淋巴细胞功能测定

免疫学技术原理与应用

PAIT-12

淋巴细胞功能测定

孙汭

中国科技大学生命科学学院

免疫学研究所

PMIT-12

淋巴细胞功能测定

一、常用的淋巴细胞功能测定

二、T淋巴细胞功能测定

三、B淋巴细胞功能测定

四、NK细胞细胞毒试验

一、常用的淋巴细胞功能测定

1、淋巴细胞增殖试验

① 3H-TdR掺入法

原理：淋巴细胞在丝裂原或特异性抗原刺激下转化为淋巴母细胞，发生转化过程中，细胞DNA合成大量增多。此时在细胞培养液中加入氚标记的胸腺嘧啶核苷（3H-Thymidine riboside, 3H-TdR), 使3H-TdR掺入新合成的DNA中，经β-液体闪烁仪检测淋巴细胞DNA 的3H-TdR掺入量，根据掺入细胞内的核素的量可判断淋巴细胞增殖的程度。

② MTT比色法

原理：MTT（（3-4，5-二甲基-2-噻唑）-2，5-二苯基溴化四唑）是一种黄色可溶性噻唑盐，掺入细胞后，在细胞活化增殖时通过线粒体能量代谢过程，MTT代谢形成蓝色的甲攒（Formazan)沉积于细胞内或细胞周围，形成甲攒的量与细胞增殖程度成正相关。甲攒经异丙醇溶解后呈紫蓝色，借助酶标仪测定OD值，可反映细胞增殖水平。

2、细胞毒试验

①51Cr释放法

原理：用放射性核素51Cr（Na251-CrO4，鉻酸钠）标记靶细胞，51Cr即可进入靶细胞，与胞浆蛋白结合。然后将51Cr标记的靶细胞与待检细胞（效应细胞）共同孵育一定时间后，若待检细胞能够杀伤靶细胞，则51Cr从靶细胞释放出来。51Cr的释放量与效应细胞的活性成正相关。即靶细胞被杀伤的越多，释放到上清中的游离51Cr越多，用γ计数仪测定上清中51Cr的放射活性（cpm）值，以计算待检细胞的细胞毒活性。

（一）、用51Cr铬酸钠标记靶细胞

1．用RPMI-1640培养液洗涤107靶细胞，去上清液。用0.5ml不含碳酸氢钠的完全培养液悬浮细胞。加入100μCi（3.7MBq）51Cr铬酸钠，37℃水浴中标记1小时，每5～10分钟摇晃一次，混匀细胞。

2．用RPMI-1640培养液洗涤细胞3次，每次400×g 10分钟。用50ml RPMI-1640培养液悬浮细胞，置37℃水浴30分钟，以减少非特异的自然释放。

3．离心200×g 10分钟，去上清液。小心用RPMI-1640培养液悬浮细胞，尽量减少振荡引起的细胞损伤以降低靶细胞的自然释放率，将细胞浓度调整为0.5×105或1×105/ml备用。

（二）、4小时51Cr释放实验

1．在96孔圆底细胞培养板中加入51Cr标记的靶细胞，每孔加100μl。

2．向各孔加100μl效应细胞，效应细胞与靶细胞的比例（效靶比，E：T）根据要求而定，通常为5：1～50：1。阴性对照孔（自然释放）不加效应细胞只加100μl完全培养液，阳性对照孔（最大释放）中加100μ1％NP40（或2％SDS，1mol/L盐酸）。每个实验置三个复孔。

3．稍稍离心培养板（100×g 3分钟）后，置37℃5％ CO2的二氧化碳培养箱中培养4小时。

（三）、放射性活性（cpm) 测定和特异性杀伤活性的计算

离心培养板200×g 10分钟，每孔吸出100μl上清液置一次性使用的检测管中，在γ－计数仪上（或液闪计数仪上）测定上清液中的每分钟放射性活性（cpm值）。

（实验组cpm－自然释放组cpm）

细胞毒性（％）＝×100％

（最大释放组cpm－自然释放组cpm）

② CFSE法

原理：根据CFSE 和PI两种荧光染料的光谱不重叠的特性，用流式细胞仪在FL1 和FL3 通道可区分CFSE 和PI (碘化丙碇)，用CFSE 和PI 同时标记靶细胞, CFSE标记靶细胞, PI 标记DNA，如果细胞膜已破坏的靶细胞则PI+。E：T为50∶1～25∶1 ,共孵育2～4h,流式细胞仪收集1 000 个靶细胞并测定被杀细胞的百分率。

优点：CFSE/PI 双荧光染料标记的流式细胞分析检测细胞毒具有多个优点:避免放射性试剂的应用,敏感性增强,单细胞水平的分析。

（一）、用CFSE标记靶细胞

YAC-1 cells was used as targets. Target cells were labelled with CFSE for 10 min at 37 ?C and 5% CO2 using a final concentration of 7.5mmol/l. After labelling, the cells were washed once and diluted in CM.

