5.04: A. The Immune System - Antibodies, B- cells, T-cell receptors and T-cells

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Learning Goals (DeepSeek, 1/29/25)

1. Differentiate Innate and Adaptive Immunity

   • Describe the components of the innate immune system (e.g., macrophages, neutrophils, natural killer cells) and their mechanisms for recognizing pathogens.
   • Explain the role of the adaptive immune system, emphasizing the function of B cells and T cells in targeted antigen recognition and response.

2. Explain Antigen Recognition and Binding Mechanisms

   • Identify the various types of foreign molecules (antigens) that the immune system must recognize, including proteins, glycans, nucleic acids, and non-biological substances.
   • Discuss how binding interactions are central to immune recognition and the subsequent cellular responses.

3. Describe the Structure and Function of Antibodies (Immunoglobulins)

   • Outline the basic structure of an IgG antibody, including the arrangement of heavy and light chains, variable (V) and constant (C) domains, and the significance of the Y-shaped structure.
   • Explain the roles of the complementarity-determining regions (CDRs) in antigen binding and how structural features like disulfide bonds and beta-sheet arrangements contribute to antibody stability and function.

4. Understand the Generation of Antibody Diversity

   • Analyze the genetic mechanisms (e.g., V(D)J recombination, exon splicing, somatic mutation) that create a vast repertoire of antibodies, enabling the recognition of a wide array of antigens.
   • Connect how these molecular processes contribute to the specificity and affinity maturation of antibody responses.

5. Examine T Cell Receptor (TCR) Structure and Antigen Presentation

   • Describe how TCRs recognize antigen fragments only when presented by Major Histocompatibility Complex (MHC) proteins, highlighting differences between MHC Class I and Class II pathways.
   • Illustrate the structural differences between the TCR complex and antibodies, noting the role of co-receptors (e.g., CD4 and CD8) in modulating immune responses.

6. Discuss the Role of the Immunological Synapse and Co-signaling in Immune Activation

   • Explain the formation and function of the immunological synapse in T cell activation, including the involvement of co-stimulatory signals (e.g., CD28-B7 interaction) and cytokine receptors.
   • Evaluate how the “double-key” mechanism for immune activation serves as a safeguard against inappropriate or autoimmune responses.

7. Apply Structural and Functional Concepts to Immunotherapy

   • Compare traditional T cell receptors with engineered chimeric antigen receptors (CARs), focusing on their structural modifications and therapeutic implications.
   • Discuss how insights into antigen recognition and immune cell activation inform the design of vaccines and cancer immunotherapies.

8. Integrate Knowledge of Immune Tolerance and Autoimmunity

   • Analyze the mechanisms by which the immune system distinguishes self from non-self to avoid autoimmune diseases, including the role of MHC presentation and co-receptor signaling.
   • Discuss examples of autoimmune diseases arising from failures in immune tolerance and the potential biochemical targets for therapeutic intervention.

Each goal is intended to encourage a deeper exploration into the molecular details, structural biology, and functional mechanisms that underlie immune recognition and response, providing a robust foundation for advanced study and research in immunology and biochemistry.

Introduction to the Immune System

Now, let's consider the daunting task faced by the immune system - to recognize all possible "foreign" molecules and react to them, either by targeting them for elimination or, paradoxically, to recognize them but not react to them (a process called tolerance). The same can be said of "self-molecules, and the immune system must recognize them but not respond to them. Otherwise, autoimmune disease might arise in which the body's powerful immune system targets itself.

It is impossible to describe the immune system in depth in a short section. Our goal is to illustrate how the immune system recognizes such a vast number of molecules. We will briefly cover the innate and adaptive immune system and their differences, and how some cells (macrophages in particular) in the innate immune system and cells (B and T cells) in the adaptive immune response recognize and respond to target molecules and cells. Finally, we'll discuss how the immune system can respond to similar molecules by recognizing common molecular patterns. Emphasis will be given to recognition. Ways to simplify the complexities of the immune system have been presented in a fantastic book written by Lauren Sompayrac, How the Immune System Works. (2003, Blackwell Publishing. ISBN: 0-632-04702-X) and adopted here.

