Human Immune Response to DNP-Modified Autologous Cells after Treatment with a DNP-Conjugated Melanoma Vaccine

Immunizing patients with metastatic melanoma by injection of autologous tumor cells modified by DNP induces inflammatory responses in metastatic masses, which is sometimes associated with tumor regression. To elucidate this phenomenon, we studied the immune response to DNP-modified cells in these patients. All developed DTH to DNP-modified autologous lymphocytes (mean ± SE: 13.3 ± 1.3 mm), but not to TNP-modified lymphocytes. Larger responses (21.9 ± 3.6 mm) were elicited by DNP-modified autologous melanoma cells. In 8/11 patients tested, PBL proliferated in vitro when stimulated by autologous DNP-modified lymphocytes, and in 5 patients the stimulation resulted in production of interferon-γ. DNP-modified autologous melanoma cells elicited lymphocyte responses as well. PBL from 1 patient were expanded by culture in IL2 and repeated restimulation with DNP-modified B lymphoblastoid cells. This T cell line proliferated and produced interferon-γ but not IL4, when stimulated by autologous DNP-modified lymphocytes or melanoma cells. Both CD4⁺ and CD8⁺ subpopulations responded as determined by panning experiments and by testing of phenotypically homogeneous cultures obtained by limiting dilution. Studies of a stable CD8⁺ subline of the expanded T cells indicated that the response to DNP-modified cells was MHC-restricted, since it was blocked by antibody to class I determinants. Moreover, these T cells were able to respond to allogeneic DNP-modified stimulators that were matched at one or both HLA-A loci, but not to stimulators that were HLA-A mismatched. Finally, the CD8⁺ subline killed DNP-modified autologous melanoma cells, but not an HLA-A mismatched allogeneic melanoma, in a 6-hr ⁵¹Cr-release assay. These results may have significant implications for understanding the pathogenesis of drug-induced autoimmunity and for the development of new approaches to cancer immunotherapy.     

HLA class II-restricted recognition of common tumor epitopes on human melanoma cells by CD4+ melanoma-infiltrating lymphocytes

CD4⁺ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma-derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II-expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA-DR or HLA-DQ monoclonal antibodies. Most of these clones cross-reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4⁺ T lymphocytes.     

共刺激分子B7-1与IL-6协同诱导T细胞活化的研究

目的：探讨Ｂ７－１单独或者与ＩＬ－６联合体外诱导Ｔ细胞活化的能力。方法：在混合淋巴细胞肿瘤细胞培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数和ＣＴＬ杀伤活性。结果：Ｂ７－１＾＋的Ｂ１６细胞能够较有效地刺激淋巴细胞增殖,诱导特导ＣＴＬ杀伤活性,当与ＩＬ－６结合时效更加显著。结论：Ｂ７－１基因在肿瘤细胞中表达可增强其免疫原性在体外有效诱导Ｔ细胞活化;在此过程中ＩＬ－６与Ｂ７－１分子存在协同效应。     

Immunogenicity of the ALLAVGATK (gp10017 – 25) peptide in HLA-A3.1 melanoma patients

A T cell line recognizing autologous and allogeneic HLA-A3.1 melanomas was obtained from a disease-free melanoma patient (patient 15392). By transfection of a tumor cDNA library and in vitro sensitization experiments, the ALLAVGATK gp100/Mel17-derived peptide was found to be the epitope recognized by this melanoma-specific T cell line. The role of the ALLAVGATK peptide in the systemic immune response to melanoma of this patient was evaluated. When pulsed on the autologous peripheral blood mononuclear cells, the ALLAVGATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single restimulation in vitro was sufficient to raise gp100-specific T lymphocytes, indicating a high frequency of epitope-specific T cells. gp100-specific T cells were also induced from T lymphocytes purified from tumor-invaded lymph nodes (tumor-associated lymphocytes, TAL). TAL-derived effectors displayed lower peptide affinity and lower tumor recognition than effectors elicited from peripheral blood lymphocytes (PBL). To further evaluate its immunogenicity, ALLAVGATK was used to stimulate PBL derived from six additional HLA-A3.1 melanoma patients and seven healthy donors. After 7 weeks of peptide stimulation in vitro the generation of anti-gp100 and tumor-specific T cell lines was achieved in one out of the six patients analyzed. Taken together these data indicate that an in vivo priming leading to a systemic immunity against gp100 in HLA-A3 melanoma patients may occasionally occur and that the immunogenicity of ALLAVGATK peptide in melanoma patients is comparable to that of other HLA-A2-restricted epitopes derived from gp100/Mel 17 protein.     

