• T cells are lymphocytes that directly or indirectly eradicate pathogens.
• They respond to intracellular targets, as opposed to the B cells of the humoral arm, which respond to extracellular microbes.
• Key events in the life cycle of the T cell:
  — They arise from stem cells in the bone marrow.
  — T cells mature in the thymus (T is for Thymus): maturation involves positive and negative selection, and gives rise to naïve (non-activated) cells defined by the presence of either CD4 or CD8 proteins on their surfaces.
  — In the secondary lymphoid organs, such as the lymph nodes and spleen, naïve T cells are activated by antigens; the naïve T cells become functional effector cells.
  — After the pathogen is eradicated, most of the effector cells undergo apoptosis; otherwise, they pose a potential danger to the host cells.
  — Some of the T cells differentiate to become memory cells, which will respond if/when the host is exposed to the same antigen – thus, the secondary response to subsequent exposure can occur much faster.

MHC

Major Histocompatibility Complex (MHC) molecules present peptide antigens that activate T-cells.

Class I MHC molecules

• Present fragments of antigens that are synthesized endogenously – i.e., peptides derived from viral antigens produced within the cells.
• Class I MHC molecules are only recognized by naïve CD8+ T cells and their Cytotoxic T cell descendants.

Class II MHC molecules

• Present fragments from extracellular microbes and pathogens – i.e., peptides derived from extracellular microbes.
• Class II MHC molecules are only recognized by naïve CD4+ T cells and their Helper T cell descendants.

T CELL MATURATION

• In the histological sample, we indicate the cortex of a lobule, which is where positive selection occurs, and, the medulla, which is where negative selection occurs.
• “Thymic education” entails two selective mechanisms that eliminate T cells that would otherwise harm the host:
  — In positive selection, immature T cells are exposed to cortical epithelial cells displaying self-MHC complexes:
  The T cells that recognize the MHC complexes survive
  Those that fail to recognize the MHC complexes undergo apoptosis.
  — Then, in negative selection, T cells are exposed to MHC complexes with self-antigen:
  Those T cells that do NOT respond to the self-antigen survive
  The T cells that DO respond undergo apoptosis.
  — Thus, positive selection ensures that the surviving T cells can recognize the MHC complex, which is necessary for their activation, while negative selection ensures that self-destructive T cells are eliminated.
  — Ultimately, thymic maturation produces three main types of T cells, which we designate based on their unique cell surface proteins: CD8+, CD4+, and CD4+/CD25+.
  CD4+/CD25+ T cells are Regulatory T cells; they can suppress the activity of the other T cell types via expression of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4).
  — In addition to the CD proteins, naïve T cells also express receptors (T-Cell Receptors, TCRs) for specific antigens; binding with their specific antigen induces their activation.

T CELL ACTIVATION

• Activating Cells:
  — MHC class I molecules are displayed by all nucleated cells (in other words, most body cells except red blood cells).
  — MHC class II molecules are displayed by dendritic cells, macrophages, and B cells – because of this unique ability, these are referred to as “antigen-presenting cells.”
  However, be aware that B cells do not activate naïve T cells; they stimulate mature Helper T cells as part of their own activation (discussed in detail, elsewhere).
  — As we learned earlier, CD8+ and CD4+ T cells recognize different MHC classes; this means that they can only be activated by cells displaying the appropriate MHC molecules.
• A general example:

1. An antigen-presenting cell, such as a dendritic cell, recognizes and engulfs a microbe.
2. It digests the microbe and re-packages a peptide fragment with an MHC class II molecule on its surface.
3. The MHC- antigen complex is recognized by a naïve CD4+ cell, which is subsequently activated. Notice that if the antigen-presenting cell displayed only antigens complexed with MHC class II molecules, the CD4+ cell would not have recognized it.

Activating mechanisms in more detail:

• 2-signal activation of a CD8+ T cell, which differentiates to become a cytotoxic cell.
  — The cell surface of the CD8+ has the T-cell Receptor Complex (TCR complex), which consists of the following components:
  The T-cell receptor, which is specific to the peptide antigen displayed by the MHC molecule; CD3 proteins; and, the CD8 protein, which recognizes and interacts with the MHC class I molecule of the nucleated cell.
  — The representative nucleated cell displays the class I MHC – antigen complex.
  — The interaction between the TCR complex and nucleated cell allows for the second signal, which involves co-stimulationbetween CD28 and B7-2.
  — Activation triggers proliferation, aka, cloning, of the T cell and differentiation into the effector type – which, for CD8+ cells, is the Cytotoxic T cell.
  These processes are guided by cytokines, which are released by T cells and antigen-presenting cells.
• 2-signal activation of a CD4+ T cell, which differentiates to become a Helper T cell.
  — The dendritic cell surface displays the MHC II-antigen complex.
  — The CD4+ cell has the TCR complex: the T-cell receptor, which is specific to the antigen; the CD3 molecules; and, the CD4 protein that interacts with the MHC II molecule on the antigen-presenting cell.
  — The second signal comprises co-stimulation: interaction between CD28 on the surface of the T cell and B7-2 on the dendritic cell.
  — Activation results in proliferation and differentiation to effector cells.

Effector Cell Functions:

• Cytotoxic T cells directly kill pathogen-bearing cells via the following steps:

1. The T cell recognizes the MHC I – antigen complex.
2. Docking brings the two cell membranes in close association.
3. The T cell releases perforins, which form a pore in the infected cell’s membrane.
4. The cytotoxic cell releases granzymes, which move through the pore and trigger apoptosis of the infected cell.

• Helper T cells, the products of activated CD4+ cells, have multiple roles in both innate and adaptive responses:
  — They amplify the innate response via cytokine release and recruitment of neutrophils and macrophages.
  — They activate B cells, which mediate the humoral arm of the adaptive immune response.
  — They activate cytotoxic T cells, in part by upregulating the expression of co-stimulatory molecules on dendritic cells.
  — Superantigens, such as Staphylococcus bacteria, are super potent activators of CD4+ cells.

• 4 subsets of Helper T cells:
  — Under the influence of interferon-gamma and IL-12, cells of subset Th1 develop.
  They engage in anti-viral activity, macrophage activation, and induce cytotoxic T cell differentiation; when unregulated, they are associated with autoimmune diseases.
  This subset produces IL-2 and interferon-gamma.
  — Under the influence of IL-4, Th-2 develop.
  This subset is particularly important in defense against worms and in mobilization of eosinophils; they are associated with allergies and asthma.
  They produce IL-4, IL-5, and Il-13.
  — Tissue growth factor-beta, IL-6, IL-1, and IL-23 induce differentiation of subset Th17, which recruit neutrophils and monocytes.
  They are also associated with autoimmune disease.
  They produce IL-17 and IL-22.
  — Follicular helper T cells differentiation is thought to require interaction with B cells.
  Follicular helper T cells promote the humoral immune response and produce IL-21.

• The humoral response is mediated by B cells, which produce antibodies that act combat infectious agents.
• Class switching and somatic hypermutation provide diversity in antibody specificity and response, which protects the host from a wide variety of infections.
• Two types of antibody-mediated immunity:
  — Passive immunity occurs when an individual is given antibodies to infectious agents.
  — Active immunity occurs when an individual’s immune system produces antibodies in response to infection.

B CELL MATURATION

• B-cell maturation is antigen-independent (in other words, B cell maturation does not require antigen interactions).
  — Occurs within the bone marrow.
• Pre-B cells are characterized by μ-chains.
  — Upon stimulation by Burton’s tyrosine kinase (BTK), the pre-B cell transitions to an immature B cell; it expresses the antibody IgM (immunoglobulin M) as its B cell receptor (aka, BCR).
• Immature B cells undergo negative selection, also called clonal deletion, which removes B cells that bind with self-antigen.
• The surviving B cells become naïve mature cells, characterized by both IgM and IgD.
• These cells exit the bone marrow and travel via the circulation to the secondary lymphoid organs for activation.
  In our histology sample, we highlight the splenic nodules and lymphoid germinal centers where B cells encounter activating antigens.

B CELL ACTIVATION

• Most antigens require input from Helper T cells to activate naïve B cells; this is referred to as Thymus-Dependent activation.

1. Naïve B cells bind and internalize antigen.
2. Within the B cell, the antigen is complexed with MHC II and displayed on the B cell surface.
3. When a Helper T cell recognizes and interacts with the antigen-MHC II complex, the B cell is activated.
4. The B cell proliferates and differentiates, giving rise to memory cells, which participate in subsequent immune responses, and, plasma cells, which secrete large quantities of antibodies that circulate in the blood to fight infection.

• Thymus-independent B cell activation occurs when antigens, such as polysaccharide bacterial products, directly activate B cells. Write that thymus-independent B cell activation results in minimal class switching.

Details of B and T cell interactions:

Two key signals are required

• The first signal involves MHC recognition by the T cell, and is necessary for the second signal.
  — The B cell displays the antigen-MHC II complex, and that it is recognized by the T-cell receptor (TCR) and CD4 proteins of the Helper T cell.
• The second signal involves co-stimulation by B and T cell proteins:
  — Binding of B7-2 with CD-28 triggers cytokine activation.
  — Binding of CD40 with CD40L (L is for ligand) triggers class switching and affinity maturation.
• Cytokines released by the Helper T cell influence class switching from IgM/IgD to the other isotypes (IgA, IgG, and IgE).

ANTIBODIES

Key Functions

Antibodies do not directly kill pathogens.

• Neutralize microbes by blocking their extracellular receptors and inhibiting their attachment to host cells; antibody binding can also inhibit viral replication, stopping the spread of infection.
• Antibodies can opsonize microbes, which means they bind to them and make it easier for phagocytes to recognize and engulf them.
• Activate the complement cascade, leading to formation of membrane attack complexes (MAC) and, consequently, microbe lysis.
• Agglutination occurs when antibodies bind multiple cell-bound antigens simultaneously, causing them to clump; precipitation, which we haven’t shown, occurs when antibodies simultaneously bind multiple soluble antigens. In both cases, binding makes antigen capture and phagocytosis easier.
• Finally, show that antibody-dependent cellular cytotoxicityoccurs when antibody-coated cells are targeted by natural killer cells.

Primary and secondary humoral immune responses:

• At the time of first exposure, antibody concentration is low; exposure provokes the primary response, which is characterized by a rise in low-affinity antibody concentration.
  — When infection is cleared, antibody concertation dips back down (but notice that it is not as low as pre-exposure levels).
• Upon subsequent exposure to the same antigen, the secondary response rapidly produces a spike in high-affinity antibodies; thus, the secondary response is “primed” by the first.

Antibody Structure:

Because IgG is a the most abundant antibody, we’ll use it as an example of representative antibody structure; however, be aware that IgM and IgA look quite different.

• Antibodies, also called immunoglobulins, are glycoproteins that comprise heavy and light chains.
• They can be separated into fragments and regions:
  — Fab: F = fragment, ab = antigen-binding; notice that IgG has two Fab’s.
  Paratope binds the eptiope of a specific antigen.
  — Fc end: c = crystallizable region, but it can also be remembered because the C fragment interacts with Fc Cell surface receptors.
• Variable and constant regions; the variable regions differ across antibodies, while the constant region is constant.

Isotypes:

• IgM is the first antibody formed during B cell development; it opsonizes antigens and fixes complement.
• IgD, the second antibody type formed, can bind bacteria and activate B cells.
• IgG, which we drew above, is the predominant antibody type during secondary responses; it opsonizes bacteria, fixes complement, and neutralizes toxins.
  — Importantly, it passes from maternal to fetal blood via the placenta, and is an example of naturally acquired passive immunity.
• IgA is the predominant antibody in secretions, including breast milk (another example of passive immunity); it neutralizes antigens and blocks their adhesion to mucosal surfaces.
• IgE provides defense against parasites; it is associated with hypersensitivity and allergic reactions.

• The first line of defense comprises physical and chemical barriers that prevent pathogen entry into the body.
• The second line of defense comprises the internal cells, complement system and other circulating proteins, and pathogen recognition receptors.
• Some participants of the innate immune system also activate the adaptive arm:
  – Some pathogen receptors trigger B and T cell responses of the adaptive system; the toll-like receptors are especially important.
  – Dendritic cells and macrophages present antigens to T cells, which then participate in the cellular response to pathogens; thus, dendritic cells and macrophages are called “Antigen presenting cells.”

1ST LINE OF DEFENSE

Physical Barriers
– Keratinized squamous epithelia of the skin physically prohibits entry into the body.
Chemical Barriers
– Low surface pH of the skin, vagina, and stomach; this barrier is referred to as the acid mantle.
– Mucus creates another type of chemical barrier. It is secreted by goblet cells, and comprises antimicrobial lysozymes and sticky mucin that traps microbes and prevents binding to host cells.

• When physical and chemical barriers are breached, internal defenses activate.

INTERNAL INNATE DEFENSES

Cellular Defenses

Leukocytes

• Neutrophils and macrophages are phagocytic cells that engulf and destroy microbes; recall that they are early responders in the acute inflammatory response.
• Eosinophils and mast cells, release pro-inflammatory molecules, such as histamine.

Natural killer cells

• Often considered a specialized lymphocyte; these cytotoxic cells are regulated via inhibitory and activating signals.
  – Healthy cells display MHC I, which inhibits the natural killer cell; also, the natural killer cell’s activator receptor is unbound.
  – Virus-infected cells have diminished expression of MHC I molecules; also, NK activation receptor is stimulated, which results in destruction of the infected cell.

Dendritic cells

• Bind with antigen and trigger cytokine release; recall that cytokines mediate the inflammatory response.

Complement System

The complement system, specifically through the actions of proteins C3a and C3b, destroys microbes.
– Comprises inactive proteins that circulate in the blood; of these proteins, the products of C3 cleavage (C3a and C3b) have multifold functions.

• Three pathways lead to C3 cleavage:
  – In the classical pathway, C1 is “fixed” to antibody-antigen complexes, which initiates a cascade of events that lead to C3 cleavage.
  – The alternative pathway is triggered by spontaneously activated C3b.
  – The lectin pathway is triggered when lectins, such as, mannose-binding lectin, binds microbial sugars and marks them for phagocytosis.
• Effects of cleaved C3:
  – C3a has pro-inflammatory effects; it recruits neutrophils and macrophages.
  – C3b opsonizes microbes, which involves binding to pathogens and marking them for phagocytosis.
  – Membrane Attack Complexes (MAC): C3b combines with other complement proteins (C5b, C6, C7, C8, and C9) to form a pore in the membrane of the microbe; massive water influx through the MAC lyses the microbe.

Circulating proteins with antimicrobial effects

• Defensins are positively charged peptides that insert pores into microbe membranes and trigger lysis. Defensins are particularly active in the GI and respiratory tracts (be aware that some authors include defensins as part of the first line immune defenses, too).
• Interferons are antiviral proteins that inhibit virus replication and activate natural killer cells to enhance destruction of infected cells.
• Acute-phase proteins promote opsonization and/or activate the complement system.
  The liver is a major source of these proteins, which include:
  – C-reactive protein, serum amyloid A, and the collectins.
  – Some important examples of collectins are pulmonary surfactant proteins that fight pathogens in the lungs, and mannose-binding lectin, which, as we learned, can activate the complement system.

Pathogen recognition receptors (PRRs)

We address these individually, but be aware that they often coordinate to effectively eradicate pathogens.

• Toll-like receptors sense a wide variety of pathogens, including bacteria, myocbacteria, viruses, and fungi.
  – They are present in both cellular and endosomal membranes.
  – Upon stimulation, they trigger the release of pro-inflammatory cytokines and interferons.
• NOD-like receptors (Nucleotide-binding Oligomerization Domain-like) are cytosolic sensors that also respond to diverse stimuli:
  – Some NOD-like receptors recognize bacterial wall peptidoglycans and trigger pro-inflammatory cytokine release.
  – Some NOD-like receptors respond to microbial and non-microbial materials via inflammasomes, which are protein complexes that can trigger cell death and recruit pro-inflammatory cells.
• RIG-like receptors trigger interferon release in response to viral RNA.
• Cytosolic DNA sensors induce interferon release in response to DNA from damaged cells.

Pathogens

Disease-causing or harmful microorganisms

Antigens

Material that can evoke an immune response

TWO BRANCHES OF THE IMMUNE SYSTEM

1) Innate Branch – non-specific, fast

• Physical barriers such as skin or chemical barriers
• Chemokines are a chemical signal produced by damaged cell to alert the body to danger and act as a homing signal for immune cells
• Neutrophils are the first type of phagocytic cell to arrive
• Monocytes arrive and mature into macrophages which engulf and destroy pathogens
• Inflammation (response to tissue damage) has four clinical signs: redness, heat, swelling and pain

2) Adaptive Branch – specific, slow, systemic, memory

• Humoral immunity – B cells (matured into plasma cells) producing antibodies (Y-shaped proteins)

• Cell-mediated immunity – Cytotoxic T cells recognize infected cells and kill them while helper T cells act as the general of the immune army and release chemical signals that activate various immune cell types

GROSS ANATOMY

• The splenic artery and vein enter at the hilum.
• Blood vessels travel within the lieno-renal ligament (aka, splenorenal ligament), which connects the intraperitoneal spleen to the posterior abdominal wall.

