Rajat Singh

By Rajat Singh

1.1 Early theurgic understandings of immunity

In 61 AD, the Roman poet Marcus Annaeus Lucanus started work on his unfinished epic ‘Pharsalia’ which tells the story of the civil war between Julius Caesar and the Roman senate forces led by Pompey. In book IX, after the assassination of Pompey, Cato the Younger embarks on a journey from Dyrrhachium to Utica, where he would make one last stand against Caesar (Ridley, 1905). Lucan describes an encounter with members of the Psyllian tribe who come to their aid during the perilous trek. Here, with some artistic liberties, the great poet uses—arguably for the first time—the word ‘immunes’ to describe the famous resistance to snake venom that the tribe possessed. While Lucan has often been touted as an example of a great classical poet who stripped the role of the supernatural from historical events, where he succumbed to the allure of the magic of the Psyllian’s when he wrote

“Of all who till the earth

The Psyllians only are by snakes unharmed.

Potent as herbs their song; safe is their blood,

Nor gives admission to the poison germ

E'en when the chant has ceased. Their home itself

Placed in such venomous tract and serpent-thronged

Gained them this vantage, and a truce with death…”

Up until the 19th century, the term ‘immunitas’ and ‘immunis’, which were originally used in Latin legal texts to refer to ‘exempt status’, rarely ever alluded to incidents of resistance to poisons or plagues (Silverstein, 2009). However, the observations were inevitably noted all along. In describing the Plague of Justinian (spread of the plague described in figure 1.1), 541 AD, Procopius said

“It started from the Aegyptians who dwell in Pelusium. Then it divided and moved in one direction…it spread over the whole world, always moving forward and traveling at times favorable to it…For it left neither island nor cave nor mountain ridge which had human inhabitants; and if it had passed by any land, either not affecting the men there or touching them in an indifferent fashion, still at a later time it came back; then those who dwelt round about this land, whom formerly it had 'afflicted most sorely, it did not touch at all…” (Dewing, 1914).

Figure 1.1 Possible origin and spread of the Justinian plague as described by Procopius (Keys, 2000)

While curiosity around the phenomenon we refer to today, as ‘acquired immunity’ remained alive, another pertinent and related question was being tackled in parallel—what was the source of diseases? As one might guess, the earliest explanations were theurgic, i.e. the agency of divine or supernatural intervention was the source of all illness. In ancient Greece, Asclepius, the God of Medicine and healing was revered for centuries. Although Hippocrates (460-377 BC), the father of medicine, himself was considered to be a follower of Asclepius, he rejected the supernatural causations of ailments and established empiricism, rational thought, and science as virtues to be extolled in the search for explanations of disease (Savel & Munro, 2014). As is with family recipes passed down through generations that experience subtle evolution not through outright replacement but by the addition of newly acquired spices, the theurgic ideas made room for the Hippocratic tradition of medicine and slowly, and reluctantly (but never completely) gave way to western medicine. The blending of rational thought with a cogent belief in the power of the supernatural is the template of early medical prescriptions that can be found in historical texts of almost every culture around the world. The physicians and healers of the time walked the line between cautious prescription of strategies understood through observation and analysis and explaining everything else with the help of the mood of Mother Nature, the alignments of stars, and when all else failed, the Gods.

1.2 Early theories of immunity and disease

History does not speak of a watershed moment that may have caused a drastic paradigm shift. From Asclepius to Hippocrates, the transition had been gradual and is arguably ongoing. However, mankind had found value in empiricism and the philosophy of the Hippocratic school of thought caught on. An early theory from that period—pertinent to this discussion—advanced the idea that illnesses were a result of imbalances between the humors—blood (sanguine), phlegm (phlegmatic), yellow bile (choleric), and black bile (melancholic). These imbalances were initially thought to be in the actual amount of the humors. However, a ‘rediscovery’ of Hippocratic teachings by Galen (2nd century AD) along with a better understanding of physiology, anatomy, and pathology resulted in a shift in the paradigm from quantitative to qualitative imperfections of the humors. Between the 5th and 10th centuries AD, a gradually improving understanding of smallpox helped refine these early theories of disease. An illustrious Arab physician by the name of Rhazes, not only gave a thorough description of the clinical symptoms of smallpox but also put forward, possibly for the first time, a theory for ‘acquired immunity’ (even though he didn’t use these terms) (Abu Bekr Mohammad ibn Zakariya al-Razi, n.d.). In 1546, Girolamo Fracostoro, believed that small seeds (seminaria) were the cause of disease, that they spread from person to person, and had distinct tissue affinities. He also concluded that the differential affinities were the reason for ‘natural immunity’.