（二）、杀伤实验

Effector (E) and target (T) cells were mixed to a final volume of 200 μl in CM 10 ng rrIL-2 was added to all tubes except control samples with target cells only. The tubes were mixed and spun down at 120×g for 2 min. The samples were incubated in a water bath at 37 ?C and 5% CO2 for 18–20 h.

At the end of the incubation time, the samples were put in an ice water bath and 50 μl, 50 μg/ml, propidium iodide (PI) (Sigma, Sweden) was added for DNA labelling of dead cells. The samples were then incubated for 5 min and analysed by flow-cytometry within 60 min.

Percentm specific target cell death

dead targets in the sample (%) ? spontaneously dead targets (%) (cytotoxicity%) = × 100

100 ? spontaneously dead targets (%)

The proportion of spontaneously dead targets normally varies between 2 and 7%

二、T淋巴细胞功能测定

1、T淋巴细胞增殖试验

①形态学方法-T淋巴细胞转化试验

原理：人或小鼠T细胞表面有PHA或ConA受体，T细胞在体外受PHA或ConA的刺激后能转化为体积较大、代谢旺盛、且能进行分裂的淋巴细胞，以此测定T细胞的功能。

② CFSE

Fig. 1. CD4+CD25? T cell proliferation monitored using CFSE labeling. CFSE labeled

CD4+CD25? T cells (2×104 c/well) were cultured in the presence of autologous irradiated PBMC (feeder cells) and were either unstimulated (A) or stimulated with anti-CD3 Ab (2 μg/ml) (B). After 5 days, cells were harvested, stained for CD4 expression and analyzed by means of flow cytometry. Target cells (ratio 1:0) were gated by a combination of forward and side scatter signal (lymphocyte gate) and bright CD4+ signal (CD4+ T cell gate). Prelabeling (harvested) cells with 7-AAD demonstrated that N95% of cells in the CD4+ T cell gate were viable (7-AAD negative; data not shown). CFSE histograms from one representative HC is shown (n=5). Cells under M1 represent non-divided CFSE labeled responder cells whereas others bins (M2-M6) indicate daughter cell populations which have subsequently lost half of their CFSE signal with each division round.

Journal of Immunological Methods 322 (2007) 1–11

③ Mitogen assay

Figure 8. Effects of rhPRL on splenic T-cell mitogen responsiveness after congenic bone marrow transplantation.

C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million

C57BL/6-Ly5.2 congenic mice then received PBS or 50 μg rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and spleen cells proliferative response to ConA was measured, as described in “Materials and Methods.” Data shown are from 3 separate experiments. *P<.05; ***P <.001 compared with PBS-treated controls.

Biology of Blood and Marrow Transplantation 9:426-434 (2003)

2、抗原特异性T细胞检测

原理：抗原肽-MHC分子四聚体是根据T细胞活化的双识别原理，用生物工程技术将MHC分子在体外组装，并结合抗原表位肽，形成抗原肽-MHC分子复合物。用生物素标记的抗原肽-MHC分子复合物与荧光标记的亲和素结合，由于一个荧光标记的亲和素能结合4个生物素分子，这样将4个抗原肽-MHC分子复合物组装在一起，成为四聚体。用流式细胞技术即可确定待检标本中抗原特异性T细胞的频率。MHC I类、MHCII类分子和CD1d分子与抗原肽形成的四聚体复合物可分别鉴定表达特异性TCR的CD8+T细胞、D4+T细胞和NKT细胞。

Figure 5. Csf-2 Controls Functional Maturation of iNKT Cells in a Cell-Intrinsic Manner (A) Radiation BM chimeras were generated as indicated, and 8 weeks after transplantation, iNKT cell numbers were monitored in the thymus, spleen, and liver. Numbers within plots represent % iNKT cells among total lymphocytes. Representative of two independent experiments. Immunity 25, 487–497, September 2006

3、细胞因子检测

① ELISport

Figure 4. Administration of Csf-2 during Development but Not in the Periphery Rescues iNKT Cell Function

(B) Splenocytes from C57BL/6, Csf-2-, and CD1d1-deficient mice were stimulated with 100

ng/ml aGC+Csf-2 for 4 days. IL-4 and IFN-g secreted by activated iNKT cells were measured by ELISA after 1, 2, 3, and 4 days. Representative results from 2-day culture are shown (left). Splenocytesfrom C57BL/6, Csf-2-deficient mice were similarly stimulated for 24 hr, and secreted IFN-g was captured and detected by ELIspot assay (right). Representative of two independent experiments performed in triplicates; bars indicate mean 6 SEM.

