Saturday, February 16, 2008

History of CLL (part 2)

The Twentieth Century

Minot and Isaacs produced the first detailed description of the clinical features and natural history of a large series (80 cases) of patients with CLL, and pointed out that radiation therapy shrank the lymph node masses but did nothing for the course of the disease (Minot & Isaacs, 1924). Over the next few decades, many doctors studied CLL, but there were very few insights into its nature. Richard Doll established that it was a disease of late middle age, twice as common in men (Court Brown & Doll, 1959) The usefulness of chlorambucil (Galton et al, 1955) and corticosteroids (Shaw et al, 1961) in treatment was recognized. By the late 1960s, three great haematologists knew all there was to know about the clinical features of CLL and its natural history. The large series of patients from Maxwell Wintrobe's department at Salt Lake City confirmed the very variable survival times in this disease and suggested a means of stratifying the disease according to its clinical features (Boggs et al, 1966). David Galton described a proliferative variant that did poorly and a stable variant that did well (Galton, 1966). He also described CLL as a disease of accumulation of long-lived functionally incompetent lymphocytes, a conclusion arrived at independently by William Dameshek (Dameshek, 1967). In 1973, Mùrk Hansen published a series of 189 cases of CLL that had been followed for a long period of time (Hansen, 1973). This volume, which describes CLL in great detail, is one of my treasured possessions and has been of great value to me in preparing this review. However, because no one had a clear idea of what a lymphocyte did, further progress was inhibited.

What is a lymphocyte?

A major textbook of immunology published in the mid-1950s contains only one reference to the lymphocyte and that was to dismiss it as a serious contender as an antibody producing cell (Boyd, 1956). Although immunology had made great strides after Jenner's rather pragmatic approach to vaccination through the work of Pasteur, Ehrlich, Landsteiner and Metchnikov, it was still all about macrophages and antibodies. As a young medical student in Bristol in the 1960s, I was taught by Professor Yoffey that the lymphocyte was the precursor of the red cell (Yoffey,

Cell-mediated immunity.

The idea that the lymphocyte was in some way involved in the immune response kept surfacing. James B. Murphy, working mainly alone at the Rockefeller Institute in New York on what he thought was tumour immunity but in fact was probably transplantation immunity, assembled an impressive array of evidence (Murphy, 1926). He found that exposure of rodents to X-rays killed their lymphocytes and lowered resistance to cancer grafts. Murphy also discovered that tumours can be grafted into embryos, but are rejected if a graft of spleen or marrow from an adult is included. Finally, he found that rejection of a tumour graft is accompanied by a proliferation of lymphocytes in spleen and bone marrow and that these invade the tumour.

In the 1940s and 1950s, Peter Medawar and colleagues (Billingham et al, 1954), inspired by the horrific burns suffered by wartime fliers, worked on skin grafts. They demonstrated that the accelerated rejection of second grafts could be transferred by lymphocytes and not by antibody. Even Landsteiner, shortly before his death, had demonstrated that contact sensitivity could be transferred between animals by lymphocytes (Landsteiner & Chase, 1942). Thus, it was fairly easy to accept that the lymphocyte might beresponsible for what became known as cell-mediated immunity.


Antibody was a different proposition. Although McMaster & Hudduck (1935) had shown that most of the antibody produced in response to injection of antigen into the ear of an animal was produced in draining lymph nodes, it was clear that the antigen was picked up there by macrophages. The simplest explanation was that they also made the antibody. However, several workers noticed that such an immune response made no alteration to the macrophage, yet caused the proliferation of the lymphocytes, which became big and blastic. Ehrich and Harris (Harris et al, 1945) in Philadelphia cannulated the efferent lymphatic of a responding lymph node and demonstrated that the antibody contained in the lymphocytes that they collected was six times the concentration of that in the fluid.

The plasma cell.

