结肠癌患者自体瘤细胞对不同来源淋巴细胞功能的影响

目的：研究结肠癌患者自体瘤细胞对肿瘤浸润淋巴细胞、肿瘤引流淋巴结的淋巴细胞、外周血淋巴细胞的影响。方法：比较结肠癌患者自体肿瘤细胞对体外培养TIL、PBL、LNL细胞增殖、诱生细胞因子（IL－2、TNFα、IFNγ）水平的影响。结果：发现结肠癌患者的TIL和PBL、LNL相比，CD8^ 细胞增加，CD4^ 细胞减少。PBL和LNL对PHA、rhIL-2刺激产生的细胞增殖能力比TIL强。PBL能产生较高水平的TNFα和IFNγ，而IL－2产生水平较低；LNL产生高水平的TNFα和IL－2，但仅产生低水平的IFNγ。TIL产生的TNFα、IFNγ水平较PBL、LNL低。实验结果表明结肠癌患者自体肿瘤细胞对不同来源淋巴细胞的细胞因子分泌有不同影响，其机制有待进一步研究。     

结肠直肠癌患者PBL、LNL、TIL免疫功能的比较研究

目的 :研究结肠直肠癌患者外周血淋巴细胞、肿瘤浸润淋巴细胞、肿瘤引流淋巴结淋巴细胞的免疫学功能 ,为研究结肠直肠癌患者的过继性细胞免疫治疗积累实验资料。方法 :比较结肠直肠癌患者不同来源淋巴细胞的细胞表型、NK活性、LAK活性、特异性细胞毒活性、体外增殖反应等特征。结果 :结肠直肠癌患者PBL、LNL、TIL在免疫功能上有较大差别。从结肠直肠癌患者肿瘤原位新鲜分离的LNL和TIL的NK活性、LAK活性和特异性细胞毒活性均明显低于PBL。PBL中含有较多的NK细胞 ,LNL中含有较多的B细胞和CD4+ 细胞 ,PBL和无肿瘤转移的LNL的细胞增殖能力明显高于TIL和有肿瘤转移的LNL。结论 :一般情况较好的结肠直肠癌患者外周血淋巴细胞的免疫学指标和正常人PBL相比基本正常。     

结肠直肠癌患者PBL、LNL、TIL免疫功能的比较研究

目的：研究结肠直肠癌患者外周血淋巴细胞、肿瘤浸润淋巴细胞、肿瘤引流淋巴结淋巴细胞的免疫学功能,为研究结肠直肠癌患者的过继性细胞免疫治疗积累实验资料。方法：结肠直肠癌患者不同来源淋巴细胞的细胞表型、NK活性、LAK活性、特异性细胞毒活性、体外增殖反应等特征。结果：结肠直肠癌患者PBL、LNL、TIL在免疫功能上有较大差别。从结肠直肠癌患者肿瘤原位新鲜分离的KLNL和TIL的NK活性、LAK活性和特异性细胞毒活性均明显低于PBL。PBL中含有较多的NK细胞,LNL中含有较多的B细胞和CD4^ 细胞,PBL和无肿瘤转移的LNL的细胞增殖能力明显高于TIL和有肿瘤转移的LNL。结论：一般情况较好的结肠直肠癌患者外周血淋巴细胞的免疫学指标和正常人PBL相比基本正常。     

细胞因子诱导的杀伤细胞

正常人或患者外周血、骨髓单个核细胞/淋巴细胞中定期加入γ-干扰素(IFN-γ)、白细胞介素-1(IL-1)、白细胞介素-2(IL-2)、抗CD3抗体(CD3mAb)经2～4周的诱导可获得大量的新型肿瘤杀伤细胞——细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK),增殖旺盛及肿瘤杀伤活性高,包含大量CD3~+CD56~+细胞。有关基础及临床研究证明,CIK细胞较细胞毒性T淋巴细胞(CTL)、淋巴细胞因子活化的杀伤细胞(lymphokine-activated killer,LAK)、肿瘤细胞浸润的淋巴细胞(tumer infiltrating-lym-phocyte,TIL)更适用于肿瘤的生物治疗。本文就CIK细胞的来源与生物学特征、对肿瘤细胞的杀伤及临床应用进行综述。     

