Stem cells, tolerance and rebooting the thymus

Professor Richard Boyd is deputy director of the Monash Immunology and Stem Cell Laboratories and group leader of the Immune Regeneration Laboratory at Monash University.

He also runs the immunology platform at the Australian Stem Cell Centre and is chief scientific officer of Norwood Immunology, a subsidiary of biotech company Norwood Abbey, which was set up to commercialise his discoveries.

In October last year he received a $5.2 million grant from the NHMRC to lead a program that will combine stem cell therapies with rebuilding the thymus.

He will speak on the topic of reversal of thymic atrophy for the development of tolerance to stem cell-based therapies and the improvement of immune function in adults at the ISSCR meeting in Cairns next week.

How did you become interested in the thymus and its function?

Immunology was just taking off in the early 70s when I was starting out. Cancer was big and AIDS was on the horizon. We were still trying to figure out exactly what that the thymus did - it was Jacques Miller, a good friend and colleague, who discovered it.

The distinction between T cells from the thymus and B cells from the bone marrow was just starting in the early 70s and the question was: what are T cells and what do they do? Fortunately for me I took a different path from most - it was clear that T cells were important but it was also clear that a lot of diseases had T cells as the problem. That could be a lack of T cells or a failure of T cells in cancer or abnormal T cells in asthma, in allergies and more importantly in auto-immune diseases like multiple sclerosis, diabetes and the like.

Rather than try to tweak the T cells, which a lot of people were doing, I decided to figure out how they were produced. I thought that if we knew how to produce them, then we could figure out what goes wrong in diseases and have a better way of repairing them.

Back then we looked at it very basically and asked, how does a thymus function? We found it was because it had this very specific class of stromal cells - everyone talks about stromal cells now but back then it was pretty rare. We knew that the microenvironment made up of epithelial cells was the reason that the thymus had the exclusive right to make T cells. My lab's main claim to fame was that we mapped all of that out.

Then you began to look at the reason why the thymus atrophies?

Yes. The next point was to find out which bits did what, so we looked at disease states. We found a correlation between thymic defects and disease and then we looked at how it develops in the embryo. And here was this unusual finding that the thymus, despite its importance in maintaining health and creating immune systems, was one of the first organs that deteriorated with age. Only up to about 10 or 15 years ago it was considered a dead organ.

In evolution per se, you are designed to live two generations - in the first generation you grow up and you reproduce, you live one more generation and by that time your offspring have been able to reproduce. In all mammalian species that's the golden rule, and that would put the lifespan of humans at about 30. So perhaps it is a built-in self-destruction mechanism maintaining optimum population growth and not too much demand on the food chain. We don't really know why the thymus collapses but perhaps it's because if you've made enough T cells it stops you from making too many.

How do you go about rebooting the thymus?

First of all we wanted to regrow it. Our target disease back in the 80s was HIV. My PhD was about making epithelial cells, taking them from the thymus and proving that they can make T cells. Then it took another 20 years to figure how to do that properly.

Our clinical approach was, how can we reverse the ageing process of the thymus? We have two approaches - one was to identify thymic stem cells, which we've managed to do in an embryo. We can purify them and transplant them into an animal and grow an entire thymus. We pursue that quite vigorously - it's all mouse and mouse embryo but one of our big tasks is how that can be expanded.

The second approach was to consider what turns the thymus off during normal development and can we reverse that process. There are a number of factors but the most obvious one was steroids. Any steroid is the immune system's natural enemy, so when you are stressed you see a high level of glucocorticoids, which shut down the thymus. But the thing that became even more obvious was the sex steroid story, ridiculous as it is. As much as you need them for fun, every time you had fun your thymus got smaller.

We looked then at some old literature that suggested that if you castrated an animal you can reboot the immune system. We didn't take much notice of that but we repeated it and it certainly was the case. That in itself was fascinating science but it was never going anywhere because not many people line up to be surgically castrated.

What rescued the package was that we had beautiful data on rebooting the immune system in mice with surgical castration, and then Geoff Grigg from CSIRO alerted me to the fact that you can do this hormonally, not surgically, with LHRH (luteinising hormone-releasing hormone). LHRH turns out to be our golden gift because it is fully reversible, it has been used in the clinic for over 25 years in prostate cancer and it's a very safe drug.