Better evidence that the plasma cell was the main antibody-producing cell was emerging from Scandinavia. First, the observation that myeloma, a tumour of plasma cells, was associated with an excess of antibody globulin (Bing & Plum, 1937), then the observation that repeated immunization of rabbits led to marked increases of plasma cells in lymph nodes and bone marrow (Bjùrneboe & Gormson, 1943) and, finally, the convincing evidence of the binding of fluorescence-labelled antigen to plasma cells and not lymphocytes by the Harvard workers (Coons et al, 1955). Ehrich and Harris were forced to concede the primacy of the plasma cell, but few scientists are willing to let go of their ideas (and research grants) so easily. In a series of brilliant experiments, Harris and his wife explored the immune response in the rabbit to Shigella bacilli. They demonstrated that lymphocytes taken from a responding lymph node, washed free of antigen and macrophages, would induce the production of antibody when injected into a different animal, presumably by transmogrifying into plasma cells (Harris & Harris, 1960). They were right, but
the sceptics still needed convincing.

Lymphocyte life span.

The final obstacle to proving that the lymphocyte had anything to do with immunity was its fast disappearance time. At the beginning of the century, Davis & Carlson (1909) in Chicago demonstrated that the blood lymphocytes were replaced four times every 24 h. Of their four possible explanations, their first hypothesis (that they were rapidly destroyed) was more easily believed than their last explanation (that they escaped through capillary endothelium and then recirculated via the lymphatics), which happened to be the correct answer. The essence of immunity is memory. For the lymphocyte to play a part in immunity it must live long enough to hold a memory. James Learmonth Gowans, in the Dunn School of Pathology at Oxford, solved the conundrum. He injected radiolabelled lymphocytes into the bloodstream and collected them a few hours later from the thoracic duct (Gowans, 1959). When it became possible to examine the chromosomes of lymphocytes after stimulation by phytohaemagglutinin, it became apparent that treatment of men with ankylosing spondylitis by radiotherapy induced unstable chromosome aberrations. By studying these, cytogeneticists in Edinburgh were able to conclude that the average small lymphocyte went several years between cell divisions (Buckton et al, 1967). The lymphocyte clearly lives long enough to carry memory.

Thymus and bursa.

Beard (1900) in Edinburgh believed that the thymus was the source of all white blood cells, but that this function ceased early in life after the whole body had been seeded. After this the thymus could be removed with impunity. The obvious test of this hypothesis, to remove the thymus immediately after birth, was delayed for 60 years. Jacques Miller, working at the Chester Beatty Institute, discovered that neonatally thymectomized mice had impaired immune responses (Miller, 1961). Workers at Yale demonstrated that this deficiency involved delayed hypersensitivity and graft rejection, rather than antibody production (Arnason et al, 1962).

The bursa of Fabricius is a lymphoid organ located at the dorsal aspect of the cloaca in birds. Like the thymus, it involutes rapidly after hatching. The obvious experiment of removing it shortly afterwards occurred to immunologists rather earlier than it did for the thymus. Chicks bursectomized shortly after hatching grew up to be chickens, but when (because of a shortage of birds for teaching) they were used in a class exercise they unexpectedly failed to produce antibody against Salmonella (Glick et al, 1956). This theme of two types of lymphocyte being processed by different neonatal organs to constitute the two arms of the immune response had great power and symmetry. Encouragingly, the experimental work in rodents and poultry was mirrored by the clinical studies of Bob Good in Minneapolis on immunodeficient children (Good & Varco, 1955). Thus, we had B cells and T cells. Much time and effort has been expended looking for a non-existent `bursa equivalent' in mammals. Eventually, it was decided that the bone marrow itself fulfilled the function, but it is a mistake to think of B cells as `bone marrow derived' as both types of lymphocytes have their genesis in the bone marrow.

Recognizing B and T cells.

It is difficult to convey the excitement of the period of the early 1970s when it became possible to recognize B and T cells in the peripheral blood. Listening to Martin Raff speaking with that extraordinarily attractive accent at the British Society for Immunology meetings describing anti-theta antibodies reacting with T cells and anti-mu antibodies reacting with B cells fired me with enthusiasm for that fusion of haematology and immunology that I have practised ever since.