In the sixteenth and seventeenth centuries, two schools of medicine arose as a result of the scientific persuasions of physicists and chemists: iatrophysics—touting that all bodily functions have mechanical characteristics; and iatrochemical—whereby physiology was a product of chemical reactions. Most of the theories of acquired immunity were founded on the iatrochemical perspective (Silverstein, 2009).

By the end of the seventeenth century, Western Europe had become seriously concerned with smallpox, and in 1714 the news of the eastern practice of variolation had arrived in letters to the Royal Society of London that focused efforts on acquired immunity to the disease. These letters from Timoni and Pylarni, described the strategy of establishing a benign infection by inoculating pustule-derived crusts from ‘favorable’ cases of smallpox. What followed was the rapid popularity of this strategy that ultimately led to the first clinical trial in immunology—performed on a group of prisoners and another group of orphans in 1721. The success of these ‘Royal Experiments’ brought about renewed interest into the mechanisms of immunity facilitated by variolation. Around this time, the practice also spread to Colonial America thanks to the efforts of Cotton Mather who regularly read the Proceedings of the Royal Society of London, and learned of the letters from Timoni and Pylarni. Mather, with the help of other physicians, spread the word via published documents from the New England region and addressed concerns that were raised in objections to the practice of inoculation (Figure 1.2). The text on the title page of the document reads “Some account of what is said of inoculating or transplanting small pox / by the learned Dr. Emanuel Timonius, and Jacobus Pylarinus ; with some remarks thereon ; to which are added, a few in answer to the scruples of many about the lawfulness of this method; published by Dr. Zabdiel Boylstone.” (Mather, 1721).

Figure 1.2 Title page from ‘Some account of what is said of inoculating or transplanting the small pox. Boston: Sold by S. Gerrish at his shop in Corn-Hill.’ (Mather, 1721)

One of the main theories of the time, explaining the acquired resistance to smallpox, was the Depletion Theory which suggested that the basis of this immunity was the depletion of a particular resource specifically required by the disease-causing agent. According to this view, the reintroduction of the causal agent or seed expedited the loss of said resource. Proponents of this theory included Thomas Fuller (Fuller, 1730), James Kirkpatrick, Louis Pasteur (Pasteur L., Chamberland C., 1880), and even Paul Ehrlich who extended the idea to tumor immunity.

An inflection point in the history of disease and immunity came from the work of Louis Pasteur and Robert Koch. They provided experimental evidence for bacterial agents as the root cause of infectious diseases and a rubric for establishing them as such. Critical discoveries were made in the ten years from 1880, when Pasteur published his experiments on acquired immunity to fowl cholera via attenuated organisms (Pasteur, 1880), to 1890 when von Behring and Kitasato discovered anti-toxins against diphtheria and tetanus (Behring & Kitasato, 1890). However, the investigators of the time ignored the role of the host in establishing the immunity, therefore producing a list of ‘passive theories’ as classified by Sauerback (Sauerbeck, 1909). The time for ‘active theories’ was on the horizon and was bolstered by the discovery of the antibody and complement.

1.3 Humoralists versus Cellularists

Towards the end of the 19th century, there were two major debates within the field of immunology that split the scientists of the time in two factions. The first issue pertained to the role of inflammation—was it a physiological response that was ultimately beneficial to the organism or was it a deleterious side effect that contributed to the pathology of illness. The second debate, which was more of a battle, had to do with the mechanisms that explained the basis of innate and acquired immunity—was it humoral or cellular.

