Functional analysis of mononuclear cells infiltrating into tumors: lysis of autologous human tumor cells by cultured infiltrating lymphocytes

Lymphocytes separated from surgically resected tumor tissue, uninvolved lung tissue, and peripheral blood of lung cancer patients were investigated for cytotoxic potential and analyzed for their phenotypes at the time of surgery and after having been propagated for 4 to 5 wk in the presence of interleukin-2. Most of the tumor lymphocyte infiltrates examined were shown to have a shift in favor of T8 subsets from those found in peripheral blood. No natural killer activity and low cytotoxicity against the autologous tumor were found to characterize the tumor-derived lymphocyte population. Propagation of lymphocytes from the different tissues of the cancer patient in the presence of interleukin-2 preparation induced widespread lytic activity against K562 cells, autologous and allogeneic tumors, but not autologous normal lung or lymphoblasts. However, cytotoxic activity against autologous tumor cells exerted by cultured tumor-infiltrating lymphocytes was found to be significantly higher than the activity of cultured lymphocytes isolated from peripheral blood or uninvolved lung tissue of the same patient. The elevated lytic activity of cells derived from the tumor tissue indicates the accumulation at the tumor site of precursors of natural killer-like cells and specifically stimulated antitumor effectors. Our results suggest the coexistence of two types of anti-autotumor cytotoxic lymphocytes at the tumor site: natural killer-like and specific cytotoxic T-cells.     

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.     

Immunosensitization of resistant human tumor cells to cytotoxicity by tumor infiltrating lymphocytes

Most anti-cancer therapies induce apoptotic cell death, but a major barrier to long-term cancer treatments is the generation of apoptosis-resistant tumor cells. Tumor cells that become resistant to one therapy are usually cross-resistant to subsequent therapies, including those with different cellular/molecular targets, suggesting that resistant tumor cells acquire modifications of the general apoptotic pathway. Most solid tumors are characterized by infiltration of lymphocytes (tumor infiltrating lymphocytes, TIL), which may serve as a basis for new strategies to generate tumor specific lymphocytes. However, TIL frequently are unable to kill autologous tumor cells suggesting that they are anergic/tolerant. It is possible that the TIL are functional but the tumor cells are resistant to TIL-mediated apoptotic pathways. Previous findings revealed that resistant tumor cells can be sensitized with cytokines or subtoxic concentrations of chemotherapeutic drugs and restore killing by cytotoxic lymphocytes. In this study, we examined whether TIL can kill autologous and allogeneic tumor cells following sensitization with chemotherapeutic drugs. Renal and prostate cancer-derived TIL were cytotoxic to chemosensitized resistant tumor cells. Killing by TIL was found to be perforin-dependent and perforin-independent. These findings demonstrate that combination drug and immunotherapy may be able to overcome tumor cell resistance to killing by TIL. Further, in vivo sensitization of drug-resistant tumor cells by subtoxic doses of sensitizing chemotherapeutic drugs may result in tumor regression by the host immune system.     

树突细胞对肿瘤浸润淋巴细胞抗乳腺癌免疫活性影响的研究

背景与目的:树突细胞(dendriticcell,DC)又称树突状细胞,是目前已知的功能最强的抗原呈递细胞,它可以在体内、外向T淋巴细胞呈递抗原,并诱发细胞毒T淋巴细胞(cytotoxicTlymphocyte,CTL)反应。本研究旨在探讨DC激活的肿瘤浸润淋巴细胞(tumorinfiltratinglymphocytes,TIL)体外对乳腺癌细胞的杀伤活性。方法:从乳腺癌患者外周血获取DC,应用粒/巨噬细胞集落刺激因子(granulocyte/macrophagecolonystimulatingfactor,GM-CSF)、白细胞介素-4(interleukin-4,IL-4)和肿瘤抗原激活DC,然后用DC激活TIL,观察TIL在体外对自体乳腺癌细胞和Bcap-37乳腺癌细胞的杀伤活性。结果:DC激活的TIL对自体乳腺癌细胞具有很强的杀伤活性,杀伤率为(85.76±2.93)%,明显高于未经DC激活的TIL、DC激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体乳腺癌细胞的杀伤率犤分别为(52.11±1.48)%、(51.35±1.46)%和(3.59±0.25)%犦。而它们对Bcap-37乳腺癌细胞的杀伤活性则相对较低犤分别为(40.03±1.29)%、(22.09±0.87)%、(21.66±0.85)%和(1.76±0.14)%犦。结论:乳腺癌患者外周血DC能诱导TIL产生高效而特异的抗乳腺癌免疫活性。     

