Topic 6.1 Accessory Molecules Involved in T-Cell Activation and Co-Stimulation

MODULE 6

Topic 6.1 Accessory Molecules Involved in T-cell Activation and Co-stimulation

T-cell activation (priming) is strictly dependent upon direct interaction and stimulation by antigen presenting cells. Furthermore, the antigen presenting cells must also be appropriately activated before they are capable of stimulating T-cells. APC activation requires stimulation via PRRs /DRRs upon uptake of antigen. At this point it might be helpful to go back and review this concept of pathogen and damage recognition that is covered in module 1. Figure 8.1 illustrates how dendritic cells in the tissues pick up antigens and migrate to local lymph nodes where they can potentially activate T-cells.

As described in module 4, the processing and presentation pathways of antigens depend upon where the antigens originate. Generally, exogenous antigens are processed by the endoctyic pathway and presented on MHCII to Th cells; while endogenous antigens are processed by the cytosolic pathway and presented on MHCI to Tc cells.

However, in order for Tc cells to become activated, and capable of killing, they do first require activation by a professional APC such as a dendritic cell or a macrophage. This creates a particular challenge if the intracellular pathogen does not replicate inside the APC. By a still poorly understood mechanism, APCs have been shown to be capable of presenting such antigens on MHCI even if they gather these antigens from outside sources. This phenomenon is called "cross presentation". See figure 8.3.

The encounter between a naïve T-cell and an activated APC occurs in the secondary lymphoid tissues such as in a lymph node as illustrated in figure 8.5. Note that the APC first picks up the pathogen in inflamed tissue, which leads to activation and migration of the APC to the secondary lymphoid organ. Meanwhile the naïve T-cells are in constant circulation through the blood and the lymphatic vessels. The chance meeting of activated APCs presenting antigens to pathogen specific T-cells causes them to proliferate and differentiate (clonal selection).

The physical interface between the APC and the T-cell, referred to as the immunological synapse, has been highly studied at the molecular level. In addition to the TCR/MHC interaction previously described in module 4, several other cell-cell contacts are required for an effective activation of T-cells. The binding affinity of CD4 or CD8, along with the TCR for the MHC/peptide complex is actually not strong enough to adequately anchor the interaction of a T-cell with an APC. Therefore, there are several adhesion molecule interactions that serve to stabilize this interaction. See figure 8.8 and figure 8.9. In particular, LFA-1 (CD11a/CD18) on the T-cells, binds to ICAM-1 (CD54) on the APC; and CD2 on the T-cell and LFA-3 (CD58) on the APC contribute significantly to this interaction.

Additionally, activation of the T-cell requires co-stimulatory signals from other cell surface protein interactions between the T-cell and the APC. In particular, a T-cell molecule called CD28 interacts with B7 (CD80 or CD86) on the surface of an APC to provide required additional stimulation. CD28 is expressed on the surface of all T-cells, while B7 is expressed only on the surface of previously activated APCs. See figure 8.10 and figure 8.13.

Activated T-cells express a cell surface protein called CTLA-4 (CD152) which also binds to B7. In fact it binds even more tightly to B7 than does CD28. Surprisingly, knock-out mice with deletions in CTLA-4 displayed a lethal hyper proliferation response amongst T-cells, which revealed the role of CTLA-4 as an inhibitor of T-cell activation. Thus T-cells have built in self inhibiting mechanisms to finely regulate their levels of activation. Several new pharmaceutical drugs have been designed to take advantage of these interactions for modulating the immune system in treatments for both autoimmune diseases and cancer, as will be discussed later in modules 9 and 10.

If a T-cell binds to antigen presented by an APC in the absence of co-stimulation, the T-cell becomes anergic and will not undergo clonal proliferation or differentiation. This is an important regulatory feature that contributes to peripheral tolerance which will be considered again in module 9. See figure 8.18.