淋巴细胞功能亢进与造血组织损伤

越来越多的证据表明,淋巴细胞功能亢进与造血组织损伤的关系非常密切。现就该方面研究作一综述。1　淋巴细胞及其功能亢进所致血液系统疾病　1990年Ro magnami等[1 ] 研究证实人类辅助性T细胞(Th)存在辅助性T细胞1(Th1细胞)、辅助性T细胞2 (Th2细胞)两种亚型,分别分泌Ⅰ类细胞因子和Ⅱ类细胞因子。Th1细胞优势分泌γ干扰素(IFN γ)及IL 2 ,以分泌IFN γ为主要特征,主要介导细胞免疫,活化细胞毒性T淋巴细胞(CTL)或激活CD8 T细胞,进而产生大量凋亡配体[如肿瘤坏死因子(TNF)等];Th2细胞主要产生IL 4、IL 6、IL 10 ,以IL 4的分泌…     

白介素12诱导肝癌肿瘤浸润淋巴细胞的实验研究

目的比较IL-12诱导肝癌肿瘤浸润淋巴细胞(TIL)与常规用IL-2诱导的肿瘤浸润淋巴细胞的特异性杀伤力、增殖能力、细胞因子和细胞表型的改变.方法将20例原发性肝癌手术切除的癌瘤进行体外分离,分别用常规含IL-2的培养液和含IL-12的培养液以及IL-12+IL-2的培养液培养,诱导TIL的生长.培养10～14d测定细胞杀伤率、细胞因子、增殖能力及用流式细胞仪检测淋巴细胞亚群CD+3、CD+4、CD+8、CD+56的表达.结果①IL-12+IL-2组和单用IL-12组对自体肝癌细胞的细胞杀伤率比IL-2组明显增高(P＜0.001;P＜0.01),均具有极显著差异;②IL-12+IL-2组较IL-2组表达CD+3、CD+4、CD+8明显升高(P＜0.01);CD+56增多不明显,无显著差异(P＞0.05).③IL-12+IL-2组比IL-2组扩增能力明显增强(P＜0.05).④IL-12+IL-2组或IL-12组较IL-2组细胞因子IFN-γ和TNF-α水平明显增高.(P＜0.05).结论白介素12作为诱导剂诱导肝癌TIL可明显增强TIL细胞的特异性细胞毒作用且增殖能力明显增强.     

科研新闻速递

细胞毒性T细胞和自然杀伤细胞活化在脓毒症和多器官功能障碍中的作用最近,瑞士和荷兰学者研究了活化的细胞毒性细胞直接标志物(端粒酶A和B)和间接标志物(细胞因子)在严重脓毒症和脓毒性休克中的作用。研究共纳入脓毒症患者32例及脓毒性休克患者8例,分别检测了端粒酶A和B活性及肿瘤坏死因子α(TNFα)、γ干扰素(IFNγ)、白细胞介素12p70(IL12p70)、IL12p40等细胞因子水平。结果显示:58%的脓毒症患者IL12p40升高,仅少数患者可检出TNFα、IFNγ和IL12p70。此外,端粒酶A和B活性分别升高42.5%和22.5%,而IL12p40与脓毒症病情呈负相关…     

细胞因子诱导的杀伤细胞

正常人或患者外周血，骨髓单个核细胞/淋巴细胞中定期加入γ-干扰素（IFN-γ），白细胞介素-1（IL-1），白细胞介素-2（IL-2），抗CD3抗体（CD3mAb)经2-4周的诱导可获得大量的新型肿瘤杀伤细胞-细胞因子诱导的杀伤细胞（cytokine-induced killer,CIK)，增殖旺盛及肿瘤杀伤活性高，包含大量CD3^ CD56^ 细胞，有关基础及临床研究证明，CIK细胞较细胞毒性T淋巴细胞（CTL），淋巴细胞因子活化的杀伤细胞（lymphokine-activted killer,LAK)，肿瘤细胞浸润的淋巴细胞（tumer infiltrating-lym-phocyte,TIL）更适用于肿瘤的生物治疗，本文就CIK细胞的来源与生物学特征，对肿瘤细胞的杀伤及临床应用进行综述。     

