The function of antibodies

Summary

Antibodies have multiple biological activities. They can both recognize and act on specific antigens. They can protect against and cause serious diseases, enhance and inhibit antibody responses, enable survival, and threaten life. Which among their many, often antagonistic properties explains that antibodies were selected half a billion years ago and transmitted to mammals across millions of generations? In other words, what is the function of antibodies? Here I examine how their structure endows antibodies with unique cognitive and effector properties that contribute to their multiple biological activities. I show that rather than specific properties, antibodies have large functional repertoires. They have a cognitive repertoire and an effector repertoire that are selected from larger available repertoires, themselves drawn at random from even larger virtual repertoires. These virtual repertoires provide the adaptive immune system with immense, constantly renewed, reservoirs of cognitive and effector functions that can be actualized at any time according to the context. I propose that such a flexibility, which enables living individuals to adapt to a rapidly changing environment, and even deal with an unknown future, may provide a better selective advantage than any particular function.

1. INTRODUCTION: ANTIBODIES FOR BETTER AND FOR WORSE

With the very first Nobel Prize in Physiology or Medicine awarded to Emil von Behring in 1901 “for his work on serum therapy,” antibodies were hailed as the heroes of the new science of immunity. Indeed, serum anti‐diphtheria toxin saved thousands of children from the “Strangling Angel” at the beginning of the 20th century, ¹ and serum anti‐tetanus toxin saved hundreds of thousands of wounded soldiers during World War I. ² One century later, antibodies keep being used as therapeutic agents. Anti‐CD20 ³ or anti‐HER2 antibodies ⁴ paved the way for cancer immunotherapy. Anti‐PD1 or anti‐PDL1 antibodies, the so called “immune checkpoint inhibitors,” opened new avenues for the treatment of malignancies with a poor prognosis. ⁵ Anti‐TNF‐α antibodies are used in inflammatory diseases ⁶ ; anti‐IgE antibodies in allergies. ⁷ Intravenous injections of serum immunoglobulins (IVIG) pooled from thousands of healthy patients, which contain antibodies against multiple antigens including autoantigens, proved beneficial in autoimmune diseases such as thrombocytopenic purpura, dermatomyositis or Guillain–Barré syndrome, ⁸ and inflammatory diseases such as inflammatory demyelinating polyneuropathy. ⁹ Last but not least, with the notable exception of BCG, antibodies account for the protective immunity conferred by vaccines, whether against bacteria and their toxins, or against viruses. Antibodies protect.

In 1902, however, Paul Portier and Charles Richet reported that dogs previously injected with a toxin undergo a deadly shock if they are injected again with the same sublethal dose of toxin. ¹⁰ Five years later, Richet showed that this “anaphylactic” sensitivity can be transferred to naive dogs by the serum of sensitized dogs. ¹¹ In 1913, the Nobel Prize in Physiology or Medicine was awarded to Richet “for his work on anaphylaxis.” Accidents of serum therapy, due to “anaphylactins” and “precipitins,” that is, antibodies to horse serum found in high concentrations in the serum of treated patients were soon reported. ¹² Antibodies are not always protective. They can indeed cause life‐threatening anaphylactic reactions. IgE antibodies were named for their ability to cause “Erythema‐wheal” reactions in the skin. ¹³ Antibodies play prominent roles in allergies ¹⁴ and in inflammatory diseases the incidence of which has increased dramatically in the last 50 years. ¹⁵ , ¹⁶ They are responsible for or contribute to autoimmune diseases such as myasthenia gravis, ¹⁷ rheumatoid arthritis, ¹⁸ systemic lupus erythematosus, ¹⁹ idiopathic thrombocytopenic purpura, ²⁰ anti‐phospholipid syndrome. ²¹ IgA nephropathy ²² was recently found to involve IgA autoantibodies. ²³ Alloantibodies mediate acute graft rejection ²⁴ and heteroantibodies serum sickness. ²⁵ Maternal anti‐RhD antibodies are responsible for neonatal hemolytic anemia. ²⁶ Yet, an injection of IgG anti‐RhD antibodies to RhD⁻ pregnant women before delivery prevents the disease in RhD⁺ fetuses. ²⁷

How can antibodies exert so many effects including antagonist effects? How could such double‐edged molecules which can exert deleterious if not lethal effects, enhance chances of survival and be selected during evolution? If indeed, “the function of a trait is the effect for which it was selected,” ²⁸ what is the function of antibodies? I will argue that antibodies do not have a function but two functional repertoires: a cognitive repertoire and an effector repertoire. I will then discuss how actual repertoires are selected by the context from available repertoires, and how available repertoires are themselves drawn at random from virtual repertoires. I will conclude that virtual cognitive and effector repertoires provide living organisms with always renewed sources of responses that enable them to constantly adapt to unpredictable contexts while they are alive.

2. “X IS THERE BECAUSE IT DOES Z"

Biologists need functions to understand how living beings live. A function indeed provides a biological object with explanations ²⁹ : It explains not only “how,” but also “why” and “what for” this object functions. It provides a cause for its existence. This cause is a special cause, it is an end cause. The problem is that end causes violate the principle of antecedence that rules causality in science and states that causes must precede effects and not the contrary. In biology, an effect (a function fulfilled by a biological object) explains a cause (the existence of the biological object that fulfills that function). The eyes were made for seeing. In spite of this temporal inversion, biologists continue to use functions, and finding the functions of myriad previously unknown molecules has become the grand challenge of post‐Human Genome Sequencing biology. ³⁰

“The function of X is Z means: (a) X is there because it does Z; (b) Z is a consequence (or result) of X's being there.” Based on these statements, an etiological theory of function that aimed at addressing the issue of teleology (from “τέλος” in Ancient Greek, “end,” “purpose”) was elaborated by Larry Wright. ³¹ As discussed above, a function is explained by the existence of a biological object that has this function, and the existence of a biological object is explained by its function: X explains Z and Z explains X. But Wright adds: saying X is there because it does Z explains X's being there not only by the ability of X to do Z in beings that live today, it implies that X did Z already in phylogenetic ancestors and that X was selected because doing Z conferred these ancestors some survival advantage. Living beings have an intrinsic history, in the depth of which biological functions can be traced.