For a good concise overview of antigen presentation, cross presentation, and the role of CD28 co-stimulation see:

For a more detailed description about the role of co-stimulation and the T-cell response see:

Topic 6.2 T-cell Activation Signaling Pathways

One of the most exciting advances in cellular biology over the past few decades has been our increased understanding of intracellular signaling events that follow cell surface activation. The elucidation of signaling pathways is enabling us not only to understand how a cell interprets and responds to information from the outside, but this detailed knowledge is enabling scientists to develop new and powerful targeted therapeutic drugs. The complexity of signaling pathways is confounding. Biologists are teaming up with computer scientists to help interpret the networks of signaling pathways that cells must balance out in order to respond appropriately to outside stimuli.

Here is a link to a good overview of this concept of cell signaling:

In this section we focus on three well studied signaling pathways that follow from the interaction of Th cells with an APC. The initiating events are illustrated in figure 8.14. The specific binding of TCR to MHC+peptide signals the activation of a kinase, Lck that is associated with CD4. Phosphorylation of ITAMs (immunotyrosine activation motifs) in the cytoplasmic tails of CD3 allows for the docking and activation of another kinase called ZAP70.

This activation cascade ultimately branches out in to three parallel pathways. The textbook outlines these three pathways in figure 8.16. Pivotal to these pathways is the cleavage of a membrane phospholipid called PIP2 into two by-products: DAG, which remains in the membrane, and IP3, which diffuses into the cytosol.

What is the end result of the pathways? All together, the end result of these activation cascades will initially cause the Th cell to do two things (See figure 8.17):

1. Secrete IL-2 - The cytokine IL-2 is an important T-cell growth factor.
2. Proliferate - This is one of the distinguishing aspects of clonal selection that is central to adaptive immunity.

Here is a link to a mini-lecture from the Howard Hughes Medical Institute that focuses on the IP3 signaling pathway of Th cell activation. The movie is accompanied by a transcript and a legend for the cartoon images that appear in the movie:

Topic 6.3 Helper T-cell Subsets and Cytokines

Following clonal proliferation, activated T-cells differentiate further so as to carry out regulatory functions. Th cells carry out their functions by making direct cell-cell contacts and by secreting cytokines. In particular, Th cells influence the action of macrophages, Tc cells and B-cells. Although there is tremendous variation in the characteristics of activated Th cells, there are two distinct differentiation pathways, Th1 and Th2, which have been well studied and identified to occur in vivo. See figure 8.19 and figure 8.27.

1. Th1 cells: In general, Th1 cells secrete the cytokines IL-2 and IFN-γ. IL-2 activates Tc cells, and IFN-g activates macrophages. Thus, Th1 cells favor an overall "cell mediated response" and are most effective for handling intracellular pathogens. See figure 8.21, figure 8.28, figure 8.34, and figure 8.36.
2. Th2 cells: In general, Th2 cells secrete the cytokines IL-4 and IL-5. Th2 cells are particularly significant for the activation of humoral responses, which would enable antibody production for dealing with extracellular pathogens. See figure 8.37. Th2 responses pre-dominate in allergic hypersensitivity reactions as described in module 8.

Note that Th1 and Th2 responses represent extremes. In reality, responses are not so clear cut. A typical immune response will likely lead to activation of Th cells that secrete a spectrum of cytokines that bias the overall response towards Th1 or Th2 characteristics. Although this makes for what might seem like a "sloppy soup" of cytokine mixtures, it is the sloppiness which allows for fine tuning of immune responses within small micro-environments of immune activation. Simple "on-off" switches, while easier to understand, are not as powerful in terms of in vivo regulation. How do Th cells "decide" which differentiation pathway to take? The instructions come in the form of cytokines secreted by the APCs which are involved in their activation. See figure 8.20.

In addition to the well described Th1 and Th2 cell types, Th cells can differentiate into Treg cells that inhibit responses. We will consider these cells again in more detail in topic 6.5. Other sub-types of functional Th cells are also being studied; including Th17 cells which activate inflammatory responses and Tfh cells which contribute to B-cell activation in lymph nodes.

For a nice concise summary of the various Th cell sub-sets see:

Here is a link that provides a table of some key cytokines, the cells types that produce them, and their key effects:

Topic 6.4 Cytotoxic T-cells

The principal effector function of Tc cells is to kill self cells that are infected or diseased. This includes cells infected with viruses, intracellular bacteria or intracellular parasites, and cancer cells.

