Dissertation on The Evolution of B Cells in Immunity

Abstract

Evolution of life is related to adaptation of the organism to constantly changing environmental conditions. The succession of organisms during evolution is achieved due to the development of mechanisms for control of mutational processes in somatic cells and improvement of systems for coordination and regulation of various forms of essential activity, including the resistance of the organism to various foreign antigens.

The present-day huge repertoire of Antibodies in the human immunity system, is affected by major events in the evolution of B cells. Homologs genes of rearranging T cell receptors and Immunoglobulins (B cell receptor and antibodies) are present in jawed vertebrates, but have not been identified in any other animal groups. Even though the genes are not present in other groups, major group of transcription factors that are vital for developmental process of B and T Cells in mammals belong to multi-gene families that are represented by members in most of the metazoans, providing a potential understanding of pre-vertebrate ancestral roles. The structure and regulation of homologs genes of the specific transcription factors known to regulate mammalian T and B cell development in one of the earliest diverging gnathostome, the clearnose skate (Raja eglanteria). The Skate orthologs of the mammalian transcription factors for development of B and T cells have been characterized and shown that they co-express with each other in combinations, that, for the most part, correspond with a known mouse B and T cell patterns, supporting the theory of conservation of function in jawed vertebrates.

If we go back in the “evolutionary tree” we can identify lymphocyte-like cells in the typhlosole of the sea lamprey, Petromyzon marinus. The purified cells of the lamprey were morphologically similar to mammalian lymphocytes. In addition, finding and characterizing of closely linked pair of genes, SpRAG1L and SpRAG2L, from an invertebrate, the purple sea urchin (Strongylocentrotus purpuratus) with similarity in both genomic organization and sequence to the vertebrate RAG1 and RAG2 genes. Together these experiments have uncovered an unanticipated and complexed evolution of developmental plasticity of B cells, which may help us understand the secret of the amazing human immunity.

Table of contents

Introduction 3

The adaptive immunity system  3

B cell development 7

Evolution of the development of B cells within the gnathostomes 7

Identification of adaptive Immune System in Agnathans  8

RAG genes in purple sea urchin 10

Discussion and Conclusions 12

References 13

Developmental plasticity in evolution of B cells

Introduction

Evolution of life is related to adaptation of the organism to constantly changing environmental conditions. The succession of organisms during evolution is achieved due to the development of mechanisms for control of mutational processes in somatic cells and improvement of systems for coordination and regulation of various forms of essential activity, including the resistance of the organism to various foreign antigens1. Darwin (1859) understood during his travels, that organisms must adapt to their environment to survive. He looked at the morphology of Animals and concluded that a successful organism is a long process which is affected by changes in the environment and competition. But Darwin still didn’t know that the inner environment as an equal or even greater effect on the evolution of species, thus making the immunity system one of the most important systems that affected the evolution and survival of organisms.

We can go back in evolution and find traces of several innate-immunity-like systems in bacteria, such as abortive infection, receptor mutation, and restriction-modification. Likewise, Vertebrates with jaws (gnathostomes) possess both an innate immunity, that is based on protective strategy of the organism to constitutively produce generic receptors that recognize conserved patterns on different classes of pathogens to trigger an inflammatory response that limits pathogen invasion, as well as an adaptive immune system that can recognize and initiate a specific protective response against potential pathogens, including bacteria, fungi, viruses and parasites. Our adaptive immune system also “remembers” previous pathogen encounters and can either prevent a second invasion or quickly eliminate the recurrent invader by mobilizing a more efficient and faster immune response 2.

In the last several years, we examine the recently discover of the characterized Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system that has been described as an adaptive immune system in bacteria and archaea, which provides specific and acquired immunization against exogenic mobile genetic elements 3.