We realize we have not yet reached the chapters on carbohydrates, membrane proteins, and nucleic acids. Nevertheless, we present the material in this section to organize it in one location. Users can revisit this page after studying subsequent chapters.

Before we start, think of the variety of chemical species that the immune system should recognize as foreign:

• a bacterial glycan or glycolipid on the outside of the cell
• a viral surface protein, such as the spike protein of the SARS-CoV-2
• bacterial dsDNA (and not host dsDNA)
• viral dsRNA (which is not common in host systems
• a self-protein that has been modified in a tumor cell
• a crystal of urea
• extracellular ATP (a place where it is not usually found)
• a silica particle found in particles like asbestos.

How would you design an immune system to bind each of the "enemy" targets above? That is what we will explore in this section - the binding interactions. What happens after the binding is beyond the scope of this section and falls in the field of signal transduction - how binding events at the cell surface are transferred into intracellular responses.

Three lines of defense protect us from the "enemies," foreign substances (bacteria, viruses, and their associated proteins, carbohydrates, and lipids) collectively called antigens.

• physical barriers of cells that line our outside surface and our respiratory, GI tract, and reproductive systems.
• the innate immune system (IS) that all animals have. It is composed of scavenger cells like macrophages (MΦ), neutrophils, dendritic cells, and natural killer cells (NK) that can move around the body through the blood and lymph systems and burrow into tissues to meet the enemy where they can engulf and destroy bacteria and "cellular debris." Macrophages start as immature circulating monocytes, which enter tissues by slipping through blood vessel walls. They differentiate into macrophages. There they lie in waiting, ready for the enemy.
• the adaptive immune system, which, as its name implies, can change and adapt to new molecular threats. This branch is better at dealing with viruses, which do their damage inside host cells. The adaptive IS consists of B cells that make and secret protein antibodies that recognize specific foreign molecules and T cells.

In a world that has experienced the most deadly pandemic (COVID) of the last 100 years, and with more to come, immune recognition must be an important part of any biochemistry text. This chapter section could be a whole chapter, but we'll leave it as a very long section. Let's start with the adaptive immune system, which we can co-opt to make vaccines for our major threats.

B Cells and Antibodies

B cells and their differentiated forms (B memory and plasma cells) make antibodies. Antibodies bind to foreign molecules (proteins, glycans, lipids, etc), which might neutralize their effects. For example, an antibody can bind to the hemagglutinin molecule of the influenza virus and prevent its entry into cells. We are all familiar with the utility of vaccines that create antibodies to recognize the spike protein of the acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibodies also bind to foreign cells like bacteria, which signals other host immune proteins and cells to come in for the kill. Antibodies are secreted by B cells, which also have a membrane-bound form of the antibody on their surface. This antibody acts like a receptor that binds antigens and, through a signal transduction process, helps to activate the B cell. Mature B cells (those that have previously seen antigens) can secrete lots of antibodies quickly. Surface and secreted antibodies can recognize and bind to almost any molecule.

There are many forms of antibodies, also called immunoglobulins (Ig). These include IgA, IgG, IgM, and IgD. We will concentrate on the structure of IgG. It consists of 4 chains (a tetramer), two light chains, and two heavy chains. The light chains form disulfide links with the heavy chains, and disulfides also link the heavy chains. Effectively, it's one big protein molecule (about 160 K). Figure \(\PageIndex{1}\) shows a spacefill, secondary structure, and geometric cartoon rendering of a mouse IgG protein (pdb ID 1IGT).

Figure \(\PageIndex{1}\): Renderings of an IgG antibody

The antibody is shaped like a Y. Foreign molecules (antigens) bind at the end of the top tips of the Y, with both chains contributing to antigen binding. Antiparallel beta sheets dominate both structures.

Each chain consists of a single N-terminal variable domain (V_L or V_H), which participates in antigen recognition. The light chains have an additional constant domain (C_L), while the heavy chains have three constant domains (C_H1-C_H3). The constant domains are not involved in antigen recognition. Still, they are involved in effector functions (such as binding other immune molecules like complement proteins) to the antigen-bound antibody heavy chain constant regions. Each of the domains is about 100 amino acids. Figure \(\PageIndex{2}\) shows a cartoon showing the domain structures.

