Effects of Interleukin-12 on the Induction of Cytotoxic T Lymphocytes from the Regional Lymph Node Lymphocytes of Patients with Lung Adenocarcinoma

Lung cancer-specific cytotoxic T lymphocytes (CTL) were induced by repeated stimulations of regional lymph node lymphocytes (RLNL) in lung cancer patients with either autologous or HLA-A-locus-matched tumor cells. To investigate the effect of interleukin-12 (IL-12), IL-12 was added during the stimulation of RLNL from HLA A24 / adenocarcinoma patients with either autologous tumor cells or HLA A24-positive adenocarcinoma cells (PC-9) in combination with, or instead of interleukin-2 (IL-2), and then the cytotoxic activity, cytokine production and populations of the lymphocyte subsets were examined. The addition of IL-12, or the substitution of IL-2 by IL-12 was found to enhance the cytotoxic activity and the cytokine production (IFN-γ, GM-CSF) of the CTL as compared with IL-2 alone. The cytotoxic activity and cytokine production were both partially inhibited by anti-MHC-class I monoclonal antibody. The CTL thus induced by IL-12 had a higher proportion of CD3⁺/CD56⁺ cells than the CTL induced with IL-2 alone. The positively selected CD8⁺/CD56^– lymphocytes showed PC-9-specific cytotoxic activity, because the population did not show any cytotoxicity to K562 or A549 (HLA-A26/A30). However, the CD3⁺/CD56⁺ lymphocytes were cytotoxic to both PC-9 and K562. In conclusion, IL-12 is considered to be a useful cytokine for both the induction of lung-cancer specific CTL and the augmentation of non-MHC-restricted cytotoxicity against tumor cells, and may be applicable for adoptive immunotherapy using CTL.     

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.     

Human CTL epitopes prostatic acid phosphatase-3 and six-transmembrane epithelial antigen of prostate-3 as candidates for prostate cancer immunotherapy

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.     

A HLA-A2-restricted CTL epitope induces anti-tumor effects against human lung cancer in mouse xenograft model

Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes. In this report, we evaluated human CTL epitope-induced anti-tumor effects in human lung cancer xenograft models. The tumor associated antigen L6 (TAL6) is highly expressed in human lung cancer cell lines and tumor specimens as compared to normal lung tissues. TAL6 derived peptides strongly inhibited tumor growth, cancer metastasis and prolonged survival time in HLA-A2 transgenic mice immunized with a formulation of T-helper (Th) peptide, synthetic CpG ODN, and adjuvant Montanide ISA-51 (ISA-51). Adoptive transfer of peptide-induced CTL cells from HLA-A2 transgenic mice into human tumor xenograft SCID mice significantly inhibited tumor growth. Furthermore, combination of CTL-peptide immunotherapy and gemcitabine additively improved the therapeutic effects. This pre-clinical evaluation model provides a useful platform to develop efficient immunotherapeutic drugs to treat lung cancer and demonstrates a promising strategy with benefit of antitumor immune responses worthy of further development in clinical trials.     

Characterization of factors regulating successful immunotherapy using a tumor-specific cytotoxic T lymphocyte clone: Role of interleukin-2, cycling pattern of lytic activity and adhesion molecules

Adoptive immunotherapy against cancer has met with varying degrees of success, the reasons for which remain unclear. The present study characterizes factors that regulate successful immunotherapy of mice bearing a syngeneic T-cell lymphoma, designated LSA, using a tumor-specific cytotoxic T lymphocyte (CTL) clone, PE-9. Adoptive transfer of PE-9 cells afforded significant protection in normal but not in nude mice against LSA tumor. However, the PE-9 cells could protect the nude mice when injected along with normal CD4⁺ T cells. Administration of IL-2 along with PE-9 cells failed to enhance tumor immunotherapy. IL-2 therapy was toxic inasmuch as injection of the CTL clone PE-9 + IL-2, but not PE-9 or IL-2 alone, for 5 days into irradiated mice caused vascular leak syndrome (VLS). PE-9 cells cultured with high doses of rIL-2 in vitro also caused TCR-independent and MHC-unrestricted lysis of SV40-transformed endothelial cells. Furthermore, PE-9 cells cultured in vitro for 24-96 hr with IL-2 exhibited cycling pattern of tumorspecific cytotoxicity with maximum cytotoxicity demonstrable at 48 hr and virtually no cytolytic activity at 96 hr of culture or thereafter. The loss of cytotoxicity correlated with downregulation of several adhesion molecule expressions on PE-9 cells, particularly the αβ-TCR, as well as the mRNA for TNF-α, IFN-γ and perforin, although the levels of granzyme A were not altered. Interestingly, the outcome of immunotherapy by PE-9 cells depended on the cycling pattern of cytotoxicity. Our data suggest that successful immunotherapy against cancer using a CTL clone depends on several factors, such as the cycling pattern of lytic activity, density of adhesion molecules, levels of cytokines expressed and the ability of IL-2 and CTL to trigger VLS.     

Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤小鼠肿瘤生长的影响

目的 探讨Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤(DLBCL)小鼠肿瘤生长的影响.方法 采用Mini MACS免疫磁珠分离纯化BALB/c小鼠脾来源的CD4+ CD62L+初始T细胞,采用"转化生长因子(TGF-β)、白细胞介素6(IL-6)、干扰素γ抗体(anti-IFN-γ)、白细胞介素4抗体(anti-IL-4)、白细胞介素23(IL-23)"因子组合体外诱导小鼠Th17细胞分化,采用ELISA法测定Th17细胞产生IL-17水平;采用DLBCL细胞株SUDHL-4接种SCID小鼠建立DLBCL荷瘤小鼠模型;选择0、8、18d(3组,5只小鼠/每组)对荷瘤小鼠进行Th17细胞过继免疫治疗,计算肿瘤体积,观察荷瘤小鼠生存期.结果 小鼠在接种SUDHL-4细胞8d左右均形成瘤结节,成瘤率为100％.与对照组小鼠相比,3个过继免疫治疗组小鼠肿瘤体积均明显缩小(P＜0.05),荷瘤小鼠生存期均显著延长(P＜0.05).结论 Th17细胞过继免疫治疗对DLBCL荷瘤小鼠具有抗肿瘤作用.     

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy.     

构建人类肾上腺皮质腺癌细胞系并用其验证过继免疫细胞治疗效果

 目的　建立人类肾上腺皮质腺癌（ACC）细胞系ACC-LWL,研究其表型及肿瘤相关抗原表达,并以此模型初步探讨过继免疫细胞治疗ACC的可行性。方法　手术获得的ACC的新鲜肿瘤组织经体外原代和传代培养至稳定生长,分析其生物学特征,包括癌细胞集落形成、染色体及成瘤性,经流式细胞术分析细胞系细胞表型,RT-PCR检测MN／CA9和HLA-A2基因表达。体外用IL-2（200 U／ml）和ACC-LWL冻融抗原（20 μg／ml）共同刺激异体人单个核细胞（PBMC）产生细胞毒性T淋巴细胞（CTL）,通过流式细胞术分析异体人CTL的CD3、CD4、CD8表达水平,检测CTL对ACC-LWL肿瘤细胞的杀伤作用。结果　ACC-LWL表现恶性肿瘤细胞的生物特性,能于裸鼠体内成瘤,细胞高表达MHC-I,低表达Her-2／neu和MHC-Ⅱ,不表达CD80和CD86。ACC-LWL表达MN／CA9和HLA-A2基因。从HLA-A2+和HLA-A3+供体获得的CTL均显示杀伤ACC-LWL,未见HLA限制性杀伤。结论　成功建立ACC细胞系ACC-LWL,可提供用于研究人类ACC的细胞及动物模型,为过继免疫细胞治疗在ACC中的应用提供初步的实验依据。     

The Induction of Cytotoxic T Lymphocytes against HLA-A Locus-matched Lung Adenocarcinoma in Patients with Non-small Cell Lung Cancer

To induce cytotoxic T lymphocytes (CTL) against non-small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA-A locus-shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA-A2/A24-positive NSCLC cell line (PC-9), and thereafter the cytotoxic activity was examined by ⁵¹Cr-release assay. In patients with HLA-A24/ adenocarcinoma, anti-PC-9 cytotoxicity was induced in all 6 patients tested. Anti-PC-9 cytotoxicity was induced in 2 out of 5 patients with HLA-A2 (A24⁻)/adenocarcinoma, in 2 out of 4 patients with HLA-A24/squamous cell carcinoma, and 1 of 2 patients with HLA-A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC-9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti-MHC class I antibody, but not by anti-MHC class II antibody. The PC-9-specific CTL produced γ-interferon in response to autologous tumor cells. These results indicated that the anti-PC-9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA-A2 and/or HLA-A24-positive NSCLC.     