联合应用白介素2和α干扰素对肝癌术后患者免疫机能的影响

目的 :通过在白介素 2 (IL - 2 )和α-干扰素 (IFN -α)治疗原发性肝癌术后患者过程中测定血清白介素 12 (IL - 12 )水平和外周血淋巴细胞表型的变化 ,评价其对患者免疫机能的影响。方法 :将 5 0例原发性肝癌术后患者随机分成两组 :一组给予IL - 2和α -IFN、另一组为对照组。用药前、后分别测定血清白介素 - 12以及流式细胞仪测定淋巴细胞的表型。结果 :用药后血清白介素 - 12及CD3+ 、CD4+ 、CD8+ 、CD5 6 + 比用药前明显升高 (P <0 .0 5 )。结论 :IL - 2和α -INF治疗后内源性IL- 12分泌增加可诱发抗肿免疫 ,改善机体免疫机能。     

食管癌患者外周血CD3+T细胞分泌细胞因子的水平变化及其临床意义

目的探讨食管癌患者外周血CD3~+T细胞分泌细胞因子的水平变化及其临床意义。方法选取2016年1月至2018年1月在西安交通大学第二附属医院接受治疗的28例食管癌患者为研究组,28例健康体检者为健康对照组。比较两组患者的外周血T细胞、外周血CD3~+T细胞分泌因子白细胞介素(IL)-2、IL-4、IL-10、IL-12表达水平、外周血CD3~+T细胞分泌因子肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达水平。结果研究组CD3~+/CD4~+、CD4~+/CD8~+表达水平低于对照组,研究组CD3~+/CD8~+、CD3~+表达水平高于对照组,差异有统计学意义(P0.05)。研究组IL-2、IL-10、IL-12表达水平低于对照组,研究组IL-4表达水平高于对照组,差异有统计学意义(P0.05)。研究组TNF-α表达水平高于对照组,研究组IFN-γ表达水平低于对照组,差异有统计学意义(P0.05)。结论对于食管癌患者,食管癌患者外周血CD3~+T细胞分泌细胞因子CD4~+/CD8~+、CD3~+/CD4~+表达水平显著增高,说明患者的免疫耐受力与免疫逃逸存在较明显的关系。     

Immunoreactivity of lymphocytes from draining lymph nodes, peripheral blood and tumor infiltrates from oral cancer patients

Lymphocytes from metastatic (met) and nonmetastatic (non-met) regional lymph nodes, LNL peripheral blood (PBL) and tumor infiltrating lymphocytes (TIL) of patients with squamous cell carcinoma of the oral cavity and healthy donors were investigated for CD3, CD4, CD8 and HNK-1 phenotypes, Natural Killer (NK) cell Activity, Antibody Dependent Cellular Cytotoxicity (ADCC) and proliferative response to mitogen (PHA). Modulation of NK cytotoxicity with recombinant Interferon alpha (IFN-alpha) was also investigated in some cases. Lymphocytes from met and non-met lymph nodes showed no variation in the percentages of CD3+, CD4+ and CD8+ cells, when compared with each other and with PBL of oral cancer patients. TIL showed significantly less proportion of CD3+ and CD4+ cells. The percentage of HNK-1+ cells was significantly lower in LNL and TIL when compared to PBL of oral cancer patients. The mitogen responses of met and non-met LNL were comparable to each other and better than that of PBL from the same patients, while, TIL showed significant impairment in mitogen responses. The NK cytotoxicity and ADCC of PBL from oral cancer patients were comparable to healthy donors which could be augmented by rIFN alpha. LNL and TIL showed almost negligible NK and ADCC activities and NK activity could not be modulated by rIFN alpha. The results thus demonstrate that in oral cancer patients, lymphocytes from three compartments viz. PBL, LNL and TIL showed differential effector functions. The metastatic status of LN did not affect the immunoreactivity of LNL.     