At the beginning of the debate there was a prevailing bias towards the humoral school of thought. After all, the history of the humors and their perceived role could be traced back to the early tradition of empirical medicine starting from Hippocrates. However, in 1858, Rudolph Virchow challenged the humoral basis of disease when he claimed that all pathology was a result of cellular malfunction and not that of maladjusted humors (Virchow, 1858). While his work gradually gained acceptance, the events that followed and the allegiances that scientists picked were peculiar. In the late 1870s, Robert Koch’s work on the etiology of wound infections further bolstered the germ theory of disease (Koch, 1876). In 1884, Ilya (Elie) Metchnikoff proposed the phagocytic theory (Metschnikoff & Freund, 1884), which initially, had more to do with the questions surrounding inflammation—a question that was primarily within the purview of pathologists. While this might make it seem that both Koch and Metchnikoff would have held similar ideas, you would be gravely mistaken in that assumption

Metchnikoff, who was by training a zoologist, had an early interest in the digestive processes of invertebrates. While studying ‘mobile cells’ in starfish he formulated the idea that those cells could provide protection to the organism when challenged with a foreign, invasive object or organism. He performed a pilot experiment, in which he inserted a splinter into the starfish and then waited overnight to see how the mobile cells would respond. What he noticed was the cells (phagocytes) moving into the region of the injury. He postulated that the role was protective, and that idea was the foundation of what would become the cellular basis of inflammation. This was also the beginning of a protracted attack on his cellularist viewpoint which lasted decades. His early assertions from the platform of the phagocytic theory pertained to the nature of inflammation, which he claimed was not a deleterious response. This met with strong opposition from pathologists who viewed macrophages to be associated with purulent discharge and inflammation associated with poor prognoses. Such was the reaction to his ideas that Rudolph Virchow during a visit in 1883 cautioned Metchnikoff against advancing his theories at the time (Metchnikoff, 1921).

As one might imagine, the phagocytic theory had immunological implications and soon came under attack from the humoralists. The tale that followed is multilayered and complex and had serious implications for the trajectory of research in immunology. The debate that should have remained within the hallways of scientific discussion bled into political and nationalistic ideologies of the factions—the humoralists were primarily German and generally took their cues from Robert Koch, while the cellularists were mostly French who followed the lead of Metchnikoff (Silverstein, 2009). A long-held divide between the nations had come to a head during the Franco-Prussian war of 1870, which the French lost to the Germans, and led to beliefs and biases that compromised objectivity in the assessment of science.

Pasteur, who had received an honorary M.D. from the University of Bonn, returned the degree in the aftermath of the war. Ten years later, Koch and Pasteur engaged in an unpleasant debate over the etiology and pathogenesis of anthrax and other diseases, which eventually lost all sense of civility. This enmity between the two countries played out once again in the form of a scientific argument between Jules Bordet and Paul Ehrlich many years later (Zinnser, 1914).

A number of observations came in quick succession in the last two decades of the 19th century from both sides, which in retrospect, hinted at the importance of both mechanisms. However, at the time, this possibility was forcibly overlooked and, in the end, the cellularists lost traction as a number of papers showing that cell-free fluids of normal and immunized animals could kill bacteria started surfacing. The humoralists held the view that the phagocytes that did show up were there to clean up debris and not actively involved in protective activities. The most important discovery that stalled the development of Metchnikoff’s ideas, was the discovery that antibodies conferred immunity against diphtheria and tetanus exotoxins (Behring & Kitasato, 1890). Another study showed that this serum could be passively transferred to another organism to confer protection against diphtheria without the involvement of any cellular components. This led Koch to proclaim the end of the phagocytic theory. Subsequent work from Paul Ehrlich showing the titration of anti-diphtheria antibodies (Ehrlich, 1897) emphasized the nature of antibodies as an entity that could be studied ex-vivo in the test tube—a convenience that immunologists readily found an affinity for—and heralded the age of antibody and complement research, a major step in the evolution of immunology, albeit at the cost of ignoring the cellular basis of immunity to the chagrin of many modern immunologists.