How are haemopoietic stem cells involved?

We proved that we could reproduce everything that we did with surgical castration with hormone therapy in mice. Basically all mammalian species have this phenomenon. We worked out that it didn't have any side-effects, because a big question was, if you rebuild an old thymus do you make an abnormal one? The answer was no. Does a new thymus mean that you get new T cells in the blood stream? The answer was yes.

It's like a new for old insurance policy: you replace your old cells with new ones. And another very important thing that we found was that the thymus needs to feed on something and the feed is provided by the bone marrow in the form of haemopoietic stem cells. So we showed that rebuilding the thymus required these haemopoietic stem cells. That was a good thing because it meant that it gave us a way of not only rebuilding the thymus but steering it the way we wanted it to go.

We've just finished a trial here in Melbourne and we are just writing it up and submitting the data. It is asking the question: would it be useful if we could rebuild a thymus in chemotherapy patients and the answer was, that would be fantastic. At least if you have new T cells you may defend against opportunistic infections which are a big killer in cancer patients.

We started a clinical trial and asked one simple question - in a cancer patient, with chemotherapy and a bone marrow or haemopoietic stem cell transplant - if we give them this hormone, do they get new T cells? And the answer was yes. As a result of that pilot study in Melbourne we are now running two US trials, one on bone marrow or haemopoietic stem cell transplant patients and the other is giving melanoma patients who receive a vaccine this hormone to see if they will have a stronger immune response to the vaccine.

What is the potential of this approach for organ transplant patients?

An important application is the finding that the thymus takes in haemopoietic stem cells. The stem cells go through the blood, they go into the thymus and one stem cell turns into millions of these new T cells. Each of those new T cells has an individual specifity - every new T cell could either react with a virus or bacteria and it could also react against you.

What the thymus does, for every hundred cells it produces, it eliminates 95 per cent, because either they are not formed properly or they are deemed to be self-reactive. Here's the rule - you have an active thymus and you are making T cells and those T cells see a self-antigen in the thymus. If they are able to see it and bind strongly to it they will be killed by a sort of suicide pathway. And that's the reason we don't self-destruct, because all the cells that could destroy us are killed before they leave the thymus.

So if we know that rule and we look at organ transplants, we have to do two things. We have to have an active virus and we've got to have, somehow or other, donor material inside the thymus. So how do you do that? The first problem is that old people don't have a thymus so we reactivate that with the LHRH.

Next, if we give those patients a haemopoietic stem cell of the donor type - they go into the thymus and form into donor cells inside the thymus and now you are producing T cells that are being screened for reactivity against self and against donor. So now, for every 100 cells that we produce we may only release three into the blood stream, but now we don't have any that react against the donor or the host.

Our approach to the organ transplant is first of all to eliminate the immune system and then rebuild a new one by reactivating the thymus, giving a haemopoietic stem cell from the same donor as the kidney or the liver. That becomes a fundamental change in the way organ transplants are done.

What will you be telling the stem cell researchers at the ISSCR conference?

If you have a stem cell and turn it into a cell for treating diabetes, then that pancreatic beta cell, if you inject it into the patient, it will be rejected. What if we go back a step? Here we have an embryonic stem cell: on one side we push it to become a beta cell to treat someone with diabetes. On the other side we push that same embryonic stem cell to become a haemopoietic stem cell. Now we give the patients a dual transplant. We have the LHRH to activate the thymus, the embryonic stem cell-derived haemopoietic stem cell to create tolerance, and the beta cell to get them over the diabetes.

Then there is multiple sclerosis too. We also use the same principle to recover the immune system after chemotherapy and another application would be in HIV: if you can introduce a gene into the haemopoietic stem cell that blocks HIV infection and you inject that haemopoietic stem cell into a patient whose thymus has been activated by LHRH, every new T cell that that patient produces will be HIV-resistant. And then you have a really smart way of dealing with HIV. There are a lot of tricks you have to around it but the principles are fairly simple.

The immune system gets damaged by so much - HIV, ageing, chemotherapy, radiation, viral infections. So we need to rebuild it and we can do that with haemopoietic stem cells. Then we can reboot it with thymic stem cells. It's a very nice marriage.