Recognition of breast cancer-associated peptides by tumor-reactive, HLA-class I restricted allogeneic cytotoxic T lymphocytes

Strategies to identify tumor-associated antigens rely on the paradigm that tumor-associated peptides presented in the context of HLA-class I are recognized by the cellular immune system. Approaches to isolate tumor-specific cytotoxic T lymphocytes (CTL) from tumor-infiltrating lymphocytes are difficult because long-term growth of the CTL requires autologous tumor cells and lymphocytes (PBL) as feeder cells. In this study, a CTL line (BL.HBL-100 CTL) was generated from PBL from a normal healthy donor by stimulating with irradiated, HLA-class I partially matched breast cancer cell line HBL-100. Activated T lymphocytes generated expressed TCR alpha/beta+ with a predominant CD8+ population after 12 stimulations (98.54% CD8+ vs. 0.18% CD4+). These CTL lysed HLA-A1+, but not HLA-A1-, breast cancer cell lines. Moreover, HLA-A1+, non breast cancer cell lines were not recognized. The lytic activity of BL.HBL-100 CTL against HLA-A1+ breast cancer cell lines was blocked by monoclonal antibodies (MAbs) to HLA-class I and CD8, but not by anti-HLA-class II and CD4. Recognition of HLA-A1+ breast cancer cells by the CTL was dependent on peptides associated with HLA-class I since the lysis was inhibited by acid elution of HLA bound peptides. HBL-100 tumors were grown in severe combined immunodeficient (SCID) mice. Immunohistochemical staining of the HBL-100 tissue harvested from SCID mice demonstrated human breast cancer cells. HLA-class I molecules were affinity purified from the HBL-100 harvested from the SCID mice; class I bound peptides were eluted and separated by RP-HPLC. Pooled HPLC peptide fractions were tested for reconstituting antigenic epitopes recognized by the BL.HBL-100 CTL and found to reside within fraction 40. Our results show that a tumor reactive, HLA-class I restricted CTL was produced by stimulating normal PBL against an HLA-class I matched breast cancer cell line. We also provide evidence for a breast cancer-associated, HLA-class I bound peptide antigen(s) that reconstitutes the antigenic epitope(s) recognized by these CTL.     

Generation of Melan-A/MART-1-specific CD8+ cytotoxic T lymphocytes from human naive precursors: Helper effect requirement for efficient primary cytotoxic T lymphocyte induction in vitro

This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4⁺ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4⁺ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-γ, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols. Int. J. Cancer 72:987–994, 1997. © 1997 Wiley-Liss, Inc.     

Autologous Tumor-specific Cytotoxic T Lymphocytes in a Patient with Lung Adenocarcinoma: Implications of the Shared Antigens Expressed in HLA-A24 Lung Cancer Cells

Human lung adenocarcinoma-specific cytotoxic T lymphocytes (CTL) were generated by multiple stimulations with autologous tumor cells (named A110L) from regional lymph node lymphocytes and tumor-infiltrating lymphocytes expanded by solid-phase anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2. The CTL lysed A110L but failed to kill either autologous B lymphocytes immortalized by the Epstein-Barr virus or K562. The killing activity of the CTL against autologous A110L was inhibited by anti-MHC class I mAb (W6/32), but not by anti-MHC class II mAb. The CTL produced interferon-γ and GM-CSF in response to A110L and the production was completely blocked by the addition of anti-MHC class I mAb. The HLA type of the CTL was HLA-A2/A24, B52/B54, Cw1/-. Allele-specific deletion of HLA-A2 molecules was observed in A110L by staining with anti-HLA-A2 mAb. A partial blocking effect on the cytokine production from the CTL was also obtained with anti-CD8, and anti-HLA-A24 mAbs, but not with anti-MHC class II, anti-CD4 and anti-HLA-A2 mAbs. To analyze further the mechanism of antigen recognition by the CTL, the cross reactivity of the CTL against several HLA-A locus-matched (HLA-A24+) and mismatched allogeneic tumor cells (HLA-A24-) was investigated. The A110L-specific CTL showed a weak but significant cytotoxicity against some HLA-A24 positive lung cancer cell lines, such as Sq-1 (HLA-A11/A24, squamous cell carcinoma) and PC-9 (HLA-A2/A24, adenocarcinoma), but failed to kill HLA-A locus-mismatched allogeneic tumors. This cross reactivity of the CTL against Sq-1 and PC-9 was blocked by anti-MHC class I mAb. These results thus demonstrate that shared common tumor antigens might exist among lung cancer cells in the context of HLA-A24.     