树突状细胞对肿瘤浸润性淋巴细胞杀伤自体肝癌细胞活性影响的研究

目的　探讨树突状细胞 (DC)激活的肿瘤浸润性淋巴细胞 (TIL )体外对自体肝癌细胞杀伤活性。方法　从肝癌患者外周血获取 DC,应用粒 /巨噬细胞集落刺激因子 (GM- CSF)、白细胞介素 - 4 (IL - 4 )和肿瘤抗原激活 DC,然后用 DC激活 TIL ,观察 TIL在体外对自体肝癌细胞的杀伤活性。结果　 DC激活的 TIL对自体肝癌细胞具有很高的杀伤活性 ,杀伤率为 (89.39± 3.0 5 ) % ,明显高于未经 DC激活的 TIL、DC激活的 T淋巴细胞和未经 DC激活的 T淋巴细胞对自体肝癌细胞的杀伤率。其杀伤率分别为 (5 5 .2 3± 1.5 3) %、(5 4 .89± 1.4 8) %和 (3.6 5± 0 .2 6 ) %。结论　肝癌患者外周血 DC能诱导 TIL产生高效而特异的抗肝癌免疫     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and tumor-infiltrating lymphocytes activated by PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10-100 micrograms/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN) alpha or IFN gamma. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with alpha-chain or beta-chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractionation experiments revealed that CD3-CD16+ large granular lymphocytes (LGL) and/or CD3+CD16- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.     

Enhanced susceptibility of adriamycin-treated human renal cell carcinoma cells to lysis by peripheral blood lymphocytes and tumor infiltrating lymphocytes

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P<0.05). This ADR-induced enhancement of susceptibility of RCC cells to lysis by PBL was also observed when freshly derived TIL were used as effector cells (P<0.05). ADR up-regulated the expression of leukocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1), which are critical in the binding and killing of CTL against cancer cells. Of the five fresh RCC cell cultures treated with ADR, LFA-3 was increased in all and ICAM-1 was increased in three of them, respectively (P<0.05). Up-regulation of LFA-3 and ICAM-1 was also observed in ACHN cells treated with two derivatives of ADR, epirubicin and pirarubicin. ADR further significantly increased the bindings of PBL to RCC cells (P<0.05). These findings suggest that treatment of RCC patients with low doses of ADR may sensitize the RCC cells to killing by PBL and TIL and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant RCC. The inducing of LFA-3 and ICAM-1 by ADR may be involved in the enhancement of susceptibility of PBL and TIL-mediated cytolysis in human RCC cells.     

肿瘤浸润淋巴细胞的体外培养及临床应用研究

目的:研究肺癌患者胸腔积液中肿瘤浸润淋巴细胞(tumorinfiltratinglymphocytes,TIL)对瘤细胞的杀伤活性及其表型变化,并对其回输后毒副反应及患者免疫功能进行观察。方法:应用贴壁法分离恶性胸腔积液中TIL,并经rIL2诱导培养;用间接免疫荧光法测定CD3+、CD4+和CD8+比例;应用3HTdR释放法测定TIL对自体瘤细胞和801D细胞的杀伤作用;用活化的自体TIL注入患者胸腔内,观察胸腔积液变化。结果:第21天时CD3+和CD8+比例明显提高,而CD4+比例和CD4+/CD8+比值变化不大;对自体瘤细胞和801D细胞的杀伤力明显提高;40例自体回输治疗胸腔积液的患者,有效率为72.5%,且一般状况好,毒副反应小,辅助检查无异常改变,免疫功能增强。结论:肺癌TIL过继免疫治疗对恶性胸腔积液患者是一种安全、有效的免疫治疗方法。     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and pleural effusion lymphocytes activated by OK432

