Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins (HSPs) isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195–314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.     

Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor

 In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2K^d-restricted NP(147–155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147–155)-specific CTL response within 2 weeks after the boost. When challenged with 10⁴ P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4⁺ or CD8⁺ T cells and then challenging with 10⁴ P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8⁺ CTL-mediated antitumor response. Received: 8 May 1997 / Accepted: 28 August 1997     

Stable expression of a retrovirally transferred adhesion molecule in a human melanoma-specific cytotoxic T lymphocyte clone

 The adoptive transfer of in vitro generated tumor-specific cytotoxic T lymphocytes (CTL) is considered a promising perspective in cancer therapy. One possible drawback lies in the inappropriate homing of in vitro cultured lymphocytes, which could be circumvented by introducing the appropriate targeting molecules. Here we describe a protocol that allows a rapid and stable transfection of cytotoxic T cell clones. As a model system we used a CTL clone specific for the melanoma-associated antigen gp100 and a cDNA encoding for murine CD14 containing the variant exen v10 which is supposed to facilitate lymphocyte homing towards the skin. CD44v10 cDNA was ligated into the retroviral vector pMV-7, which was used to transfect the ecotropic GP-E-86 and the amphotropic PA317 cells. After several cycles of transduction to increase the viral titre, supernatants of the amphotropic PA317-CD44v10 line were used for transduction of CD44v10 into a human CTL clone. After three cycles of transduction at 12-h intervals, low but stable expression of CD44v10 was observed throughout the culture period of 10 weeks. The phenotype of the transduced CTL clone was unaltered and the cytotoxic potential was only slightly reduced as compared to the parental clone. The efficiency of stable transduction within a period of 1 week makes the protocol well suited for the in vivo transfer of transduced cells and, in the special case, should guarantee appropriate homing of the transduced CTL clone. Received: 14 August 1997 / Accepted: 20 November 1997     

Synthetic β-endorphin-like peptide immunorphin binds to non-opioid receptors for β-endorphin on T lymphocytes

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [¹²⁵I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K_i = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K_i = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K_i = 1.0 nM) possessed the ability to inhibit specific binding of [¹²⁵I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K_d values characterizing the specific binding of ¹²⁵I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice

 In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86⁺C1300, expressing a high level of mouse CD86 on the cell surface. While CD86⁺C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86⁺C1300-rejecting mice with CD86⁺C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86⁺C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86⁺C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86⁺C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors. Received: 18 November 1996 / Accepted: 3 March 1997     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

T cell activation and retargeting using staphylococcal enterotoxin B and bispecific antibody: An effective in vivo antitumor strategy

 The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor×anti-CD3 F(ab′)₂ bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 γg intraperitoneal) with or without bsAb (5 μg i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P=0.02 and P<0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97^– parental cell line, K1735(P=0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and nontargeted tumor antigens. Accepted: 14 October 1997     

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats

 Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression. Received: 16 October 1997 / Accepted: 8 January 1998     

Cellular immune responses in acute leukaemia patients with severe chemotherapy-induced leucopenia; characterization of the cytokine repertoire of clonogenic T cells

 T lymphocytes are important both for the host defence against infections and probably also as antileukaemic effector cells in patients with acute leukaemia. To investigate the T lymphocyte cytokine repertoire of clonogenic T lymphocytes, CD4⁺ and CD8⁺ T lymphocyte clones were prepared from acute leukaemia patients with chemotherapy-induced cytopenia (leucocytes <0.5×10⁹/l). A majority of both CD4⁺ and CD8⁺ clones secreted detectable interleukin-2 (IL-2), IL-10, IL-13, granulocyte/macrophage-colony-stimulating factor and interferon γ (IFNγ) in response to phytohaemagglutinin + accessory cells (Epstein-Barr-virus-transformed B cell line, 80-Gy-irradiated). The CD4⁺ clones showed significantly higher levels of IL-10 secretion than the CD8⁺ clones. Decreased levels of IL-2, IL-13 and IFNγ were observed when acute myelogenous leukaemia (AML) blasts were used instead of cells from the B cell line as accessory cells during phytohaemagglutinin activation, but the differences in IL-13 and IFNγ levels were reversed by addition of exogenous IL-2. On the basis of these results we conclude: (i) the remaining clonogenic T lymphocytes derived from acute leukaemia patients with therapy-induced leucopenia can respond to activation with a broad cytokine response, and T-cell-derived cytokines may then contribute to cytokine responses during complicating infections in these patients; (ii) although T cells can modulate AML blast functions and mediate antileukaemic effects, the leukaemia blasts will also modulate T cell functions and alter the cytokine profile of activated T lymphocytes. Received: 6 November 1997 / Accepted: 5 March 1998     

HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions

 The DNA from human papillomavirus (HPV) can be detected in 90% of cervical carcinomas. To address whether patients infected with HPV can mount efficient T cell responses to this pathogen we examined the cytotoxic T lymphocyte (CTL) response of peripheral blood mononuclear cells (PBMC) from patients with abnormal genital epithelial cells. PBMC from 11 HLA-A2⁺ patients were stimulated with CaSki, a cervical carcinoma cell line that is HPV 16⁺ and HLA-A2⁺. The CTL were screened for reactivity to the cervical carcinoma cell line C33A (HPV^–, HLA-A2⁺) transfected with the HPV 16 E6 or E7 genes or the plasmid without insert. The CTL of 1 patient showed particularly strong CaSki and HPV E6 or E7 protein-specific cytotoxicity in a HLA-A2-restricted fashion. In contrast, these CTL lysed neither a vector-only transfectant, the natural killer cell (NK) target, K562 nor the lymphokine-activated killer cell (LAK) target, Daudi. HLA-A2 restriction was demonstrated by the lack of recognition of a HLA-A2^– CaSki cell line developed in our laboratory. The CTL line was cloned and 99 clones were harvested and screened; 51 clones lysed CaSki, of which 17 did not lyse the A2^– CaSki. Of these HLA-A2^– restricted clones, 8 did not lyse C33A transfectants, 6 lysed all C33A transfectants, 3 lysed C33A-E7 only and none lysed C33A-E6 only. These data imply that, within the bulk CTL line, HLA-A2-restricted recognition of antigens was restricted to CaSki antigens, antigens common to cervical carcinoma (CaSki plus C33A), or HPV-16-E7-derived antigen on the clonal level. The E7-restricted clones were negative for recognition of known HLA-A2-binding peptides from E7. Received: 16 November 1995 / Accepted: 15 January 1996     

Taxol-mediated changes in fibrosarcoma-induced immune cell function: Modulation of antitumor activities

 The anticancer drug taxol (paclitaxel) inhibits tumors through multiple cytotoxic and cytostatic mechanisms. Independently of these mechanisms, taxol induces distinct immunological efficacy when it acts as a second signal for activation of tumoricidal activity by interferon-γ(IFNγ)-primed murine normal host macrophages. We reported that tumor-distal macrophages, which mediate immunosuppression through dysregulated nitric oxide (NO) and tumor necrosis factor α (TNFα) production, are differentially regulated by taxol. Because taxol influences tumor cell growth dynamics and activates immune cell populations, we assessed the ex vivo immunosuppressive and antitumor activities of taxol-treated normal host and tumor-bearing host (TBH) macrophages. Pretreatment of such cells with taxol partly reconstituted T cell alloantigen reactivity, suggesting that taxol mediates a limited reversal of TBH macrophage immunosuppressive activity. Taxol-treated TBH macrophages significantly suppressed the growth of fibrosarcoma cells (Meth-KDE) through soluble effector molecules and promoted direct cell-mediated cytotoxicity, indicating that taxol enhanced tumor-induced macrophage antitumor activities. Tumor-induced helper T cells, however, showed a higher sensitivity to direct taxol-induced suppression. These data demonstrate that taxol exerts pleiotropic effects on antitumor immune responses with the capacity to abate the immunosuppressive activities of macrophages and promote macrophage-mediated antitumor activities simultaneously, but also directly modulating T cell reactivity. Collectively, these studies suggest that the antineoplastic drug taxol may impart antitumor activity through an immunotherapeutic capacity. Received: 31 December 1996 / Accepted: 1 July 1997     

Generation of specific antitumor reactivity by the stimulation of spleen cells from gastric cancer patients with MAGE-3 synthetic peptide

The induction of cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using MAGE peptide has been investigated in order to use MAGE antigens immunotherapeutically. We therefore developed a simplified method for inducing peptide-specific CTL that kill tumor cells expressing MAGE from the PBMC of either healthy donors or even cancer patients. Since the spleen is a major lymphoid organ, we used a simple method to examine the capacity of spleen cells to generate MAGE-specific CTL by in vitro stimulation with MAGE peptide in gastric cancer patients. The CTL responses could thus be induced from unseparated spleen cells in HLA-A2 patients with gastric carcinoma expressing MAGE-3 by stimulating these cells with autologous spleen cells pulsed with HLA-A2-restricted MAGE-3 peptide as antigen-presenting cells and by using keyhole limpet hemocyanin and interleukin-7 for the primary culture. The induced CTL were thus able to lyse HLA-A2-positive carcinoma cells transfected with MAGE-3 and expressing MAGE-3, as well as the target cells pulsed with the peptide, in an HLA-class-I or -A2-restricted manner. Since MAGE-specific CTL could be induced from the spleen cells of gastric cancer patients, the spleen appears to play an important role in either clinical tumor vaccination or the treatment of cancer patients by adoptive immunotherapeutic approaches using the MAGE peptide. Received: 3 December 1998 / Accepted: 30 March 1999     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996