Figure \(\PageIndex{2}\): IgG antibody and fragments

Two other features are depicted in the above figure. In each variable region of both light and heavy changes, there are hypervariable regions that contribute to the unique binding features of a given antibody. The regions are also called complementarity-determining regions (CDRs). Membrane-bound forms of antibodies that serve as "receptor" proteins have additional domains, now shown in the figure above. The binding site on the antigen recognized by the antibody is called the epitope. The corresponding binding site on the Y-shaped ends of the antibody that recognizes the antigen is called the paratope.

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Recently Updated:  11/3/24

In Figure \(\PageIndex{2}\), you can see that the intact, full IgG molecule has 12 variable V and constant C domains, each an example of an immunoglobulin domain (IGD) or immunoglobulin fold (IGF). Each has about 110 amino acids in length, two layers of β-sheets that face each other, with each sheet containing 3-5 antiparallel β-strands with a disulfide bond connecting the two layers.  Beta-hairpins connect strands in a given sheet while "beta-arches" connect the two sheets. 

The immunoglobulin fold (IGF) and domain (IGD) are ubiquitously found in nature in all domains of life, and proteins with them are part of the Ig Superfamily (IgSF). It's estimated that about 2% of all proteins contain the domain.  They are found in the cytoplasm, membranes, and secreted proteins. The domain is often found on cell membrane proteins and the proteins that bind to them, making them part of a complex IGF "interactome". They are especially abundant in eukaryotes in systems (immune, muscle, and nervous, for example), for which intracellular and extracellular communications are critical. There are over 200 IGD in one of the largest human proteins, titin.  Figure \(\PageIndex{3}\) shows the topology of immunoglobulin-like domains.

Figure \(\PageIndex{3}\):  Topology of immunoglobulin-like domains.  Chidyausiku, T.M., Mendes, S.R., Klima, J.C. et al. De novo design of immunoglobulin-like domains. Nat Commun 13, 5661 (2022). https://doi.org/10.1038/s41467-022-33004-6.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

Panel a: Three-dimensional cartoon representation of an Ig structure formed by seven β-strands (left) and backbone hydrogen bond patterns (annotated thin lines) between paired β-strands along the sequence (right). Cross-β interactions have higher sequence separation (and higher contact order) than β-hairpins, slowing folding. 

Panel b: β-arches of the cross-β motif belong to two contiguous and distinct Greek key motifs: with two β-strands in each β-sheet (left) and with three β-strands in one β-sheet and one β-strand in the other (right). From the folding and design perspective, the main limiting factor for correctly assembling the Ig structure is the formation of the cross-β motif since the three β-hairpins can form independently.

IGDs in eukaryotes are divided into four main variants, IgI (I set), IgV (V set), IgC1 (C1 set), and IgC2 (C2 set), based on the immunoglobulin structure (for example, V stands for variable).  They differ in topology and secondary structure but generally resemble two stacked β-sheets or a β-sheet sandwich barrel (with the sheets being the "bread").  A new structural and numbering scheme for the strands shows the Ig domain can contain nine strands, A, B, C, C’, C’’, D, E, F, and G (Caesar Tawfeeq et al.)

Figure \(\PageIndex{4}\) shows  interactive iCn3D models of the IgG2a monoclonal antibody (1IGT) shown in Figure 1 above. The left panel shows the Ig domains of just one variable chain (which contains two domains), while the right panel shows all the IgDs of the full antibody.  The Ig strands (A, B, C, C’, C’’, D, E, F, and G) are colored as shown below.

1IGT Light Chain. Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...xpZS1JFPX99yeA

1IGT Full antibody.  Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...zTPnVZhjmnAQa6

Figure \(\PageIndex{4}\): Ig domain structure of the IgG2a monoclonal antibody (1IGT) light chain (left) and complete antibody (right). (Copyright; author via source). 