Co-expression of B7-1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response

Although the transfection of B7-1 cDNA into a few mouse tumor cell lines can induce anti-tumor T cell immunity, its expression alone is ineffective in many other tumor cell lines tested. We were interested to study what factors limit B7-1 co-stimulatory activity, and decided to investigate whether B7-1 requires the cooperation of ICAM-1 to provide the minimal co-stimulatory signal for establishing an efficient anti-tumor immunity. We show that the transfection of B7-1 cDNA into three ICAM-1⁺ (plasmocytoma J558L, T lymphomas EL-4 and RMA), but not into two ICAM-1^? tumor cell lines (adenocarcinoma TS/A and melanoma B16.F1), is sufficient to induce their complete rejection in syngeneic mice. The expression of ICAM-1 is necessary for the rejection of the B7 expressing tumors, since the primary response elicited by B7-1⁺ EL-4 and RMA clones expressing reduced levels of ICAM-1 is severely reduced. Furthermore, super-transfection of ICAM-1 cDNA into B7-1⁺ adenocarcinoma and melanoma clones optimizes their primary rejection. Histologic examination of transfected tumors reveals that B7-1 and ICAM-1 exert a potent pro-inflammatory activity. The intra-tumor infiltration is composed of both eosinophils and lymphomono-cytes, and is already massive 5 days after the tumor challenge. The primary rejection of the B7-1⁺ ICAM-1⁺ tumors depends critically on CD8⁺ T cells, natural killer cells and granulocytes, but is independent of CD4⁺ T cells. Remarkably, in addition to its effects on the early phases of the immune response, the co-expression of ICAM-1 and B7-1 on tumors is also necessary for the efficient induction of a memory response. In fact, only the primary challenge with B7-1⁺, ICAM-1⁺ tumor cells protects the majority of the mice from a second injection of parental tumor cells. Collectively, our findings indicate that B7-1 and ICAM-1 are fundamental components for triggering the primary rejection of tumors and establishing a protective memory response. These findings may help to define new strategies for the rational application of co-stimulation in tumor immunotherapy.     

Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors

Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngeneic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic. The expression of the B7-1 co-stimulatory molecule on immunogenic tumors can further increase their capacity to induce a T cell-dependent anti-tumor immunity, whereas it has limited effects on nonimmunogenic tumors. Recently, B7-2, a second molecule with an apparently similar co-stimulatory activity, has been cloned. In this report, we compare the efficiency of nonreplicating cells from one immunogenic and two nonimmunogenic mouse tumors transfected with B7-1 or B7-2 in the induction of protective and curative anti-tumor immunity. Immunogenic lymphoma cells expressing B7-1 or B7-2 are equally effective in both protecting against a subsequent challenge and curing established tumors. By contrast, nonimmunogenic adenocarcinoma and melanoma cells expressing B7-2 provide superior protective immunity, and only B7-2⁺ adenocarcinoma cells induce an efficient curative immunity. CD8⁺ and polymorphonuclear cells, but not CD4⁺ T cells, are critically involved in the rejection of the adenocarcinoma elicited by both B7-1⁺ and B7-2⁺ vaccines. These data indicate that B7-1 and B7-2 are not redundant co-stimulatory molecules and that, in these experimental models, B7-2 is superior to B7-1 in the induction of an efficient immunity when the immunogenicity of a tumor is a limiting factor.     