In vivo trafficking of adoptively transferred interleukin-2 expanded tumor-infiltrating lymphocytes and peripheral blood lymphocytes. Results of a double gene marking trial.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and IL-2 appears to produce dramatic regressions in patients with metastatic melanoma and renal cancer. However, the in vivo mechanism of TIL function is not known. We conducted an UCLA Human Subject Protection Committee, Recombinant DNA Advisory Committee, and FDA-approved clinical trial using genetically-marked TIL to test the hypothesis that these cells have unique, tumor-specific in vivo trafficking patterns. TIL and PBL (as a control effector cell population) were isolated and expanded in parallel in vitro in IL-2-containing medium for 4-6 wk. During the expansion, TIL and PBL were separately transduced with the amphotropic retroviral vectors LNL6 and G1Na. Transduced TIL and PBL were coinfused into patients and their respective numbers measured in tumor, peripheral blood, and normal tissues; integrated provirus could be quantitated and distinguished by DNA PCR. Nine patients were treated (six melanoma, three renal) and received between 4.5 x 10(8) and 1.24 x 10(10) total cells. Both "marked" TIL and PBL could be detected circulating in the peripheral blood, in some patients for up to 99 d after infusion. Marked TIL and/or PBL could be detected in tumor biopsies in six of nine patients as early as day 6 and as late as day 99 after infusion. No convincing pattern of preferential trafficking of TIL vs. PBL to tumor was noted. Moreover, concurrent biopsies of muscle, fat, and skin demonstrated the presence of TIL/PBL in comparable or greater numbers than in tumor in five patients. The results of this double gene marking trial provide interesting insights into the life span and trafficking of adoptively transferred lymphocytes, but do not support the hypothesis that TIL specifically traffic to tumor deposits.     

再生障碍性贫血患者细胞免疫功能与造血细胞因子的研究

目的：进一步探讨再生障碍性贫血（再障）的免疫发病机制，阐明细胞免疫、造血细胞因子在再障患者中发生变化的基础与临床意义。方法：用单抗试剂盒，采用改良ＡＰＡＡＰ法及酶联免疫试剂盒，采用ＥＬＩＳＡ法对３８例再障患者及２０名正常人外周血Ｔ细胞亚群，ＨＬＡ－ＤＲ抗原表达以及外周血单个核细胞（ＰＢＭＮＣ）培养上清诱生Ｇ－ＣＳＦ、ＩＬ－６、ＴＮＦα、ＩＦＮα及ＩＬ－８水平进行测定。结果：再障患者外周血ＣＤ４细胞减低、ＣＤ８细胞增高、ＣＤ４／ＣＤ８降低或倒置，ＨＬＡ－ＤＲ抗原表达率增高。再障患者ＰＢＭＮＣ培养上清中Ｇ－ＣＳＦ阳性率减低，ＩＬ－６、ＴＮＦα、ＩＦＮα及ＩＬ－８水平增高。相关分析发现Ｇ－ＣＳＦ与ＣＤ４细胞及ＣＤ４／ＣＤ８呈正相关，而与ＩＦＮα呈负相关；ＩＬ－６与白细胞数及ＣＤ４细胞呈负相关；ＴＮＦα与ＣＤ８细胞呈正相关而与ＣＤ４／ＣＤ８呈负相关；ＩＬ－８与ＣＤ８细胞及ＨＬＡ－ＤＲ呈正相关。结论：细胞免疫功能异常及细胞因子网络失调在再障发病中起一定的作用。     

Natural killer (NK) cell stimulatory factor increases the cytotoxic activity of NK cells from both healthy donors and human immunodeficiency virus-infected patients

Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and IL-2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5- NK cells and does not confer cytotoxic activity to CD16-CD5+ T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumor-derived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV-infected patients with NKSF and/or IL-2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon gamma in response to NKSF and/or IL-2, although at levels 5- or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/IL-12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.     