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When we discussed domain structure, we indicated that proteins with multiple binding domains can often be selectively cleaved with a protease, with cleaved fragments often retaining binding and other functional properties. The same is true with antibodies. Cleavage with the proteases pepsin or papain forms fragments with binding activities, as illustrated in the figure above. Selected protease digestion was used to clarify structure/function relationships in antibody recognition.

When antibodies targeting different antigens were sequenced, it was clear that much variability was found among antibodies in the variable domains of both the light and heavy chains. In those domains, there were also hypervariable regions. The variability and hypervariability originate mostly from an extremely large number of gene segments (also exons) in the gene encoding the variable domains. The exons can be spliced together at the DNA and RNA levels to produce many different DNA/RNA sequences. These are decoded into the variable and hypervariable regions of the light and heavy chain proteins. Somatic mutations are also enhanced in this region.

In-depth: Generation of Antibody Diversity

We mentioned above that DNA and RNA splicing occur as B cells mature to become antibody-secreting cells (plasma cells). Splicing for primary RNA transcripts should be no surprise for those who have studied the Central Dogma of Biology. Surprisingly, the DNA genome of B cells changes on their maturation due to the splicing of multiple exons within the variable chain genes to produce unique coding sequences for each B cell clone. There are sets of exons (V, D, and J) or segments within the genes for the variable chain. As the immune cells terminally differentiate, a unique combination of a VDJ segment forms in the DNA genome, so each terminally differentiated B cell is different. When needed (i.e., when their unique antigen binds to membrane forms of the antibody), the cell secretes a monoclonal antibody.

Figure \(\PageIndex{5}\) shows how the different segments become linked in the DNA and can be uniquely spliced in the RNA to produce a unique, monoclonal antibody.

Figure \(\PageIndex{5}\) Generation of Antibody Diversity. Oliver Backhaus DOI: 10.5772/intechopen.72818. Creative Commons Attribution 3.0 License,

The first antibodies produced by the immune system are often of low affinity. Over time, high-affinity (low K_D) antibodies are produced. What differentiates high and low affinity binding at the molecular level? Do high-affinity interactions have many intramolecular H-bonds and salt bridges (ion-ion interactions), or are hydrophobic interactions most important? Crystal structures of many antibody-protein complexes were determined to study the basis of affinity maturation of antibody molecules. Clones of antibody-producing cells with higher affinity are selected through binding and clonal expansion of these cells. Investigators studied the crystal structure of four different antibodies that bound to the same site or epitope on the protein antigen lysozyme. Increased affinity was correlated with increased buried apolar surface area and not with increased numbers of H bonds or salt bridges, as described in Table \(\PageIndex{1}\) below.

Antibody	H26-HEL	H63-HEL	H10-HEL	H8-HEL
Kd (nM)	7.14	3.60	0.313	0.200
Noncovalent Interactions
H bonds	24	25	20	23
VDW contacts	159	144	134	153
salt bridges	1	1	1	1
Buried Surface Area
ΔASURF (A2)	1,812	1,825	1,824	1,872
ΔASURF-polar (A2)	1,149	1,101	1,075	1,052
ΔASURF-apolar (A2)	663	724	749	820

Table \(\PageIndex{1}\): Characteristics of Antibody:Hen Egg Lysozyme Complexes (HEL). Data from Y. et al. Nature: Structural Biology. 6, pg 484 (2003)

Many crystal structures of antibody:antigen complexes have been determined. Especially interesting are those in which the antigen is a protein. It is important to understand antibody:protein antigen interactions to develop vaccines against key epitopes in proteins such as the spike protein of SARS-CoV-2. Let's look at the antibody that binds to hen egg white lysozyme (HEWL). The crystal structures of many different IgG antibodies that bind HEWL are known. One recognizes a discontinuous epitope on lysosome consisting of the following amino acids: H15, G16, Y20, R21, T89, N93, K96, K97, I98, S100, D101, G102, W63, R73, and L75. Most of these amino acids are polar, and five are charged.

Figure \(\PageIndex{6}\) shows the interaction of part of the Fab fragment of an antibody that binds to the HEWL epitope just mentioned (3hfm). The light chain is shown in magenta, the heavy chain in dark blue, and the antigen lysozyme in gray. The amino acids' side chains in the HEWL epitope are shown in sticks. Note the complete complementarity of HEWL and Fab surfaces. Water is excluded from the interface.