CD8<Superscript>+</Superscript>, HLA-unrestricted,cytotoxic T-lymphocyte line against malignant melanoma

A CD8⁺ cytotoxic T lymphocyte (CTL) line was derived from the peripheral blood mononuclear cells of a patient with primary melanoma. The CD8⁺ CTL line specifically lysed the autologous primary melanoma cells and not the natural killer cell-sensitive K562 cells or lymphokine activated killer cell-sensitive DAUDI cells. When a large panel of human leukocyte antigen (HLA)-matched and -unmatched allogeneic melanoma, glioma, breast and colorectal carcinoma cells was tested as targets in cytolysis assays, 4 HLA-matched and two HLA-unmatched allogeneic metastatic melanoma lines were lysed by the CD8⁺ CTL. Lysis of autologous and allogeneic melanoma cells was dependent on the effector-to-target cell ratio. Lysis of autologous melanoma cells was not blocked by anti-HLA class I or class II antibodies, confirming that the cytolytic activity of the CD8⁺ CTL was HLA-unrestricted. CTL lysis of autologous melanoma cells was CD3 (T cell receptor) dependent and FAS-FAS-L, and CD1 independent. Identification of the melanoma-associated antigen recognized by the HLA-unrestricted CTL may provide a vaccine for a broad population of melanoma patients.     

B-1 cells promote immunosurveillance against murine melanoma in host absence of CCR5: New perspective in autologous vaccination therapy

Autologous vaccination with tumor-primed dendritic cells increases immune response against tumor, which seems to be improved in host absence of CCR5. Because B-1 lymphocytes modulate the activity of different immune cells, we decided to study their influence in the resistance against murine B16F10 melanoma in a CCR5 deprived environment. Adoptive transfer of peritoneal B-1 CCR5^+/+ lymphocytes to CCR5^−/− animals inhibited the establishment of lung metastasis and melanoma cell growth, in comparison to saline-treated CCR5^−/− mice. In loco cell analysis demonstrated that the adoptive transfer of B-1 CCR5^+/+ lymphocytes to CCR5 deficient host was associated with a more intense influx of T CD8⁺ to tumor site, indicating that the presence of CCR5^+/+ B-1 cells in the tumor environment induces the migration of T CD8 CCR5^−/− cells to the implantation site. To corroborate this idea, CCR5^−/− mice were injected with non B-1 peritoneal cells from wild type (WT) mice before B16F10 inoculation. In this regimen, CCR5^−/− mice were not protected from tumor growth reinforcing the idea that, in host absence of CCR5, B-1 cells are essential to confer tumor resistance. This work indicates that, in the host absence of CCR5, naive B-1 cells may activate CD8T lymphocytes thereby promoting tumor resistance. Our results strongly suggest that autologous vaccination with B-1 lymphocytes in combination with CCR5 antagonists can be an alternative approach to tumor therapy.     

Potent Systemic Antitumor Immunity Induced by Vaccination with Chemotactic-Prostate Tumor Associated Antigen Gene-Modified Tumor Cell and Blockade of B7-H1

We previously reported that several DNA fragments from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA) genes were selected and fused to create a novel hPSM-mPAP-hPSA fusion gene (named 3P gene), and human secondary lymphoid tissue chemokine (SLC), 3P, and human IgG Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. In this report, to establish a more efficient treatment for immunotherapy against prostate cancer, the construct was transfected into B16F10 to generate gene-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). In poorly immunogenic B16F10 mouse melanoma model, the immunization with B16F10-SLC-3P-Fc resulted in a strong antitumor response and 50% of tumor-bearing mice achieved long-term survival (>120 days). In vivo depletion of lymphocytes indicated that CD8⁺ T cells were involved in the direct tumor killing, whereas CD4⁺ T lymphocytes were required for the induction of CD8⁺ CTL response in B16F10-SLC-3P-Fc-immunized mice. Splenocytes from B16F10-SLC-3P-Fc-immunized mice specifically recognized and lysed PSM, PAP, PSA, and 3P expressing tumor cells. The combined therapy of B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further enhanced the immune response. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. These observations suggest pSLC-3P-Fc-modified tumor cells could serve as a vaccine against prostate cancer, and the therapy combined with anti-B7-H1 MAbs further enhanced the antitumor immune response.     

