Lectures List

The foundations of Immunology

In the preceding lecture two points have been discussed: that the overall impact of medicine on population and health is more limited than commonly believed and that the impact that medicine has had on human health has been largely through vaccination. This lecture covers the time comprised between the 1880s and the early 1960s and will focus on a different issue, ie the emergence of Immunology as a science towards the ends of the 19th century building on the science of emerging microbiology (initially bacteriology) and the the gigantic steps that both L Pasteur and R Koch made toward the development of the germ theory of disease.

It was made very clear in the earlier lecture that vaccination emerged and for many years remained an empirical practice. Only later it acquired the features of a true scientific procedure. Unlike E Jenner, L Pasteur had achieved control of the process of attenuation with the anthrax and rabies vaccines but, just as E Jenner, he had little or no idea of the mechanisms by which vaccination induced immunity. Towards the end of the 19th century, however, the study of immunity acquired a mechanistic foundation. It did so through two separate and for many years seemingly conflicting lines of research: the study of humoral immunity and the study of cellular immunity. I will discuss these separately and, in the following lecture, I will discuss the evidence that led to the view that one cell type, the lymphocyte, is responsible for both types of immunity.

In vertebrates the immunity mediated by lymphocytes and known as adaptive immunity exists alongside a more ancient type of immunity, the so -called innate immunity, that is the main type of immunity of invertebrate organisms and that, in turn, consists of both soluble and cellular defence mechanisms. The discovery of several and essential mechanisms of innate and adaptive immunity is outlined below.

The concept of humoral immunity developed from the analysis of serum components with anti-bacterial activity. In 1888 G Nuttall described the bactericidal properties of blood and a year later H Buchner demonstrated that this was due to a serum activity (alexin) which was later redefined complement by Paul Ehrlich. We now know that the complement is a complex system of plasma proteins able to induce cell lysis. It will be discussed in detail later in this Course.

During the same years, the bacteria that cause diphteria and tetanus were isolated. The bacterium that caused diphtheria was first described in 1883 by Edwin Klebs, a former associate of Rudolph Virchow, and was grown as pure culture by Robert Koch's associate Friedrich Loeffler in 1884, who identified C diphtheriae as the agent of the disease. In the same year, Loeffler obtained evidence that C diphtheriae produced a soluble toxin, and in 1888, Emile Roux and Alexandre Yersin demonstrated the presence of the toxin in cell-free cultures of C diphtheriae. These cell-free preparations, when injected into suitable lab animals, caused diphtheria. From antitoxin to serotherapy In 1890 Emil von Bhering and Shibasaburo Kitasato succeeded in immunising guinea pigs with a heat-attenuated form of the toxin and demonstrated that the sera of immunised animals contained an antitoxin that could neutralise the activity of the toxin, was capable of protecting other susceptible animals against the disease and could transfer immunity to non immune animals although this modified toxin could not be used as a vaccine in humans because it induced severe local reactions. Within a few years antitoxin was prepared commercially, rigorous titration methods were developed in 1897 by Paul Ehrlich and antitoxin was used for the treatment of diphteria with significant results. This led to the emergence and widespread use of serotherapy, namely the transfer of protective immunity by means of serum or their active components (the antibodies). Serotherapy thus constitutes the most important form of passive artificial immunity.
Protective humoral immunity

Antitoxin was thus the first major success of therapeutic Immunology. A number of several other immune phenomena were described towards the end of the 19th century and in the early years of this century. These included lysis of bacterial or red blood cells in the presence of complement, agglutination (clumping) of bacterial or red blood cells, precipitation reaction from cell-free cultures of bacterial cells and enhanced phagocytosis (see below). These phenomena are collected in the Table The discovery of humoral immunity. I. Protective and, together with the earlier findings on the complement system, they represent the basis for the discovery of humoral immunity .

All these activities were found to be specific (they occurred only with the agent that had caused infection or had been used for immunisation) and were grouped under the general term of antibodies, that is substances produced in response to foreign bodies (anti foreign bodies). The substances that induced the formation of antibodies in immune animals were defined antigens (or antibody generators). Athough the relationship between the different types of antibodies remained unclear at the time, the specificity of these substances in serum became the major tool for establishing immunity and the presence of a previous state of infection. This became the basis for serological diagnosis or serology.