Figure \(\PageIndex{6}\): Surface interactions between a hen egg white lysozyme epitope and an IgG Fab antibody fragment

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the same HEWL:Fab complex (3hfm). Lysozyme is shown in black.

Figure \(\PageIndex{7}\): Hen egg white lysozyme:Fab complex. (3hfm) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...agQkuAKhgfs177

Here is an external link to an interactive iCn3D model showing a detailed view of the multiple interactions (salt bridges, hydrogen bonds, pi-cation)

Abstract:  Despite the central role of antibodies in modern medicine, no method currently exists to design novel, epitope-specific antibodies entirely in silico. Instead, antibody discovery currently relies on immunization, random library screening or the isolation of antibodies directly from patients^1. Here we demonstrate that combining computational protein design using a fine-tuned RFdiffusion^2 network with yeast display screening enables the de novo generation of antibody variable heavy chains (VHHs), single-chain variable fragments (scFvs) and full antibodies that bind to user-specified epitopes with atomic-level precision. Bennett, N.R., Watson, J.L., Ragotte, R.J. et al. Atomically accurate de novo design of antibodies with RFdiffusion. Nature 649, 183–193 (2026). https://doi.org/10.1038/s41586-025-09721-5.

T Cells

What happens if a virus makes it into a cell? Antibodies can not bind to them anymore to prevent their entry. Something must be able to recognize a virally infected cell and eliminate it. What about a cancer cell? Wouldn't it be nice if something could recognize a tumor cell as foreign and eliminate it before it divides too much and metastasizes? Those "somethings" are T cells. There are many T cells in a person and many different kinds, including T helper cells (Th), cytotoxic lymphocytes (CTL), and even suppressor T cells. They express different subsets of proteins that differentiate them and their functions.

T cells also recognize antigens, but unlike B cells, these antigens can only be protein fragments. The membrane proteins that recognize protein fragments are called T-cell receptors. In addition, they don't recognize protein antigens in isolation. They must be bound to a protein on the surface of an "antigen" presenting cell (such as a macrophage or dendritic cell). The T cell receptor recognizes and binds simultaneously to the foreign protein fragment and the self "antigen-presenting" protein on the surface of the antigen-presenting cells. The self-protein that binds and presents the foreign protein fragments (peptides) is called a Major Histocompatibility Complex (MHC) protein.

Antigen-presenting cells like macrophages and dendritic cells have MHC Class II molecules on their surface. These bind protein fragments from engulfed bacteria (for example) and present them on the surface. T cell receptors bind to the peptide:MHC II complex. All cells in the body have MHC Class I proteins on their surface. If a cell is infected with a virus, protein fragments from the virus end up bound to the MHC Class I protein on the surface. Now, a T cell can bind through its T cell receptor to the peptide:MHC Class I complex. By displaying a viral protein fragment on the surface, the immune cell can recognize a virally infected cell without getting inside the cell where the virus is. Sompayrac describes MHC molecules as looking like a hot dog bun. In the groove of the bun lies the peptide fragment, like the hot dog. The T cell receptor recognizes both the bun and the hot dog!

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of a MHC Class I heavy chain complexed with a peptide fragment (i.e. the antigen) of the vesicular stomatitis virus nucleoprotein (2VAA). 

Figure \(\PageIndex{8}\): MHC Class I heavy chain - vesicular stomatitis virus nucleoprotein peptide (antigen) complex (2VAA). (Copyright; author via source). Click the image for a popup or use this external link https://structure.ncbi.nlm.nih.gov/i...NPS9QfLeghCmo8

 

In-depth: Generation of T-Cell Receptor Diversity

We described above how an undifferentiated B cell has the potential to produce an incredible diversity of different antibodies from a starting genetic sequence. This occurs through both DNA and RNA splicing. The same processes occur with the alpha and beta chains of T-cell receptors. This is illustrated in Figure \(\PageIndex{9}\). Note that the alpha chains have no D (diversity) coding sequences.

