Suppression of Anti-tumor CD4+ T Cell Responsiveness in the Tumor-bearing State and Its Recovery in in vitro Culture Free of Tumor Burden

We investigated whether the responsiveness of anti-tumor CD4⁺ T cells suppressed in the tumor-bearing state is reversed in conditions free of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and IL-4 upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4⁺ T cells and antigen-presenting cells (APC) that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. However, spleen cells from late (8–10 wk) tumor-bearing stages produced reduced levels of lymphokine production despite the presence of comparable proportions of CD4⁺ T cells. Because APC in these cell populations exhibited enhanced capacities to present tumor antigens, reduced responsiveness was ascribed to the dysfunction of CD4⁺ T cells themselves. When spleen cells from early tumor-bearing mice were preincubated for 1–2 days and recultured in fresh medium, the magnitude of lymphokine production by these cells was not changed. In contrast, the same protocol of preincubation and reculture for cells from late tumor-bearing mice resulted in the recovery of anti-tumor lymphokine-producing capacity. The recovered capacity was comparable to or slightly higher than that expressed by cells from early tumor-bearing stages. Since the CD4⁺ T cell content did not significantly differ before and after preincubation, enhanced lymphokine production was due to the recovered responsiveness of anti-tumor CD4⁺ helper T cells. The recovery of anti-tumor responsiveness was also induced in vivo by tumor removal at the late tumor-bearing stage: spleen cells from mice 2–4 wk after tumor resection efficiently produced IL-2 and IL-4. These results indicate that the immunodysfunction of anti-tumor CD4⁺ T cells induced in the tumor-bearing state is reversible because release from tumor burden either by preincubation in vitro or by tumor removal in vivo results in almost complete recovery of the potent anti-tumor responsiveness initially expressed.     

Mediation of in vivo Tumor-neutralizing Activity by Lyt-2+ as Well as L3T4+ T Cell Subsets*1

The present study reexamines the cell surface nature of T cells mediating in vivo protective tumor immunity with the use of anti-L3T4 and -Lyt-2 antibodies. C3H/HeN mice hyperimmune against syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated challenges with viable tumor cells. Spleen cells from these mice were fractionated into L3T4⁺ or Lyt-2⁺ T cell subset by treatment with anti-Lyt-2 or -L3T4 antibody plus complement (C). Winn assays performed by utilizing such fractionated T cells have revealed that both L3T4⁺ and Lyt-2⁺ T cell subsets from hyperimmune mice produced complete tumor protection. Flow microfluorometry study illustrated that the treatment with anti-L3T4 or -Lyt-2 antibody plus C resulted in the complete isolation of L3T4^? Lyt-2⁺ (Lyt-2⁺) or L3T4⁺ Lyt-2^? (L3T4⁺) T cell subset, respectively. This contrasted with the failure of treatment with anti-Lyt-1 antibody plus C to isolate all T cells expressing Lyt-2 marker. It was further demonstrated that each subset of T cells exerted its anti-tumor effect in a tumor-specific way and without a requirement for the other alternative subpopulation of unprimed T cells. These results indicate that Lyt-2⁺ T cell subset can be successfully isolated by treatment with anti-L3T4 but not with anti-Lyt-I antibody plus C, and that each single subset of Lyt-2⁺ and L3T4⁺ T cells can function as in vivo effector T cells.     

Transforming Growth Factor-β CD4+ T cells Tumor-bearing state Immunosuppression Enhanced Production of TGF-β and a Progressive Increase in TGF-β Susceptibility of Anti-tumor CD4+ T Cell Function

The present study deals with the effect of transforming growth factor-β (TGF-β) on anti-tumor immune responsiveness at various stages of the tumor-bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and macrophage-activating factor (MAF)/interferon-γ (IFN-γ) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4⁺ T cells and antigen-presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The IL-2-producing capacity of CD4⁺ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor-bearing stages. In contrast, the capacity of CD4⁺ T cells to produce MAF/IFN-γ was not affected but was maintained at high levels even late in the tumor-bearing state. The addition of recombinant TGF-β (rTGF-β) to cultures of spleen cells from various tumor-bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF-β-induced suppression varied depending on which tumor-bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1–3 wk) tumor-bearing stages but increased in cells from donor mice at later tumor-bearing stages. Thus, spleen cells from late tumor-bearing stages with weak but significant IL-2-producing and considerable MAF/IFN-γ producing capacities failed to produce these lymphokines when rTGF-β was present in cultures. A progressive increase in the TGF-β susceptibility was also observed for IL-4-producing Th2 as well as IL-2/MAF-producing Th1 cells. In addition, increased levels of TGF-β were detected in plasma from tumor-bearing mice at late stages. Taken together, these results indicate that tumor-bearing mice exhibit enhanced production of TGF-β as well as a progressive increase in the susceptibility of anti-tumor CD4⁺ T cells to TGF-β-induced suppressive mechanisms.     