The adaptive immunity system

The immunity system is divided into two main branches, an innate immunity and an adaptive immunity. The innate immunity, which is inherent feature of all living organisms, from unicellular organisms to vertebrates, is not specific to a certain antigen or invader and based on recognition of pathogen associated molecular patterns. For instance, in vertebrates, the innate immunity involves an inflammatory reaction and the complement system. Furthermore, toll receptors and toll like receptors are a major part of the innate immunity and were found in both invertebrates and vertebrates4. In contrast to the innate immunity, the adaptive immunity is based on huge repertoire of receptors and immunoglobulins (Igs) that enables us to react in a specific and precise way against a wide verity of antigens.

In gnathostomes, two major lineages of lymphocytes are generated in the thymus and the bone marrow or the avian bursa of Fabricius. These are called T (for thymus-derived) and B (for bursa or bone-marrow-derived) lymphocytes. Like other types of blood cells, the early progenitors of T and B lymphocytes are derived from multipotent hematopoietic stem cells. During their early developmental stages, T and B lymphocyte progenitors rearrange different sets of prototypic Ig. Igs are Y shaped glycoproteins that are found in various forms both freely in our body’s tissues and blood system and on the surface of B and T cells. Igs are made from two light chains and two heavy chains, both constructed from a variable (V) region and a constant (C) region. The gene segments to generate the antigen binding sites of T cell receptors (TCRs) and B cell receptors (BCRs) and the “free” antibodies (Abs), are Variable (V), diversity (D), and joining (J) in a process known as V(D)J recombination 5.

Additional to the V(D)J process, further modifications are achieved by somatic hyper mutations, gene conversion and class-switch recombination, in some organisms, and can generate an approximate 1014 different option to react with almost any antigen in the environment. The V(D)J rearrangement form of somatic recombination is mediated by recombination-activating gene 1 (RAG1) and RAG2, which function in a lymphocyte- and site-specific recombinase complex and are supported by ubiquitous DNA repair factors5. The V(D)J recombination is the base of the adaptive immunity system in all gnathostomes and has led to increased diversity, specificity, and affinity of Igs, which has thus fine-tuned adaptive immune responses against almost any pathogen. This evolutionary trend may have been fueled, at least partially, by the emergence of warm-blooded vertebrates on earth 6.

B cell development

Hematopoietic stem cells (HSCs) in the bone marrow can develop into all blood cells types by differentiating via multipotent progenitors into common myeloid progenitors (CMPs) or a common lymphoid progenitor (CLPs), with their characteristic B, T, and NK cell potential. These uncommitted progenitors express selected genes of different lineage programs by a process known as transcriptional lineage priming and are thus able to respond to various inductive cues because of the presence of multiple signaling pathways.  Entry of CLPs into the B cell lineage critically depends on signaling of the IL-7 receptor and expression of the three transcription factors E2A, EBF1, and Pax57.

Maturation of B cells is dependent on Growth factor and V(D)J recombination. All stages of maturation have some sort of a check point of which they must present a certain progress of the creation of Igs, until a full presentation of IgMs and IgDs as BCRs on the B cell membrane and become naïve B cells. During the process in the bone marrow, B cells go through two different types of selection to guarantee the correct development of the cells. Positive selection which occurs by antigen-independent signaling that involvs both the pre-BCRs and the BCRs. If these receptors will not bind to the proper ligand, B cells will not receive the correct signals and the development process will stop. Binding of self-antigen with BCRs is the negative selection of the cells, If a BCR can bind strongly enough to self-antigen, produced by the organism itself, then the B cell enters one of four options: clonal deletion, receptor editing, anergy, or ignorance (B cell ignores signal and continues development) 8.

To complete their development, immature B cells migrate to the spleen and lymph nodes, where they would be fully matured and be activated. The activation occurs when a BCR on a naïve B cell binds to a specific antigen, an event that will result in a chain of reactions to make plasma cells that will produce a great number Igs with high affinity to a specific antigen that invaded the body. The activation process includes some Activation-Induced (Cytidine) Deaminase (AID) dependent processes such as somatic hypermutation and class switching, both of them help to insure higher affinity of the Ig to the antigen.