However, saying both “(a) X is there because it does Z; (b) Z is a consequence of X's being there” and “The function of X is Z means (a) and (b)” implies that the function Z of X is both the cause and the consequence of X's being there. Biological functions are, as Wright says, “causal consequences.” ³¹ End causes are replaced by causal consequences. The difference is subtle. By anchoring functions in phylogeny, the etiological theory nevertheless excludes intentions, purposes, or other “intelligent designs.” It integrates them within the Modern Synthetic Theory of Evolution. ³² , ³³ Thus, if “X” is “antibodies” in Wright's statement, what is “Z”? Which, among their many effects, explain(s) that antibodies are there because they were selected ±500 million years ago ³⁴ and were conserved across phylogeny?

3. THE FAB AND THE FC PORTIONS OF ANTIBODIES

3.1. The cognitive and the effector properties of antibodies

Antibodies can both “recognize” specific antigens and activate effector systems. Stricto sensu, antibodies can recognize nothing. They nevertheless have an affinity for those molecules that engaged the BCRs of the B cells whose differentiation into plasma cells led to the production of these antibodies. Antibodies thus “re‐cognize” the same molecule that was previously “cognized” by B cells.

Antibodies recognize antigen via their two Fab portions. Each Fab contains the amino‐terminal variable sequences of a light chain and of a heavy chain, which together form one antigen‐binding site. Antibodies activate effector systems via their Fc portion. The Fc portion consists in the two or three carboxy‐terminal domains of the two heavy chains, which characterize immunoglobulin isotypes (classes and subclasses). The cognitive and the effector properties of antibodies are thus mediated by distinct parts of antibodies. These parts are encoded by genes that undergo unique somatic rearrangements in naive B cells.

Rearrangements of genes that encode variable sequences occur first. In the human genome, 34–38 V_κ genes and 5 J_κ genes encode the variable domains of κ light chains; 29–33 V_λ genes and 5–6 J_λ genes encode the variable domains of λ light chains; and 38–46 V_H genes, 23 D_H genes, and 6 J_H genes encode the variable domains of heavy chains. ³⁵ One variable gene from each set is drawn at random and they associate in a B cell: one V gene and one J gene for the κ and λ light chains; one V gene, one D gene, and one J gene for the heavy chains. The rearranged VJ and VDJ complexes subsequently undergo somatic point mutations that increase their diversity. Heavy chains then associate either with a κ light chain or with a λ light chain. The specificity of all the B‐cell receptors (BCRs) expressed by a B cell is therefore determined (1) by the stochastic association of five variable genes, two of which encode the variable sequences of the light chains, and three of which those of the heavy chains, and (2) by the pairing of a heavy chain with a κ or with a λ light chain. As variable gene rearrangements occur at random, different B cells express BCRs with different variable sequences that recognize different antigens. When engaged by a specific antigen, BCRs activate B cells which (1) proliferate and form clones, and (2) differentiate into plasma cells that secrete antibodies with the same specificity as that of the BCR.

As proposed previously, B cells possess several repertoires of specificities ³⁶ , ³⁷ : an available cognitive repertoire, whose size is limited by the number of naive B lymphocytes is thus randomly drawn from an immense virtual cognitive repertoire made of all possible combinations of variable genes in heavy and light chains and all possible associations of heavy and light chains. The estimated size of the virtual repertoire of each individual is of the order of 2 × 10¹². ³⁵ As anticipated by Burnet's Clonal Selection Theory, ³⁸ a small actual cognitive repertoire resulting from antigen‐driven clonal expansion is subsequently selected from the available repertoire. Antigens do not shape antibodies on themselves as it was proposed once by instructive theories, ³⁹ but they shape the actual repertoire of antibodies.

A second series of rearrangements occurs in B cells that have already acquired a specificity. ⁴⁰ The rearranged VDJ genes lie 5′ to the Cμ gene that encodes the heavy chains of IgM. They are sequentially translocated next to one of the 8 other genes that encode the constant domains of heavy chains: Cδ, Cγ1, Cγ2, Cγ3, Cγ4, Cε, Cα1, and Cα2. This Class‐Switch Recombination thus enables the production of 9 isotypes of antibodies: IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2, which have the same Fab portions, hence the same specificity, but different Fc portions, hence different effector properties (Figure 1A).

FIGURE 1.

The FcR‐mediated effector repertoire of human antibodies. (A) Rearranged genes that encode the variable domains of the heavy chains of BCRs are sequentially translocated next to genes that encode the constant domains of the nine isotypes of immunoglobulin heavy chains. This class recombination switch enables the formation of nine classes and subclasses of antibodies that have the same specificity for antigen, but different Fc portions. Heavy chains of different isotypes are shown in different colors. (B) Antibodies in complex with antigen engage complement and Fc receptors (FcRs). For the sake of simplicity, FcRs only are shown in this cartoon. Arrows show the relative specifcities of antibodies of the nine isotypes for the various FcRs. (C) FcRs of the various types are differentially expressed by myeloid (and lymphoid) cells. Lines show the tissue distribution of FcRs. (D) Different cell types have different functional repertoires. Horizontal bars show the biological activities induced by the engagement of FcRs expressed by the various cells. Both FcR expression and the biological properties of effector cells also depend on their differentiation state and their location in the body.

