Cell Mediated Immunity

EFFECTOR T CELLS
Once a T cell has differentiated into an armed effector cell, further encounter with its
specific Ag results in immune attack without the need for co-stimulation.
This change in requirement applies to both CD8 and CD4 T cells. Its importance is easy
to demonstrate in the case of CD8 T cytotoxic cells which must be able to act on any cell
infected with a virus regardless of whether or not that cell can express co stimulatory
molecules. Effector CD4 cells can also interact with B cells and MOs that have taken up
Ag but are not expressing enough co stimulatory activity. In this way any cell displaying
appropriate Ag can trigger an appropriate T cell response.
In addition to reduced stringency of their activation requirements, all armed
effector T cells have an increased sensitivity to activation. This is due to increased
numbers of adhesion molecules on their surface (especially LFA-1 and CD2)
which allows more effective adhesion to target cells.
Naive CD8 T cells emerging from the thymus are already predestined to become
cytotoxic T cells even though they are not yet differentiated into effector cells. However,
CD4 T cells can become either inflammatory TH1 cells or TH2 cells and the final
decision on which fate the cell will follow is made during its first encounter with Ag. As
CD4 T cells differentiate, they are thought to go through an intermediate stage known as
Th0. Th0 cells express some differentiated effector functions of both the inflammatory
and helper T cells.
The factors that determine whether a proliferating CD4 T cell will differentiate into an
inflammatory T cell or a helper T cell are not fully understood. Cytokines elicited by
infection especially IL12 and IL4, the accessory molecules used to drive the response,
and the nature of the peptide:MHC ligand all have an influence.
The difference of the outcome of infection depending on which Th cell is activated can
be seen in leprosy.
*Remember- Th1 cells lead to CMI and Th2 cells provide humoral immunity
Mycobacterium leprae causes leprosy and the organism grows in MO vesicles. Effective
host defense requires MO activation by inflammatory (Th1) T cells.
In patients with tuberculoid leprosy in which inflammatory T cells are
preferentially induced, few live bacteria are found, little Ab is produced, and
although skin and peripheral nerves are damaged by the inflammatory responses
associated with MO activation, the disease progresses slowly and the patient
usually survives.

However, when T helper 2 cells are preferentially induced, the main response is
humoral, the Abs cannot reach the intracellular bacteria, and the patients develop
lepromatous leprosy in which the organism grows abundantly in MOs causing
gross tissue destruction which is eventually fatal.
CD8 T cells
Naive CD8 T cells can differentiate only into cytotoxic cells, and perhaps because the
effector actions of these cells are so destructive they require higher levels of co
stimulatory activity to activate them as compared to naive CD4 T cells.
This requirement can be met in two ways
1) they can be activated by APC such as dendritic cells which express high levels of co
stimulatory activity and
2) these APC can directly stimulate CD8 T cells to synthesize the IL2 that drives their
own proliferation and differentiation.
Moreover some Cytotoxic T cell responses seem to require the presence of CD4 T cells
during the priming of the naive CD8 T cell.
In these responses, both the CD8 and CD4 T cell must recognize Ag on the
surface of the same APC.
It is thought that the actions of the CD4 cell may be necessary to compensate for
costimulation by the APC which can not directly activate the CD8 cell. This
compensation can occur in one of two ways:
The CD4 could be an effector T cell which in turn activates the APC to express
higher levels of costimulatory activity and thus stimulate the CD8 cell.
The CD4 cell could be a naive or memory cell which the APC stimulates to begin
IL2 production which is available to the CD8 cell which needs IL2 to drive its
proliferation and differentiation.
T cell mediated cytotoxicity
All viruses and some bacteria multiply in the cytoplasm of infected cells. Once inside
cells, these pathogens are not accessible to Abs and can be eliminated only by the
destruction of the infected cells on which they depend.
The critical role of T cytotoxic cells in limiting such infections is seen in the increased
susceptibility of animals artificially depleted of these T cells or in individuals who have a
genetic defect and so lack MHC class I molecules.

Cells can die in two ways:

necrosis due to membrane damage such as is seen with Ab and C' which leads to
cell disintegration. The dead or necrotic tissue is taken up and degraded by
phagocytic cells which eventually clear the damaged tissue and heal the wound.
programmed cell death or apoptosis is a normal cellular response that is crucial
during normal growth ie during thymocyte selection. The first changes seen in
apoptotic cell death are fragmentation of the DNA, disruption of the nucleus, and
alterations in cell morphology. The cell then destroys itself from within shrinking
and degrading itself until little is left.
There is evidence that T cytotoxic cells kill their targets largely by programming them to
undergo apoptosis. When cytotoxic T cells are mixed with target cells in vitro they can
program specific target cells to die within 5 minutes- although death may take hours for
completion.
The short period required by T cytotoxic cells to program their targets to die is
believed to reflect the release of preformed effector molecules by the T cell which
activates an endogenous apoptotic pathway within the target cell.
The apoptotic mechanism may also act directly on cytosolic pathogens. The
nucleases that are activated in apoptosis to destroy cellular DNA can also degrade
viral DNA preventing the assembly of virions and thus preventing release of virus
to infect nearby cells. Other enzymes activated during apoptosis may destroy
other cytosolic pathogens.
Apoptosis is therefore preferable to necrosis as a means of killing cells because in
necrosis intact pathogens are often released.
The principal mechanism through which cytotoxic T cells act is by the release of
secretory granules upon recognition of Ag on the surface of a target cell. These secretory
granules contain at least two distinct classes of cytotoxins that are expressed selectively
in T cytotoxic cells.
Perforins-these molecules can polymerize to generate transmembrane pores in
cell membranes.
The other class comprises at least three proteases called granzymes or
fragmentins which belong to the serine protease family
The release of granules from Tc cells occurs almost immediately after receptor
ligation and requires no new protein or RNA synthesis.
This allows cytotoxic T cells to act very rapidly on their targets programming
them for eventual lysis in a matter of minutes. Polymers of perforin polymerize on