All that remained for the field to latch onto the idea of antibodies was a theoretical framework that could be the foundation of future work. To that extent, Ehrlich did not disappoint, and in a remarkable set of postulates, gave the world the idea of the side-chain theory. It should be added that while the majority of the field shifted its focus to antibodies, there was an appreciation for the cellularist’s viewpoint for a number of reasons including, of course, legitimate curiosity around the phagocyte theory but also in recognition of Metchnikoff as a relentless and brilliant scientist. This was reflected in the 1908 Nobel Prize that was jointly awarded to Metchnikoff and Ehrlich for their work on immunity (Nobelprize.org, 2014a).

From this point on, a relative hiatus in research surrounding cellularist ideas followed. Questions around delayed hypersensitivity (or bacterial allergy) were half-heartedly addressed. Landsteiner and Chase demonstrated the importance of mononuclear cells in cellular immunity in 1942 through their famous cell transfer experiments (Landsteiner & Chase, 1942) but these experiments amongst many others were coming late in terms of what was technically possible earlier in the 20th century. This half-a-century-long lull was turned around in the 1960s when questions about allograft rejection, tolerance, viral infections, immune deficiencies, and autoimmune diseases started surfacing.

1.4 Early theories of antibody formation

The next step in the antibody story starts with the academic masses huddled around the new door that had just appeared—the question of where the antibodies came from and how were they made?

On March 22, 1900, Paul Ehrlich, presented the “side-chain theory” in the Croonian lecture to the Royal Society of London. The theory, which later evolved into one of the foundational tenets of adaptive immunity, postulated that blood cells presented on their surface a variety of “side-chain receptors” that bound to toxic/infectious agents and inactivated them. He went on to suggest that the interaction between the foreign substance and the cell-bound receptor activated the cell, resulting in the large-scale production and release of the receptor with the same specificity into the bloodstream of the organism. He further noted that the specificity of the receptor for the infectious agent was pre-determined (Ehrlich, 1900). An interesting observation about Ehrlich’s work has been made which suggests that a part of Ehrlich’s lay in the pictures he drew to convey his ideas. Figure 1.3 below is one of his illustrations depicting the side-chain theory (Himmelweit, n.d.).

Figure 1.3 Ehrlich’s side-chain theory depicts the antibody-producing cell as having receptors for more than one type of antigen (black). Each antigen has a receptor with a different specificity. The antigen selects for increased production of the antibody.

The side-chain theory soon faced significant opposition from immunologists who believed in an instructional model of antibody production. According to these instructional theories, the antigen itself had a role to play in determining the specificity of the anti-toxin/antibody (Bordet, 1940).

It wasn’t until the second half of the 20th century that selective theories re-emerged. Eventually, the work of N. Jerne, D. Talmadge and F.M. Burnet resulted in the acceptance of the “clonal selection theory” as a paradigm for adaptive immunity (Goldsby, Kindt, Osborne, & Kuby, 2003). This theory confirmed most of Ehrlich’s predictions (with modifications), and in its current form states that the origin of the diverse repertoire of specific antibodies is independent of the antigen pool and each lymphocyte (both B and T cells) expresses only one kind of antigen receptor on its surface (Burnet, 1957). The antigen receptor diversity is attributed to a somatic gene rearrangement process that Burnet alluded to in 1957 when he proposed the clonal selection theory. According to him “The [clonal selection] theory requires at some stage in early embryonic development a genetic process for which there is no available precedence”. This speculative remark proved to be largely correct as was shown by a series of discoveries and the formal proposal of a somatic recombination process known as V(D)J recombination in 1976.

The history described thus far in this text has, for the sake of brevity, skipped over the minutia of the discoveries that were made up until the mid-1900s. However, a component of this story that may seem more pertinent includes the cells of the immune system that we refer to as lymphocytes, and in the next section, I will briefly recount the trajectory of work that was done that led to the discovery of lymphocytes and the distinction that was made between T and B-cells.