Mechanisms for Recognition of Tumor Antigens and Mediation of Anti-tumor Effect by Noncytolytic Lyt-2+ T Cell Subset*1

The mode of anti-tumor function in vivo of noncytolytic Lyt-2⁺ T cells from C3H/He mice hyperimmune to syngeneic MH134 hepatoma was investigated in a double diffusion chamber system which was recently established in our laboratory. C3H/He mice were implanted intraperitoneally with the double diffusion chamber unit in which each chamber contained either L3T4⁺ T cell-depleted MH134-hyperimmune spleen cells plus mitomycin C-treated MH134 tumor cells or other syngeneic X5563 viable tumor cells plus normal spleen cells as a source of macrophages. Inclusion of anti-MH134 Lyt-2⁺ T cells together with MH134 tumor cells in one chamber resulted in comparable growth inhibition of viable X5563 tumor cells in the other chamber to that obtained by unfractionated MH134-hyperimmune spleen cells. The induction in the Lyt-2⁺ T cell-containing chamber of anti-tumor effect to be delivered into the other chamber was dependent on the co-existence of la-positive adherent cells along with Lyt-2⁺ T cells. Although adherent cell-depleted Lyt-2⁺ T cells regained the inducibility of anti-tumor immunity when supplemented with splenic adherent cells, the addition of adherent cells pretreated with chloroquine failed to restore the ability of Lyt-2⁺ T cells to induce their anti-tumor effect. In addition, paraformaldehyde-treated MH134 tumor cells instead of untreated tumor cells were not capable of activating Lyt-2⁺ T cells. These results indicate that a portion of Lyt-2⁺ T cells exerts their anti-tumor effect by a mechanism distinct from direct tumor cell lysis and that their activation for mediation of this type of tumor immunity requires the recognition of tumor antigens processed and presented by la-positive adherent cells.     

Cytotoxic Activity of CD4+ T Cells against Autologous Tumor Cells

The ⁵¹Cr-release assay is mostly applied to detecting the cytotoxic activity of CD8⁺ T cells, and little is known about the activity of CD4⁺ T cells. Therefore, the correlation between the cytotoxic activity of CD4⁺ or CD8⁺ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the ⁵¹Cr-release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4⁺ and CD8⁺ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin-2 and immobilized anti-CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4⁺ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4⁺ T cells were increased 67-fold in comparison with the activities in 4 h assay (range: 5–197). In the case of CD8⁺ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4⁺ and CD8⁺ T cells was calculated as 1.5 in the 20 h assay (range: 0.2->7.2) versus 0.4 in the 4 h assay (range: < 0.1–1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h ⁵¹Cr-release assay in all eight cases. These results indicate that CD4⁺ T cells have cytotoxic activity as strong as that of CD8⁺ T cells towards autologous tumor cells.     

Bispecific Antibody-directed Antitumor Activity of Human CD4+ Helper/Killer T Cells Induced by Anti–CD3 Monoclonal Antibody plus Interleukin 2

Freshly isolated human CD4⁺ T cells can not respond to recombinant interlenkin 2 (rIL–2) because of their lack of p75 IL–2 receptor expression. However, we succeeded In inducing a marked proliferation of purified CD4⁺ T cells by activation with rIL–2 plus anti–CD3 monoclonal antibody (mAb) cross–linked to a plastic plate. The proliferated CD4⁺ T cells produced a significant amount of IL–2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4⁺ T cells activated with anti–CD3 mAb plus rIL–2 revealed a strong cytotoxic activity against Fc receptor (FcR)–positive tumor cells in the presence of anti–CD3 mAb. Moreover, the CD4⁺ T cells could lyse FcR–negative glioma cells by targeting with bispecific mAb containing anti–CD3 mAb and anti–glioma mAb. Thus, we demonstrated that rIL–2 and immobilized anti–CD3 mAb allowed the rapid generation of human CD4⁺ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Macrophage–T Cell Interaction Is Essential for the Induction of p75 Interleukin 2 (IL–2) Receptor and IL–2 Responsiveness in Human CD4+ T Cells