Evolution of the development of B cells within the gnathostomes

Cartilaginous fish are the first jawed vertebrate group within living gnathostomes and diverged from the common ancestor of other jawed vertebrates approximately 500 Mya, and they are the oldest living organism to possess B cells, and present the V(D)J recombination system6. They lack bone marrow and Lymph nodes, thus maturation of B cells occurs in different organs. Cartilaginous fish do poses thymus and spleen as lymphoid organs and have unique lymphoid tissues, such as the Leydig organ (associated with the esophagus) and the epigonal organ (a tissue connected to the gonads). Continuous transcript expression of RAG, terminal deoxynucleotidyl transferase (TdT), and T and B cell–specific transcription factors are found in thymus, Leydig organ and epigonal tissue. Thus, Leydig and epigonal organs are regarded as a primary lymphoid organ for B cells.

Unlike cartilaginous fish, the avian family have bone marrow, but it is not involved in most of the development of B cells. Likewise, they have a different organ which takes a major part in maturation of B cells, called the Bursa of Fabricius. In 1965 Cooper et al.9  demonstrated that thymus- and bursa-dependent systems are critical for cellular and humoral immunity,  respectively. Making experiments in chickens the first to reveal lymphocyte specialization for antibody production and the existence of dual arms of adaptive immunity of T and B cells. As oppose to mammals, chickens have only one functional V and J segment for both the heavy and light Ig chains and the rearrangement occurs only during a short period of embryonic development. Ig gene rearrangement begins in the yolk sac and is seen throughout embryogenesis in all hematopoietic tissues, while in humans the rearrangement occurs  long as B cells generate in the bone marrow 6.

Although we can see a big divergence in the developmental process of B cells in gnathostomes. It has been shown that in one of the earliest diverging gnathostome, the clearnose skate (Raja eglanteria), the regulation and structure of homologs genes of certain transcription factors that are known as regulators of mammalian T and B cell development. The Skate orthologs of mammalian Pax-5, Pax-6, EBF-1, GATA-3, GATA-1, Runx2, and Runx3 have been characterized and shown that they co-express with each other in combinations, that, for the most part, correspond with a known mouse B and T cell patterns, supporting the theory of conservation of function in gnathostomes. 10

Identification of adaptive Immune System in Agnathans

Many of the genes for transcription factors involved in gnathostome lymphocyte development can be found in agnathans. SPI-B, IKAROS, EBF, GATA, PAX-2/5/8, and BACH2 gene relatives have all been identified in the lamprey11. A research by Mayer et al. (2002)12 found lymphocyte-like cells. The typhlosole, an internal fold surrounding over of the lamprey intestine, was the primary tissue source for the cells in this analysis. Cells derived from typhlosole tissues were analyzed in parallel with mouse intestinal epithelial cells by automated flow cytometry. The analysis found a sub-population of cells from the lamprey typhlosole that have the same light-scatter characteristics, closely resemble those of the lymphocytes in the mouse intestinal epithelium (Fig. 1). This population of lamprey cells was isolated with an automated cell sorter for further analysis. Both light and electron microscopy reveal the purified lamprey cells to represent a fairly homogeneous population in terms of size, staining properties, and structure 12. 

Fig. 1. Light-scattering characteristics of lamprey typhlosole cells (A) mouse intestinal intraepithelial lymphocytes (IEL, B) using flow cytometry: forward (180°) and sideward (90°) light-scatter.

Similar cells in both diagrams are encircled 12.

What has yet to be found in agnathans are rearrangeable TCR/BCR V(D)J gene segments, RAG1 and RAG2. It has been demonstrated that lampreys not only possess a lymphocyte-like cells but an equivalent adaptive immune system with its own recombination system based on transcripts for leucine-rich-repeat (LRR) proteins in great abundance and these proved to have highly variable amino acid sequences13. The diversity of these variable lymphocyte receptors (VLRs) is based on the variable numbers of sequence-diverse LRR modules that are sandwiched between the capping N-terminal and C-terminal LRR units, which also display sequence variability.  The genetic basis for the VLR diversity is of a complex of recombinatorial process2. The lamprey genome contains a single VLR gene that has three coding regions separated by two large noncoding intervening sequences, which lack canonical splice sites. The germline VLR gene encodes only the signal peptide, partial N-terminal and C-terminal LRRs, and the invariant stalk region. Flanking this incomplete VLR gene are a vast number of cassettes that encode one, two, or three of the different LRR modular units. In order to assemble the complete VLR gene, the surrounding LRR coding units are randomly incorporated into the germline VLR gene via a multistep assembly process14.