Immunity 25, 487–497, September 2006

② FACS

Figure 6. Csf-2 Controls Development of Rapid Cytokine Secretion by Activated iNKT Cells

(A) Csf-2+/2 and Csf-22/2 mice were injected i.p. with 5 mg aGC (open histograms) or vehicle (gray histograms). After 2 hr, B220loCD3+ tetramer+ cells were electronically gated and intracellular IFN-g (top), and IL-4 (bottom) expression was monitored. Numbers refer to

MFI. Representative of three independent experiments. Numbers represent MFI. (B) Splenocytes from Csf-2+/2 and Csf-22/2 mice were incubated with vehicle (gray histograms) or 100 ng/ml of aGC(open histograms) for 5 days. CD3+tetramer+ iNKT lymphocytes were identified and analyzed as described in (A). Representative of two independent experiments. Numbers represent geometric MFI.

Immunity 25, 487–497, September 2006

4、信号分子检测

Figure 1. iNKT Cells Express Functional Csf-2R

(C) Purified thymic 4get iNKT cells were activated with 100 ng/ml PMA and 2 mM ionomycin, 100 ng/ml rmCsf-2, or 100 ng/ml LPS. After 15 min, total and phosphorylated IkBa were determined by immunoblot. Representative of three independent experiments.

Immunity 25, 487–497, September 2006

三、B淋巴细胞功能测定

1、B细胞Mitogen assay-3H-TdR掺入法

Figure 6. Effects of rhPRL on splenic B-cell mitogen responsiveness after congenic bone marrow transplantation.

C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million

C57BL/6-Ly5.2 congenic bone marrow cells. The mice then received PBS or 50 μg rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and spleen cells proliferative response to LPS was measured as described in “Materials and Methods.” Data shown are from 3 separate experiments. * = P<.05; ** =P< .01 compared with PBS-treated controls.

Biology of Blood and Marrow Transplantation 9:426-434 (2003)

2、抗体形成细胞测定（溶血空斑试验）

原理：溶血空斑试验（plaque forming cell assay, PFC):是体外检测B淋巴细胞抗体形成功能的一种方法。原理是将绵羊红细胞（SRBC）免疫家兔或小鼠，取家兔淋巴结或小鼠脾脏制成细胞悬液，与高浓度的SRBC混合后加入琼脂凝胶中、，其中每个释放溶血性抗体的细胞可致敏其周围的SRBC，在补体参与下，抗体形成细胞周围的SRBC溶解，在其周围形成一个肉眼可见的空斑，一个空斑代表一个抗体形成细胞，孔板的数量反映机体的体液免疫功能。

3、抗体形成细胞检测-ELISport

4、信号分子检测

四、NK细胞细胞毒试验

1、ADCC试验

ADCC (Antibody dependent cell-mediated cytotoxicity) : 选用适当的靶细胞，加相应的免疫血清，再加入效应细胞，作用一段时间后，观察细胞被破坏的程度并推测效应细胞的ADCC 作用。常用的靶细胞为鸡、鸭、羊红细胞，也可用HeLa等传代细胞，检测方法：形态学检测法或51Cr释放试验检测靶细胞是否被破坏。ADCC试验用于检测NK细胞、巨噬细胞及中性粒细胞的细胞毒作用。

2、NK细胞的细胞毒试验-51Cr释放法

细胞介导的细胞毒试验-NK细胞的细胞毒试验 : 将淋巴细胞作为效应细胞（NK）与相应的靶细胞作用，NK细胞一旦与靶细胞结合，即可被活化，发生细胞内颗粒重排，继而释放NK细胞毒因子（NKCF）、穿孔素等物质破坏靶细胞，一般历时4-6小时，可完成细胞毒作用。测定靶细胞生长情况即可判断效应细胞的活性。

测定方法：51Cr释放法

效应细胞:外周血淋巴细胞、动物脾脏淋巴细胞

靶细胞：人NK敏感细胞为：K562细胞株（人红白血病细胞）

小鼠NK敏感细胞为：Yac-1细胞株（小鼠T淋巴瘤细胞）

Fig 3. Cytotoxicity and cytolysis effector molecules of human NK-92 cells in the presence of PRL and cytokine. NK-92 cells were harvested immediately after 12 h incubation in the presence of PRL (100 ng/ml) plus IL-2 (10 U/ml) or plus IL-15 (10 U/ml). The NK cells were admixed with 51Cr-labeled K562 target cells for cytotoxicity at an effector to target (E:T) ratio of 10:1, 5:1, 2.5:1 and 1.25:1 as described in Materials and Methods (A, B).

Cell Research (2004); 14(1):67-73

3、Hepatic NK Cytotoxicity Assay

Purpose

The assay for measuring the cytotoxicity of hepatic MNCs against freshly isolated primary mouse hepatocytes by using a 4-hour AST release assay.