Effector systems are differentially activated by the Fc portions of antibodies of different isotypes, complexed with antigens. They comprise molecules and cells. Molecules include free (C5a and C3a) and antibody‐bound proteins (e.g., C3b, C4b, or C5b) of the complement system. Cellular effectors are cells that express complement receptors (CRs) and/or Fc Receptors (FcRs). CRs include C5aR, C3aR, CR1, CR2, CR3, CR4, and CRIg. Human FcRs comprise IgG receptors (FcγRI, FcγRII, FcγRIII, and FcRn), IgE receptors (FcεRI and FcεRII), IgA receptors (FcαR), and IgM receptors (FcμR) (Figure 1B). Complement receptors activate cells upon ligand binding. Most FcRs activate cells when aggregated by multivalent antigen–antibody complexes because they possess intracytoplasmic Immunoreceptor Tyrosine‐based Activation Motifs (ITAMs). ⁴¹ Members of a unique family of FcRs (FcγRIIB) inhibit cell activation when co‐engaged with activating receptors on the same cell. ⁴² Inhibition is mediated by an intracellular Immunoreceptor Tyrosine‐based Inhibition Motif (ITIM). ⁴³ FcRn do not contribute to the biological activities of antibodies. They prevent the intracellular degradation of IgG and transport them across cell barriers, ⁴⁴ which markedly extends their half‐life in vivo.

3.2. Neutralizing and blocking antibodies

By definition, antibodies bind to antigens. They can thus “mask” epitopes and prevent immune recognition, or “block” active sites and prevent biological activities of specific molecules. Such properties are exemplified by engineered IgG antibodies that bear a mutation of asparagine 297 in their CH₂ domain. This point mutation prevents glycosylation and impairs the binding to FcRs and C1q, ⁴⁵ but not to FcRn. Fab‐mediated binding to antigens has long been thought to explain most biological properties of antibodies. Antibodies, however, do not have only Fabs, and evidence accumulated supporting that, with one exception, the biological properties of antibodies are also Fc‐mediated.

Thought to prevent toxicity or infection by binding to toxin or viral epitopes involved in toxicity or infection, anti‐toxin and anti‐virus antibodies were described as “neutralizing antibodies.” Mouse IgG antibodies against anthrax toxin can indeed protect both macrophages exposed to the toxin in vitro and wild‐type mice infected with Bacillus anthracis in vivo. Antibody‐induced protection against infection was however abrogated in KO mice lacking activating FcRs. ⁴⁶ Similarly, neutralizing IgG antibodies against influenza virus hemagglutinin required FcRs to protect mice from a lethal challenge by H1N1. ⁴⁷ FcγRs contributed to the ability of neutralizing mouse‐human chimeric antibodies against the HIV‐1 envelope to block viral entry, to suppress viremia, and to protect from infection in a murine HIV‐1 infection model. Protection was enhanced with Fc‐variants of human IgG1 anti‐HIV‐1 which bound preferentially to activating FcγRs in transgenic mice all the FcγRs of which were replaced by human FcγRs. ⁴⁸ Likewise, an optimal therapeutic effect of passively administered anti‐chikungunya ⁴⁹ or anti‐SARS‐Cov‐2 antibodies required the engagement of FcγRs. ⁵⁰ These data, altogether indicate that, if binding to antigen via their Fab portions enables specific antibodies to neutralize toxins or viruses in vitro, protection from bacterial or viral infection in vivo requires that they engage activating FcRs via their Fc portion. It follows that effective in vitro neutralization may not predict in vivo protection against viral infection.

IgG anti‐allergen antibodies elicited by allergen immunotherapy in allergic patients were described as “blocking antibodies” because they were thought to compete for allergen with the patient's IgE antibodies. No correlation, however, was found between serum IgG levels and protection. ⁵¹ , ⁵² , ⁵³ Besides, IgG antibodies have long been known to induce anaphylactic reactions on their own in guinea pigs, ⁵⁴ mice, ⁵⁵ and humans. ⁵⁶ Nevertheless, peanut allergen‐induced activation of normal basophils sensitized with serum from allergic patients collected before immunotherapy was inhibited by patients' serum collected after immunotherapy, and inhibition was reproduced by peanut‐specific IgG antibodies purified from post immunotherapy serum. Noticeably, inhibition was abrogated if basophils were incubated with monoclonal antibodies against FcγRII. ⁵⁷ Human basophils express minute amounts of activating FcγRIIA but high amounts of inhibitory FcγRIIB. FcγRIIB not only prevent IgG‐induced basophil activation when co‐engaged with FcγRIIA, they also exert a dominant inhibition of IgE‐induced basophil activation when co‐engaged with FcεRI. ⁵⁸ Anti‐allergen IgG antibodies may therefore be more than blocking antibodies when they dampen food allergy symptoms in desensitized patients.

3.3. Enhancement and inhibition of antibody responses by antibodies

Antibodies have potent immunoregulatory properties: They can either enhance (more than 100‐fold) or suppress (more than 99%) antibody responses against the antigen they recognize. ⁵⁹ The outcome and the mechanism involved depend both on the class of antibody and on the antigen.

Mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgE can all enhance antibody responses, that is, they can behave as adjuvants ⁶⁰ when passively administered together with antigen to naive mice, usually intravenously. IgM antibodies enhance responses to large proteins and heterologous erythrocytes, IgG responses to large and small proteins, and IgE antibodies to small proteins only. IgM‐ and IgG3‐mediated enhancement requires neither FcμRs nor FcγRs, whether activating or inhibitory, but complement ⁶¹ , ⁶² and complement receptors (CR1/CR2). ⁶³ IgG1‐, IgG2a‐, and IgG2b‐mediated enhancement requires activating FcγRs and it is reduced by FcγRIIB. ⁶⁴ IgE‐mediated enhancement requires FcεRII (CD23) ⁶⁵ expressed by B cells, ⁶⁶ but not activating FcRs. ⁶⁴ Whatever the isotype, antibody‐mediated enhancement of antibody responses therefore depends on the ability of Fc portions to engage membrane receptors. IgM and IgG3 use complement receptors expressed by follicular dendritic cells (FDCs) and B cells. They seem to concentrate antigen–antibody‐complement complexes on FDCs in B‐cell follicles, ⁶⁷ , ⁶⁸ and thus enhance B‐cell stimulation. IgE‐antigen complexes are transported to B‐cell follicles by circulating FcεRII⁺ B cells ⁶⁹ and they are subsequently presented to T cells by dendritic cells. ⁷⁰ IgG1, IgG2a, and Ig2b use activating FcγRs probably expressed by dendritic cells, ⁶⁰ and they also seem to enhance antigen presentation. ⁷¹

Mouse IgG1, IgG2a, IgG2b, and IgG3 antibodies can also profoundly suppress antibody responses to particulate antigens such as erythrocytes. This long‐known phenomenon ⁷² was thought to depend on the coengagement of BCRs with FcγRIIB on antigen‐specific B cells, ⁷³ as FcγRIIB were shown to suppress BCR‐mediated activation of murine ⁷⁴ and human ⁷⁵ B cells in vitro, and the mechanism of this suppression was extensively investigated. In vivo suppression was however not affected by the absence of complement (in C1q^−/− and C3^−/− mice) or complement receptors (in CR1^−/− and CR2^−/− mice), by the absence of FcγRs, whether activating (in FcRγ^−/− mice) or inhibitory (in FcγRIIB^−/− mice) or by the absence of FcRn (in β2 microglobulin^−/− mice). ⁷⁶ , ⁷⁷ Antibodies therefore may not need to use their Fc portion to suppress antibody responses. A likely explanation is that, when they bind to antigens, their Fab portions mask epitopes involved in the initiation of antibody responses. Supporting this interpretation, IgG anti‐hapten antibodies suppressed the response to hapten, but not to sheep red blood cells (SRBC), while IgG anti‐SRBC suppressed the response to SRBC, but not to hapten in mice immunized against SRBC‐hapten conjugates, ⁷⁸ unless heavily haptenated SRBC were used. ⁷⁹ This unexpected result is likely to account for the widely used and markedly efficient prevention of the hemolytic disease of the newborn in the clinics. This prophylactic treatment consists in injecting IgG anti‐RhD antibodies to RhD⁻ pregnant women carrying an RhD⁺ fetus. ²⁷ If conclusions drawn from mouse models apply to humans, anti‐RhD antibodies mask RhD antigens on fetus erythrocytes and thus prevent the development of an anti‐RhD antibody response by the mother. Whether they also apply to other passive immunotherapies is not known. In any case, inhibition of antibody responses by passively‐administered IgG antibodies would be an exception to the “rule” that antibodies act by binding to antigen and by engaging CRs and/or FcRs by their Fc portion.

4. ANTIBODIES OR THE EFFECTOR PROPERTIES OF B CELLS BY PROXY

4.1. Antibodies, the adaptive antigen receptors of innate myeloid cells

CRs and FcRs are predominantly, but not exclusively, expressed by myeloid cells: CRs are also expressed by B and T cells (C3aR, C5aR, CR1, and CR2), and by NK cells (CR3, CR4); some FcRs are also expressed by B (FcγRIIB, FcεRII, and FcμR), T (FcμR) and NK cells (FcγRIIIA and FcμR). Myeloid cells comprise the majority of white blood cells. They include polymorphonuclear leukocytes of the three types and monocytes, as well as a variety of tissue‐resident cells such as macrophages, dendritic cells, mast cells, etc. (Figure 1C). Myeloid cells are effector cells of innate immunity. Unlike lymphocytes, they have no antigen receptors. When interacting with FcRs or with CRs via complement, antibodies enroll innate myeloid cells in adaptive immunity. When bound to FcRs, they also provide innate myeloid cells with bona fide antigen receptors. ⁸⁰

Myeloid cells express between a few thousands to more than one million FcRs per cell. ⁸¹ Because antibody responses are polyclonal, antibodies of many specificities are induced against one or several antigens in an immune response. As antibodies bind to FcRs whatever their specificity, every FcR‐expressing cell can carry a multitude of antibodies (via high‐affinity FcRs) or bind a multitude of immune complexes (via low‐affinity FcRs), and it can respond simultaneously to a multitude of antigens recognized by these antibodies. Antibodies provide myeloid cells with the actual cognitive repertoire of B lymphocytes.

Unlike T lymphocytes, which upon TCR‐dependent activation, differentiate into a variety of effector cells (Th1, Th2, Th3, Th17, Tfh, CTL, etc.), B lymphocytes differentiate into antibody‐producing plasma cells upon BCR‐dependent activation. Whereas regulatory and memory B cells are functional equivalents of regulatory and memory T cells, there is no effector B‐cell equivalent of effector T cells. When engaging CRs or FcRs, however, antibodies provide B cells with the whole effector repertoire of fully differentiated ubiquitous and ready‐to‐work innate myeloid cells that they enroll in adaptive immune responses. B cells thus acquire a whole array of effector functions “by proxy.”

Unlike pattern recognition receptors (PRRs) and TCRs, which enable innate cells and effector T cells, respectively, to act on what they recognize, BCRs enables B cells to recognize, but not to act on antigens. They require that antibodies secreted by B‐cell‐derived plasma cells both confer their specificity and activate myeloid cells that can act on antigen. While B cells provide myeloid cells with their own cognitive repertoire, myeloid cells provide B cells with their own effector repertoire. This dissociation between the cognitive and the effector functions of B cells enables a diversification of their biological activities. The effector repertoire of B cells is thus much wider than the effector repertoire of T cells, limited to what these cells can themselves do. It is also much more flexible.