Figure \(\PageIndex{9}\): The diversity of T-cell receptor (TCR)αβ is a result of genetic recombination and diversification mechanisms occurring at the α and β TCR chain loci. Diversity is first created in the germline via recombination of variable V, diversity D (for β chain), and joining J segments. Further diversification occurs through imprecise junctions of these gene segments (addition of P- and N-nucleotides adjacent to the D segment), and the combination of α and β chains

Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy. International Journal of Molecular Sciences 21(7):2378 (2020). DOI: 10.3390/ijms21072378. License CC BY

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the T-cell receptor alpha and beta chains binding to MHC Class 1 protein with a bound peptide (6rp9). The MHC protein complex consists of the histocompatibility antigen A-2 alpha chain and β-2-microglobulin, an 11K subunit of MHC Class I proteins, but not Class II MHC proteins. Bound to it is the nine-amino acid cancer/testis antigen 1 (shown in spacefill). The peptide is sandwiched between the MHC protein complex and the T-cell receptor α and β chains.

Figure \(\PageIndex{10}\): T-cell receptor alpha and beta chains binding to MHC Class 1 protein with a bound peptide (6rp9) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...txUWWfbNhNXwQ6

The T-cell receptor consists of two transmembrane protein chains, alpha and beta, each containing a single variable and constant Ig domain, followed by a transmembrane domain. Hence, they are less complicated than an antibody chain. They bind through their extracellular variable domains to a peptide fragment bound to a MHC Class I or Class II membrane protein on the target cell.  The alpha and beta chains of the T-cell receptor, the HLA class I chain, and the associated beta-2 microglobulin are comprised of Ig domains, just like antibodies.  Figure \(\PageIndex{11}\) below shows the Ig domains color-coded to show their A-G beta strands. The MHC Class 1 protein domain that actually binds the peptide antigen (shown in spacefill) is shown in cyan.

Figure \(\PageIndex{11}\): T-cell receptor alpha and beta chains binding to MHC Class 1 protein with a bound peptide (6rp9)

The actual functional structure in vivo is more complicated. The T cell receptor is found within the much larger T Cell receptor complex (TRC), which contains two copies of the CD3 complex, which itself consists of γ, δ, ε, and ζ chains, as shown in Figure \(\PageIndex{12}\) (A). Part A shows the variable and C domains of the α and β chains of the T-cell receptor in green and dark. The rest of the T-cell receptor complex includes two copies each of the CD3 complex, which consists of one copy of εδζ chains and one copy of εγζ chains.

Figure \(\PageIndex{12}\): T cell receptor structure. Kumaresan Pappanaicken R., da Silva Thiago Aparecido, Kontoyiannis Dimitrios P. Methods of Controlling Invasive Fungal Infections Using CD8+ T Cells. Frontiers in Immunology, 8, 1939 (2018). https://www.frontiersin.org/article/...mmu.2017.01939. DOI=10.3389/fimmu.2017.01939. Creative Commons Attribution License (CC BY).

As mentioned earlier, one of the functions of the MHC Class I molecules is to present peptides derived from tumor antigens to T-cells. This leads to activating other immune cells and, hopefully, destroying the tumor cells displaying the tumor antigen. Much work has gone into studying immune surveillance and the ultimate destruction of tumor cells to improve our immune response to cancer cells. In early work, T-cells that had infiltrated tumors were isolated from a patient, amplified in the lab by adding a cytokine (ex. interleukin 2 - IL2), a protein growth factor released by activated immune cells, and then re-infusing the tumor-specific T cells along with IL2 back into the patient. This adoptive cell transfer (ACT) therapy led to remissions in some patients, but the therapy also could be lethal.

One promising type of immune therapy is chimeric antigen receptor (CAR) T cell therapy (CAR T), in which patients are treated with modified versions of their T-cells. T cells are removed from a cancer patient's blood. A gene is constructed to mimic the V and C domains of the alpha and beta chains of the T-cell receptor and inserted into the patient's T-cell using a viral vector. The gene construct contains as its tumor antigen binding motif the V and C domains of an antibody gene constructed to recognize the tumor antigen. The receptor is, hence, a chimeric (formed from parts of different proteins) antigen receptor (CAR) with antibody and T cell parts. The genetically modified cells are amplified and re-infused back into the patient. Once the collected T cells have been engineered to express the antigen-specific CAR, they are "expanded" in vitro into the hundreds of millions.  This approach is very expensive, so some recent successes in using a virus to add the appropriate gene construct without removing cells from the patients are noteworthy. 