Fresh human CD8⁺ T cells showed a strong proliferative response to a high concentration of interleukin 2 (IL–2) in the absence of macrophages. In contrast, CD4⁺ T cells revealed no significant IL–2 responsiveness in the absence of macrophages. However, if CD4⁺ T cells were cocultured with macrophages, they showed higher proliferative response to IL–2 than CD8⁺ T cells. In accordance with the magnitude of IL–2 responsiveness, freshly isolated CD8⁺ T cells expressed significant amounts of p75 IL–2 receptor, while fresh CD4⁺ T cells did not express p75 IL–2 receptor. The expression of p75 IL–2 receptor on CD4⁺ T cells was induced by coculture with macrophages. The macrophage–induced p75 IL–2 receptor acquisition was blocked by monoclonal antibody (inAh) against class II antigen. Moreover, the addition of anti–CD4 mAb or anti–class II mAb to the culture caused a great inhibition of IL–2 responsiveness of CD4⁺ T cells. These results strongly suggest that macrophage–T cell interaction through CD4 and/or class II molecules is essential for the expression of p75 IL–2 receptor and IL–2 responsiveness in human CD4⁺, but not CD8⁺ T cells.     

CIK细胞中CD4+T细胞亚群抗肿瘤免疫活性

目的：观察CIK细胞中CD4+T细胞亚群抗肿瘤免疫活性。方法：体外大规模扩增CIK细胞，利用磁珠分离系统富集纯化CIK细胞中的CD4+T细胞亚群。采用胞内染色法分析其中Th1/Th2的比例变化； LDH法和荧光染色法比较4 h和20 h其对raji细胞的杀伤率和raji凋亡。结果：经磁珠分离法富集的CD4+CIK细胞纯度高达96%， 其中Th1/Th2的分布较PBMC有显著的改变：Th1亚群、Th0亚群明显升高，Th2亚群无显著变化。CD4+CIK细胞虽然不能在4 h之内溶解raji细胞，但可在20 h时产生同CD4-CIK细胞同样强大的杀伤活性，荧光染色可见其在4 h之内诱导raji出现早期凋亡的迹象。结论：本研究提示CD4+CIK细胞具有明显的“Th1优势”可以调节宿主免疫细胞活性；同时CD4+CIK细胞可通过诱导肿瘤细胞凋亡实现对肿瘤的抑制和杀伤。     

Natural Antigenic Peptides from Squamous Cell Carcinoma Recognized by Autologous HLA-DR8–restricted CD4+ T Cells

A large number of human tumor antigens recognized by CD8⁺ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4⁺ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC–20, was effectively lysed by HLA-DRB1·08032 (HLA-DRS)-restricted autologous CD4⁺ T cell line, TcOSC–20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC–20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321–336 of human a-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4⁺ T cells.     

Cytotoxicity of Histocompatibility Leukocyte Antigen–DR8–restricted CD4+ Killer T Cells against Human Autologous Squamous Cell Carcinoma

Although CD8⁺ killer T cells reacting against human autologous tumor cells have recently been studied in detail, little is known about the cytotoxic mechanism of CD4⁺ T cells against such tumor cells. In order to investigate this, we have established CD4⁺ cytotoxic T lymphocyte TcOSC–20 lines. TcOSC–20 showed selective cytotoxic activity against autologous OSC–20 cells, derived from a cancer of the tongue, in an HLA–DR–restricted fashion. HLA–DR8 (DRB1* 08032) is the only DR molecule expressed on OSC–20 cells, and anti–DRS monoclonal antibody could inhibit the Cytotoxicity, suggesting that HLA–DRB1^★ 08032 is the tumor rejection antigen–presenting moleculeto TcOSC–20. The Fas ligand was expressed on TcOSC–20 lines, and its expression was induced upon mixed lymphocyte–tumor cell culture of autologous peripheral blood lymphocytes. Furthermore, the Cytotoxicity of TcOSC–20 was inhibited by anti–Fas ligand antibody.These data imply that TcOSC–20 lines recognize the tumor antigenic peptide presented by HLA–DR8, and exert Cytotoxicity against autologous tumor cells via a Fas–mediated cytotoxic pathway.     

