A Selection of Editing

By Jason Socrates Bardi

Several years ago, Professor Norman Klinman of The Scripps Research Institute was surprised when he received a review copy of an article by a young immunologist he had met in Europe in the early 1980s.

This immunologist, David Nemazee, who was then working at the National Jewish Hospital in Denver, was making a bold assertion that seemed to violate the theory of clonal selection—a physiological process whereby the immune system generates a diversity of B and T cells.

The clonal selection theory had been around since the 1950s, when it was first suggested by the Australian immunologist Frank Macfarlane Burnet. Nemazee's paper did not suggest that clonal selection was wrong, but it was a significant departure from that theory, which was subscribed to by most immunologists of the time.

Clonal selection held that autoreactive B cells were merely eliminated. But in the paper, Nemazee showed that B cells have a proofreading quality-control mechanism enabling autoreactive cells to edit, modify, and reexpress the receptor to become non-autoreactive.

He called this mechanism receptor editing.

"[The B cells] seem to throw out part of the receptor and replace it, keeping the other half," says Nemazee. "The body can recycle cells that otherwise would be thrown out—we thought it was quite important."

Such a stunning claim caused Klinman to raise an eyebrow. He sent the article back to Nemazee, asking him to provide more experimental evidence.

Nemazee did the experiments Klinman suggested, and published the paper in 1993. Another group at Princeton University published a similar finding at the same time.

"It was huge," says Klinman.

Making the Most of Recombination

Nemazee, who is now a professor at Scripps Research, has always been interested in understanding tolerance (why antibodies don't attack the body's own tissues). His discovery of receptor editing came out of that interest.

The immune system faces a daunting problem in a world full of pathogens: how can it possibly recognize the myriad viral and bacterial antigens—pieces of protein or carbohydrate that stimulate an immune response? Harder still, how can the immune system anticipate new antigens from mutated viruses and bacteria that don't even exist yet?

What enables a great number of foreign antigens to be recognized by the immune system is the extraordinarily large T and B cell "repertoire" that the body produces and maintains. This diverse repertoire is generated within the body by a process that involves the rearrangement of specific genes within these B and T cells.

When a B cell develops from stem cells in the bone marrow, a process that occurs continuously throughout life, it rearranges its immunoglobin gene—that codes for both the large receptor protein that sits on the surface of the B cell to recognize antigen and the antibody that is specific for this antigen once it is recognized.

Immunoglobins and antigens have two components, a heavy chain and a light chain, which are recombined to develop an incredible diversity of antibodies.

The rearrangement of the heavy chain occurs first, and it involves bringing together three segments (termed V, D, and J for variable, joining, and diversity, respectively), which are spliced together in a process that is appropriately named V(D)J recombination. Since there are multiple copies of the V, D, and J genes in the human genome, a recombined heavy chain has over a million possible combinations.

Following recombination, the final sequence is permanently spliced together as the heavy chain so that a mature B cell will produce only one specific antibody. Then this "pro" B cell is checked with a quality control mechanism to ensure that the heavy chain is able to express, fold, and bind to its eventual light chain. Most B cells will not make this cut.

The ones that do will proliferate through a number of cell divisions (the exact number is not known in humans) in a process known as clonal expansion and become "pre" B cells.

"Cells at that stage are developmentally arrested," says Nemazee. "They are not allowed to leave until they are fully schooled."

The schooling first involves a test. The immunoglobin gene is checked to make sure that its immunoglobin receptor does not recognize self antigen, which would lead the mature B cell to produce autoreactive B cells. The B cells that are not autoreactive at this point pass the test and are positively selected and become immature B cells.

In the early 1980s, Nemazee made an in vivo model to study tolerance. The model had only autoreactive B cells, and Nemazee and colleagues tested what would happen if they introduced the autoreactive cells to the models both in the bone marrow and in the periphery.

"At that time, says Nemazee, "the thinking was that the [autoreactive B cells] should have all been eliminated."