As discussed previously, Tc cells express CD8 and interact with MHCI / peptide complexes. Naïve Tc cells are incapable of killing, as they do not yet express the "machinery" to carry out target cell destruction. Naïve Tc cells also do not express the IL-2 receptor and thus will not proliferate in the presence of IL-2 unless they are activated first. Naïve Tc cells must be activated by a dendritic cell that has itself been previously activated to express B7. This can occur by at least three possible mechanisms (See figure 8.22):

1. Infected dendritic cell expressing B7 can directly activate the Tc to express the IL-2 receptor and secrete IL-2 to drive its own proliferation (autocrine action).
2. CD4Th cell on the same APC activates the APC to express B7, which subsequently expresses IL-2 receptor and IL-2.
3. CD4Th cell on the same APC as above, but the Th cell serves as the source of IL-2 (paracrine action).

Following activation, Tc cells proliferate and differentiate into active killers. See figure 8.23. The activated Tc cells exit the secondary lymphoid tissue and move through the peripheral circulation in search of infected cells. Infected cells display pathogen specific antigens on MHCI molecules displayed on their surface. See figure 8.30.

Killing can occur by two distinct mechanisms: the perforin pathway or the FAS pathway.

1. Perforin pathway: Active Tc killer cells carry cytoplasmic vesicles called lytic granules which are loaded up with two key types of proteins: perforin and granzymes. Upon binding to a target cell, the Tc cell releases granule contents in the direction of the target cell. Perforin forms pores on the surface of the target cell and the granzymes enter through these pores into the target cell cytoplasm. The granzymes activate the apoptosis pathway which causes the target cell to undergo programmed cell death. See figure 8.29, figure 8.31 and figure 8.32.
2. FAS pathway: Activated Tc cells express a cell surface molecule called Fas ligand (CD178) which binds to a cell surface molecule called Fas (CD95). Fas is present on the surface of most cells of the body. The binding of Fas ligand on the Tc cell to Fas on the target cell also induces the target cell to undergo apoptosis.

Here is a flash animation of the perforin pathway:

Here is a flash animation of the FAS pathway:

Here is an animation of Tc induced apoptosis of a target cell:

Topic 6.5 Regulatory T-cells

A truly exciting segment of immunology research is focused on our increasing understanding of a population of CD4 T-cells whose function is to suppress immune responses. These cells are called regulatory T-cells, Treg.

For many decades immunologists hypothesized the existence of this specialized class of inhibitory cells. Due to shortcomings in the ability to convincingly distinguish them from Th or Tc cells, belief in the existence of a specialized class of suppressor T-cells fell out of favor. We now know for certain that they do exist and in fact play an incredibly important role in modulating many immune responses.

Treg cells are very similar to Th cells in that they also express CD4. Treg cells also characteristically express CD25, which is simply a component of the IL-2 receptor. Alone, these two cell surface markers are not particularly distinctive as compared to regular effector T-cells. However, only Treg cells express a particular transcription factor called FoxP3. Significantly, a rare human autoimmune disease called IPEX results from a defect in the FoxP3 gene. This disease is characterized by a multi-organ autoimmune attack, particularly to the gut, the pancreas, and the skin; thus underscoring the important role of Treg cells in modulating immune responses against self tissues.

Treg cells assert suppression particularly by counteracting the activation of Th cells. See figure 7.19.

How they do this is still a subject of intense research. Several possibilities include: outcompeting for binding to APCs, inactivation of APCs, and secretion of inhibitory cytokines such as IL-10 and TGF-β.

Treg cells arise by at least two distinct mechanisms:

1. During development in the thymus as a lineage distinct from CD4 Th and CD8 Tc cells.
2. In the periphery under the influence of the cytokine TGF-β. See figure 8.20.

At this point I highly recommend that you read the supplementary Scientific American article called Peacekeepers of the Immune System, which presents an excellent overview of regulatory T-cells.

