M. R. Irwin

Immunological Studies in Embryology and Genetics

T _ HE subject of this symposium suggests that immunology and biology are more or less distinct scientific disciplines and that there is need to show how the first may be useful in advancing knowledge of the other. Some years ago attention was called to the close familial relationship existing between immunology and one branch of biology. At the risk of boresome repetition, this relationship might be reviewed briefly. Shortly after the middle of the preceding century, there were two eggs fertilized at slightly different times in a common mother, biology. These eggs developed, respectively, into the sciences of genetics and bacteriology. A somewhat later fertilization of another egg in the same mother resulted in a third progeny, experimental embryology. Furthermore, bacteriology begat a lusty offspring known as immunology, and genetics originally claimed descent from a double fertilization by botany and zoology, although quite recently there is increasing evidence that bacteriology itself is more than a foster parent. Thus the lines of descent of these different disciplines from a common ancestor, biology, are steadily becoming more closely intertwined. (Perhaps some day some curious mind will attempt to calculate the coefficient of inbreeding of each of these disciplines.) As is so often the case among relatives, at least in the human family, acquaintance is necessary to promote and maintain sympathetic understanding. This symposium is a promising sign that steps have already been taken to encourage these relatives to become friendly, and to learn a little of each others language. Indeed, certain fundamental questions in both biology and immunology have much in common. Thus, in immunology very little insight has been gained as to how the globulins of the serum, in which all or nearly all the antibodies are found, can be changed specifically during immunization and how this change can be maintained for varying periods. Likewise, in general biology, the basic problem of how a gene reproduces itself is still a major mystery. A current hypothesis of the manner in which antibodies are formed with their well known specificities-the idea of a master molecule or template which Haurowitz has ably presented-has provided the foundation and much of the framework for proposals of the self-duplication of the gene. Boyd has effectively pointed out that one of the earliest attempts to link the technics of immunology to a general biological problem-other than in bacteriology-was the classical work of Nuttall (1904), who compared the taxonomic relationships of many species of animals with those deduced from similarities of the serum proteins of these species. Incidentally, the relationships among the species based on morphological criteria and on antigenic similarities were in fairly close agreement. Several workers have extended such studies, as Baier, Boyden, Moody, Wolfe and others. The related work in plants, particularly that by Mez and his co-workers, was reviewed several years ago by Chester (1937). In general, these studies showed that in the higher plants as in animals the relationships between species as revealed by serological tests parallel closely those determined by morphological criteria. Special attention should be called to the studies of Wells and his colleagues, who utilized particular proteins of related species of plants in their experiments on protein specificities.

STUDIES ON BLOOD GROUPS IN THE AMERICAN BISON (BUFFALO)

The antigenic characteristics of red cells provide unusual advantages for the analysis of genetic similarities and differences among related species. This opportunity has been most extensively exploited in the analysis of relationships among various species of pigeons and doves (cf. Irvin, 1953) and in ducks (McGibbon, 1944). Rather numerous studies have been made also of the occurrence of antigenic factors related to those found in human blood, as they occur in other animals (cf. Wiener, 1943). The most common experience in such studies has been to find that antigenic materials similar to, but often not identical with, the antigens segregating or fixed in a given species are either segregating or fixed in a related species. There has been little basis for evaluating the degree of such relationships in terms of the evolutionary affinities of the populations concerned. Some years ago, we undertook to capitalize on the availability of a large battery of antibody reagents distinguishing individual differences in cattle, for the investigation of this kind of problem. Several levels of taxonomic diversity were subjected to study, beginning with differ-

INTERRELATIONSHIPS AND EVOLUTIONARY PATTERNS OF CELLULAR ANTIGENS IN COLUMBIDAE

Previous papers from this laboratory have described the recognition of heritable antigenic characters which differentiate between pairs of species of Columbidae which hybridize. The qualitative differences which distinguish one species of a pair from the other have been obtained singly in backcross populations by mating the species hybrids and selected backcross hybrids to the other parental species. The presence or absence of these differentiating characters can be determined in related species of Columbidae by immunological tests. It is possible to determine whether such antigenic characters, when present in other species, are carried in these species as an indistinguishable or only as a similar character. For example, a previous paper (Irwin, 1953) described the sharing or non-sharing in over 20 species of Columbidae of antigenic specificities of the cellular antigens which differentiate Streptopelia chinensis from S. risoria. From such information inferences can be drawn concerning the relationships of the causative genes for the antigenic characters in the different species. This paper describes the occurrence or non-occurrence in over 30 species of Columbidae of antigenic specificities which reciprocally differentiate Columba guinea and C. livia. Four cellular antigens (A, B, C, and

Immuno-genetic studies of species relationships in columbidae

SummaryThe comparisons of the species-specific and homologous characters in the erythrocytes of four species of pigeons and doves, as presented in this paper, have as their foundation previously reported cellular characters peculiar toColumba guinea (not inC. livia) which have been isolated as a result of back-crosses tolivia. Comparable results, entirely confirming these, have been obtained in the comparison of the Pearlneck and Ringdove species, especially in the separation of specific Pearlneck characters in back-crosses of the species hybrids to Ring dove.The present studies show definitely that two of the specific components ofguinea, by which it is differentiated fromlivia, have homologues in both Pearlneck and Ring dove. Certain other specific characters ofguinea, as contrasted withlivia, are shared in part by either or both Pearlneck and Ring dove; the others presumably are found only inguinea.These results show that a genetic character which distinguishes one species from another may in turn be shared with still another species. Further, other reactions show that each of these four species possessed cellular substances not found in any of the others.The interrelationships of the cellular components of each species with those of the other three are shown in the diagrams. Presumably these may be taken as approximate phylogenetic relationships.