However, they noticed some of the B cells escaped elimination when they were introduced into the bone marrow. Upon closer inspection, they were startled to find that these no longer expressed an autoreactive receptor. The receptor had been "edited." This was the work Nemazee submitted for publication that Klinman reviewed.

Receptor editing allows the immune system to correct a bad receptor rather than throwing the whole cell away. Through editing, the immune system saves time and energy, getting the most antibody diversity at the lowest cost.

Once B cells are committed to their receptor, they leave the bone marrow and circulate through the blood, lymph nodes, and spleen, where they mature further. When they bind to the antigen directly or are activated by other components of the immune system, they begin the process of proliferating and secreting antibodies to mark bacteria and viruses for destruction.

Application to Disease

Now Nemazee is interested in basic questions of biochemistry and molecular biology related to receptor editing, such as how recognition of self antigen turns the genes that control recombination on and off.

He is also studying what regulates the survival of B cells, looking for the genes that are involved in B cell survival. "Your bloodstream is full of B cells that have probably been around for years and have never divided since they were produced," Nemazee says. There is very little data showing which genes are involved in keeping non-autoreactive cells alive preferentially.

Nemazee is also interested in the implications of receptor editing for human health. A number of diseases, such as lupus and rheumatoid arthritis, are caused by B cells making antibodies against self tissue. This sort of "friendly fire" leads to a number of health problems.

"Maybe a defect in this process plays a role in autoimmune diseases or in immune deficiency diseases," he says.

One hypothesis he is looking at is that if a person's immune system cannot edit autoreactive immunoglobin receptors efficiently, then immune deficiency results as the B cells that produce these autoreactive receptors are eliminated. He is looking at in vivo models that have defects in the genes that control receptor editing to see if they have an abnormal level of B cells.

Another project in Nemazee's laboratory involves a collaboration with Scripps Research Professor Dennis Burton on a grant that went into effect on April 1 and is funded by the National Institute of Allergy and Infectious Diseases, one of the components of the National Institutes of Health.

Improving HIV Vaccine

The idea is to come up with a way of improving the body's response to an HIV envelope DNA vaccine.

"As my colleague Dennis Burton has taught us, most of the antibodies that you raise against HIV are irrelevant because they see the wrong parts of the HIV envelope protein," says Nemazee. "One would like to have a vaccine that induces more antibodies to the right part of the HIV envelope protein—the CD4 binding site."

The solution that Nemazee and Burton are working on involves fusing the HIV envelope protein with a second protein that both stabilizes the normal structure of the envelope protein and stimulates better antibody production.

The approach was inspired by work on a broadly neutralizing antibody discovered in the bone marrow of a 31-year-old male who had been HIV positive without symptoms for six years. Designated b12, the antibody has a long finger-like region on its surface that penetrates the surface of the main viral glycoprotein gp120 on HIV and prevents it from causing disease. A team of scientists at Scripps Research and at the Glycobiology Institute at Oxford University in the United Kingdom elucidated the structure of the antibody a few years ago. See http://www.scripps.edu/news/press/081001.html.

Since gp120 on HIV is a protein that forms a trimer, vaccines that mimic this structure may work better.

The protein to which they are fusing gp120 is called BAFF, which is a B cell survival factor. Significantly, this may help the antibody because BAFF naturally causes a huge increase in antibody production when it is overexpressed.

Continuing the Tradition

Klinman helped the Department of Immunology at Scripps Research recruit Nemazee in 1998. Around the same time, immunologist William Weigel, one of the founders of the institute, was retiring. Weigel had pioneered research into the area of immune tolerance by showing in the 1960s and 1970s that B and T cell immune tolerance could be induced in early in vivo models, and he laid the foundations of the field by exploring the parameters of tolerance.

With Weigel retiring, Klinman wanted to recruit a faculty member who would work in the same area of immunology and carry the torch.

"This is a department that has been extremely strong in the issues of autoimmunity and tolerance," says Klinman. "It's our history—it's who we are."

Send comments to: jasonb@scripps.edu

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Scripps Research Professor David Nemazee, who discovered receptor editing, is now interested in its basic mechanisms and implications for human health.