T Cell Response to Embryonal Carcinoma F9 Cells: Induction and Characterization of T Cell Receptor αβ+ Double-negative Cytotoxic T Lymphocytes

We investigated the mechanism of T cell response to marine embryonal carcinoma F9 cells. Thy-1⁺, CD4⁻, CD8⁻ (double-negative) cytotoxic effector cells were induced in spleen cells obtained from immune A.BY mice to F9 cells, and the cytotoxic activity was major histocompatibility complex (MHC)-unrestricted. Furthermore, CD4⁺ T cells were essential for the induction of double-negative cytotoxic T lymphocytes directed to F9 cells. Most of the double-negative cytotoxic T lymphocyte lines obtained by long-term culture of the effector cells had CD3 molecule and T-cell receptor β chain on their cell surface, and the CDS molecule was found to be involved in target cell recognition. The T cell receptor αβ⁺ double-negative cytotoxic T lymphocyte line (2A5) also lysed various tumor cells in a non-MHC-restricted manner, but did not lyse concanavalin A-stimulated blasts of 129 strain, from which F9 cells had originated. These results indicate that T cell receptor αβ⁺ double-negative cytotoxic T lymphocytes induced by F9 cells recognize a common antigen(s) expressed on F9 cells and other tumor cells but not minor histocompatibility antigens.     

调节性T细胞在口腔鳞癌患者外周血及癌组织中的表达和意义

目的 探讨CD4+CD25+Foxp3+调节性T细胞（regulatory T cell,Treg）在口腔鳞癌患者外周血及癌组织中的表达和意义。方法 选取初诊的30例口腔鳞癌患者,按临床分期分为A、B两组,A组为Ⅰ、Ⅱ期患者,B组为Ⅲ、Ⅳ期患者,10例健康志愿者为对照组（C组）。分别采集三组受试对象晨起空腹外周血3 ml,采用流式细胞术测定外周血中CD4+CD25+Foxp3+调节性T细胞、CD80、CD86和HLA-DR的表达情况;取试验组及对照组手术标本,利用免疫组织化学法测定Foxp3+调节性T细胞的浸润情况。结果 CD4+CD25+Foxp3+T淋巴细胞占CD4+ T淋巴细胞比例在三组中分别为（2.80±0.90）%(A组)、（5.09±1.72）%（B组）、（0.57±0.15）%（C组） , 组间比较差异具有统计学意义（P=0.000）。A、B组外周血中单个核细胞表面共刺激分子CD80+CD86+、 CD80+HLA-DR+及CD86+HLADR+细胞的表达明显低于C组;CD4+CD25+Foxp3+调节性T细胞所占比例与CD80+CD86+、CD80+HLA-DR+和CD86+HLA-DR+表达率进行直线相关分析,相关系数分别为-0.512、-0.430、-0.461,均有统计学意义。免疫组织化学结果显示,Foxp3在口腔鳞癌组织中主要分布在黏膜固有层肿瘤间质,其中Foxp3+调节性T细胞在高分化和中分化口腔鳞癌中少,在低分化口腔鳞癌中多,两者间差异有统计学意义。结论 检测口腔癌患者外周血和肿瘤组织中CD4+CD25+Foxp3+调节性T胞,有助于对机体抗肿瘤免疫水平、病变进展情况和肿瘤预后进行评估,为临床生物治疗提供佐证。     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.     