Cells

Target Cells: Fresh hepatocytes 1x104/100ul/well (96-well round-bottom plate)

Effect cells: Hepatic MNCs

Procedure

1.Separated hepatocytes: primary mouse hepatocytes were

isolated by perfusion of mouse livers with EGTA solution and digestion with 0.075% collagenase type I solution.

2.Separated hepatic MNCs: Hepatic MNCs isolated as effectors cells.

3Placed hepatocytes 1x104 cells/100ml (105/ml) and 100 ml hepatic MNCs at various ratios into 96-well round-bottom plates cultured in RPMI1640 medium with 10%FBS (total volume of 200 ml) for 4 hours at 37oC in a 5% CO2 incubator.

E:T (50:1) 25:1, 12.5:1, 6.25:1, 3.125:1, 1.6:1

Maximum release: 0.05% triton-100 in 10% FBS RPMI1640

Spontaneous : 10% FBS RPMI1640

4. The supernatant was harvested and AST activity was measured.

5. The specific cytotoxicity (%) was calculated as

AST experimental-AST spontaneous

Specific cytotoxicity (%) = x 100%.

(AST maximum-ASTspontaneous )

Figure 1. IL-6 treatment reduces the cytotoxicity of Con A-treated hepatic MNCs and NKT cells against primary mouse hepatocytes.

(A) and (B) C57BL/6 mice were intravenously (i.v.) administrated with saline or IL-6 (2μg/g), followed 6 hours later by injection (i.v.) of Con A (15 μg/g). After 2 hours, hepatic MNCs (panel A) or hepatic NKT cells (panel B) were isolated and their cytotoxicities were tested against primary hepatocytes from C57BL/6 mice at the indicated effector-to-target (E/T) ratios as described in “Materials and Methods.”

The Journal of Immunology, 2004; 172:

Review Question

1、淋巴细胞丝裂原反应的检测方法及原理

2、细胞毒试验有几种？简述其原理

3、抗原特异性T细胞检测的方法的特点及原理

4、如果研究一种新的蛋白质或药物对机体免疫功能的影响，怎样用所学过的免疫学方法进

行体内外研究？

5、比较Figure 1和Figure 3 所用的两种杀伤方法有何不同？为什么？

6、Figure 8的实验主要检测什么免疫功能？为什么？简述原理。

7、Figure 5所用的方法与表面标志检测有何不同？所检测的阳性细胞群是什么细胞？

Figure 1. IL-6 treatment reduces the cytotoxicity of Con A-treated hepatic MNCs and NKT cells against primary mouse hepatocytes.

(A) and (B) C57BL/6 mice were intravenously (i.v.) administrated with saline or IL-6 (2μg/g), followed 6 hours later by injection (i.v.) of Con A (15 μg/g). After 2 hours, hepatic MNCs (panel A) or hepatic NKT cells (panel B) were isolated and their cytotoxicities were tested against primary hepatocytes from C57BL/6 mice at the indicated effector-to-target (E/T) ratios as

described in “Materials and Methods.”

The Journal of Immunology, 2004; 172:

Fig 3. Cytotoxicity and cytolysis effector molecules of human NK- 92 cells in the presence of PRL and cytokine.

NK-92 cells were harvested immediately after 12 h incubation in the presence of PRL(100 ng/ml) plus IL-2 (10 U/ml) or plus IL-15 (10 U/ml). The NK cells were admixed with 51Cr-labeled K562 target cells for cytotoxicity at an effector to target (E:T) ratio of 10:1, 5:1, 2.5:1 and 1.25:1 as described in Materials and Methods (A, B).

Cell Research (2004); 14(1):67-73

Figure 8. Effects of rhPRL on splenic T-cell mitogen responsiveness after congenic bone marrow transplantation.

C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million C57BL/6-Ly5.2 congenic mice then received PBS or 50 mg rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and spleen cells proliferative response to ConA was measured, as described in “Materials and Methods.” Data shown are from 3 separate experiments. *P<.05; ***P <.001 compared with PBS-treated controls.

Biology of Blood and Marrow Transplantation 9:426-434 (2003)

Figure 5. Csf-2 Controls Functional Maturation of iNKT Cells in a Cell-Intrinsic Manner (A) Radiation BM chimeras were generated as indicated, and 8 weeks after transplantation, iNKT cell numbers were monitored in the thymus, spleen, and liver. Numbers within plots represent % iNKT cells among total lymphocytes. Representative of two independent experiments.

Immunity 25, 487–497, September 2006

Reference

1. Journal of Immunological Methods 322 (2007) 1–11

2. Immunity 25, 487–497, September 2006

3. Biology of Blood and Marrow Transplantation 9:426-434 (2003)

4. The Journal of Immunology, 2004; 172: