Chapter 10: Lymphocyte Activation

Intro

Overview

Compared with innate immune responses, adaptive immune responses against newly encountered antigens initially develop slowly. Although many self-reactive cells are eliminated during development, lymphocytes undergo a further set of time-consuming checks and balances to minimize the potential for adverse immune responses. This system of checks and balances is imposed by different cell types for recognition, regulation, and effect or function.

T cells play a central role as arbiters of adaptive immune function, and because of this role, the manner in which T cells recognize and are activated by epitopes is stringently regulated. It is useful to think of the innate immune system as the gatekeeper for adaptive immune responses (Fig. 10.1). Adaptive immune system effect or responses often activate and focus cells and/or molecules of the innate system on targets selected by the lymphocytes.

Figure 10.1.Interactions of the innate and adaptive immune systems.

Interactions of the innate and adaptive immune systems.

Antigen Presentation

Phagocytes sample their environment by phagocytosis and macropinocytosis. Ingested proteins are enzymatically degraded, and some of the resulting peptide fragments are loaded into MHC class II (forming pMHC class II) molecules in a process called antigen presentation. Some pathogens avoid phagocytotic and macropinocytotic mechanisms altogether or infect cells that do not express MHC class II molecules. Such antigens are broken down, and their peptide fragments are loaded into MHC class I (forming pMHC class I) molecules (Fig. 10.2).

Figure 10.2.Antigen presentation pathways.

Antigen presentation pathways.

Extracellular antigens (e.g., bacteria, cells, and many soluble molecules) enter the cell by phagocytosis or macropinocytosis packaged in phagocytic vesicles. Inset: Phagocytic vesicles fuse with enzyme-(lysozymes) containing vesicles (lysosomes) to form phagolysosomes that enzymatically degrade the ingested material. The lysosomal enzymes proteolytically degrade the ingested material into peptides in the late phagosome. The late phagosome will fuse with vesicles containing MHC class II. Intracellular pathogens (e.g., viruses and certain bacteria) and some antigens directly enter the cell’s cytoplasm, circumventing the phagocytic apparatus. Intracellular antigens are degraded by the proteasome into peptides that are loaded into MHC class I (pMHC class I) for display on the cell surface.

A. Presentation by MHC class II

Dendritic cells located at potential microbial portals of entry (e.g., skin and mucous membranes) and in other tissues and organs serve as sentinels (see Fig. 4.5). Immature dendritic cells are voracious eaters that ingest large amounts of soluble and particulate matter by phagocytosis and macropinocytosis. Phagocytosis involves the engagement of cell surface receptors (e.g., Fc receptors, heat shock proteins, and low-density lipoprotein-binding scavenger receptors) associated with specialized regions of the plasma membrane called clathrin-coated pits (Fig. 10.3).

Receptor engagement induces actin-dependent phagocytosis and receptor internalization to form small phagosomes or endocytic vesicles. Immature dendritic cells also sample large amounts of soluble molecules as well as particles present in the extra cellular fluids by macropinocytosis, a process in which cytoplasmic projections (cytoplasmic ruffles) encircle and enclose extracellular fluids to form endocytotic vesicles (Fig. 10.4).

Macropinocytosis does not require clathrin-associated receptor engagement. Lysosomes (enzyme-containing cytoplasmic vesicles) fuse with the endocytic vesicles derived from phagocytosis or macropinocytosis (see Fig. 10.2). Within this newly formed phagolysosome, ingested material is enzymatically degraded.

Figure 10.3.Phagocytosis.

Phagocytosis.

Cells, particles, and molecules are captured by PRRs associated with clathrin-coated pits. Clathrin-associated membrane invaginates and pinches off to form a phagosome. Clathrin is recycled back to the cell membrane to help form new coated pits.

Figure 10.4.Macropinocytosis.

Macropinocytosis.

1. Cytoplasmic protrusions or ruffles engulf and surround microbes, particles, or molecules to form a cytoplasmic vesicle (2) that fuses with a lysosome (3) to form a phagolysosome. 4. Vesicles containing enzymatically degraded material fuse with vesicles containing MHC class II (see Fig. 10.6). 5. Empty phagolysosomes are recycled back to the cell membrane.

When an immature dendritic cell senses an invasive threat, it rapidly begins to mature. Threats are detected by the same cell surface receptors used by the innate immune system. Direct sensing occurs through engagement of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) on viruses, bacteria, fungi, and protozoa. Engagement of other receptors (e.g., those that detect antibodies or complement molecules that have bound to microbes) is responsible for the indirect sensing of perceived threats.

Although the mechanisms responsible for dendritic cell maturation remain to be clarified, we know that threat sensing causes the dendritic cells to migrate to nearby lymph nodes, decrease their phagocytic and macropinocytic activity, and increase their MHC class II synthetic activity. MHC class II ? and ? polypeptides, together with an invariant chain, are assembled as a complex within the endoplasmic reticulum (Fig. 10.5). Vesicles bud off from the endoplasmic reticulum to fuse with the peptide-containing, acidic phagolysosomes.