Compare the structure of the chimeric antigen receptor (CAR) in Figure XX-C with the normal T-cell receptors shown in A. The CAR contains two single-chain variable fragments (scFv) derived from combining the variable domains of the light (V_L) and heavy (V_H) chains of an antibody recognizing the tumor cell. These domains are connected by a linker peptide (10-25 amino acids) enriched in glycines, which confers flexibility and serines/threonines for hydrogen bonding interactions. This is attached to an F_C fragment and other intracellular effector domains to create the receptor. Figure \(\PageIndex{13}\) shows the scFv structure. We'll discuss the addition of the cytoplasmic CD28 domain in a bit.

Figure \(\PageIndex{13}\): Single-chain variable fragment structure used in CART

You can imagine this whole T-cell receptor complex involved in the binding of a tumor peptide antigen presented by a MHC I transmembrane protein on a tumor cell, as illustrated in Figure \(\PageIndex{14}\) (in different colors). The entire interacting structure is called the T cell immunological synapse.

Figure \(\PageIndex{14}\): T cell immunological synapse of T cell with a cancer cell. Zhao Lijun, Cao Yu J. Engineered T Cell Therapy for Cancer in the Clinic. Frontiers in Immunology, 10, 2250 (2019) https://www.frontiersin.org/article/...mmu.2019.02250. Creative Commons Attribution License (CC BY).

Protection Against Autoimmune Recognition - Coreceptors

How can the immune system recognize and bind to any foreign molecule but not self-molecules? The subject of immune tolerance is too specialized to include here, but there are a few features we will discuss.

The MHC Class I proteins present "self" peptides in their binding pockets. Self-proteins are also cleaved into peptides in the cell by proteasomes. However, the T cell receptor does not recognize and bind to the self-peptide fragment bound to the MHC Class 1 protein. Hence, T cells do not recognize self and turn against their own cells. Once in a while, they do, however, and autoimmune diseases like MS, rheumatoid arthritis, and lupus result.

B cells and T cells must be activated before they can respond. It is important to regulate the "on" switch. If the cells were activated without need, they might turn against self. In addition to T cell receptor complex binding to foreign peptides, MHC complexes for immune cell activation must bind another protein on the antigen-presenting cell.

In the case of T helper cells, the T cell protein CD28 must also bind the B7 protein on an antigen-presenting cell, like a macrophage expressing an MHC II protein:foreign peptide complex. Hence, there is one specific signal (the peptide:MHC complex binding to the T cell receptor complex) and a nonspecific signal (B7 binding CD28). Why are two signals needed for activation? Again, Sompayrac has a great analogy. A safety deposit box at a bank takes two keys: a specific key (which you have) and a "nonspecific" key (which the bank uses for all boxes) to open the box. Think of it as double security. You don't want to activate immune cells for killing unless you need to do so.

CD4 and CD8 T cells

Yet other proteins are involved to ensure correct T-cell activation. We'll consider T-cells expressing either the proteins CD4 or CD8. T-cells expressing these expand after antigen stimulation (infection or immunization). It depends on the subtype of T-cell. Let's consider two here:

T cells expressing the protein CD4: After initial simulation, they differentiate and proliferate into the T helper cells named T_H1, if they produce the cytokine interferon (IFN)-γ, and T_H2, if they produce the cytokine IL4. CD4 is an integral membrane protein and acts as a co-receptor for MHC Class II:peptide complex found on cells like macrophages. These "present" foreign antigens and antigens that are part of microbes are taken up into phagosomes in antigen-presenting cells like macrophages.  Hence, they protect phagosomal spaces, similar to the role of antibodies, which bind to and protect from antigens in extracellular spaces.  Traditional vaccines targeted to foreign proteins, such as the spike protein of the SARS-CoV-2 virus, mainly elicit antibodies that bind to antigens in the blood and other intracellular spaces.  Such vaccines are ineffective against antigens such as the malaria parasite in the blood since the parasite stays in circulation for only an hour before it is internalized in liver cells.