Role of normal adherent cells in the regulation of the autologous mixed lymphocyte reactions in humans

The effect of normal adherent suppressor cells on the blastogenesis of human T lymphocytes in the mixed lymphocyte reaction (MLR) was studied in both allogeneic and autologous combinations. Non-T cells and Ia⁺ T lymphocytes were used as stimulator cells in both allogeneic and autologous MLR. The addition of adherent cells to the stimulators inhibited blastogenesis of T lymphocytes in both types of MLR when the stimulator population was made up of non-T lymphocytes but did not interfere with blastogenesis when Ia⁺ T lymphocytes were used as stimulator cells. The present data indicate that the T lymphocytes able to respond to Ia⁺ T cells (in the MLR, autologous or allogeneic) may be different from those which respond to non-T lymphocytes or may be less sensitive to the regulatory function of normal adherent cells.     

Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines

Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell-deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in singlegene-transfected tumors. However, T cells in B7 and IL-7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28⁺ and CD25⁺, while in IL-7-transfected tumors, T cells were mainly CD28^? and CD25⁺. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28⁺ and CD25⁺. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.     

CD8high(CD57+) T cells in normal,healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines

We have previously identified two subsets of CD8⁺, CD57⁺ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8^high(CD57⁺)] and ii) natural killer cells expressing low levels of surface CD8 [CD8^low(CD57⁺)]. We investigated the cytotoxic and suppressive function of CD8^high(CD57⁺) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8^high(CD57⁺) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8^high(CD57⁺) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8^high(CD57⁺) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8^high(CD57⁺) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8^high(CD57⁺) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8^high(CD57⁺) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8⁺ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.     

Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumor-infiltrating lymphocytes

Three melanoma-specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor-infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA-A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA-A2, but by a B or C HLA antigen. HLA-A2-restricted CTL clones M17-1 and M17-2 lysed 5 and 12 out of 15 HLA-A2⁺ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein-Barr virus B cells, HLA-A2.1-transfected P815 cells,13 HLA-A2⁺ non-melanoma tumor cell lines and 10 HLA-A2^? melanomas, these clones appeared specific for melanoma-restricted epitopes presented by the HLA-A2.1 molecule. We then tried to determine why a few HLA-A2⁺ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA-A2 genes, we show that failure to lyse did not result from low expression or polymoprhism of the HLA-A2 molecule, or from deficient expression of the adhesion molecules ICAM-1 and LFA-3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA-A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA-A2⁺ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.     

Antitumor cytotoxic T-lymphocyte response in human lung carcinoma: identification of a tumor-associated antigen

Summary: We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a lung carcinoma of a patient with long survival. These clones showed a CD3⁺, CD8⁺, CD4^–, CD28^– phenotype and expressed a T‐cell receptor (TCR) encoded either by Vβ8‐Jβ1.5 or Vβ22‐Jβ1.4 rearrangements. Functional studies indicated that these clones mediated a high human leukocyte antigen (HLA)‐A2.1‐restricted cytotoxic activity against the autologous tumor cell line. Interestingly, TCRβ chain gene usage indicated that CTL clones identified in vitro were selectively expandedin vivo at the tumor site as compared to autologous peripheral blood lymphocytes (PBL). These findings provide evidence that an immune response may take place in non‐small cell lung carcinoma and that effector T cells may contribute to tumor regression. Further study indicated that the CTL clones recognized the same decamer peptide encoded by a mutated α‐actinin‐4 gene. Using tetramers of soluble HLA‐A2 molecules loaded with the mutated antigenic peptide, we have derived several anti‐α‐actinin‐4 T‐cell clones from patient PBL. These CTL, recognizing a truly tumor‐specific antigen, may play a role in the clinical evolution of this lung cancer patient. Adoptive transfer of CTL clones in a SCID/NOD mice model transplanted with autologous tumor supported their antitumor effect in vivo.     

Defective lymphokine production by most CD8+ and CD4+ tumor-specific T cell clones derived from human melanoma-infiltrating lymphocytes in response to autologous tumor cells in vitro

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-γ (IFN-γ) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-γ. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-γ secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4⁺ and CD8⁺ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.     

T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the ‘dominant’ rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TlL-5). These IL-2-propagaled TIL cell lines had a CD8⁺ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3⁺, CD4^?, CD8⁺ and expressed the α/β TCR. From TIL-1.27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK, -negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TiL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectimediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs, Of the 34 TlL-1 clones analysed, 15 shared a rearranged TCRβ EcoR1 restriction fragment of approximately 9 5 kb with the bulk culture. Clones sharing the EcoR1 10 5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCRβ rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response lo autologous tumour cells were also detected among those contributing to the dominant rearrangement; (ii) some clones that share an apparently identical rearranged band different from the “dominant” rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour. Further study of the TCR gene rearrangements should help to clarify how selection at this level can benefit future immunotherapeutic approaches.     