The invariant chain disintegrates in the acidic environment of the newly formed vesicle, allowing phagolysosome-derived peptides to occupy the peptide-binding groove of the MHC class II molecule. The pMHC class II complex is transported to the cell surface for display and possible recognition by CD4+ T cells. MHC class II molecules make no distinction between peptides of self and nonself origin. Self antigens displayed on the phagocyte surface usually go unrecognized because most self-reactive CD4+ T cells have been eliminated during development.

Figure 10.5.Presentation of extracellular antigens.

Presentation of extracellular antigens.

Antigens of extracellular origin (left side of diagram) or of self origin (right side) are degraded within phagolysosomes. MHC class II ?? heterodimers together within variant chain are assembled within the endoplasmic reticulum. Vesicles containing MHC class II + invariant chain bud off from the endoplasmic reticulum to fuse with peptide-rich vesicles that bud off from the phagolysosome. The acidic environment of the fused vesicle causes the invariant chain to disintegrate, allowing peptides to occupy the peptide-binding groove of the MHC class II molecule. Invariant chain-lacking MHC class II molecules that do not bind a peptide disintegrate in the acidic environment of the vesicle. The exocytotic vesicle containing pMHC class II fuses with the cell’s plasma membrane, and the pMHC class II molecules are displayed on the cell surface for recognition by TCRs of CD4+ T cells.

B. Presentation by MHC class I

Not all antigens enter cells by phagocytosis or macropinocytosis. Some pathogens avoid phagocytes and endocytic vesicles entirely. Intracellular microbes and viruses bind to cell membranes and directly enter the cytoplasm of the host cell (Fig. 10.6). These pathogens are processed differently.

Figure 10.6.Presentation of intracellular or cytoplasmic antigens.

Presentation of intracellular or cytoplasmic antigens.

Cytoplasmic proteins of both self and nonself origin may be marked for destruction by the covalent attachment of ubiquitin, which targets them for proteolytic degradation by the proteasome enzyme complex. Proteasome-generated peptide fragments within the cytoplasm are transported into the endoplasmic reticulum by gatekeeper TAP-1 and TAP-2 heterodimers. Calnexin, a chaperone molecule, binds to newly synthesized MHC class I molecules to allow ?2 microglobulin to form a MHC class I:?2 complex. Calnexin is replaced by another chaperone molecule, calreticulin. A third chaperone molecule, tapasin, associated with the TAP heterodimer, assists possible loading into the peptide into the MHC class I:?2 complex. The MHC class I:?2 complex rapidly disintegrates if a suitable peptide is not loaded. Exocytotic vesicles containing the newly formed pMHC class I complexes bud off from the endoplasmic reticulum and are transported for display on the cell surface by CD8+ T cells with the appropriate TCR.

Nucleated cells normally degrade and recycle cytoplasmic proteins. Both self and nonself cytoplasmic proteins targeted for destruction are covalently tagged with ubiquitin, a highly conserved 76-amino-acid protein. The selection mechanisms for protein ubiquination are not known. Binding of one or more ubiquitin molecules to a protein selects it for destruction by the proteasome, a large proteolytic enzyme complex within the cytoplasm.

Proteasome-generated peptides of 6 to 24 amino acids are transported to the endoplasmic reticulum by the transporter associated with antigen processing (TAP-1 and TAP-2). The TAP heterodimer allows peptides to load into MHC class I (pMHC class I). These then move to the Golgi. Special transport exocytic vesicles containing pMHC class I bud from the Golgi and are rapidly transported to the cell surface for display and recognition by the appropriate CD8+ T cells.

MHC class I molecules make no distinction between peptides of self and nonself origin. However, self peptides displayed on the phagocyte surface usually go unrecognized because CD8+ T cells that are potentially reactive to self peptides are removed during thymic selection.


MHC restriction

A useful memory device to remember CD4/CD8 MHC restriction is the “Rule of eight:”

1

2

T cells of the ?? lineage often express neither CD4 nor CD8, and their restriction is unclear.

Intracellular pathogens

I just read that the TCRs of CD4+ T cells recognize pMHC class II complexes of exogenous origin. How can a peptide derived from an intracellular pathogen that circumvents phagolysosome vesicles load into a class II molecule?

To avoid detection by the adaptive immune system, some pathogens employ a “stealth mechanism” by circumventing phagolysosome vesicles altogether. Others may enter the cell in phagosomes but are able to leave them and enter the cytoplasm. But their ruse is not perfect, as some infected cells die, prompting dendritic cells to take up dead cells and cellular debris by either phagocytosis or macropinocytosis. The proteolytic peptides are then displayed in class II molecules. Mystery solved.


T Cell Activation

T cells largely direct the adaptive immune response. Unlike innate immune system receptors and BCRs, TCRs cannot recognize soluble molecules. T cells recognize only peptides presented by MHC class I or class II molecules that are displayed by antigen-presenting cells (APC). The nature of the adaptive immune response is strongly influenced by how epitopes are presented by APCs. The interface between APC and a previously unactivated (nai?ve) T cell is called the immunologic synapse.