For an excellent animation about Treg cells see:

Topic 6.6 B-cell Activation

We finish this module by looking at how B-cells are activated to become antibody producing plasma cells. We begin with the signal transduction events that follow recognition of antigen, and then examine the role that Th cells play in promoting B-cell activation and differentiation.

As you become familiar with the B-cell signaling events take note of how similar the pathways are to those described in topic 6.2 for T-cells. Initiating events of B-cell activation are illustrated in figure 9.1 and figure 9.2. Cross-linking of surface antibody molecules, referred to as the B-cell receptor (BCR), bring about activation of three key receptor associated kinases called Blk, Fyn and Lyn. These kinases then phosphorylate ITAMs on the cytoplasmic tails of the Igα/Igβ molecules. The phosphorylated tails now provide a suitable docking site for another kinase called Syk. Activation of Syk ultimately results in a signaling cascade that branches out into three parallel signaling pathways as previously described for T-cells! See figure 9.20.

Like T-cells, B-cells require secondary co-stimulatory signals to become fully activated. For most antigens, this second co-stimulatory signal is provided by helper T-cells; and these are referred to as T dependent antigens (TD). Some antigens do not require T-cell help in order to activate B-cells; and these are referred to as T independent antigens (TI). We will consider activation by T independent antigens first, and then conclude with T dependent antigens.

B-cell activation in response to T-cell independent antigens involves additional signaling through the B-cell co-receptor (co-BCR). See figure 9.3, figure 9.4 and figure 9.5. The co-receptor binds to a complement protein that is on the surface of the targeted pathogen.

There are two types of T independent antigens: TI-1 and TI-2.

• TI-1: In addition to engaging the BCR and the co-BCR, TI-1 antigens also activate B-cells through PRRs (pathogen recognition receptors) such as the TLRs. See figure 9.6.
• TI-2: The TI-2 antigens bring about B-cell activation by extensive cross-linking of BCRs and co-BCRs as shown in figure 9.7. This ability to extensively cross link is possible for an antigen that has a highly repetitious epitope such as in the polysaccharide cell wall of the bacterial pathogen, Steptococcus pneumoniae.

Here is a link to another animation illustrating T-cell independent (TI-2) activation of B-cells (remember to turn on your sound!):

As stated above, most B-cell antigens are dependent on T-cell help. In fact, there are many shortcomings to B-cell activation by the T independent mechanisms just described. First of all, the B-cells will not give rise to memory cells, and thus there are no secondary immune responses to T independent antigens. Second, the responses will always yield IgM antibodies, as the B-cells will not be induced to switch isotypes, and there will be no affinity maturation. Therefore, a "full" B-cell response that results in memory, isotype switching and affinity maturation requires help from T-cells.

Let us turn now to the cooperation between Th cells and B-cells in response to T dependent antigens. The response is initiated in secondary lymphoid tissue such as in a lymph node as illustrated in figure 9.8. Here, Th cells which have first been activated by APCs can subsequently interact with B-cells whose BCRs have bound to an antigen.

How does the T-cell "know" which B-cell to activate? This is accomplished because the B-cell can act as an APC and presents peptide antigens to a Th cell. Subsequently the T-cell delivers co-stimulatory signals through direct cell-cell contact and by cytokine secretion. This is described step by step below. See also figure 8.38, and figure 9.10.

• BCR binds to antigen.
• B-cell internalizes BCR + antigen and processes the antigen along the endocytic pathway for presentation on MHCII.
• Th cell binds to peptide loaded MHCII. Note here that the T-cell epitope is not necessarily, and in fact unlikely to be the same as that which is recognized by the BCR. The T-cell epitope can be derived from any portion of the antigen that had been bound by the surface antibody receptor.
• Co-stimulation occurs in both directions via B7 on the B-cell and CD28 on the T-cell; and via a molecule called CD40 on the B-cell and CD40L (CD154) on the T-cell.
• The T-cell secretes cytokines that further promote the proliferation and differentiation of B-cells into antibody secreting plasma cells. See figure 8.37.

Here are two animations illustrating these steps to T-cell dependent activation of B-cells. The first animation shows BCR + antigen internalization and processing by MHCII; and the second one shows B-cell / T helper cell interaction.