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CBR Institute's Klaus Rajewsky Discusses miRNA-155 and the Germinal Center Response

Name: Klaus Rajewsky
Position: Senior investigator, CBR Institute for Biomedical Research, Professor, pathology/pediatrics, Harvard Medical School
Background: Professor, molecular genetics, University of Cologne — 1970-2001
EMBO senior fellow, National Institute for Medical Research, London — 1969
Research assistant, Institute of Genetics, University of Cologne — 1964-1966
Postdoc, Institut Pasteur — 1962-1963
MD, Institute of Chemistry, University of Frankfurt/Main — 1962

This week, researchers from the CBR Institute for Biomedical Research, the FIRC Institute for Molecular Oncology, Regeneron Pharmaceuticals, Brigham and Women’s Hospital, and the Max Delbruck Center for Molecular Medicine published data in Science showing microRNA-155 plays a key role in the mammalian immune system.
Specifically, it was found to regulate “T cell helper differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response,” according to the paper’s authors.
“These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo,” they added.
This week, RNAi News spoke with Klaus Rajewsky, senior author on the Science paper, about the findings.
Let’s start with an overview of your lab.
What we are mostly doing is using mouse genetics and conditional gene targeting [methods] we have developed to study gene function, mainly in the immune system, with a special emphasis on B cell antibody-producing cells and B cell lymphomas.
When did microRNAs make their way into your research?
I came [to Harvard] in 2001 … and that was right at the time when RNA interference, microRNAs, the pathways required to produce microRNAs, and so forth came onto the table. We started almost right away with experiments addressing some of the basic components of the RNAi machinery.
But then we decided to focus on individual microRNAs also because that is what one really has to understand — whether these molecules, like transcription factors, control specific functions in a biological context.
You talk about specific microRNAs, and your latest paper in Science focuses on microRNA-155. Did you have this microRNA in mind before you started the experiments or did it come out of the work?
It’s actually both. In the case of microRNA-155, it has a history because [it] is produced from the RNA encoded by a particular gene that was identified some years ago as being involved in lymphomagenesis.
I had been working on lymphomagenesis a lot before and I am still working on it, in particular in the case of Hodgkin’s disease, which is a special focus of my group. In Hodgkin’s disease, the RNA from which microRNA-155 is generated is actually very highly expressed.
It also turns out that this microRNA is very specifically expressed in immune cells, and we focused our work on microRNAs that are selectively expressed in the immune system and, if possible, at selected … stages of differentiation. We thought these would be the most likely candidates to answer the question of whether these things are playing distinct physiological roles.
To do the experiments [detailed in the paper], you created a knockout mouse?
We did two things: We generated a knockout mouse and we generated a mouse that is constitutively expressing microRNA-155 in the cells of interest. For that purpose, we used a conditional targeting approach we have developed so we can actually induce gene expression specifically in particular cells at particular stages of differentiation.
Can you describe this conditional targeting method?
The conditional targeting method uses a recombination system, in this case a bacterial-derived recombination system, which consists of two components: One is a site-specific DNA recombinase. The second are the target sites for this recombinase.
What you do is put the target sites for the recombinase, which are called loxP, around the gene of interest or around a stop signal that you put in front of the gene of interest. Then you express the recombinase transgenically in a cell type-specific way.
Then the recombinase, which is called Cre, is expressed in a cell type-specific fashion — it cuts out in a cell type-specific fashion either the gene of interest that you want to delete or the stop [signal] in order to get the gene expressed.
Can you talk about the experiments covered in the paper and the results?
The experiments in the paper provide evidence that [microRNA-155] is really expressed just [for the] short term after B or T cell activation. In a particular environment in the immune system, these so-called germinal centers, the cells are chronically activated. It turns out that this microRNA is predominantly expressed in these germinal centers.
The germinal center is a key structure in the immune system because it is a place where, after contact with antigens or pathogens, antibody responses are programmed. The B cells that are responding to the antigen are proliferating and are, by processes called somatic hypermutation and class-switch recombination, changing their antibodies such that they would better fit to the antigen in question. Out of the germinal center reaction you get high-affinity antibody-producing cells and memory cells, which carry immunological memory and which are able to produce these high-affinity antibodies.
So it’s a key structure in the generation of high-affinity antibodies and also the memory response.
The second aspect of this germinal center reaction, which brings us back to lymphomas, is that because there is cell proliferation together with processes introducing … mutations into the DNA, things can sometimes go wrong. So you have a sort of carcinogenic micro-environment from which most of the known B cell lymphomas in humans arise — most B cell lymphomas in humans are derived from B cells in the germinal center reaction or those that have passed through the germinal center reaction.
It turns out that microRNA-155 is not only expressed in the germinal center but is actually required for an optimal germinal center response. That is what the paper really shows.
Are there going to be experiments following up on this? Where do you go from here?
The main thing at the moment is that we want to understand how, mechanistically, [miRNA-155 is functioning]. That is where, in this particular case, there is a lack of knowledge.
We found a very interesting phenomenon in [our experiments], namely that in the microRNA-155-deficient cells, there is a deficiency in the production of certain cytokines that are known to be essential for an optimal germinal center response. One of them is called TNF, or tumor necrosis factor, and the other is called lymphotoxin alpha.
Somehow the microRNA is interfering with the proper expression of these cytokines by the B cells undergoing the germinal center response. We have some idea about how that might work, but that is far from established. So we really want to find out the mechanism behind it.
Also, we hope that as soon as we understand the mechanism, we will get a better idea about how deregulated expression of this microRNA is actually involved in lymphomagenesis.

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