A. Immunologic synapse

The immunologic synapse is initiated by TCR recognition of pMHC (Fig. 10.7). The weak interaction of TCR with pMHC is stabilized by the interaction with CD4 or CD8 molecules that bind to the “constant” nonpeptide-binding portions of pMHC class II and class I, respectively. Formation of the pMHC:TCR:CD (4 or 8) complex provides a first signal though the TCR-associated CD3 complex to the T cell.

This first signal is necessary but not sufficient to stimulate a nai?ve T cell to proliferate and differentiate. A second signal (or more properly, a group of signals) provided by one or more costimulatory molecules is also required for T cell activation. The first and second signals initiate intracellular signaling cascades activating one or more transcription factors leading to specific gene transcription. Without costimulation, T cells either become selectively unresponsive, a condition known as anergy or undergo apoptosis.

Figure 10.7.Immunologic synapse.

Immunologic synapse.

Extracellular antigens are displayed (presented) by MHC class II molecules by APC. TCRs of circulating CD4+ T cells that recognize peptide and MHC class II (pMHC class II) form a weak bond that is stabilized by the noncovalent interaction of the T cell’s CD4 molecule with the nonpeptide-binding portion of MHC class II. Inset. Adhesion molecules expressed by T cells (leukocyte function antigen-, LFA-1, or CD11a/CD18) interact with ICAM-1 (immune cell adhesion molecule-1 or CD54) on APC. LFA-1:ICAM-1 complexes move away from the pMHC:TCR:CD4 complex. At the same time, CD2:LFA-3 (CD2:CD58) and costimulatory complexes (e.g., CD28:CD80/86) move toward the pMHC:TCR:CD4 complex.

B. T cell signal transduction

The immunologic synapse stabilizes T cell-APC interaction and promotes the migration of adhesion molecules within the T cell membrane. Cytoplasmic tails of some of these molecules contain immunoreceptor tyrosine–based activation motifs (ITAMs) that initiate a signaling cascade when brought into close proximity (Fig. 10.8). The cytoplasmic tails of CD3 complex molecules (CD3?, ?, ?, and CD247 ?) bear ITAMs.

In contrast, the cytoplasmic tails of the TCR lack ITAMs. The first signal for T cell activation is provided by the signals transduced following TCR engagement of a peptide presented by pMHC II and tyrosine phosphorylation of ITAMs on the cytoplasmic tails of CD3 complex molecules (Fig. 10.9). Costimulatory molecules provide the second signal for T cell activation (Fig. 10.10).

Figure 10.8.Immunoreceptor activation motifs (ITAMs).

Immunoreceptor activation motifs (ITAMs).

Ligand engagement leads to polypeptide dimerization, activation of tyrosine kinases, and the phosphorylation of tyrosine residues within specialized intracellular portions of receptor or accessory polypeptides. These ITAMs contain four amino acids (indicated as two Xs flanked by tyrosine (Tyr) and lysine (Lys). Multiple ITAMs are located at 10- to 12-amino acid intervals along the cytoplasmic tail.

Figure 10.9.Details of T cell “first signal” transduction.

Details of T cell “first signal” transduction.

1. The T-cell receptor (TCR) engages a peptide presented by MHC class II (pMHC II). 2. CD4 stabilizes this complex by binding noncovalently to the nonpeptide-binding region of MHC class II, causing (3) the LCK tyrosine kinase to phosphorylate immunoreceptor activation motifs (ITAMs) on the cytoplasmic tails of CD3 complex molecules (CD3 ?, ?, and ? and the CD247 ?-? homodimer). 4. ZAP-70 tyrosine kinase “docks” on the phosphorylated ITAMs and phosphorylates the remaining CD247 ?-? ITAMs and phosphorylates and activates phospholipase C-? (PLC-?). 5. PLC-? cleaves phosphatidyl inositol 4,5-bi(s) phosphate (PIP2) into diacylglycerol (DAG) and inositoltri(s) phosphate (IP3). 6. IP3 promotes release of calcium from intracellular stores, and calcium together with DAG activates protein kinase C (PKC) and the protein phosphatase, calcineurin. 7. PKC phosphorylates I?B (inhibitor of nuclear factor kappa B, NF?B) causing the inhibitor to dissociate from NF?B. Likewise, calcineur in dephosphorylates and activates nuclear factor of activated T cells (NFAT). Both transcription factors (NF?B and NFAT) migrate to the nucleus, where they activate genes. 8. ZAP-70 also phosphorylates the linker of activation for T cells (LAT), which activates the guanine nucleotide exchange factors (GEFs), ras and rac. 9. Ras and rac initiate phosphorylation cascades (see Figs. 6.10 and 6.11) to activate the AP-1 family of transcription factors.

Figure 10.10.The second signal: Costimulation.

The second signal

A second signal (costimulation) is required for T cell activation. Upon formation of the immunologic synapse (see Fig. 10.9), the common leukocyte antigen (CD45) dephosphorylates and activates Fyn kinase. Both CD28 (costimulatory molecule) and Fyn associate with inositol tri(s)phosphate kinase (IP3K) to activate Ras and initiate a phosphorylation/activation cascade (see Figs. 6.10 and 6.11).