Question

Study Guide: Topic 10 Functions of Effector T cells:

1)   Explain why classical activation of macrophages by T_H1 cells is needed in order to clear out some pathogens.

2)   Describe how perforin and granzymes released by CTLs activate apoptosis in target cells. Be detailed and specific in your response.

Answer 1

1) The production of cytokines and membrane-associated molecules by armed CD4 T_H1 cells requires new RNA and protein synthesis.

Within minutes of the recognition of specific antigen by armed effector cytotoxic CD8 T cells, directed exocytosis of preformed perforins and granzymes programs the target cell to die via apoptosis. In contrast, when armed T_H1 cells encounter their specific ligand, they must synthesize de novo the cytokines and cell-surface molecules that mediate their effects. This process requires hours rather than minutes, so T_H1 cells must adhere to their target cells for far longer than cytotoxic T cells.
Recognition of its target by a T_H1 cell rapidly induces transcription of cytokine genes and new protein synthesis begins within an hour of receptor triggering. The newly synthesized cytokines are then delivered directly through micro-vesicles of the constitutive secretory pathway to the site of contact between the T-cell membrane and the macrophage. It is thought that the newly synthesized cell-surface CD40 ligand is also expressed in this polarized fashion. This means that, although all macrophages have receptors for IFN-γ, the macrophage actually displaying antigen to the armed T_H1 cell is far more likely to become activated by it than are neighboring uninfected macrophages.

Activation of macrophages by armed T_H1 cells promotes microbial killing and must be tightly regulated to avoid tissue damage.

T_H1 cells activate infected macrophages through cell contact and the focal secretion of IFN-γ. This generates a series of biochemical responses that converts the macrophage into a potent antimicrobial effector cell. Activated macrophages fuse their lysosomes more efficiently to phagosomes, exposing intracellular or recently ingested extracellular microbes to a variety of microbicidal lysosomal enzymes. Activated macrophages also make oxygen radicals and nitric oxide (NO), both of which have potent antimicrobial activity, as well as synthesizing antimicrobial peptides and proteases that can be released to attack extracellular parasites.
2) Both perforin and granzymes are required for effective cell killing. The separate roles of perforin and granzymes have been investigated in a cell system that relies upon similarities between the lytic granules of T cells and the granules of mast cells. Release of mast cell granules occurs on cross-linking of the Fcε receptor, just as release of lytic granules from CD8 T cells occurs on cross-linking of the T-cell receptor. The mechanism of signaling for granule release is thought to be the same or similar in both cases, as both the Fcε receptor and the T-cell receptor have ITAM motifs in their cytoplasmic domains, and cross-linking leads to tyrosine phosphorylation of the ITAMs.

When a mast-cell line is transfected with the gene for perforin or for granzyme, the gene products are stored in mast cell granules, and when the cell is activated through its Fcε receptor, these granules are released. When transfected with the gene for perforin alone, mast cells can kill other cells, but large numbers of the transfected cells are needed as the killing is not very efficient. By contrast, mast cells transfected with the gene for granzyme B alone are unable to kill other cells. However, when perforin-transfected mast cells are also transfected with the gene encoding granzyme B, the cells or their purified granules become as effective at killing targets as granules from cytotoxic cells, and granules from both types of cell induce DNA fragmentation. This suggests that perforin makes pores through which the granzymes can move into the target cell.

The granzymes are proteases, so although they have a role in triggering apoptosis in the target cell, they cannot act directly to fragment the DNA. Rather, they must activate an enzyme, or more probably an enzyme cascade, in the target cell. Granzyme B can cleave the ubiquitous cellular enzyme CPP-32, which is believed to have a key role in programmed cell death in all cells. CPP-32 is a caspase and activates a nuclease, called caspase-activated deoxyribonuclease or CAD, by cleaving an inhibitory protein (ICAD) that binds to and inactivates CAD. This enzyme is believed to be the final effector of DNA degradation in apoptosis.

Cells undergoing programmed cell death are rapidly ingested by nearby phagocytic cells. The phagocytes recognize some change in the cell membrane, most probably the exposure of phosphatidylserine, which is normally found only in the inner leaflet of the membrane. The ingested cell is then completely broken down and digested by the phagocyte without the induction of co-stimulatory proteins. Thus, apoptosis is normally an immunologically ‘quiet’ process; that is, apoptotic cells do not normally contribute to or stimulate immune responses.

The importance of perforin in this process is well illustrated in mice that have had their perforin gene knocked out. Such mice are severely defective in their ability to mount a cytotoxic T-cell response to many but not all viruses, whereas mice that are defective in the granzyme B gene have a less profound defect, probably because there are several genes coding for granzymes.