Dendritic cell infiltration in colon cancer

We quantitatively evaluated dendritic cell (DC) infiltration in primary colorectal cancers from 44 patients and metastatic colorectal tumors from 13 patients using immunohistochemistry for the DC marker CD83, HLA-DR, and the DC activation molecules CD40 and CD86. Nearly all CD83+ cells were also HLA-DR+, CD40+, and CD86+, indicating that the DCs that infiltrate colon cancer in vivo express the activation and costimulatory molecules associated with a mature DC phenotype. The density of DCs in colorectal cancer primaries was three times lower than that seen in normal colonic mucosa (0.29 versus 0.84 CD83+ cells/ high-power field (hpf), p < 0.001). Dendritic cells were rarely observed in metastatic tumors: DC density in metastases was sixfold lower than in colorectal primary tumors (0.05 versus 0.29 CD83+ cells/hpf, p < 0.001). Because cytokines have been shown, in vitro, to exert potent effects on DCs, we also evaluated the relationship between intratumor DC density and the expression of cytokines by tumor-infiltrating lymphocytes (TILs) and tumor cells. Expression of interleukin-10 and transforming growth factor beta by either TIL or tumor cells was not associated with decreased DC density or decreased expression of CD40 or CD86 on DCs. Tumor expression of vascular endothelial growth factor was associated with a more than twofold increase in DC density (p = 0.01). Patients who had a high proportion of TILs expressing tumor necrosis factor (TNF) had a greater intratumor mature DC density than patients with a low proportion of TNF + TIL (0.54 versus 0.21 CD83+ cells/hpf, p < 0.01).     

同种输血对食管癌患者围手术期细胞因子的影响

目的　探讨同种输血对食管癌患者细胞因子生成的影响及相互关系。方法　食管癌患者手术常规输血18例 ,去白细胞输血 14例 ,采用生物素 亲和素系统测定技术检测患者围手术期常规输血和去白细胞输血后血清中IL 10、IFN γ和TNF α的浓度。结果　常规输血后第 1天与输血前相比血清中IL 10、IFN γ和TNF α浓度升高 ,且以IL 10、IFN γ变化尤为显著 ;输血后第 5天IFN γ和TNF α降低接近输血前水平 ,并明显低于去白细胞输血组 ,IL 10仍明显高于输血前水平。去白细胞输血后不同时间IL 10无显著性改变。结论　食管癌患者围手术期同种输血后血清中IFN γ和TNF α的降低与IL 10升高有关 ,而IL 10升高可能是患者同种输血后免疫抑制的最重要原因 ,输注去白细胞血液可减轻或去除这一作用。     

人外周血NKT细胞体外扩增及其分泌细胞因子的研究

目的　建立人外周血自然杀伤T细胞 (NKT)体外扩增及检测NKT细胞所分泌的细胞因子的方法。方法　分别采用单加α 半乳糖神经酰胺 (α Galcer组 ) ,单加树突状细胞 (DC组 )以及同时加α Galcer、DC(α Galcer、DC组 )的方法从人外周血单个核细胞 (PBMC)和纯化T淋巴细胞中扩增TCRVα2 4+ /TCRVβ1 1 + NKT细胞。采用细胞内细胞因子流式细胞术检测手段测定NKT细胞中IL 4 ,IFN γ、TNF α阳性细胞比例。结果　PBMC中的α Galcer组 ,DC、α Galcer组和纯化T淋巴细胞中的DC、α Galcer组 ,NKT细胞扩增 1 9d后分别占淋巴细胞的 (2 5 .5± 7.2 ) %、(1 8.2± 8.2 ) %和 (2 4 .8± 5 .5 ) % ,NKT细胞分别扩增了 (1 5 .1± 9.1 )× 1 0 3 倍、(2 1 .8± 1 7.3)× 1 0 3 倍和 (2 3.0± 1 6 .7)× 1 0 3 倍 ,与其它各组差异有显著性意义 (P <0 .0 1 )。扩增过程TCRVβ1 1+ NKT细胞中分泌IL 4 ,IFN γ和TNF α的细胞比例均高于TCRVβ1 1 T淋巴细胞 (P <0 .0 5 )。结论　α Galcer具有体外大量扩增NKT细胞的能力 ,同时α Galcer的激活能力受CD1d分子的限制。由于PBMC中的单核系细胞也表达CD1d分子 ,因此直接从PBMC扩增NKT也能获得良好的效果     

