
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. Marzec, Q. Zhang, A. Goradia, P. N. Raghunath, X. Liu, M. Paessler, H. Y. Wang, M. Wysocka, M. Cheng, B. A. Ruggeri, et al. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1) PNAS, December 30, 2008; 105(52): 20852 - 20857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Rodig, J. Ouyang, P. Juszczynski, T. Currie, K. Law, D. S. Neuberg, G. A. Rabinovich, M. A. Shipp, and J. L. Kutok AP1-Dependent Galectin-1 Expression Delineates Classical Hodgkin and Anaplastic Large Cell Lymphomas from Other Lymphoid Malignancies with Shared Molecular Features Clin. Cancer Res., June 1, 2008; 14(11): 3338 - 3344. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wei, J.-T. Li, X.-P. Zhang, Y. Tang, J.-X. Wang, B. Zhang, and Y.-Z. Wu Identification of an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope in rotavirus VP6 protein. J. Gen. Virol., November 1, 2006; 87(Pt 11): 3393 - 3396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhou, D. Xu, X. Li, H. Li, M. Shan, J. Tang, M. Wang, F.-S. Wang, X. Zhu, H. Tao, et al. Screening and Identification of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific CTL Epitopes J. Immunol., August 15, 2006; 177(4): 2138 - 2145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kasprzycka, M. Marzec, X. Liu, Q. Zhang, and M. A. Wasik From the Cover: Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces the T regulatory cell phenotype by activating STAT3 PNAS, June 27, 2006; 103(26): 9964 - 9969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chen, J. Hou, X. Jiang, S. Ma, M. Meng, B. Wang, M. Zhang, M. Zhang, X. Tang, F. Zhang, et al. Response of Memory CD8+ T Cells to Severe Acute Respiratory Syndrome (SARS) Coronavirus in Recovered SARS Patients and Healthy Individuals J. Immunol., July 1, 2005; 175(1): 591 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wang, H. Chen, X. Jiang, M. Zhang, T. Wan, N. Li, X. Zhou, Y. Wu, F. Yang, Y. Yu, et al. Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein Blood, July 1, 2004; 104(1): 200 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J J Oudejans, R L ten Berge, and C J L M Meijer Immune escape mechanisms in ALCL J. Clin. Pathol., June 1, 2003; 56(6): 423 - 425. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
(IFN-
) and 25:1
(
) using as targets T2 cells pulsed with 5 µM cognate peptide or
irrelevant influenza matrix-derived peptide (FLU) (A) and
NPM/ALK+ lymphoma cell lines SU-DHL1 and SUP-M2 (B).
Results of 1 representative experiment of 2 performed are
shown.
). Results of 1 representative experiment of at least 3 performed are shown.
