Clarence Cook Little (1888–1971): The Genetic Basis of Transplant Immunology

Abstract

Clarence Cook Little was born just outside of Boston in 1888 into an old Boston family descended from Paul Revere (1, 2). He became the captain of the track team at Harvard College, received his PhD from Harvard University, and then joined its faculty. His first (of 15) publications in Science appeared when he was 21 years old. He had been hired (and resigned) as the president of two important universities by the age of 40. His passion was genetics and his ambition was to find the genetic causes of cancer, but his greatest contribution to cancer research was the discovery of an important nongenetic cause (the murine mammary tumor virus). For 15 years, starting in 1929, he was the managing director of the American Cancer Society. After that he was the scientific director of the Tobacco Industrial Research Committee, distributing money for research, but insisting for years that the evidence did not prove that there was a relationship between smoking and lung cancer. By all accounts, he was charismatic, strong-minded, charming, and brilliant. None of this, however, describes C. C. Little's two most important achievements. First, he elucidated the genetic basis for transplant rejection – leading to the discovery of the MHC – from early experiments that he and others performed and from his insights into their results. Second, he founded the Jackson Laboratory, which has been so instrumental in advancing transplantation as well as numerous other scientific fields. Despite his importance, C. C. Little's name is less well known today than that of George Snell, Jean Dausset, Peter Medawar, or others of later generations who went on to win Nobel prizes for their work. But while Little never received a Nobel Prize, he truly laid the groundwork for those who followed. His contributions deserve to be remembered. While an undergraduate, Little came under the influence of W. E. Castle, one of the leading geneticists of his era (3, 4). Genetics was then largely an agricultural field, but after the rediscovery of Mendel's laws in 1900 there was a growing interest in determining how they applied to mammalian inheritance. Castle selected rabbits, guinea pigs, rats, and mice and assigned the mice to Little (2). Little's earliest genetic studies were on the inheritance of mouse coat colors (5-10). This turned out to be a fortunate selection, as multiple genes determine coat color expression. Little soon realized that if the inheritance of a particular color required coexpression of several alleles, then its inheritance, in aggregate, would occur less frequently than would be predicted by the application of simple Mendelian laws, even though the individual genes behaved according to Mendel's rules (10). For example, assume that a given color is determined by the coexpression of A, B, and C dominant alleles. If a mouse that was ‘ABC’ at these loci were bred with a mouse that was ‘abc’, then the F1 generation would be ‘Aa, Bb, Cc’. Therefore the F1 mice would continue to express the given coat color, as the trait was dominant. However, if members of the F1 generation were bred together to generate an F2 generation, multiple combinations of alleles would emerge, such as (AA, BB, CC) (AA, Bb, CC) (AA, Bb, Cc) (AA, Bb, cc) (AA, bb, CC), etc. Little performed the calculations to show that only 27/64 (less than half) of the mice in the F2 generation would express at least one A, B, and C allele (11). This was much less than 3/4 of the mice in a F2 generation that would inherit a dominant trait by Mendelian laws if it were determined by a single gene. Actually, this example is oversimplified because it assumes that the mice in the parental generations were homozygous (AA or aa) at the three loci. Little apparently realized almost immediately that genetic studies of the sort that he had in mind would depend enormously on the development of inbred mouse strains in which homozygosity could be assumed. Fortunately for him, nature had provided some help in this process, as wild mice tend to live in isolated colonies with significant inbreeding. In addition, a group of people known as ‘mouse fanciers’ had carried the process further after they had realized that some of the prized traits among their captured mice could be maintained by regular brother-sister matings. One example was the Japanese Waltzing Mouse, which Little used for some of his early studies, and which he continued to inbreed in his laboratory (12). (Waltzing mice had a neurologic disorder that caused them to turn rhythmically around in their cages in a way that suggested that they were dancing.) An especially important source of mice at this time was from Abby Lathrop of Granby, Massachusetts (13). A former schoolteacher, Miss Lathrop had discovered that she could actually make a living breeding unusual mice for the mouse fanciers (such as creamy buffs or ruby-eyed yellows) and she eventually also became the supplier for