干扰肿瘤微环境对细胞毒性T淋巴细胞瘤体内归巢和杀伤活性的影响

目的 探讨应用低剂量环磷酰胺（cyclophosphamide, CYC）干扰荷瘤小鼠体内微环境对改善过继回输的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）瘤体内归巢和杀伤活性的作用。方法 建立C57BL/6荷瘤小鼠模型并分为对照组、CYC注入（CYCI）组、0.9%NaCl溶液注入（NSI）+CTL组和CYCI+CTL组,每组40只,分别给CYCI组、CYCI+CTL组每只小鼠腹腔注入CYC （20mg/kg）,NSI+CTL组小鼠注入等量0.9%NaCl溶液。检测注射前后不同时间小鼠瘤体内调节性T细胞（Treg）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）水平变化。于注射后第4天回输CFSE-CTL,每只0.2 ml,含5×l0⁶个细胞。流式检测对比NSI+CTL组和CYCI+CTL组瘤体内CFSE-CTL比例。结果 NSI+CTL组小鼠Treg、IL-10、TGF-β水平随着瘤体增长而逐渐增高;CYCI+CTL组比NSI+CTL组回输的CFSE-CTL在肿瘤内归巢明显增加和持久（P<0.05）。各组肿瘤体积除对照组和CYCI组外,其余各组间差异均有统计学意义（P<0.05）。结论 提前应用低剂量CYC干预荷瘤小鼠体内微环境,可降低瘤体内Treg、IL-10和TGF-β水平,使过继回输的CTL在肿瘤中的归巢聚集明显增强,杀伤肿瘤效率提高。     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.     

Induction of tumor-specific cytotoxic T lymphocytes from regional lymph node lymphocytes of human breast cancer

Background  In this study we activated breast cancer-specific cytotoxic T lymphocytes (CTL) from regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with breast cancer. Melthods  Freshly isolated RLNL were stimulated with solid phase anti-CD3 monoclonal antibody followed by expansion with recombinant interleukin-2. Subsequently, the RLNL were stimulated with an irradiated HLA 0201 breast cancer cell line, MCF-7, at a responder/stimulator ratio of 10/1 once a week for 2 weeks. Results  The cultured RLNL exhibited specific lysis against MCF-7 in all 5 HLA-A2-positive patients tested, but not in 2 HLA-A2-negative patients. Cytotoxicity against MCF-7 was substantially inhibited by addition of anti-HLA-A2 mAb. In 3 of 5 HLA-A2-positive patients, anti-MCF-7 CTL also exhibited a substantial level of reactivity against PC-9, an HLA-A0206-positive lung adenocarcinoma cell line. Conversely, anti-PC-9-specific CTL were inducible by multiple stimulations of RLNL with PC-9 cells in 2 of 3 patients. Conclusions  These results suggest that several common tumor antigens might exist among HLA-A2-positive breast cancers, some of which may be shared with lung adenocarcinomas.     

Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment

Background  

Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro -generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear.     

The induction of enhanced antitumor effect against a nonimmunogenic tumor by highly immunogenic variants obtained by mutagen treatment

Nonimmunogenic 1767-3 fibrosarcoma was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, and stable variant cell clones (M-clones) were obtained that were able to elicit an immunological rejection response in syngenic C3H mice. Mice immunized with some M-clones were protected against a challenge from the original nonimmunogenic fibrosarcoma. Furthermore, when spleen cells of immunized syngenic mice were restimulated in vitro with M-clones, cytotoxic T lymphocytes (CTL) were obtained that were able to lyse not only M-clones but also the original nonimmunogenic tumor. These in vivo and in vitro results demonstrate the immunogenicity of M-clones and the existence of a singular antigenic specificity between the original nonimmunogenic tumor and M-clones. For the purpose of application of this mutagen treatment to cancer therapy, we combined it with lymphokine-activated killer (LAK) adoptive immunotherapy (AIT). With interleukin 2 and in vitro stimulation with highly immunogenic variant clones, we tried to induce transfer cells that had not only nonspecific LAK cells but also CTL with specific immunity against the original nonimmunogenic tumor. Successful results were obtained in the LAK AIT models. These findings indicate that an immunotherapy of human cancers that are thought to be weakly or nonimmunogenic may be possible by the application of this approach to LAK AIT.     