RAG genes in purple sea urchin

Even though we cannot find any evidence of V(D)J recombination system in any invertebrate, studies have shown that we can find RAG1/2-like genes within the genome of purple sea urchin (Strongylocentrotus purpuratus). These genes are co-expressed in adult tissues and during the development of larvae. A stable complex form from the proteins recombinant versions. They also interacted with RAG1 and RAG2 proteins from several gnathostomes15. The coding regions of both sea urchin genes are encoded in multiple exons, Unlike the RAG1 genes in most gnathostomes and all the RAG2 genes. The S. purpuratus RAG1/2- like (SpRAG1/2L) proteins role in sea urchins is still unknown, but there is an assumption that they may facilitate somatic rearrangement in yet to be identified genes within the sea urchin genome. Alternative assumption suggests that these genes might perform a more basic function. In order for the RAG complex to function there is a necessity for a stable interaction between RAG1 and RAG2, Fugmann et al. (2006)15 tested whether SpRAG1L and SpRAG2L share this property. The genes were co-expressed as strep- and GST-fusion proteins in 293T cells and carried out pull-down experiments with the respective cell lysates (Fig. 2)15. Surprisingly, the assay showed that SpRAG1 strongly interacts with RAG2 from sandbar shark (Carcharhinus plumbeus, Cp) (Fig. 2a, lane 4), while it also interacted with SpRAG2 (Fig. 2a, lane 5), suggesting that at least some features of the RAG1–RAG2 interaction domains are well conserved Throughout the evolution.  Another assay was preformed to co-express SpRAG2L with SpRAG1L as well as RAG1 from various gnathostomes to perform pull-down experiments with streptactin Sepharose, an affinity resin for the strep-tag (Fig. 2b). The second assay re-confirmed that SpRAG2 interacts with SpRAG1L (Fig. 2b, lane 4) and even interacted with RAG1 from another kind of shark (Carcharhinus leucas, Cl, Fig. 2b, lane 3) 15.

Fig. 4. Interaction of SpRAG1L, SpRAG2L, and vertebrate RAG1/2. The experiment was done as described in Fugmann et al. (2006)15. Affinity of Strep SpRAG1 test (a) with PHD (lane 2), MmRAG2 (lane 3), CpRAG2 (lane 4), SpRAG2 (lane 5), and a control group (lane 1). Affinity of GST- SpRAG2 test (b) with MmRAG1 (lane 2), CIRAG1 (lane 3), SpRAG1 (lane 4), and a control group (lane 1)15.

Discussion and Conclusions

Since the discovery of the immunity system researches are trying to understand the marvelous mechanism of this unique system, by examining the effects of different factors, but also by looking back on the evolution of the immunity system in general and specifically the adaptive immunity. This work has given a glimpse about some of the evolutionary events we have discovered so far.

Throughout the process of vertebrate immunity evolution, we can also put a finger on the fact that the change in life’s environment from water based life to land based one, have resulted in an endless variety of new pathogens, that only organisms that had the ability to fight with each invader with a specific response, and respond to diseases they never seen before, were able to survive.

The real question of how the adaptive immunity and especially B cells, as we know them today, were created during the evolution process is still a mystery. But given the proofs of existing genes and transcriptional factors of the magnificent developmental plasticity of Igs and B cells, prior to the appearance of vertebrate’s adaptive immunity, an assumption is that the developmental plasticity of B cells and the appearance of lymphocytes are two different evolutionary events, that co-evolved together. In addition, we understand that BCR, TCR and antibodies are a result of more than one evolutionary event. Never the less in order to expose to true evolution of B cells we would have to investigate every organism that lived on the planet, an impossible mission, but we keep on trying.

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