several academic laboratories, including that of Little (14). At her peak she was ordering a ton and a half of oats each month to feed the mice housed in her barn and she was employing the children of her town at 7 cents/h to help maintain the colony (14). Here and elsewhere, mammalian inbreeding had been in practice long before C. C. Little, but he was the first to apply the process rigorously for scientific purposes. Little's ‘dilute brown agouti’ (d. br. a.) strain was started while he was a student at Harvard (the origin of today's DBA mice) and he also generated the C57BL strain (the origin of some of the most widely used mouse strains in modern science). Both of these strains were derived from mice provided by Abby Lathrop (14). Some have considered Little's work generating inbred mouse strains to be among his most significant achievements (1). Whether that is true or not, his experience with inbreeding, plus his early work with coat colors, prepared him for his most important insight about transplantation. Ironically, it was not the insight that he was looking for, or even one that seemed to interest him particularly during the rest of his life. After college, Little started working on his doctoral degree. He soon became interested in the genetics of cancer susceptibility and resistance. This became his primary scientific focus from that time on. Ernest Tyzzer at Harvard had already been conducting studies on the genetic basis of cancer susceptibility for several years (15). Little joined the effort, bringing his highly inbred mice to the undertaking. What Tyzzer had found was that if malignant cell lines that had been developed in one mouse strain were injected into mice of that same strain, then they would grow vigorously and cause death. On the other hand, if they were injected into other strains, then the tumors would usually fade away, allowing the recipient to survive. Tyzzer had already described a puzzling feature of this genetic trait: that while susceptibility to death from the tumor existed in the parental strain, and was maintained in a F1 generation that was derived from the parental strain, it completely disappeared in an F2 generation. When Little joined these studies, it was unclear how Mendelian laws could explain a trait which persisted in the F1 generation (therefore indicating that it was dominant), but which disappeared in the F2 generation. In 1914, C.C. Little, alone, published a two-page article in Science that many think was his most brilliant contribution (11). It was entitled, ‘A Mendelian explanation for the inheritance of a trait that has apparently non-Mendelian characteristics’. The article uses the inheritance of coat colors to illustrate the point that a trait determined by the coexpression of several dominant alleles will be inherited in a F2 generation at a rate lower than 3/4. It then elaborates the mathematics of what inheritance would look like, according to Mendelian principles, if it required that a large number of dominant alleles be inherited together. Although the article did not present the formula in precisely these terms, what Little showed was that under those circumstances, a trait would appear in the F2 generation at the rate of (3/4)n, where n = the number of genes that determined the trait. Little pointed out that if ‘n’ were large enough, a dominant trait might seem to disappear completely in the F2 generation even though it was inherited according to Mendel's laws. It is hard to know, nearly a century later, exactly what the origins of this article were. On the one hand, the mathematics were clearly an extension of Little's early coat color studies with Castle. On the other hand, by this time Little was working with Tyzzer on the studies dealing with cancer susceptibility – the studies that had produced the puzzling outcome regarding the F2 generation. In effect, Little's 1914 article seems to have been a statement of his hypothesis to explain Tyzzer's earlier results. Little's analysis suggested that it might require a really large number of F2 progeny, derived from carefully inbred parental strains to see the expression of a trait determined by a large number of genes. Perhaps these numbers and conditions exceeded those of Tyzzer's earlier experiments. Two years later, Little and Tyzzer, together, published an article reporting that Little's 1914 manuscript did indeed explain the cancer susceptibility results (16). Their new data showed that when they used highly inbred strains, and transferred tumors between them and to their progeny, they found that the rate of acceptance was essentially 100% in the original parental strain, essentially 100% in the F1 generation, and 1.6% in the F2 generation. The effort required to detect this 1.6% finding in the face of background noise must have been huge, but it is clear that by this time Little knew what he was looking for. Another manuscript in 1916 calculated that there were approximately 12 genes responsible for cancer resistance (17). Although