Paul Ehrlich had postulated the existence of preformed receptors on the outer surface of cells that could interact specifically with foreign substances (Ehrlich, 1900). He suggested that when these were bound to the receptor the cell would be switched to producing more of the receptor that would be shed into the surrounding medium as antibody. McFarlane Burnet postulated that each lymphocyte was different, genetically predetermined to synthesize only one type of antibody molecule (Burnet, 1959). Thus, after contact with antigen, only those cells preprogrammed to produce an antibody with a complementary structure would be stimulated to proliferate and produce antibody-producing progeny, in effect a clone of the antigen-recognizing cell. Sell & Gell (1965) in Birmingham had first shown that antibodies against immunoglobulin could induce blast transformation in some lymphocytes, implying that immunoglobulin was located on the surface of some lymphocytes, presumably as a receptor for antigen. In Ave Mitchison's laboratory at Mill Hill, Martin Raff and Roger Taylor demonstrated by immunofluorescent staining that this was indeed the case (Raff et al, 1970). It was the common belief at the time that immunoglobulin, or perhaps a portion of the molecule, was also the antigen receptor in T cells. A simple experiment by Raff demonstrated the error. The theta antigen (later renamed Thy-1) is present in the brain, thymus cells and thymus-derived cells in the spleen and lymph nodes of mice (Reif & Allen, 1964; Raff, 1969). Raff (1970) showed that all lymphocytes expressed either theta or immunoglobulin, but never both.

This was all in mice, of course. Then, towards the end of 1971, a flurry of papers appeared. In successive weeks of October The Lancet published first a paper from Gus Nossal from Melbourne using iodine 125 labelled antibody (Wilson & Nossal, 1971) and then one from John Holborrow from the Canadian Red Cross Memorial Hospital at Taplow in Middlesex using direct immunofluorescence (Papamichael et al, 1971) Both studies reported that B cells could be detected in human peripheral blood. Both series included patients with CLL among their subjects. In contrast with normals in whom only approximately 7% of lymphocytes expressed surface IgM, in CLL an average 89% of lymphocytes carried surface immunoglobulin. These studies were marred by non-specific binding of IgG and by the assumption that the T-cell receptor must also be immunoglobulin, but, nevertheless, it had at last been established that the CLL cell was a B cell. The Australian paper also showed that CLL cells had far less surface immunoglobulin than normal B cells.

Who got there first? Was this to be 1845 all over again? There were other contenders. Grey et al (1971a) demonstrated surface immunoglobulin on the cells of 20 CLL patients only a month later in a paper published in the Journal of Clinical Investigation, and this paper was certainly submitted before either of the Lancet papers. Moreover, it had been published in abstract form five months earlier (Grey et al, 1971b). Both Seligmann's (Preud'homme et al, 1971) and Pernis's (Pernis et al, 1971) papers were published in December and a more comprehensive study from Paris (Preud'homme & Seligmann, 1972) appeared 12 months later. It was probably Eva Klein who should be given the priority, as her single case report appeared the previous year (Johansson & Klein, 1970). What this shows is how pointless such squabbles are. This was an idea whose time had come. As the subject of much gossip and speculation on the conference circuit, any one of a dozen laboratories could have found it first.

For humans there was no theta antigen. But the remarkable property of human T cells forming rosettes with sheep red blood cells (Lay et al, 1971) formed a surrogate assay until monoclonal antibodies were developed. There then developed a fashion for rosetting that has now passed. The most useful discovery was the property of CLL cells of forming rosettes with mouse red blood cells (Stathopoulos & Elliott, 1974). The immunophenotype of CLL was quickly defined. As well as IgM, most cells also carried IgD (Fu et al, 1974; Preud'homme et al, 1974). Surface immunoglobulin density was much lower than for normal B cells (Chen & Heller, 1978). Paradoxically, an antigen initially regarded as T-cell specific and later designated CD5 was recognized on the surface of CLL cells by the monoclonal antibodies RFT-1, Leu-1 and OKT-1 (Caligaris-Cappio et al, 1982).

1 comment:

Anonymous said...

It's hard to believe the discovery of B and T lymphocytes came really just recently. We certainly knew more about the moon and Mars than we did about our own blood.

Hard to believe.

I can remember my own excitement when I read of stem cells in 'Scientific American' I believe in 1980. I had never heard of them, but it solved the problem of where blood cells come from.