Lymphocytes from peripheral blood (PBL) and from pleural effusions (PEL) of cancer patients were tested for cytotoxicity against tumor cells freshly isolated from carcinomatous pleural effusion of the same patient. Significant lysis of autologous tumor cells was recorded for 4 of 28 PBL samples and for 5 of 28 PEL cases when investigated in a 4-hour 51Cr release assay. In vitro treatment of lymphocytes for 20 hours with the streptococcal preparation OK432 resulted in an induction or augmentation of cytotoxicity against autologous tumor cells in 21 of 28 PBL and PEL specimens. OK432-induced cytotoxicity required active cell metabolism, RNA and protein syntheses, but not DNA synthesis of lymphocytes. Supernatants of OK432-stimulated lymphocytes, and interferon and interleukin 2 failed to induce autologous tumor killing. Nylon wool-nonadherent lymphocytes were involved in both spontaneous and OK432-induced lysis of fresh autologous tumor cells. OK432-activated lymphocytes from normal donors and cancer patients caused lysis of fresh allogeneic tumor cells and also K562 cells.     

Lysis of fresh human tumor cells by autologous large granular lymphocytes and T-lymphocytes: two distinct killing activities induced by coculture with autologous tumor

The specific and natural killer (NK)-restricted nature of autologous tumor killing by blood lymphocytes was studied in patients with carcinomatous pleural effusions. Large granular lymphocytes (LGL) and small T-lymphocytes were isolated by centrifugation on discontinuous Percoll density gradients. Tumor cells freshly isolated from pleural effusions of cancer patients were classified according to their susceptibility to purified LGL from normal donors in a 4-hour 51Cr release assay. Of 15 NK-sensitive tumors, 14 were lysed by fresh autologous LGL, whereas only 2 were killed by T-cells. Neither LGL nor T-cells were cytotoxic to NK-resistant autologous tumor. LGL and T-cells were then cultured in vitro with autologous tumor cells for 6 days. In 13 of 15 autologous mixed lymphocyte-tumor cultures (MLTC) NK-sensitive tumor-cultured LGL maintained their autotumor killing activity, whereas LGL cultured alone lost the activity. Depletion of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL resulted in an enrichment of effector cells. LGL from autologous MLTC were able to kill NK-susceptible allogeneic effusion tumor and K562 as were fresh LGL. No lysis of NK-resistant autologous tumor was observed with cultured LGL. In contrast, activation of T-cells in autologous MLTC resulted in the generation of autotumor killer cells in 10 of 15 NK-sensitive and 3 of 6 NK-resistant tumor samples. However, cultured T-cells were incapable of killing allogeneic tumor and K562. In autologous MLTC T-cells proliferated in response to autologous tumor, whereas no proliferation was observed in the culture of LGL. The enrichment of blasts from cultured T-cells on discontinuous Percoll gradients induced an augmentation of autotumor cytotoxicity, with no reactivity in blast-depleted, small, resting T-lymphocytes. These results indicated that 2 distinct types of autotumor-recognizing lymphocytes, LGL and T-cells, are present in the peripheral blood of cancer patients.     

Ligand-mediated cytolysis of tumor cells: use of heregulin-zeta chimeras to redirect cytotoxic T lymphocytes

New specificities may be engrafted onto lymphocytes by the transfer of genes for chimeric receptors that combine antigen recognition and signal-transducing elements. We have engineered and evaluated a new class of chimeric receptors that use the natural ligands of receptors found to be frequently overexpressed by cancer cells. The heregulin molecule, a ligand for Her3 and Her4 receptors when fused with the CD3 zeta-chain, was capable of redirecting T lymphocytes to recognize and respond to cancer cell lines that overexpress these receptors. Thus, CD8+ T lymphocytes were isolated from a healthy individual and transduced to express the chimeric heregulin-zeta receptor. These modified effector cells acquired the ability to specifically lyse a breast cancer cell line that overexpresses Her3 and Her4. A new class of chimeric receptors, such as heregulin-zeta, endowing anti-cancer effector cells with the potential to recognize and eliminate tumor targets, are likely to increase the effectiveness of adoptive immunotherapy for the treatment of cancer.     