Sea anemone

It is of interest that during the same years several phenomena were also described that questioned the notion that the invariable outcome of immunity was beneficial to the host. In 1902 Paul Portier and Charles Richet, during studies on the toxin produced by the tentacles of Actiniaria (sea anemone), found that dogs who had survived small doses of the toxin and were kept for several weeks before being used for subsequent investigations, became extremely ill (with intense dyspnea, diarrhoea and vomiting) and rapidly died upon reinjection of small amounts of toxin. This reaction was called anaphylaxis in order to contrast it with the common state induced by immunity, ie the state of prophylaxis (protection). The following year (1903) Maurice Arthus described another puzzling phenomenon when he reported that daily intracutaneous injection of horse serum in rabbits induced no obvious local response for the first few days but subsequently produced foci of oedema, inflammation and eventually tissue necrosis.

Although there was uncertainty at the time about the relationship between these responses and those that led to protective immunity (hence the introduction of the general term allergy in order to distinguish the two), the similarities were also obvious and became even clearer when in 1921, C Praustnitz and H Kustner showed that allergic reactions could be transferred by serum just as protective immunity. The mechanisms of the major forms of allergy will be discussed in details later in the Course but their subsequent understanding is rooted in these early and critical observations (see Table The discovery of humoral immunity. 2. Allergy).

I have deliberately avoided so far to discuss the issue of the cells of immunity and have given the impression that immunity is the result of circulating proteins (antibodies) being produced in response to immunisation with bacterial or bacterial derivatives. In parallel with the search for the substances responsible for humoral immunity, however, other studies at the end of the 19th century addressed the quest of the cells of immunity.

A cellular mechanism for immunity had been suggested in 1884 by Ilya Metchnikov, a Russian working successively in Russia, Italy and France.

Starfish larva (right)

Metchnikoff discovered that the transparent starfish larva contained cells that could efficiently surround and ingest foreign material, including microorganisms. Subsequent experiments indicated that similar cells existed in the blood of vertebrate animals and this led Metchnikoff to propose that the cells responsible for this process (phagocytosis) constituted the basis of immunity. Phagocytosis is a process in which specialised cells of the body known as phagocytes first migrate towards foreign organisms or components and subsequently engulf and destroy the agents in questions. Several cell types are capable of phagcytosis and these include neutrophils as well as monocyte-derived macrophages. Metchnikoff was profoundly convinced of the importance of phagocytosis in immunity but his proposition clearly failed to attract acceptance given the remarkable successes of the concomitant studies on humoral immunity and their successful applications in diagnosis and therapy. During this time, nevertheless, several experiments were conducted that later proved to be critical for the foundations of what is now known as cellular immunity.

The first such experiment was reported by Robert Kochin 1891 and is known as Koch's phenomenon (see below). Koch had identified the tubercle bacillus (M tuberculosis ) in 1882 and announced at the International Congress of Medicine of 1890 the basis of a cure for tubercolosis in the form of a culture extract defined tuberculin (a glycerine broth culture of the tubercle bacillus maintained for 6-8 weeks, concentrated by boiling and filtered). Understandably, Koch hoped that this extract would serve him in the way in which toxin extracts of cultures of diphteria and tetanus had served von Bhering. Koch's expectation unfortunately met with no success. Immunisation with tuberculin failed to protect animals from subsequent infection and its use in patients with tubercolosis led to severe local and systemic reactions and occasionally to death.

The so-called Koch's phenomenon is the following: if a normal guinea pig is inoculated with a pure culture of tubercle bacillus, the wound closes rapidly and heals in the first few days. After 10-14 days, however, a firm nodule appears at the site of injection which opens forming an ulcer. This typically persists until the animal dies. However, if the same culture of tubercle bacillus is injected in a tuberculous guinea pig (an animal infected 4-6 weeks earlier) it yields a different result. A peculiar change occurs at the inoculation site on the first or second day: the tissue surrounding the site of injection becomes indurated and dark-coloured and in the next few days it becomes necrotic. It finally sloughs leaving a shallow ulcer which usually heals quickly and permanently without infection of the local lymphnodes. Koch also established that killed bacilli or even tuberculin could substitute live bacilli and result in the same local tissue reaction.