4.2. Antibodies as immune checkpoint activators and inhibitors

Most myeloid cells co‐express activating and inhibitory FcRs. When co‐engaged by antibodies, their ITAMs, and their ITIM are tyrosyl‐phosphorylated by Src kinases, ⁸² which enables the recruitment of SH2 domain‐containing cytosolic kinases ⁸³ and phosphatases, ⁸⁴ respectively. As a consequence, the same antibody can both activate and inhibit cell activation and its biological consequences.

Thus, an IgG1 monoclonal antibody in complex with specific antigen can induce a mild anaphylactic shock in wild‐type mice, no shock in KO mice lacking ITAM‐containing FcγRIIIA, and a dramatic shock in KO mice lacking ITIM‐containing FcγRIIB (P. Bruhns & M. Daëron, unpublished). Likewise, the interplay between activating and inhibitory FcγRs was demonstrated to control the development of collagen‐induced autoimmunity in a murine model of rheumatoid arthritis. ⁸⁵ Anti‐tumor antibodies can lead to a significant reduction of tumor mass when injected in tumor‐bearing wild‐type mice. ⁸⁶ The anti‐tumor effects of these antibodies was lost in mice lacking activating FcRs, ⁸⁷ but markedly enhanced in mice lacking inhibitory FcRs. ⁸⁸ On the contrary, IgG antibodies against antigens expressed by tumor cells can enhance tumor growth in murine experimental models. ⁸⁹ , ⁹⁰ Antibody‐induced enhancement was abrogated in FcγRIIB‐deficient mice (H.T. Duc, B. Heyman & M. Daëron, unpublished). Antibodies can thus both activate or inhibit immune checkpoints that control systemic anaphylaxis, autoimmunity and anti‐tumor immunity.

Antibody responses are pluri‐isotypic, and individual myeloid cells express FcRs for several isotypes of antibodies. Hetero‐aggregates of receptors, rather than homo‐aggregates therefore form when antigen–antibody complexes engage FcRs on cell membranes. The composition of FcR aggregates is not predetermined. It depends on antibodies that happen to engage FcRs on a cell. FcRs with different signaling properties cooperate to transduce compound intracellular signals generated at random by extracellular ligands. Antibodies can thus finely tune immune checkpoints that control the responses of myeloid cells.

4.3. The biological activities of antibodies are contextual

Myeloid cells can exert a variety of biological activities that primarily depend on the cell type (Figure 1D). When engaging CRs and/or FcRs, antibodies can therefore trigger a variety of cellular responses, such as chemotaxis, cell adhesion, phagocytosis, endocytosis, exocytosis, cytokine secretion, cytotoxicity, etc. These responses may contribute to a variety of biological processes involved in immune functions such as leukocyte extravasation, clearance of pathogens and immune complexes, neutralization of toxins and viruses, antigen capture at the initiation of antigen presentation, killing of infected and transformed cells, inflammatory responses and wound repair, positive and negative regulation of antibody responses, etc. The biological activities of antibodies are not predetermined. Rather than on antibodies themselves, they depend on the biological activities of myeloid cells used by antibodies under various conditions.

Myeloid cells are found in different locations. After having differentiated from progenitor cells in the bone marrow, some remain in the blood and circulate, others settle in tissues where they terminate their differentiation under the influence of locally produced growth factors. Thus, skin and peritoneal mast cells—referred to as connective tissue‐type mast cells—differ phenotypically and functionally from gut and lung mast cells—referred to as mucosal‐type mast cells. They exhibit different transcriptomic and proteomic signatures, ⁹¹ they express different receptors, contain different mediators, secrete different cytokines and are involved in different biological responses upon activation via FcRs. ⁹² , ⁹³ Unlike human skin mast cells ⁹⁴ but like human basophils, ⁵⁸ human mast cells of the gastro‐intestinal tract express FcγRIIB that negatively regulate allergen‐induced, IgE‐dependent systemic anaphylaxis. ⁹⁵ This differential expression of inhibitory FcRs by mast cells found in different tissues may explain why not all individuals with specific IgE are allergic. It also explains that patients successfully treated by allergen immunotherapy may become non‐responsive to oral challenge while remaining responsive to skin test with the same allergen. ⁹⁶ It follows that the same antibodies may have different biological activities, depending on the location where they meet antigen. Context‐dependent cell differentiation shapes antibody‐induced cell responses. This might be considered when trying to engineer vaccines against infections that are systemic or develop in tissues.

5. THE EFFECTOR REPERTOIRE OF ANTIBODIES AS A SELECTIVE ADVANTAGE

5.1. Advantageous, deleterious, and neutral mutations

“Nothing in biology makes sense except in the light of evolution” claimed the evolutionary biologist Theodosius Dobzhansky. ⁹⁷ Charles Darwin did not use the term “evolution” in The Origin of Species, but “descent with modification through natural selection.” ⁹⁸ Selection is the natural process^* that retained the modification and enabled its transmission to descendants. Evolution therefore implies selection.

Antibodies were selected neither for better nor for worse. Their deleterious effects are unlikely to provide a selective advantage and, depending on the context, antibodies can always do both. When mutation‐bearing individuals are submitted to the challenge of life, the sentences of Darwinian selection are extreme: survival or death. Advantageous mutations are transmitted to the progeny of survivors, and their frequency rapidly increases in the population because they keep being transmitted to descendants if they keep being advantageous in the environment. Deleterious mutations remain in the genome of those who did not reproduce before dying, and they rapidly disappear from the progeny's pool of genes. Darwinian selection ignores neutral mutations, that is, mutations that do not significantly affect survival.