Induction of interferon alpha from human lymphocytes by autologous, dengue virus-infected monocytes

Human monocytes actively replicate dengue virus. To dissect the primary immune responses to dengue virus-infected monocytes (DV-monocytes), we analyzed the interaction between autologous DV-monocytes and the peripheral blood lymphocytes (PBL) of dengue nonimmune donors. Interferon (IFN) activity was detected when PBL were cultured with DV-monocytes. Cell contact between PBL and DV-monocytes was required for IFN production; however, MHC compatibility between PBL and monocytes was not necessary. DV-monocytes fixed with paraformaldehyde or glutaraldehyde, which produced no infectious virus, also induced high levels of IFN from PBL. The ability of DV-monocytes to induce IFN correlated with the appearance of dengue antigens. The PBL that produce IFN were characterized by FACS sorting using monoclonal and polyclonal antibodies. HLA-DR+ and T3- cells produced high titers of IFN, while HLA-DR- and T3+ cells produced very low or undetectable levels of IFN. Moderate titers of IFN were produced by cells contained in B cell fractions (surface immunoglobulin-positive, B1+, and Leu-12+), and cells contained in natural killer cell fractions (Leu-11+ and OKM1+). Therefore, IFN-producing cells are heterogeneous, and the predominant producer cells are characterized as HLA-DR+ and non-T lymphocytes. The IFN produced was characterized by RIA using mAbs to IFN-alpha and IFN-gamma. The IFN-alpha was the predominant IFN produced; in addition, a low level of IFN-gamma was also detected in some experiments. The culture fluids obtained from PBL exposed to autologous DV-monocytes, which contained high IFN activity, completely inhibited dengue virus infection of monocytes. These results suggest that IFN-alpha produced by PBL exposed to DV-monocytes may play an important role in controlling primary dengue virus infection.     

Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma

T cell-mediated immune response against autologous melanoma cells was analyzed, at population and clonal levels, in 31 patients with recurrent and/or metastatic disease. Fresh PBL and lymph node lymphocytes (LNL) from melanoma-involved nodes were not cytotoxic against the respective melanoma cells. When activated in in vitro coculture (IVC) against the autologous melanoma cells in the presence of IL-2, a majority of the activated PBL and LNL became cytotoxic against the autologous targets. The activated effector cells were cloned in limiting dilution microcultures, and growing clones were phenotypically defined and were functionally characterized for cytotoxicity and for potential regulatory function. Functional T cell clones were obtained from 15 of 31 cases. Of these, CTL responses exhibiting cytotoxicity restricted against the autologous melanoma were seen in four cases. All four CTL clones were CD3+, CD8+, and CD4-. Three of these four CTL clones were studied extensively. All three of these CTL clones expressed MHC class I-restricted cytotoxicity. mAb anti-CD3 blocked cytotoxicity in two and enhanced cytotoxicity in the other. Neither autologous sera nor autologous nonactivated fresh PBL modulated the cytotoxic functions of the CTL clones at the effector phase. T cell lines exhibiting regulatory function were obtained in 11 cases. The regulatory T cell lines were CD3+, CD4+, and CD8-. In three cases CD4+ clones amplified the cytotoxic response in the PBL in coculture, while in eight other cases the T cell lines downregulated the cytotoxic responses. Such T cell-mediated down-regulations were either restricted to the autologous system, induced by D/DR antigens expressed by the autologous or allogeneic melanoma cells, or induced by stimulus other than D/DR antigens. Taken together, these findings clearly demonstrate the existence of T cell-mediated cytotoxic and regulatory responses against human melanoma.