CD4+T细胞在抗肿瘤过继性免疫治疗中作用的研究

目的：观察CIK细胞中CD4^＋T细胞中Th1／Th2亚群分布情况。方法：体外大规模扩增CIK细胞，磁珠法纯化其中CD4^＋T细胞．采用胞内染色、ELISA和荧光定量RT—PCR法检测培养前后Th1／Th2细胞亚群的比例、Th1／Th2类细胞因子的分泌量和基因表达的变化。结果：富集CD4^＋细胞纯度达96％，CIK中Th1亚群和Th0亚群的比例较PBMC显著升高（P〈0．05），而Th2亚群无显著变化。CD4^＋CIK细胞中IL-2和IFN-γ等Th1类细胞因子释放量增高（P〈0．01），而IL－10和TGF-β等Th2类细胞因子释放量降低（P〈0．05）。但CD4^＋CIK细胞中除IFN-γ／的mRNA显著升高外（P〈0．05）．IL-2和IL-4的mRNA变化不明显。结论：CIK细胞中的CD4^＋T细胞具有明显的Th1优势。     

Direct Activation of Human CD8+ Cytotoxic T Lymphocytes by Interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8⁺ T cells present after 28 days in the induction culture. When purified CD8⁺ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8⁺ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

Selective suppression of the generation of anti-tumor L3T4+ but not of Lyt-2+ T cell-mediated immunity in the tumor-bearing state

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2+ and L3T4+ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2+ and L3T4+ T subsets from hyperimmune mice, only the Lyt-2+ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4+ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4+ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2+ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4+ but not of Lyt-2+ T cell function in the tumor-bearing state.     

CD4+CD25+调节性T细胞与CD4+T、CD8+T细胞在结直肠癌组织中的分布

 目的 分析CD4+CD25+ FOXP3+调节性T细胞（Treg）与CD4+T、CD8+T在结直肠癌（colorectal carcinoma, CRC）组织中的分布及其与临床病理特征之间的关系。方法 收集42例CRC新鲜手术标本,应用冰冻切片、免疫组织化学SP法检测肿瘤组织和癌旁组织中FOXP3+、CD4+T和CD8+T阳性细胞数。结果 CRC患者肿瘤组织中FOXP3表达水平显著升高,与癌旁组织相比差异有统计学意义（P<0.01）;中低分化组Treg细胞数明显高于高分化组（P<0.01）;淋巴结转移组Treg细胞数明显高于无淋巴结转移组（P<0.05）;癌巢内CD4+、CD8+T细胞数及CD4+/CD8+值显著低于间质（P<0.01）;Ⅲ+Ⅳ期、淋巴结转移组癌巢内CD4+/CD8+比值显著低于Ⅰ+Ⅱ期及无淋巴结转移组（P<0.05）;CRC中Treg数量与癌巢内CD4+/CD8+比值显著负相关（r=-0.605, P<0.01）。结论 CRC的发生发展可能与其癌组织局部微环境中Treg数量变化相关,肿瘤局部Treg数量的增多与T淋巴细胞亚群比例失调可能成为肿瘤免疫逃逸的机制之一。     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

Cryptotanshinone Induces Inhibition of Breast Tumor Growth by Cytotoxic CD4+ T Cells through the JAK2/STAT4/ Perforin Pathway

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salviamiotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibittumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Usinga mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo withpolarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation,and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes.Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogatedby concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumorgrowth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggestedthat CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin.We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, anddemonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.     

Expression of Fas/FasL in CD8+ T and CD3+ Foxp3+ Treg Cells - Relationship with Apoptosis of Circulating CD8+ T Cells in Hepatocellular Carcinoma Patients

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, includinglymphocyte apoptosis. Several studies showed that Foxp3+T cells take part in inducing this process by expressingFasL in tumor patients. However, the relationship between apoptosis, CD8+T cells and Foxp3+T cells in HCCpatients is still unclear. The present study was designed to investigate the correlation between apoptosis levelsand Fas/FasL expression in CD8+T lymphocytes and Foxp3+T cells in patients with HCC. Methods: CD8+T cellsand CD3+Foxp3+T cells were tested from peripheral blood of HCC patients and normal controls and subjectedto multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas inCD8+T cells and FasL in CD3+Foxp3+T cells were evaluated. Serum TGF-β1 levels in patients with HCC weremeasured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, aswell as FasL expression in CD3+Foxp3+T cells was then evaluated. Results: The frequency of CD8+T cells bindingannexin V and Fas expression in CD8+T cells, were all higher in HCC patients than normal controls and theproportion of apoptotic CD8+T cells correlated with their Fas expression. Serum TGF-β1 levels correlated inverselywith CD3+Foxp3+T cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of CD8+T cellsand reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis inductionin Fas+CD8+T cells in vitro are required.