Video在线视频

-大家好
-Hello

欢迎来到巴斯德研究院关于结核病的慕课
and welcome to the Institut Pasteur's MOOC on tuberculosis.

我是Darragh Duffy
My name is Darragh Duffy,

是巴斯德研究院的免疫学家
I am an immunologist at the Institut Pasteur.

我正在与Milieu Intérieur联盟合作
I am working with the "Milieu Intérieur" consortium

这个联盟旨在认识和定义
whose aim is to understand

健康免疫应答
and define a healthy immune response

并了解确定健康免疫应答的范围
and understand the determinants,

及其决定因素
the genetics and the environment,

包括遗传因素和环境因素
that determine the boundaries of a healthy immune response.

这种方法可以用于许多不同的疾病
This approach can be applied to many different disease settings.

我们这样做是基于盖茨基金会
We are doing so in the context of tuberculosis with the Gates foundation,

和南非的SATVI的结核病背景
and SATVI, in South Africa.

在接下来的十分钟内
In the next ten minutes,

我将重点介绍
I will focus on the standardised immunomonitoring tools

在我们与Milieu Intérieur开发的
that we developed within Milieu Intérieur

用于免疫应答研究的标准免疫监测工具
for the study of immune responses.

免疫应答是高动态的
Immune responses are highly dynamic.

由于这种动态性
Because of this dynamism,

我们研究刺激后诱导的免疫应答
we study induced immune responses following stimulation.

一个相关的例子
One pertinent example of this

是QuantiFERON-TB Gold分析法
is the QuantiFERON-TB Gold assay,

它使用TB抗原刺激和干扰素γ的值
that uses a stimulation with TB-antigen and an interferon gamma readout

来确认患有结核病的人
to identify individuals who have experienced tuberculosis.

在更多以研究为基础的情况中
In more research-based settings,

常用的常规方法
the conventional approach

是刺激外周血单核细胞（PBMC）
that is often used is the stimulation of PBMCs.

在密度梯度分开后分离PBMC
PBMCs are isolated following density gradient separation,

例如将全血在聚蔗糖上分层
for example, layering whole blood onto Ficoll and then,

然后离心
performing centrifugation.

之后
After this step,

在这个中间层获得这一代白细胞
you get this generation of the leukocytes in this middle layer,

然后用移液管或注射器抽出
that you then draw off with a pipette or a syringe.

然而
However,

标准化问题尚未解决
this can be challenging to standardise

并且可能在下游免疫表型方法中
and can introduce technical biases

引入技术偏差
in downstream immuno-phenotyping approach.

另一种方法是全血刺激
An alternative is to use whole blood stimulation.

难点在于血液凝固和样品处理
This can also be challenging due to blood clotting and sample handling.

但是
However,

这种方法有一个优点
it does have an advantage.

它含有多核细胞 血小板和血浆蛋白
It contains the coenocytes and also the platelets and the plasma proteins

这些物质在从PBMC中去除的
that can be important for induced immune responses

诱导性免疫应答中发挥重要作用
that are removed from PBMCs.

关键是对于临床研究
The key point to make is that for clinical research,

检测标准化至关重要
assay standardisation is very critical.

在Milieu Intérieur
Within Milieu Intérieur,

我们使用的是TruCulture
we have been using TruCulture.

这是标准化免疫监测的全血采集
This is a whole blood collection and incubation system

和培养系统
for standardised immunomonitoring.

你能看到这是基于注射器的方法
It is a syringe-based approach that you can see here.

在将其附着到患者或供体的
Your stimuli of interest are already in the tube

蝶形注射器之前
before you attach it

刺激物已经在试管中
to the butterfly syringe of the patient or the donor.

血液进入管内
The blood goes into the tube.

收集到合适的数量后
And the plunger breaks away

柱塞会脱落
when you have collected the right amount.

然后缓慢地混合三次
You then mix gently three times.

将其放到加热块中
And add it to a heating block where you can incubate

在常规氧气空气中孵育一定时间
at a defined time in conventional oxygen air.

在标准化的刺激时间之后
After a standardised time of stimulation,

细胞开始从血浆上清液中沉淀下来
the cells have begun to settle away from the plasma supernatant.

但它也带有这个塑料分离器
But it also comes with this plastic separator

您可以很简单地组装
that you can assemble very simply.

放入管中
Place into the tube

向下推细胞
and push down on the cells

使其与上清液分离
to separate them form the supernatant.

您现在可以从细胞沉淀中提取上清液
You can now extract the supernatant from the cell pellet,

稳定后供后续转录组分析
which you can stabilise for later transcriptomic analysis.

我们最近完成了一个多中心临床研究
We recently completed a multi-centre clinical study

直接比较TruCulture和传统的PBMC刺激
to directly compare TruCulture and conventional PBMC stimulation.

我们与七个FOCIS卓越中心合作
We worked with seven FOCIS centres of excellence.

这是一个临床免疫学学会的联合组织
It is a federation of clinical immunological societies.

并且与TruCulture技术的所有者
And in partnership with Myriad Rules-Based Medicine,

Myriad Rules-Based Medicine合作
the owners of the TruCulture technology.