T-cells expressing CD8: These cells produce cytokines (IFN-γ and tumor necrosis factor (TNF)-α) or secrete protein, which form pore-forming complexes on foreign cells, leading to the lysis of cells such as pathogens or tumor cells. The CD8 protein has an alpha and a beta subunit. They serve as co-receptors for MHC Class I:peptide complex found on tumor cells, for example. MHC Class I proteins are found on most body cells (see below).  Cytotoxic T-cells (a type of T-cell) express CD8.  Vaccines that elicit a robust CD8 response might be more effective in the defense against intracellular pathogens that cause malaria, tuberculosis, and acquired immune deficiency syndrome (AIDS).    Memory CD8 cells protect against intracellular infections, so more effort is being made to develop CD8 T-cell vaccines.

Figure \(\PageIndex{15}\) shows the multiple co-signals that are required to activate the CD4 T-cell (blue sphere), which has the T-cell receptor complex, the co-receptor CD4, and the CD28 protein. It also displays a cytokine receptor, which binds cytokines released by the antigen-presenting cells (macrophage shown in pink). This leads to the proliferation and differentiation of the activated T-cell.

Figure \(\PageIndex{15}\): Co-signals that are required to activate the CD4 T-cell (blue sphere) Salmonella as a Model for Non-Cognate Th1 Cell Stimulation. Frontiers in Immunology 5(621):621 (2014) DOI: 10.3389/fimmu.2014.00621. CC BY 4.0

Sompayrac asks another interesting question. Why is antigen presentation by MHC proteins necessary at all? B cells don't need presentation since they can bind antigens with membrane antibody molecules. Why do T cells need it? He gives different reasons for Class I and Class II presentations:

Class I MHC (found on most body cells): T cells need to be able to "see" what is going on inside the cell. When virally infected cells bind foreign peptide fragments and present them on the surface, they can be "seen" by the appropriate T cell. It's a way to get a part of the virus, for example, to the surface. They can't hide out in the cell. T cells don't need to recognize extracellular threats since antibodies from B cells can do that. Presentation is also important since viral protein fragments found outside of the cell might bind to the outer surface of a noninfected cell, targeting them for killing by the immune system. That wouldn't be good. It also helps that peptide fragments are presented on the surface. This allows parts of the protein that are buried and not exposed on the surface, which would be hidden from interaction with outside antibodies, to be used in signaling cell infection by a virus.

MHC Class II (found on antigen-presenting cells like macrophages): Two different cells (the presenting cell and the T helper cell) must interact for a signal for immune system activation to be delivered to the body. Again, it is a safety mechanism to prevent the nonspecific activation of immune cells. Also, as in the case above, since fragments are presented, more of the foreign "protein" can contribute to the signal to activate the immune system.

Summary

This chapter provides an in-depth look at the adaptive immune system, focusing on the molecular and cellular components that drive antigen recognition and immune response. Key topics include:

• Antibody Structure and Function:

  • Exploration of the molecular architecture of antibodies, highlighting the roles of variable and constant regions in antigen binding and immune effector functions.

• B-Cell Development and Activation:

  • Detailed discussion on the processes involved in B-cell maturation, activation, and differentiation, including the generation of antibody diversity through somatic recombination and class switching.

• T-Cell Receptors and Antigen Recognition:

  • Examination of the structural features of T-cell receptors (TCRs) and their interaction with peptide-MHC complexes, emphasizing the specificity and mechanisms of T-cell activation.

• T-Cell Function and Immune Regulation:

  • Overview of T-cell subpopulations and their roles in orchestrating the immune response, including the coordination between cell-mediated and humoral immunity.

Overall, the chapter integrates biochemical principles with immunological concepts, equipping junior and senior biochemistry majors with the foundational knowledge needed to understand how antibodies, B-cells, and T-cells function together to protect the body against pathogens.