The Induction of Enhanced Antitumor Effect against a Nonimmunogenic Tumor by Highly Immunogenic Variants Obtained by Mutagen Treatment

Nonimmunogenic 1767-3 fibrosarcoma was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, and stable variant cell clones (M-clones) were obtained that were able to elicit an immunological rejection response in syngenic C3H mice. Mice immunized with some M-clones were protected against a challenge from the original nonimmunogenic fibrosarcoma. Furthermore, when spleen cells of immunized syngenic mice were restimulated in vitro with M-clones, cytotoxic T lymphocytes (CTL) were obtained that were able to lyse not only M-clones but also the original nonimmunogenic tumor. These in vivo and in vitro results demonstrate the immunogenicity of M-clones and the existence of a singular antigenic specificity between the original nonimmunogenic tumor and M-clones. For the purpose of application of this mutagen treatment to cancer therapy, we combined it with lymphokine-activated killer (LAK) adoptive immunotherapy (AIT). With interleukin 2 and in vitro stimulation with highly immunogenic variant clones, we tried to induce transfer cells that had not only nonspecific LAK cells but also CTL with specific immunity against the original nonimmunogenic tumor. Successful results were obtained in the LAK AIT models. These findings indicate that an immunotherapy of human cancers that are thought to be weakly or nonimmunogenic may be possible by the application of this approach to LAK AIT.     

Differential effect of high doses versus low doses of interleukin-12 on the adoptive transfer of human specific cytotoxic T lymphocyte in autologous lung tumors engrafted into severe combined immunodeficiency disease-nonobese diabetic mice: relation with interleukin-10 induction

BACKGROUND: Interleukin (IL)-12 can enhance the development of effective immune responses against tumors as well as against certain infectious agents. It is therefore a potential candidate for therapeutic use in cancer therapy and in design of vaccines against several infectious diseases. METHODS: The authors have established a specific cytotoxic T-cell line (TIL-Heu) from lymphocytes infiltrating a human large cell carcinoma of the lung (LCC). In the current report, the authors have investigated the in vivo effect of recombinant human IL-12 (rhIL-12) on the adoptive transfer of TIL-Heu cells in autologous tumor (Heu-n) engrafted into severe combined immunodeficiency disease-nonobese diabetic (SCID-NOD) mice. RESULTS: Initial in vitro experiments indicated that rhIL-12 increased the cytotoxic potential of TIL-Heu cells in a dose-dependent manner. Heu-n tumors transplanted into SCID-NOD mice were injected with TIL-Heu cells, resulting in a significant tumor growth inhibition. When low doses of rhIL-12 were injected intratumorally after TIL-Heu transfer, a clear increase in tumor growth suppression was observed. Surprisingly, higher doses of rhIL-12 had no effect on cytotoxic T lymphocyte (CTL)-induced prevention of tumor growth. Further in vitro experiments revealed an inhibition of tumor cell lysis after incubation with supernatant of TIL-Heu cells stimulated with high doses of rhIL-12, strongly suggesting that an immunosuppressive factor secreted by the high dose IL-12-stimulated CTL may be responsible for the tumor escape observed in vivo. CONCLUSIONS: The authors' data indicate that IL-10 may play a critical role in the lack of effect of high dose IL-12, by mediating tumor cell resistance to CTL killing. Therefore, understanding the cross-talk between immunoregulatory and immunosuppressive cytokines ultimately may provide new approaches to improve cytokine-mediated cancer immunotherapy.     

Human melanoma therapy in the SCID mouse: in vivo targeting and reactivation of melanoma-specific cytotoxic T cells by bi-specific antibody fragments

The adoptive transfer of tumor-specific cytotoxic T cells (CTL) offers a promising perspective in cancer immunotherapy. However, the ex vivo-generated T lymphocytes are mostly IL-2-dependent. Here we explored the possibility of circumventing the requirement for IL-2, known for severe side effects in the patient, and of simultaneously targeting the CTL towards the tumor by the use of 2 bi-specific antibody fragments. As a model system, we used SCID mice bearing an s.c.-implanted human melanoma line (BLM-gp100) and in vitro-generated CTL specific for the gp100-derived immunogenic peptide YLEPGPVTA, which were injected i.v. with delay. To maintain the cytotoxic potential of the transferred CTL, 2 bi-specific antibody (biAb) fragments were generated which bound with one arm either CD3 or CD28, a combination known to support the activation of CTL. For targeting the CTL, both biAbs contained the F(ab') part of HD-Me13, an antibody recognizing p97, a non-immunogenic melanoma-associated surface molecule. In vitro and in vivo, the addition of the 2 biAbs increased the cytotoxic potential of the gp100-specific CTL and supported their clonal expansion in the absence of IL-2. Correspondingly, significantly higher numbers of CTL were recovered from melanoma-bearing SCID-mice that received the 2 biAb than from mice treated with the CTL only. In animals treated with CTL plus both biAbs, the primary tumor did not grow, and none of the mice developed metastases. Thus, this set of bi-specific antibody fragments was proved to target effector cells in the tumor-bearing host and to efficiently support in vivo clonal expansion and cytolytic activity of in vitro-generated CTL.