An Embryo Specific Red Cell Antigen in a Dove Species, Streptopelia Risoria*

The erythrocytes of all embryos tested to date of a dove species, Streptopelia risoria, have carried an “embryo antigen” during the first seven days of incubation. It gradually disappeared and was not observed beyond the fourth day after hatching. Peculiarly, this embryo antigen was detected by the use of antisera produced against the cells of backcross hybrids carrying one of the antigenic characters (ch‐4) that differentiate S. chinensis from risoria. One possible explanation is that the antibodies were directed against a contrasting character in risoria to ch‐4, and this character is expressed on the cells of the embryos, not on those of the adults.

Antigens, antibodies and genes.

This article is a survey of a few pertinent findings about antigens as they occur in one species of bacteria, the pneumococcus, and in the cells and organs of mammals and birds. The main points of general biological interest emphasized are as follows: (1) The fact that carbohydrates, not proteins, are responsible for the specificity of the different types of pneumococci points the way to a comparable concept of specificity in organisms other than bacteria. That is, substances other than proteins, if linked to proteins, may account for some part of known specificities. (2) Changes within each type of pneumococcus may take place from a smooth, capsulated form to a rough, non‐capsulated form, and vice versa. Under certain conditions a rough form of one type may be changed to the smooth form of an entirely different type. The activating principle in one such change has been found to be a nucleic acid. (3) The antigens of the blood cells of man are gene‐determined. There are, however, antigens of the blood cells, tissues or organs of other species which have not yet been explained genetically. (4) It has been possible by immunological techniques to make a sharp distinction between the cells or sera of closely related species. Reagents prepared by absorbing the antisera to the cells or serum of one species by the corresponding antigens of the other were reactive with the homologous antigen, but not with that used in the absorption. From these results it may be deduced that each species possesses ‘species‐specific’ antigens in comparison with a related species, as well as those common to the two species. (5) In bird species which produce viable hybrids the antigens have been found to be gene‐controlled. (6) The cellular antigens are generally thought to be the more or less direct products of their causative genes. However, a few instances are known of cellular antigens which are the result of complementary action of genes. Also, within the group of genes which act directly on the respective antigens there appear to be some which singly are capable of effecting the same combination of antigens as two (or more) alleles may produce.

Evolution of cellular antigens in Columbidae.

Experiments performed in this laboratory have revealed the complex serological interrelationships of the cellular antigens of a number of species of pigeons and doves (Irwin, 1951; 1953). The studies have shown that red blood cells from the respective species share antigens in common and that they also possess antigens to the exclusion of those found in the cells of one or more other species. It has been demonstrated that the cellular antigens which contrast the different species include several serologically distinct components each of which is determined by a single gene or two or more closely linked genes (Irwin and Cole, 1936; Irwin and Cumley, 1947; Irwin, Cole and Gordon, 1936). The cellular antigens of a particular species may be serologically related to, or identical with, the antigens of one or more other species. The red blood cell antigens ch-1 of Streptopelia chinensis, hu-1 of St. humtilis, or-1 of St. orientalis, se-1 of St. senegcalensis and E of Colutmbia guinea comprise a serologically cross-reactive group (Irwin and Cole, 1940; Stimpfling and Irwin, 1960 and Irwin, unpublished data). In previous papers ch-1 was called d-1, and se-1 was s-2. Evidence has been obtained for the antithetical relationship of the antigens ch-1, or-1, se-1 and probably hu-1

Differentiation of Sera of Two Species of Doves and Their Hybrid.

Summary The serum proteins of two dove species, Pearlneck and Ring dove, and their hybrid were indistinguishable by direct pre-cipitin-tests; i.e. the three kinds of serum reacted at the same dilution with antisera for each of the two species. Following absorption, however, of the antiserum to one species by the serum of the other, a differentiation of the serum-proteins was readily made. The serum of the species-hybrid appeared to possess a combination of the pre-cipitinogens of both parental species.

Parabiotic Twins as a Means of Determining Cellular Individuality.

It has been shown 1 that a division of species-specific substances of the erythrocytes of Pearlneck (Spilopelia chinensis), not present in those of Ring dove (Streptopelia risoria), is found in the progeny of male hybrids backcrossed to Ring dove. Individual differences in the cells of these backcross birds make reasonable the conclusion that many different heritable cellular components distinguish Pearl-neck from Ring dove. It seemed opportune to test whether or not a successful “take” of parabionts in backcross hybrids depends, at least in part, upon the relative number of such Pearlneck substances in one member of the pair as contrasted with their absence in the other. The appearance of antibodies would presumably be due to reciprocal immunization, since to date no isoagglutinins have been observed within the Ring dove species nor in the backcross hybrids. Accordingly, several unions of backcross hybrids, of various ages and in different backcross generations, were made by one of us, (R.T.H.). Kozelka 2 made parabiotic pairs of very young chicks by a metatarsal union. In our experiments a considerably different technique was used. If the birds to be operated on were under a week old, a thorough chilling in an ice box proved an excellent anesthetic, which reduced blood flow to a minimum, and allowed a rapid recovery. In older birds (young adults) sodium amytal, supplemented with ether, was used as an anesthetic. The operative procedure herein described was for the older birds, being nearly the same for young ones.

Probable loss of tolerance and simulation of autoimmunization in induced chimeras in doves

Chimeras were induced in doves (Streptopelia) by making parabionts of embryonating eggs that carried genes for erythrocyte antigens, which were readily identified. The parabiotic pairs were chosen so that new antigenic specificities would appear if somatic cell mating took place. However, no evidence of somatic cell mating was noted. Erythrocytic chimerism was no longer. detectable in some birds after varying periods of time. In a few others tolerance was presumably lost, since their plasma contained antibodies against cellular antigens that either were present, or had been present, in the birds circulation.