in retrospect we now know that Little's papers of 1914 and 1916 revealed that a large number of histocompatibility loci determine the acceptance of allogeneic tissues, his interpretation at the time was quite different. Little believed that the loci he was detecting encoded ‘factors’ produced by the recipient that promoted the survival and growth of particular tumors (16). Although he discussed ‘immunity’ to the tumors, he assumed that all mice (in the parental, F1, and F2 generations) had equal immunity to cancer and that this natural resistance was overcome only if the recipient provided all of the critical cancer survival factors for a particular tumor (16). Despite his primary interest in cancer genetics, Little did perform some experiments transplanting nonmalignant tissues between mice of different strains. His initial report of these experiments was published in 1922 and included new data showing that while splenic tissue transplants from a parental strain were accepted by members of the F1 generation (like tumors), transplants from the F1 generation into the parental generation were rejected (18). Despite this new information, Little still interpreted the results as he had previously: that the recipient must provide factors that matched those required by the donor tissue. In 1924, however, Little wrote his only other paper that was specifically about nonmalignant tissue transplantation. In it he analyzed the results of an expanded series of experiments with quite a different interpretation (19). When considering the rejection of the F1 transplants by the parental strains, Little suggested, for the first time, that the loss of tissue transplants might be due to the expression of something harmful by the donor rather than by the absence of something beneficial in the recipient. He wrote, ‘These facts bring out the point that the host[sic] is the agent which discriminates in favor of, or against the implanted tissue. If the host recognizes in a physiological way the implanted bit of tissue as foreign to itself, it will eliminate the implant. If on the other hand, the implant has no foreign or antagonistic elements the host does not eliminate it …. Furthermore, without actually using the term, Little also realized from his results that the genes encoding the elements responsible for transplant rejection must give rise to codominant expression because: ‘the first generation hybrids arising from a cross between two homogeneous races [strains] which differ from one another, have the ability to grow the tissue of both parent races[sic]. Finally, Little suggested in this paper that one could use linkage analysis to identify some of the particular genetic factors responsible for the transplantation outcomes. Today, introductory transplantation courses often mention ‘five laws of transplantation immunology’: (1) Strain A transplants to Strain A recipients succeed; (2) Strain A transplants to Strain B recipients fail (and vice versa); (3) Strain A (or B) transplants to (AxB)F1 recipients succeed; (4) (AxB)F1 transplants to Strain A (or B) fail; (5) Strain A (or B) transplants to members of an F2 generation almost always fail, but occasionally succeed. These ‘laws’ were formulated by George Snell in 1948 (20-22). However, these were not really laws, but rather experimental observations made by C. C. Little (and others) between 1909 and 1924. Little, however, essentially alone, provided the fundamental insight to explain these observations: that tissue rejection depends on the codominant expression of products encoded by a large number of loci, such that expression by the donor tissue of the products of these loci will lead to rejection if they are not also expressed by the recipient. Little had also provided a rough guess as to the number of these loci (his data indicated approximately a dozen) and he had suggested a way of investigating individual loci by linkage analysis. All of this had been accomplished by 1924 when he was 36 years old, but Little essentially never again wrote about transplantation immunology during the remaining 47 years of his career. However, he did do much more that was important for the field. If one reads anything about or by C. C. Little, it quickly becomes apparent that he had broad interests and enormous energy. As a young member of the Harvard faculty, he served as President Lowell's secretary and as assistant dean of the college (1). His administrative responsibilities increased and in 1922, at the age of 33, he became the president of the University of Maine – the youngest university president in the country. Little's causes included a strong commitment to enhancing the welfare of his students, a major interest in enhancing education for women, and an insistence on speaking publicly about the importance of birth control. Some of his other positions, however, were less popular with his students, such as when he vehemently resisted both the use of student automobiles and the use of alcohol at fraternity