As a consequence, mutations that have no or a mild effect whether advantageous or deleterious, are selected neither positively nor negatively, and they accumulate in the lineage's germinal genes. Thus, genome‐wide association studies identified thousands of genetic variants associated with asthma. Most affect non‐coding regions but some are associated with Th2 responses and inflammation. ⁹⁹ Neither a Th2 response nor inflammation is pathogenic per se, but both contribute to allergy. Genetic variations may therefore not cause but predispose to allergic asthma. Allergies are not genetic diseases; They are nevertheless familial diseases. Besides, even mutations that are truly pathogenic may provide a selective advantage under some conditions. Protection against severe Plasmodium falciparum malaria conferred to heterozygotes by a mutation of a gene responsible for sickle cell anemia in homozygotes is a well‐known example. This mutation has persisted for more than 7000 years since it appeared in Africa. ¹⁰⁰ This apparently paradoxical evolution has been formalized as the “Constructive Neutral Evolution” theory. ¹⁰¹ , ¹⁰² Genes that encode what is necessary to make antibodies may thus have been conserved for half a billion years, even though some may predispose to disease, even though antibodies may, themselves, have deleterious effects under some conditions.

Antibodies have two functional repertoires: a cognitive and an effector répertoire (Figure 2). Altogether, they can recognize an immense number of antigens. They recognize antigens borne by pathogens and their products, as well as antigens borne by commensals and innocuous molecules such as food and self‐antigens. They can trigger a large number of biological responses. Some responses are protective, and indeed, patients with antibody deficiencies such as Bruton's agammaglobulinemia suffer of recurrent life‐threatening infections in the absence of treatment ¹⁰³ ; some contribute to pathogenicity such as in severe COVID‐19 ¹⁰⁴ ; some such as allergic responses are nothing but pathogenic. Which, among these many properties, provided some sharks or other jawed vertebrates capable of making antibodies, ³⁴ with such a survival advantage that this ability was selected and transmitted to mammals across millions of generations?

FIGURE 2.

The two functional repertoires of antibodies. The central curved arrows schematize an antibody response: An antigen stimulates cells of the adaptive immune system; These respond by the production of antibodies that can recognize the antigen; Antibodies in complex with antigen can activate CR‐ and/or FcR‐expressing effector innate myeloid cells that can act on antigen‐bearing structures. The cognitive repertoire (in blue) enables antigens to be recognized by B cells. The effector repertoire (in red) enables antibodies to act on antigens. The virtual cognitive repertoire is made by all the possible combinations of the genomic variables genes that encode the N‐terminal variable sequences of the light and heavy chains of immunoglobulins. The available cognitive repertoire results from the stochastic rearrangements that brings together one V and one J gene encoding the variable sequences of a light chain, and one V, one D, and one J gene encoding the variable sequences of a heavy chain, and determines the specificity of BCRs in naïve B cells. The actual cognitive repertoire results from the selection of B cells by antigens that engaged their BCRs and induced their clonal expansion and differentiation into antibody‐producing plasma cells. It is thus that of antibodies. The virtual effector repertoire consists in all the possible interactions between effector cells generated in the bone marrow and antibodies of the nine classes and subclasses generated during the immune response. The available effector repertoire consists in the (mostly) myeloid cells that differentiated in various locations and express CRs and FcRs that can be engaged by the antibodies produced by plasma cells that differentiated from B‐cell clones resulting from the clonal selection and underwent class switch recombination. The actual effector repertoire consists in the myeloid cells that were enrolled into the adaptive response by antibodies via CRs and FcRs at a given place and at a given time, and the biological responses resulting from their activation in the presence of antigen.

5.2. The emergence of antibodies

Two types of combinatorial adaptive immune systems appeared within a relatively short period of time in vertebrates that already possessed PRR‐based innate immune systems like other eukaryotes. An activation‐induced cytidine deaminase (AID)‐dependent gene conversion‐like mechanism generated genes encoding variable lymphocyte receptors (VLRs) expressed by lymphocyte‐like cells in jawless vertebrates such as lampreys and hagfishes, while a recombinant activating gene (RAG)‐dependent V(D)J recombination mechanism generated genes encoding the variable domains of TCRs and BCRs expressed by T and B lymphocytes in jawed vertebrates. ¹⁰⁵

In germline configuration, VLR genes are incomplete and nonfunctional. They are rearranged in lymphocyte‐like cells to form genes that encode three types of leucine‐rich glycosylphosphatidyl‐anchored antigen receptors with different cellular expression. VLRAs and VLRCs are expressed by T‐like cells, whereas VLRBs are both membrane receptors expressed in B‐like cells and soluble antibody‐like molecules. ¹⁰⁶ VLRB antibodies are globular‐shaped multimers of VLRBs assembled through hydrophobic clustering in hagfishes, or IgM‐like decamers (or octamers) assembled through disulfide bounds in lampreys. ¹⁰⁷ These antibody molecules thus present themselves as multimers of variable antigen‐binding domains. They have agglutinating properties, and they can activate heterologous C1q or a C1q‐like endogenous complement component, and lyse antigen‐bearing target cells.

Unlike in jawless vertebrates, several genes encoding the constant domains of heavy chains (C_H) appeared together with genes encoding the variable domains of heavy and light chains in jawed vertebrates. These C_H genes enable the formation of several classes of antibodies through class switch recombination. Cartilaginous fishes possess IgM, IgW, and IgNAR, bony fishes IgM, IgD, and IgT, amphibians, IgM, IgD, IgY, IgX, and IgF, reptiles IgM, IgD, IgY, IgA, and IgD2, birds IgM, IgY, and IgA, mammals IgM, IgD, IgG, IgE, and IgA. ¹⁰⁸ Nothing is known of antibody effector functions in cold‐blooded vertebrates. One can, however, speculate that, like mammals but unlike jawless vertebrates, these animals enjoyed having a wide effector repertoire. The emergence and diversification of FcRs indeed accompanied the gradual increase in numbers of immunoglobulin isotypes during vertebrate evolution. ¹⁰⁹ Here might reside a selective advantage that antibodies could confer to jawed vertebrates: rather than stereotyped biological properties like agglutination and activation of the classical pathway of complement, they can use by proxy the wide effector repertoire of innate myeloid cells.