HISTOLOGY

Capsule

• Invaginates into the parenchyma as trabeculae, which divide the tissues into lobules; although omitted here for simplicity, blood vessels pass through the trabeculae.
• Comprises collagen, elastic, and smooth muscle fibers.
• The mesothelium, which is the outermost layer, forms the trabeculae.

Red pulp

• Is highly vascularized; this tissue is responsible for filtering of damaged red blood cells and other particles.
• Stroma comprises reticular cells and fibers, plasma cells, and macrophages.
• These cells and fibers constitute the splenic cords (of Billroth), which are interspersed between the sinusoids.
• Sinusoids comprise elongated endothelial cells on a discontinuous basement membrane; though not visible here, reticular fibers also encircle the sinusoids.
  • Blood flow through the spleen is complicated, and authors disagree on whether circulation is open and/or closed. In closed circulation, the blood flows from arterial vessels directly to the sinusoids; in open circulation, arterial vessels open into the red pulp, through which the blood percolates towards the sinusoids.

White pulp, which comprises nodules of lymphoid aggregations; this is the immune component of the spleen.

• A nodule comprises lymphocytes, primarily B cells, and antigen-presenting cells; recall that B cells participate in the immune response against blood-borne antigens.
• Perilymphoid red pulp surrounds the nodule.
• Germinal center is at center of the nodule; its size diminishes with age, and that inter-individual variation exists.
• Mantle and marginal zones form rings of darker-staining areas.
• A central artery passes through each nodule; it is a branch of the trabecular artery, and, in humans, is located peripherally in the nodule.
  • As it passes through the white pulp, T-cells surround the vessel, forming the peri-arteriolar lymphoid sheath (PALS).
  • Though not shown here, the central artery exits the white pulp and terminates in macrophage-sheathed capillaries, of the red pulp. These capillaries may either drain directly into the splenic sinusoids or, instead, into the spaces of the red pulp.

Notice that the spleen does NOT have a cortex and medulla.

Be aware that we’ve simplified some aspects of splenic histology; there is a great deal of intertextual variation regarding the details of splenic tissue structure and function.

Furthermore, although rodent models are often applied to studies of human anatomy and physiology, they are not viable in the case of the spleen because there are significant differences in the rodent and human spleens.

LYMPH NODES

• Secondary organs of the immune system. These small bean-shaped structures are found where blood and lymph vessels converge, such as the axillary and groin areas.

Overview of Structures:

Capsule

• Gives rise to trabeculae, which divide the node into sections and provide passage for blood vessels.
  Afferent lymphatic vessels
• Pierce the capsule to deliver lymphatic fluid to the node; valves promote unidirectional flow.
  Hilum
• Area of indentation of the node; the efferent lymphatic vessel drains lymph from the node at the hilum.
• Blood vessels also enter and exit at the hilum.
  Medulla
• Open to the hilum
  Cortex
• Lies just beneath the capsule.

Stroma

Reticular fibers and cells

Lymphatic tissues of Lymph Node

• Lymphatic tissues are responsible for filtering and processing antigens present in the lymphatic fluid as it travels from the afferent vessels to the efferent vessel.
• Lymphatic tissues are densely packed in the cortex
• Reside more loosely in the medulla as the medullary cords.

Cortex

• Outer cortex
  • Aggregations of B cells form primary follicles.
  • When the B cells proliferate, they produce secondary follicles, which comprise a lighter-staining germinal center and the darker-staining mantle.
  • The light and dark areas reflect lymphocyte size: the germinal centers comprise active medium-sized loosely organized lymphocytes, and the mantle comprises smaller lymphocytes with condensed chromatin.
  • In addition to lymphocytes, macrophages and follicular dendritic cells (FDC) reside within the follicles;
    Dendritic cells are the primary antigen-presenting cells (APC); they present microbial antigens on their surfaces to trigger T cell activation.
• Inner cortex
  • T cells and dendritic cells

Sinuses and Medullary cords

• Macrophages
• Additional lymphocytes

Route of lymph fluid through Sinuses

• Sinuses are lined by endothelial cells

1. Lymph fluid passes through the afferent lymphatic vessel into the sub-capsular, aka, marginal sinus
2. Travels through the cortical, aka, trabecular or para-trabecular sinus, to the medullary sinus
3. Exits medullary sinus through efferent lymphatic vessel.
4. From there, it is transported in the lymphatic vessels to other lymph nodes and eventually returned to the blood.