Koch's phenomenon was difficult to interpret (although this did not prevent its use for the diagnosis of tubercolosis early this century) but, when anaphylaxis was described in 1902, Koch's phenomenon was wrongly interpreted as an antibody-mediated reaction. Subsequent research indicated a critical difference between anaphylaxis and Koch's phenomenon: the former was shown to be transferable by serum (as already outlined) but similar attempts to transfer the tissue response of Koch's phenomenon failed until in 1945 M Chase showed that the reaction could, instead, be transferred with peripheral blood cells. Thus a basic difference had been established between serum-mediated immunity and anaphylaxis, on the one hand, and the immunity of Koch's phenomenon, on the other.

Early studies on transplantation

A second line of work contributed in an important fashion to the experimental foundations of cellular immunology. This stemmed not from the studies of the immunity to infectious agents but from a seemingly unrelated line of work: the attempt to propagate 'spontaneous' tumours arising in mice. Several workers had collected numerous observations on the transplantability of such tumours and the intense efforts of approximately fifteen years of research at the turn of the century were collected in 1912 in a book by G Schone (Heteroplastic and Homoplastic Transplantation). The general conclusions drawn in the book are listed in the Table Conclusions of early transplantation experiments.

These rules emerged from experiments with transplantable tumours but it was established at the same time that normal tissues would undergo the same fate. In a further review of the field (published in 1916 by EE Tyzzer) these concepts were extended and it was stated explicitly that the degree of immunity which developed depended on the foreigness of the immunising cells. It was also noted that accelerated rejection required a first contact with living cells from the donor (either normal or tumour). It is remarkable that our description of the phenomenology of tissue grafting has changed little since these early reports although, fortunately, our understanding of the mechanism of rejection and our ability to control rejection have made considerable progress.

In 1912 a formal mathematical treatment of the genetic effects of brother-sister mating predicted that after 20 generations all progeny would be greater than 99% identical and homozygous at all genetic loci. This set the basis for the development of a number of inbred strains of laboratory mice (including most of the lines in current use) which became increasingly used for experiments such as tumour transplantation. Tyzzer himself by 1916 had already used an inbred line and had been able to establish that the tissue rejection involved between 12 and 14 multiple independently inherited unit factors (genes).

Cellular immunity

In the 1930s P Gorer , a young English scientist, while studying blood group antigens in mice approximately 30 years after the discovery of the AB0 system in humans by Karl Landsteiner, discovered that serum from humans with type A erythrocytes would react with antigens on red cells from some inbred strains but not others and that the mouse antigen was heritable in a simple Mendelian fashion. Rabbit antisera raised against erythrocytes from several strains allowed him to define three antigens (I, II and III). In subsequent experiments he demonstrated that the rejection of albino tumours by black mice was due to the presence of antigen II on the tumour cells and that anti-II antibodies were present in the serum of black mice. He later showed, however, that although anti-II antibodies could kill the tumour cells in vitro, they had no effect on tumour growth when injected in tumour bearing animals. These experiments provided the foundations for the concept of histocompatibility antigens, whose study was put on a very strong genetic foundation by George Snellin the USA. Remarkably, the killing of Gorer's tranplantable tumour in vivo, like the tissue reaction of Koch's phenomenon, could not be transferred by antibodies. In 1954 N Mitchison was able to prove beyond reasonable doubt that tissue graft rejection could also be transferred by cells. These findings, which span nearly a century of research are the basis for the discovery of cellular immunity .

  • The evidence for humoral immunity
  • Serology and serotherapy
  • Richet and Portier's anaphilaxis
  • Arthus's phenomenon
  • The evidence for cell-mediated immunity
  • Koch's phenomenon

[1 ] Silverstein AM. A history of Immunology. Academic Press, 2nd ed (2009)

[2] Mazumdar PMH. Species and Specificity. Cambridge University Press (2002)

[3] Von Behring E. Serum therapy in therapeutics and medical science. Nobel Lecture (1901)

[4] Ehrlich P. Partial cell functions. Nobel Lecture (1908)

[5] Richet C. Anaphylaxis. Nobel Lecture (1913)

[6] Mechnikov I. On the present state of the question of immunity in infectious diseases. Nobel Lecture (1908)

[7] Koch R. The current state of the struggle against tuberculosis. Nobel Lecture (1905)