Most myeloid cells indeed co‐express (1) various sets of CRs that can mediate a variety of biological activities; (2) FcRs for several classes of antibodies which can bind a multitude of antibodies with a variety of specificities; and (3) ITAM‐containing and ITIM‐containing FcRs. Antibodies therefore engage several types of receptors with different signaling properties on any given cell, leading to the formation of receptor hetero‐aggregates. The composition of receptor aggregates thus generated depends (1) on receptors that are expressed by a given cell; (2) on the respective local concentration of antibodies of the different classes; and (3) on the nature and on the concentration of antigen present in the environment. Cellular responses induced by compound signals transduced by heterogeneous receptor complexes with non‐predetermined structures are further diversified (1) by the composition of cell populations present and/or recruited locally; (2) by their state of differentiation; and (3) by the effector repertoires of these cells.

Such properties meet the definition of dispositions invoked by the proponents of another theory of functions referred to as the “systemic theory of functions.” ¹¹⁰ Fragility and solubility are two classical examples of dispositions. A glass is fragile. This property is not apparent unless the glass falls on the floor, for instance, and breaks. A sugar cube is soluble but this property does not manifest in the sugar box. It will dissolve if I put it in my coffee. Dispositions are “latent” properties that belong to objects (the glass will not dissolve in water and the sugar cube is unlikely to break if it falls down), and which actualize if and only if they are placed under appropriate conditions. They are conditional properties. ¹¹¹ The biological properties of antibodies are conditional.

5.3. Natural selection of a third kind

Antibodies therefore have a repertoire of dispositions. Like their cognitive repertoire, the effector repertoire of antibodies can be subdivided in three parts. The virtual effector repertoire consists of all possible biological properties of effector cells and molecules that could be triggered by antibodies under all possible conditions, including those that do not exist but may have existed in the past or may exist in the future. The available effector repertoire consists of all biological activities of the effector cells and molecules that antibodies can interact with under specific existing conditions. These conditions are not the same in different places and at different times. The actual effector repertoire consists of those biological activities of effector cells and molecules that antibodies do or did induce under a given condition in time and space. The actual effector repertoire of antibodies is selected by the actual molecular and cellular contexts. Initially restricted to Darwinian selection, natural selection includes not only Burnet's clonal selection, but also a molecular selection.

Darwinian natural selection acts on individual organisms: individuals live or die and those who live can transmit their genes to their progeny. As a consequence, parental genes have a higher frequency in the offspring population, and the phenotypic traits of the parents become more prevalent in the species. They are positively selected. Selecting agents are competition between individuals, for example, for food, in a given environment, or in the presence of pathogens, for example, Yersinia pestis during the Black Death which killed one third or the European population in the Middle Age, and selected immune genes in survivors. ¹¹² The selecting process is reproduction. Selected objects are germinal genes, and they are transmitted to the offspring. The level of selection is phylogeny and the time pace depends on the reproduction rate of organisms. It takes generations.

Natural selection can also act at the cellular level. Clonal selection operates in B cells that have rearranged the variable genes of their BCR and acquired a specificity. The selecting agent is antigen that can activate lymphocytes that express specific BCRs. The selecting process is clonal proliferation. Selected objects are somatic genes that encode variable genes, and they are transmitted to daughter cells. The level of selection is ontogeny and the time pace is determined by the proliferation rate of lymphocytes. It is in the order of days. Except that it operates at the level of cells instead of organisms, clonal selection resembles Darwinian selection. It remains a “descent” (the formation of a clone of lymphocytes) “with modification” (the antigenic specificity acquired through stochastic somatic mutations) “through natural selection” (performed by the specific antigen), to use Darwin terminology.

Natural selection can also act at the molecular level. Membrane receptors expressed by a given cell select which antibodies, among the many that are present in the environment, this cell will respond to. Reciprocally, antibodies present in the environment select which receptors, among the many that are expressed on the membrane, will trigger intracellular signals in that cell. Context‐dependent molecular interactions thus select both which antibodies and which receptors will activate the cell. This molecular selection affects protein–protein interactions. Selecting agents are available molecules that can interact with each other in a given context. The selecting process is molecular interactions that actually occur. Interactions are determined by the reciprocal affinity of ligands and receptors. Selected objects are functional properties of the cells. The effects of selection are short‐lived. The consequences of selection bear on biological responses. The time pace depends on the stability of molecular interactions. It is in the order of seconds or minutes.

Context‐dependent molecular selection bears neither on germinal genes as in Darwinian selection nor on somatically‐generated genes as in clonal selection. It may therefore not be transmitted to descendants. However, genes that were previously transcribed upon cell activation by extracellular stimuli in myeloid cells were found to be transcribed faster and more strongly when their expression is induced again. ¹¹³ This epigenetic reprogramming of myeloid cells ¹¹⁴ was referred to as “trained immunity” when induced by immunological stimuli. It endows innate immunity with an immune memory, like adaptive immunity.