parties. Reading Little's articles, one senses that he enjoyed a fight – perhaps even more than the outcome – and some of his writings sound arrogant in a way that those who knew him insist that he never was (21, 22). Little, who was described as ‘brilliant but tactless’, left the University of Maine after a 3-year ‘stormy term’ (1). However, the important event during those years was not connected to the University, it was Little's decision to establish a small laboratory for summer research at Bar Harbor on Mt. Dessert Island. The island was the site of summer homes for some of America's wealthiest families, but whether that was what attracted him is not clear. It is clear, however, that Little demonstrated an exceptional talent for raising money throughout his career. Immediately after leaving Maine in 1924, Little became the president of the University of Michigan. Once again his tenure ended after just 5 years when he resigned under pressure, but once again it was not the university but the location that was important. While in Michigan, Little became friends with members of some of the wealthy families of the young automobile industry, including some of the Fords, and including Roscoe B. Jackson of the Hudson Motor Car Corporation (2). Some of these people also summered on Mt. Dessert Island where Little continued to pursue his own research during the summer months. It has been suggested that Little was looking for an excuse to leave the University of Michigan in 1929 because he had already determined that his real interest was in creating a full-time laboratory in Maine for the study of genetics (1, 2). Little raised the money to create his new laboratory and to hire a small group of young geneticists from his wealthy Michigan friends. When Roscoe Jackson died at just this time, the laboratory was named for him. (The name was later shortened to just The Jackson Laboratory.) However, while the prospects for this venture might have seemed bright early in 1929, they must have seemed very bleak by that end of the year after the stock market had crashed. A full history of The Jackson Laboratory is well beyond the scope of this short article. However, the important point is that the essence of the laboratory came almost entirely from C. C. Little, or ‘Prexy’ as he became known to all who were associated with the laboratory. Little remained the director until 1954. Inbred mice from his own colonies were among those that were used initially to stock the laboratory. Somehow Little managed to finance its early years during the depression, although scientists from those years recall growing their own food in order to survive. When the economy improved, he raised the funds for new buildings and to enlarge the staff. In 1947, after 18 years of breeding mice, a fire destroyed the entire laboratory and killed almost every single animal. Little brought his staff together and told them they were going to rebuild and start over, which they did (23). Within a year almost every mouse strain was breeding again, thanks to an extraordinary response by scientists around the world, who sent back breeding pairs from strains that had originated at The Jackson Laboratory. In 1941, George Snell edited the first edition of a book called ‘Biology of the Laboratory Mouse’ with chapters written by the scientists at The Jackson Laboratory (24). Little wrote a chapter called ‘The Genetics of Spontaneous Tumor Formation’ and another called ‘The Genetics of Tumor Transplantation’. In both he focused almost entirely on cancer susceptibility, almost as if he had forgotten his own transplantation paper of 1924. Less than one page of more than 50 was devoted to the transplantation of nonmalignant tissues. Little wrote: ‘Little [sic] has been said in this chapter on the bearing of tumor transplantation to the genetics of normal tissue transplants. This omission is not due to the fact that the subject lacks importance or interest. It results from the somewhat extraordinary fact that so little work has been done in this field – with properly controlled material – that it remains practically an open door for experimentation. Whether these words were an invitation to George Snell to pick up where Little had left off is unknown, but that is what happened (25). Over the next 10 years Snell introduced the term ‘histocompatibility loci’ (tissue compatibility) to describe the multiple ‘factors’ that Little had realized were responsible for tissue rejection (20). Snell devised a breeding strategy (based on Little's original insight) that enabled him to isolate individual histocompatibility loci by generating ‘isogenic resistant’ strains (by repeatedly identifying members of F2 generations that were resistant to death by tumor transplantation) (20). By 1948, Snell had identified one particular locus that appeared to be especially important in determining transplantation outcomes and (as Little had predicted) he had found a marker (the fused

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