Immunologic character of tumor infiltrating lymphocytes in ovarian carcinoma

Tumor-infiltratinglymphocytes(TIL)wasdirectlyisolatedfrompatient'stumortissues.ByrIL2activationandexpansioninvitro,TILwasagainimportedintothesamepatient'sbodytotreattumor,withapparenteffectsofanti-tumorandcomparativelessside-effect,withoutkillingothertumorcellsandnormalcells,allofwhichhavemadeitaeffectivewayoftreatingtumoratanadvancedstage.Inrecentyears,someresearchersfoundthattheeffectsofTIL'treatmentwerenotsofarapparent,withcomparativelyapparentdifference,whichmightbecloselyrelatedtothef…     

肿瘤浸润淋巴细胞的抗肿瘤免疫困境

体外肿瘤浸润淋巴细胞(TIL)具有一定的抗瘤活性,过继免疫治疗却未表现出理想效果,研究显示恶性肿瘤患者免疫系统处于系统性缺陷或耐受状态.在诱发阶段树突状细胞(DC)诱导TIL发生免疫偏倚,有的则因DC严重不足或状态不佳使TIL几无功能.肿瘤细胞的生理变化及肿瘤局部微环境也使TIL的杀伤活性受到严重抑制,甚至被反杀伤.体内TIL自身变化同样造成抗瘤免疫力低下.因此TIL的抗瘤免疫受到多方面困扰.     

Immunophenotypic features of tumor infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates

Background  

The immune system plays an important role in the multifactorial biologic system during the development of neoplasias. However, the involvement of the inflammatory response in the promotion/control of malignant cells is still controversial, and the cell subsets and the mechanisms involved are poorly investigated. The goal of this study was to characterize the clinical-pathological status and the immunophenotyping profile of tumor infiltrating lymphocytes and their association with the animal survival rates in canine mammary carcinomas.     

肿瘤浸润淋巴细胞与lncRNA在肿瘤发展中作用的研究进展

肿瘤浸润淋巴细胞在肿瘤微环境中扮演着重要的角色,不同的细胞亚型在肿瘤的进展中发挥抗肿瘤或促肿瘤作用.肿瘤浸润淋巴细胞主要包括T淋巴细胞、B淋巴细胞和自然杀伤细胞,这些细胞可在多种因素的刺激下发生活化成熟及产生效应,参与肿瘤的免疫应答.长链非编码RNA(long non-codingRNA)是一类多效的调控因子,可参与机...     

Adoptive immunotherapy using autologous lymphocytes sensitized with HLA class I-matched allogeneic tumor cells

A 29-year-old female breast cancer patient with multiple bone metastases (HLA-A2) was treated with adoptive transfer using autologous peripheral blood mononuclear cells (PBMCs) activated with the HLA-A2-matched allogeneic GC022588 gastric cancer cell line and interleukin-2 plus an immobilized anti-CD3 antibody culture system. The relief of bone pain in parallel with a decrease of serum carcinoembryonic antigen levels was obtained just after the administration of GC022588-activated effector lymphocytes, and a good quality of life was accomplished for 4 months. The GC022588-activated effector lymphocytes included 44% CD4+, 77% CD8+, and 26% CD4+CD8+ phenotypes, and expressed 25% killing activity against GC022588 stimulator cells at an E/T ratio of 50:1. T cell receptor (TCR) usage analysis for the effector cells showed oligoclonal expression of TCRVbeta1, 3, 9, and 11, especially TCRVbeta5.2, 12, 13.1 and 17, and their killing activity was significantly inhibited in the presence of anti-TCRalphabeta antibody and anti-TCRVbeta12 antibody. SSCP analysis revealed clonotypic bands of TCRVbeta12. These results suggest that shared antigens exist between breast and gastric adenocarcinomas. Allogeneic tumor cells can stimulate PBMCs to generate effector cells with selected TCRCDR3 usages that recognize tumor antigens. These effector lymphocytes may be good candidates for the adoptive immunotherapy of cancer.