Unlike adaptive immunity memory that involves long‐lived genetic alterations of BCR and TCR genes, innate immunity memory was thought to be rapidly lost. It was however found that, compared to F1 individuals born from non‐infected mice, F1 individuals born from male mice that survived a sublethal infection were more resistant to the same infectious agent. Their myeloid cells exhibited functional (an enhanced activated phenotype) and transcriptional changes (an enhanced transcription of genes involved in immune functions and in myeloid differentiation), and the sperm of trained parental mice showed epigenetic changes (methylation alterations at genes encoding transcription factors involved in myeloid cell regulation). ¹¹⁵ If indeed innate immune traits can be either acquired or inherited, and allow a population to adapt to changing environment, ¹¹⁶ molecular selection may also promote a “descent with modification” via transmission of epigenetic changes across generations. Natural selection may thus operate not only in evolutionary biology based on ultimate causes, but also in functional biology based on proximal causes, as defined by Ernst Mayr. ¹¹⁷

6. CONCLUSION: ANTIBODIES OR NATURAL SELECTION HIC ET NUNC

All the above being considered, what is the function of antibodies? Obviously, one cannot give a straight answer as one could do if the questions was about hemoglobin or insulin receptors for instance. Antibodies have two immense virtual repertoires generated by different mechanisms. The virtual cognitive repertoire has an intrinsic diversity that stems from the combinatorial diversity of variable genes that encode the antigen‐binding sites of antibodies. Any antigen that exists today, has existed in the past or will exist in the future may thus be recognized by BCRs and induce the production of specific antibodies. The virtual effector repertoire has both an intrinsic and an extrinsic diversity. The intrinsic diversity is that of the nine isotypes of antibodies with the same specificity but different biological properties resulting from class switch recombination. The extrinsic diversity is that of the many biological properties of the various types of the many myeloid cells that circulate in the blood or reside in tissues, that is, anywhere in the body. The virtual effector repertoire of antibodies consists in all the effects they might induce if they activated all the effector cells able to respond because they express the necessary receptors.

A subset only of these repertoires is however available in an organism. A combination of variable genes drawn at random in individual B cells determines the specificity of their BCRs, and the BCRs of all B cells together constitutes the available cognitive repertoire of this organism. Because all the BCRs of a B cell have the same specificity, this available repertoire is limited by the number of naive B cells in the body. Altogether, the available cognitive repertoires of all the living members of a species tend to resemble the virtual cognitive repertoire of this species. This makes it more likely that a high enough number of individuals of the species can respond to any antigen, including antigens borne by emerging pathogens that may arise in the future, and survive. The available effector repertoire of antibodies is determined by the cell types that are available and the biological activities they can exert in response to a stimulation by antibodies and antigen. Individuals vaccinated against a pathogen are equipped with specific antibodies that they do not use yet, but are ready to use in case the pathogen appears. Likewise, allergic patients have developed antibodies and they possess effector cells that express corresponding receptors, which can induce a whole spectrum of allergic reactions if they are exposed to allergen, but that they do not use if they are not exposed. The available effector repertoire is that of immunized people. We are all immunized against something.

Subsets of available repertoires are ultimately selected and form actual repertoires. The actual cognitive repertoire is selected by antigens, as anticipated by Burnet's Clonal Selection Theory. B cells whose BCRs are engaged by antigen proliferate, undergo a clonal expansion, differentiate into antibody‐producing cells and long‐term memory cells. The actual cognitive repertoire thus not only enables an efficient antibody response, it also keeps a memory of the individual's antigenic experiences. Likewise, the actual effector repertoire is selected by specific antigens and the context. It is further diversified by the phenotypic and functional variations of myeloid cells in different locations. Thus, the cell composition of ectopic tertiary lymphoid organs determines the actual effector repertoire of antibodies in inflammatory sites. Likewise, whether an allergen induces a local reaction, such as a rhinitis, a regional reaction, such as an asthma crisis, or a systemic reaction, such as an anaphylactic shock, depends on the location where antibodies meet the allergen. Like lymphocytes that underwent a clonal selection and expansion keep a memory of their experience, myeloid cells that were actually engaged in the response keep an epigenetic memory of their stimulation and, noticeably, this memory acquired by innate cells can also be transmitted from parents to their offspring.

Rather than a particular response, that is rather than a specific function, evolution may thus have selected repertoires of dispositions, reactivity rather than performance, multiplicity rather than specificity. The actual cognitive and effector repertoires enable immune systems to respond to anything in the current context, whatever it is. The available cognitive and effector repertoires enable actual repertoires to adapt rapidly to a changing context. The virtual cognitive and effector repertoires provide available repertoires with a reservoir of novel specificities and reactivities that are continuously renewed as hematopoiesis generates new lymphoid and myeloid cells. Such an ability to respond whatever the response, to act whatever the action or the result of the action, an ability to respond to the unpredictable ¹¹⁸ and, as proposed long ago for the cognitive repertoire, ¹⁰⁵ , ¹¹⁹ , ¹²⁰ to anticipate the unknown, might confer a better selective advantage than any specific function.

If indeed immense virtual repertoires are reservoirs for available repertoires, and if large available repertoires of dispositions are ready to be selected by an unpredictable future context, non‐selected traits may paradoxically be more useful than the selected traits of actual repertoires. Natural selection could thus operate on always renewed available repertoires in a living individual, rather than across generations on hypothetic descendants bearing uncertain mutations possibly advantageous in an unknown future. Natural selection could operate hic et nunc, and already tomorrow.

CONFLICT OF INTEREST STATEMENT

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. It was supported by no funding. Marc Daëron, MD, PhD, is an Inserm emeritus scientist at the Centre d'immunologie de Marseille‐Luminy, an invited scientist at Institut Pasteur, and an associate member of the Institut d'histoire et de philosophie des sciences et des techniques.

Daëron M. The function of antibodies. Immunol Rev. 2024;328:113‐125. doi: 10.1111/imr.13387

DATA AVAILABILITY STATEMENT

Data sharing not applicable‐no new data generated.