1.5 The discovery of B and T lymphocytes

It is clear now that before the identification of lymphocytes, we had an understanding of the nature of the antibody, and its role in immunity had gradually been unraveled. It was in 1948 when Astrid Fagraeus showed that antibody formation correlated with the appearance of plasma cells in the spleen (Fagraeus, 1948). The source of antibodies being plasma cells was subsequently confirmed via immunofluorescence microscopy (Coons, Leduc, & Connoly, 1955). Antibodies had already been shown to be gamma globulins through electrophoretic studies (Tiselius & Kabat, 1939), and between 1952-1955, immunodeficient patients were identified as lacking gamma globulins (Bruton, 1952), and more specifically not having germinal-centers and plasma cells (Good & Varco, 1955).

A curious observation of great implication was reported by Bruce Glick, when he showed that the removal of the bursa of fabricus from newly hatched chicks led to a defect in antibody formation. In retrospect, it is clear that the observation was initially overlooked, in part due to the publication of his work in Poultry Science, a journal of little consequence to immunologists (Glick, Chang, & Jaap, 1956).

While the clonal selection theory was being proposed to the world, a better understanding of the composition of the antibody molecule was emerging—it was shown to possess 4 chains—paired heavy and light chains that were connected via disulphide bridges (Edelman, 1959; Porter, 1959). It was also shown that the heavy and light chains had a homogenous, crystallizable fragment (Fc) and a variable, non-homogenous fragment (Fab). New questions about the genetic makeup of higher organisms and the manner in which the variable and constant regions could potentially arise in an antibody molecule were coming up and in 1965, Dreyer and Bennett postulated that different variable and constant region genes could combine to make a specific light chain or heavy chain of the antibody (Dreyer & Bennett, 1965).

The idea of the thymus as an organ critical for immune function had also been emerging. Jacques Miller noticed that thymectomized mice had severe immunodeficiencies. Independently, Good, who had studied agammaglobulinemia picked up clues from Glick’s work and combined them with the observations made in patients with thymoma. He also thymectomized mice and came up with similar defects in lymphocyte development. The observations made, initially projected the idea that the thymus gave rise to the cells that became lymphocytes, and these populated various lymphoid tissues—the single lineage model. However, this model did not hold water as different phenotypes emerged from the removal of the bursa and the thymus. In 1966, Max Cooper showed that the specific ablation of the thymus in conjunction with whole-body irradiation of chicks left them lymphopenic, with impaired cell-mediated immunity, and also had problems in their ability to reject grafts. These animals had lower antibody titers but still had plasma cells and formed germinal centers. On the other hand, when the bursa was removed along with whole-body irradiation the inverse phenotype was observed (Cooper, Raymond, Peterson, South, & Good, 1966). This clarified the distinct roles of the two organs and also paved the way for the two-lineage model—lymphocytes were B and T-cells.

In 1974, a number of publications showed that the mammalian equivalent of the avian bursa of fabricus was the bone marrow (and fetal liver) (Owen, Cooper, & Raff, 1974; Ryser & Vassalli, 1974; Stocker, Osmond, & Nossal, 1974). In 1976, Susumu Tonegawa and Nobumichi Hozumi heralded the new era of immunology when they published their findings on the somatic rearrangement of variable and constant genes in the process of forming an antibody light chain (Hozumi & Tonegawa, 1976), work for which Tonegawa was awarded the Nobel Prize in 1987 (Nobelprize.org, 2014b).

The discovery of VDJ-recombination by Tonegawa seems to be an appropriate place to conclude the historical introduction to the field of immunology. This is in part due to the idea that the discoveries made since the late 1970s fall under the category of modern molecular immunology. While the subject matter of this thesis falls within the purview of epigenetic mechanisms involved in the regulation of gene expression between two specific stages of B-cell development, there is, yet again, a need for context around B-cell biology. To this extent, the next chapter will include descriptions of VDJ recombination and the manner in which it is tied to early B-cell development. I will then switch to the particular stage of B-cell development that is relevant to my graduate thesis work—Germinal Center B-cells.

Chapter 1 References

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