Tumour-specific cytotoxic T lymphocyte activity in Th2-type Sézary syndrome: its enhancement by interferon-gamma (IFN-γ) and IL-12 and fluctuations in association with disease activity

Sézary syndrome (SzS) is the leukaemic variant of cutaneous T cell lymphoma (CTCL), whose malignant T cells are of the Th2 type in most cases. In this study we investigated the tumouricidal activity of cytotoxic T lymphocytes (CTL) present in peripheral blood of a patient with Th2-type SzS, focusing on the effect of IL-2, IFN-γ and IL-12 on their cytotoxic activity, and the relationship between their lytic capacity and the patient's clinical course. At four different time points during a 2-month clinical period, CD4⁺ CD7^? Sézary cells and CD8⁺ cells were separated from the patient's circulating cells. CD8⁺ cells were cultured with chemically attenuated, purified Sézary cells in the presence of IL-2 to develop specific cytotoxicity. The CD8⁺ cells thus cultured exhibited lytic activity against autologous Sézary cells. Concomitant addition of IFN-γ or IL-12 exerted a synergistic cytolytic effect with IL-2 on the tumour cells. Cytotoxicity inhibition studies using MoAbs revealed that the cytotoxicity operated in MHC class I-, CD8- and αβ T cell receptor-dependent manners. Furthermore, eight CD8⁺ T cell clones generated from cultured CD8⁺ cells exhibited a strong cytotoxicity against Sézary cells in an MHC class I-restricted fashion. During the clinical course, the activity of generated CTL and the number of CD8⁺ cells were inversely correlated with disease activity as assessed by the serum level of lactate dehydrogenase. These findings suggest that CTL down-regulate the growth of malignant T cells in this long-standing disease. Since Th2 cytokines such as IL-4 down-modulate CTL activity, CTL are assumed to be usually suppressed in SzS, whose malignant T cells are of Th2 type. It is likely that the administration of IFN-γ normalizes this Th2-skewing state, activates CTL, and thus exerts the therapeutic effectiveness in the treatment of CTCL.     

Vaccination with B7-1+ Tumor and Anti-Adhesion Therapy with RGD Pseudo-Peptide (FC-336) Efficiently Induce Anti-Metastatic Effect

We have previously shown that expression of costimulatory ligand B7-1 on MHC class I⁺ tumor cells (B16-BL6 melanoma) resulted in marked reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice and led to induction of immunity to the challenge by the parental B7-1 negative tumor. Here we investigated the effectiveness of irradiated B7-1 transfected tumor cells as a vaccine on established tumor metastasis and whether or not expression of B7-1 molecule on tumor cells in combination with administration of anti-adhesion peptide FC-336 can augment the antimetastatic efficacy. Immunization with X-irradiated B7-1 transfectants after i.v. injection of B7-1⁻ parental B16-BL6 cells was effective in inhibiting lung metastasis. We also found that vaccination with irradiated B7-1 transfectants after excision of primary tumor on day 21 resulted in significant inhibition of spontaneous lung metastasis by intrafootpad injection of viable parental B16-BL6 melanoma, as compared with the untreated control. However, immunizing twice with mock transfectants did not affect inhibition of spontaneous lung metastasis of wild-type tumors. On the other hand, multiple administration of a pseudo-peptide of RGD sequence (FC-336) after tumor inoculation inhibited spontaneous lung metastasis through the interference of tumor invasion, migration and adhesion. Combined treatment of B7-1 transfected tumor vaccine and anti-adhesive therapy with FC-336 led to the augmentation of the antimetastatic effect in both experimental and spontaneous metastasis models, as compared with either treatment alone. B7-1- and FC-336-mediated inhibition of tumor metastasis may be mediated by different mechanisms at various steps of metastasis, based on the regulation (promotion or inhibition) of tumor interaction with host cells and components. This revised version was published online in August 2006 with corrections to the Cover Date.