the cell surface and form a cylindrical structure that is lipophilic on the outside
and hydrophilic down a hollow center. This structure can insert into lipid bilayer
forming a pore. *Perforin exhibits some sequence homology with C9.
This pore allows the entry of fragmentins into the target cell which are responsible for
inducing apoptosis. *The fragmentins are proteases and cannot therefore be directly
responsible for the fragmentation of DNA. They are thought to act by inducing the cells
endogenous apoptotic program perhaps by mimicking the action of an endogenous
protease.
Granzyme B is closely related to IL1b converting enzyme (ICE) and ICE is an
endogenous protease that initiates apoptosis by cleaving an unidentified substrate
leading to DNA degradation
Granzyme B either activates ICE or acts in its place
The release of granule contents accounts for most of the cytotoxic activity of CD8 T cells
but granule mediated killing is strictly Ca++ dependent ( Ca++ induces a
conformational change in perforin allowing it to insert into the membrane). Yet some
cytotoxic action of CD8 T cells survives Ca++ depletion. Also some CD4 cells are also
capable of killing other cells yet do not contain granules (no perforins or fragmentins). So
there must be a 2nd independent pathway of cytotoxicity.
The 2nd mechanism is believed to involve the activation of the Fas receptor in the target
cell membrane. The Fas receptor (also called APO-1) is known to induce apoptosis when
activated by Abs against it. Fas receptor ligation also transmits a signal which induces
ICE activation leading to DNA degradation/apoptosis.
CTLs express a cell surface protein with sequence homology to TNF that binds to
FasR a Fas Ligand- which belongs to the TNF family of cytokine receptors, this
induces a signal in the target cell that activates the endogenous apoptotic pathway.
Fas ligand only expressed on effector T cells with CTLs >>>TH1 but the Fas
ligand is what accounts for reports of CD4 Tcell cytotoxicity.
When cytotoxic T cells are offered equal mixtures of 2 target cells-one bearing specific
Ag and the other not- they kill only the target cell with Ag. The innocent bystander cell
and the T cell are not killed. This is surprising considering that effector molecules
released by cytotoxic T cells lack any specificity for Ag. The explanation lies in the
highly polar release of the effector molecules.
Cytotoxic T cells orient their secretory apparatus to focus it on the point of
contact with a target cell. (Storage granules reorient within the cytoplasm of the
CTL becoming concentrated near the junction of the target cell).

The narrowly focused action of the CD8 T cells allows them to kill
single infected cells in a tissue without creating widespread tissue
damage and is of critical importance in tissues where cell
regeneration does not occur such as neurons in the CNS.
Ligation of the Tcell receptor similarly induces de novo synthesis
of perforin and granzymes so that the supply of lytic granules is
replenished. This makes it possible for a single T cell to kill many
targets in succession.
Although the secretion of perforin and fragmentins is known to be a major mechanism by
which CD8 T cells eliminate infection, with expression of Fas playing a lesser role, most
cytotoxic CD8 T cells also release IFNg and TNFa which contribute to host defense in
several other ways.
IFN g directly inhibits viral replication and also induces increased expression of
MHC class I thus increasing the chance that infected cells will be recognized as
target cells for cytotoxic attack.
IFNg also activates MOs recruiting them to sites of infection both as effector cells
and as APCs. The activation of MOs by IFNg is a critical component of the host
immune response to intracellular protozoan pathogens. IFNg has a secondary role
in reducing the trytophan concentration within treated cells and thus can kill
intracellular parasites by starvation.
TNF is produced by CD8 T cells and they can synergize with IFNg in killing
some target cells by a cytokine mediated pathway as well as in MO activation.
Overall we can say:
CD8 T cells act in a variety of ways to limit the spread of cytosolic pathogens.
CTL killing is Ag specific
CTL killing requires cell contact
CTL are not themselves injured because
they have a membrane protein "protectin" that inactivates perforin either
by preventing its insertion into the CTL membrane or by preventing its
polymerization there. (No protein has been isolated however)
perforin is not released in a soluble form but rather is released within
small membrane bound vesicle. Once vesicles bind to the specific target
cell, perforin is released and forms pores as described. (Vesicles seen by
EM).

CTLs can interact with another target cell