7个参与中心每个都接受标准化的方案
Each of the 7 participating centres received standardised protocols,

等分的刺激试样
aliquots of stimuli,

从健康供体那里采集血液
collected blood from healthy donors and stimulated the whole blood

并刺激同一供体的全血和PBMC
and the PBMCs from the same donors.

一段时间后
After a while,

收集上清液
the supernatants were collected,

冷冻起来
frozen,

并在CLIA认证的实验室中
and analysed by Luminex technology

通过Luminex 技术进行分析
in a CLIA certified lab.

我将展示这项研究的两个例子
I am going to show you two examples from that study.

首先是LPS诱导的IL-6应答
Initially, the LPS-induced IL-6 response.

左边是PBMC
On the left, you have PBMC.

右边是TruCulture
On the right, you have TruCulture.

我向大家展示IL-6诱导的反应
I am showing you the IL-6 induced response

单位是皮克/毫升
in picograms per millilitre.

沿X轴的是每一个研究中心
Along the X axis is each individual centre,

每个点是一个供体
and each dot is a unique donor.

如果我们首先看PBMC
If we look first at PBMCs,

你可以看到每个中心的内部变异是合理的
you can see that the intra centre variability is reasonable.

约是半对数
It is about half a log.

但是在不同中心的反应中段
But in the median response across the different centres,

可以看到从一个中心到另一个中心
you can see that it is highly variable

变化很大
from one centre to another.

相反
In contrast,

如果我们现在看TruCulture
if we now look to TruCulture,

我们可以看到在中心内部
we can see a very tight intra centre

存在非常紧密的变异反应
variability response.

这一对比至关重要
But one crucial for this comparison.

如果我们追踪不同中心的中线
If we trace the median line across the different centres,

可以看到
we can see

在欧洲不同地点的不同中心之间
they are much more reproducible and easier to compare between the different centres

中线具有更高的可重复性并且更易比较
that were in different sites across Europe.

我们可以将此扩展到IL-6应答以外
We can extend this beyond the IL-6 response

并针对中心之间的差异进行方差分析（ANOVA）
and perform the ANOVA test for differences between centres.

这样做之后
If we do that,

PBMC系统中35个蛋白质中
29 out of the 35 proteins in the PBMC system were different

有29个在具有收敛性的中心之间是不同的
between centres with astringent.

具有统计学意义的截断值
A significant cut off,

采用的是Q值小于0.01
a Q value of less than 0.01.

这是一个错误值
It is a false discovery rate

只是一个p值
and just a p-value.

相反
In contrast,

在TruCulture中
with TruCulture,

35个蛋白里
only 4 out of the 35 proteins

只有4个在各中心之间有明显不同
were significantly different between centres.

为什么我们认为PBMC方法很难标准化
Why do we think that PBMC approach is so hard to standardise

并给出了这样的结果
and gives such variable results?

一个原因是在非刺激背景下
One clue is in the null background.

这里展示的是无刺激的IL-6水平
This is showing you the non-stimulated IL-6 levels.

同一种图形
Same kind of graph.

纵观PBMC的反应
Looking at the PBMC response

我们可以在几乎所有的中心看到
we can see very variable levels of IL-6 in nearly

差异很大的IL-6水平
all of the centres

除了一个关键中心看起来像是异常点
but one key individual centre looks like an outlier.

事后证明是内毒素污染
It turned out later to be an endotoxin contamination.

通过TruCulture
With TruCulture,

我们可以看到无刺激控制的理想情况
we can see what you would expect from your non-stimulated control.

未显现IL-6
No presence of IL-6.

除了一些供体可能是真正的生物学变异
Apart for some individual donors, which now might be real biological variability.

如果我们进行ANOVA检验
If we run the ANOVA test,

在各中心35个蛋白质中有29个是不同的
29 out of 35 of the proteins were different across the centres.

如果我们删除中心7的离群值
If we remove the outlier of centre 7,

在35个蛋白质中
we still have six out of 35 proteins

仍然有6个在非刺激背景控制下是不同的
that were different in the non-stimulated background control.

相比之下
In contrast,

在TruCulture中
with TruCulture,

35个蛋白里
none of the 35 proteins

没有一个在非刺激对照中有明显不同
were significantly different in the non-stimulated control.

这足以说明TruCulture比PBMC更标准化
This convinces you that TruCulture is more standardised than PBMCs.

TruCulture可以适用任何类型的刺激
And you can adapt TruCulture for any stimuli of interest.

在Milieu Intérieur
Within Milieu Intérieur,

我们已经开发了29种不同的刺激条件
we have developed 29 different stimuli conditions that we described

在几年前《Immunity》上的一篇论文中
in a publication a couple of years ago

有所描述
in "Immunity ".

这些刺激可以由微生物组成
Those stimuli can consist of microbes.

包括细菌 病毒和真菌
We have bacteria, viruses and fungi.

也可以是卡介苗
We also have the BCG,

即结核病分枝杆菌疫苗
the vaccine mycobacteria for tuberculosis .

在激活免疫细胞受体的
At the level of the MAMP,

微生物组分（MAMP）的层面
the components of the microbes that activate immune cell receptors,

我们已经很全面地覆盖了整个领域
we have pretty extensively covered that space.

已经包括了许多临床相关的细胞因子
We have included many cytokines,

尤其是结核病
clinically relevant cytokines,

有IL-1β和1型干扰素
and in particular for TB,

这些在免疫应答中很重要
we have IL-1 beta and type 1 interferons that are important in immune responses.

也有两种刺激T细胞的方法
We also have two ways of stimulating T-cells.

直接的CD3 / CD28和肠毒素细胞
A direct CD3/CD28, and an enterotoxin cell.

在这篇论文中
In this initial publication,

我们描述了25个供体中
we described the protein response following the stimulation

所有这些刺激后蛋白质的反应
with all of these stimuli in 25 donors.

在这里仅向您展示这25个供体的
I am just showing you one result,

主成分分析（Principal components analysis,PCA）的结果
a principal component analysis on those 25 donors,

包括7个TLR激动剂和11个蛋白质的读数
for the seven TLR agonists and an 11-protein readout.

PCA将所有这些数据压缩到三维中
The PCA is condensing all of that data into 3 dimensions,

如在PCA图中所得到的
as captured in this PCA plot.

在这里
Here,

主成分1中
driving PC 1,

你可以看到蓝色部分的LPS引起的反应
you can see the LPS-induced response, coloured in blue.

主成分2中
Driving the 2nd principal component,

从顶部到底部
going from the top to the bottom,

我们可以看到它是由聚 (I:C)反应引起的
we can see it has been driven by the poly (I:C) response,

这和其他TLR反应非常不同
which is a very different response to the other TLRs.

然后
Then,

主成分3是ODN反应
the third principal component is the ODN response.

这触发了TLR 9
This is triggering TLR 9.

在这之中用oR848
In-between those,

和Gardiquimod来刺激TLR7和TLR8
we have oR848 and Gardiquimod to stimulate TLR 7 and TLR 8.

然后是FSL和鞭毛
And then FSL and flagellum,

基于本分析上很难分开
which are harder to separate based on this analysis.

这个PCA表现的是
But what this PCA captures is

我们可以启动一个独特的诱导免疫应答
that we can initiate a unique induced immune response

取决于使用哪种TLR激动剂来刺激
depending on which TLR agonists we use to stimulate.

除了蛋白质
As well as proteins,

我们可以用它来研究转录组学的反应
we can use this to study transcriptomic response.

这在我们几年前的于《Cell Reports》上
This, we described in a paper in "Cell Report s",

发表的一篇论文中描述过
a couple of years ago.

我将展示一些主要的结果
I am showing you some key results

关于应用于纳米串杂交阅读时
showing the robust evaluation independent of RNA extraction

独立于RNA提取的稳健评估
when applied to a nanostring hybridization read-over.

在X轴上
In the X axis,

可得到完整的RNA提取
you have the full RNA extraction

从而获得大于8的良好RIN
which gives you a good RIN of greater than 8.

在Y轴上
On the Y axis,

是RIN小于6的无氯仿信号阶梯提取
a chloroform-free signal step extraction with a RIN of less than 6.

不管提取方案如何
Irrespective of the extraction protocol,

我们获得了可重复和标准化的RNA反应
we get a very reproducible and standardised RNA response.

我们也做了一些可重复的测试
We also did some reproducible testing.

这里的四个生物学重复的例子
This is showing example of four biological replicates,

同一个供体在超过一个月期间的
so the same donor at four time points

四个时间点
over a one month period,

用LPS
stimulated with either LPS,

或干扰素α刺激作为实验组
or interferon alpha

和无刺激为对照组
and the Null control

展示的是聚类分析热图聚类分析图说明
and showing you a hierarchical clustering heat map,

在这个供体中诱导的免疫应答是可重复的
which captures the concept that the induced immune response in this donor is reproducible,

而且与刺激时间无关
irrespective of the time of stimulation.

总而言之
In conclusion,

诱导免疫应答的研究需要刺激免疫细胞
I showed you that the study of induced immune responses

但这是难以标准化的
requires stimulation of immune cells which can be hard to standardise.

然而
However,

刺激全血可以消除
stimulation of whole blood can remove

许多与PBMC相关的技术偏差
many technical biases associated with working with PBMCs.

TruCulture为刺激全血的研究
TruCulture provides a robust and reproducible tool

提供了一个强大和可重复的工具
for the study of stimulated whole blood.

它可以适应多种免疫刺激
It can be adapted to multiple immune stimuli.

也可以从相同的供体和相同的
You can also study the proteomic

刺激平行研究蛋白质组学
and transcriptomic response in parallel

和转录组学的反应
from the same donor and the same stimuli.

文献中描述了其他全血刺激方法
Other whole blood stimulation approaches have been described in the literature.

所有这些方法的关键点在于
The key point from all of these approaches is

对这种研究采取标准化方法是很重要的
that it is important to have a standardised approach for such kind of studies.

感谢您的关注
Thank you for your attention.