BIO/MI 494G

Fall 2008
T-cell Mediated Cytotoxicity

I. Introduction
1. Intracellular pathogens, e.g. viruses, certain bacteria
2. CD8 cytotoxic T-lymphocytes (CTL)
3. Activation of naïve CD8 T-cell

II. Mechanism of activation of naïve CD8 T-cell

1. Three means of activation

III. Steps in CTL mediated lysis of target cells

1. Initial binding
2. Conjugated formation
3. Delivery of lethal hit
4. Detachment of CTL
5. Target cell death

IV. Cellular mechanism of killing

1. Apoptosis

V. Polarization of CTL towards target cell

1. Formation of supramolecular activation complex (immunological
synapse)

VI. Biochemical mechanism of CTL induced target cell apoptosis

1. Lytic granules
a. Perforin
b. Granzymes
c. Granulysin
2. Mechanism of action of lytic granules
3. Fas/Fas ligand induced apoptosis
 Antigen Lymphocyte
recognition activation Differentiation

Activation of
macrophages,
B cells, other
cells

Killing of
infected
".""'"'~ "target cells";
macrophage
activation

Memory
CD8+
Teell
Lymphoid organs Peripheral tissues
FIGURE 9-2 Phases of T cell responses. Antigen recognition by T cells induces cytokine (e.g., IL-2) secretion, clonal expansion as a result
of IL-2-induced autocrine cell proliferation, and differentiation of the T cells into effector cells or memory cells. In the effector phase of the
response, the effector C04+ T cells respond to antigen by producing cytokines that have several actions, such as the activation of
macrophages and B lymphocytes, and C08+ CTLs respond by killing other cells. APC, antigen-presenting cell.
Fig. 1.27 Mechanism of host defense
against intracellular infection by
viruses. Cells infected by viruses are
recognized by specialized T cells called
cytotoxic T cells, which kill the infected
cells directly. The killing mechanism
I.

**

involves the activation of enzymes known

as caspases, which contain cysteine in
their active site and cleave after aspartic
acid. These in turn activate a cytosolic

*
nuclease in the infected cell, which
cleaves host and viral DNA. Panel a is a
transmission electron micrograph
showing the plasma membrane of a virus
cultured CHO cell (the Chinese hamster
ovary cell line) infected with influenza
virus. Many virus particles can be seen
budding from the cell surface. Some of infected cell killed infected cell
these have been labeled with a
monoclonal antibody that is specific for a
viral protein and is coupled to gold
particles, which appear as the solid black
dots in the micrograph. Panel b is a
transmission electron micrograph of a
virus-infected cell M surrounded by
cytotoxic T lymphocytes. Note the close
apposition of the membranes of the virus­
infected cell and the T cell (T) in the upper
left corner of the micrograph, and the
clustering of the cytoplasmic organelles in
the T cell between its nucleus and the
point of contact with the infected cell.
Panel a courtesy of M. Sui and A.
Helenius; panel b courtesy of N. Rooney.

Fig. 1.32 Cytotoxic C08 T cells

recognize antigen presented by MHC
class I molecules and kill the cell. The
peptide:MHC class I complex on virus­
infected 6ells is detected by antigen­
specific cytotoxic T cells. Cytotoxic T
cells are preprogrammed to kill the cells
they recognize.
 CDS T cells: CD4 T cells:
peptide + MHC ctass I . peptide + MHC class II

Cytotoxic (killer) T cells

Fig. 8.27 There are three classes of
effector T cell, specialized to deal
with three classes of pathogen. COB
kill
cytotoxic cells (left panels) kill target
cells that display peptide fragments of
cytosolic pathogens, most notably
viruses, bound to MHC class I
molecules at the cell sur1ace. TH1 cells
(middle panels) and TH2 cells (right
panels) both express the CD4 co­
receptor and recognize fragments of
virus·infected antigens degraded within intracellular
cell
macrophage vesicles. displayed at the cell sur1ace by
MHC class II molecules. TH1 cells
;:::=:=~ sz==~ . activate macrophages, enabling them to
destroy intracellular microorganisms
more efficiently. They can also activate
B cells to produce strongly opsonizing
antibodies belonging to certain IgG
subclasses (lgG1 and IgG3 in humans,
and their homologs IgG2a and IgG2b in
the mouse). TH2 cells, in contrast, drive
B cells to differentiate and produce
immunoglobulins of ~II. other types, and
are responsiblefor initiating B-cell
responses by activating naive B cells to
proliferate and secrete IgM. The various
apoptotic cell types of immunoglobulin together make
B lymphoblast up the effector molecules of the humoral
immune response.

T cell

L... R_E_COG_N_IT_10_N ~> IPROLIFERAT~RENTIATION> I E_F_F_EC_T_O_R_F_U_N_CT_ION ~

Fig. 8,22 Armed effector T cells can respond 10 their target differentiation of the T cells to anned effector cell stalus. Once
cells without co-stimulation. A naive T cell that recognizes the cells have differentiated into effector T cells, any encounter
antigen on the surface of an antigen-presenting cell and with specific antigen triggers their effector actions without the
receives the required two signals (arrows 1 and 2, left panel) need (or co-stimulation. Thus, as illustrated here, a cytotoxic
becomes activated, and both secretes and responds to IL-2. T cell can kill targets that express only the pepMe:MHC ligand
IL-2·drlven clonal expansion (center panel) is followed by the and not co-stimulatory signals (right panel).
 'Dendritic eell~'§ip'r.~r,~igh 'levels of B7 ,Apc slimulates effcc1oi'CD4 T eel~ APC'aclivates CD4 T cell 10 make IL-2 and naive
~nd "can' ·~c1lvat~ . .naive. CDS T .ce!ls Whi,h In,fum_elivates ttle Apc , ' CDS T cellle'express It~2 receptors '

• •
• APC

Il-2 seC(ete~byaetivated ~D4,~ cell' ,

':', ", is DOundb(CD,8 T<elf',' ' ,

• • • •

• • •
Figure 6,22 Three ways of activating a naive COB T cell. secreted by the CD4 T cell to improve the co-stimulation of the
The left panels show how a naive COB T cell can be activated antigen-presenting cell, for example by the induction of B7
directly by a virus-infected dendritic cell. The center and right expression (center panels). A second way is for cytokines secreted
panels show two ways in which antigen-presenting cells (APC) by the CD4 T cell, for example It-2, to act directly on a
that offer suboptimal co-stimulation can interact with a CD4 neighboring COB T cell (right panels).
T cell to stimulate a naive COB T cell. One way is for cytokines

Tli~kiiti~1 jrter3.~MA:~~;i~ilw<itt;~·rg;,\' Fig. 8.28 Interactions ofT cells with

their targets Initially Involve
does not carry the specific antigen, the
T cell disengages (second panel) and
.Is made by ~nonspet,l11c '~h!'Slon molecules
.:::. nonspecific adhesion molecules. The can scan other potential targets until it
major initial interaction is between LFA-1 finds the specific antigen (third panel).
on the T cell, illustrated here as a Signaling through the T-cell receptor
cytotoxic CD8 T cell, and ICAM-1 or increases the strength of the adhesive
ICAM-2 on the target cell (top panel). interactions. prolonging the contact
This binding allows the T cell to remain in between the two cells and stimulating
contact with the target cell and to scan its the T cel' to deliver its effector molecules.
surface for the presence of specific The T cell then disengages (bonom
peptide:MHC complexes. If the target cell panel).

Cross­
presentation
Dendritic cell
Infected cells
and viral
antigens picked
... up by host APCs
====:>
, Aniigeri-speelfje ;i-eC~gni~Qn.:,;~blepairing Virus,

ah,d~~~rele"se of ,effector '11)0leeules

infected cell

Figure 5-7 Cross-pres~ntationof antigens to CDS· T cells. . .

Cells infected with intracellular microbes, such as viruses, are captured by profeSSional antigen·
presenting cells (APCs), particularly dendritic cells, and the antigens o( the infectious microbes are
broken down and presented in association with the MHC molecules of the APCs. T cells recognIZe
the microbial antigens and costimulalors expressed on the APCs,. and th~ T cells are activated.
This example shows COB· T cells recognizing class I MHC.... ssoclated antlgem; the same c~oss­
presenting APC may display cia" II MHC .... ssociated anligens (rom the microbe (or recogntliOn
by C04' helper T cells,
CTL recognizes and binds ClL programs target for death, Fig. 8.34 Cytotoxic CD8 T cells can
CTL migrates to new target Target cell dies by apoptosis induce apoptosis in target cells_
vlrus-Infecled cell Inducing D.NA fragmentation
Specific recognition of peptide:MHC
complexes on a target cell (top panels)
by a cytotoxic COB T cell (CTL) leads to
the death of the target cell by apoptosis.
Cytotoxic T cells can recycle to kill
multiple targets. Each killing requires the
same series of steps, including receptor
binding and directed release of cytotoxic
proteins stored in lytic granules. The
process of apoptosis is shown in the
micrographs (bollom panels), where
panel a shows a healthy cell with a
normal nucleus. Early in apoptosis
(panel b) the chroma~n becomes
condensed (red) and, although the cell
sheds membrane vesicles, the integrity
of the cell membrane is retained, in
contrast to the necrotic cell in the upper
part of the same field. In late stages of
apoptosis (panel c), the cell nucleus
(middle cell) is very condensed, no
mitochondria are Visible, and the cell
has lost much of its cytoplasm and
membrane through the shedding of
vesicles. Photographs (x 3500) courtesy
of R. Windsor and E. Hirst.

Figure 6.28 When cytotoxic T cells

recognize specific antigen the
delivery of cytotoxins is aimed
directly at the target cell. As shown
in the panels on the left, initial adhesion
to a target cell has no effect on the
location of the lytic granules (LG) (top
panel). Engagement of the T-cell
receptor causes the T cell to become
polarized: the cortical actin cytoskeleton
at the site of contact reorganizes,
enabling the microtubule-organizing
center (MTOe). the Golgi apparatus
(GA), and the lytic granules to align
towards the target cell (center panel).
Proteins stored in lytic granules are then
directed onto the target cell (bottom
panel). The photomicrograph in panel a
shows an unbound. isolated cytotoxic
T eel/. The microtubules are stained
green and the lytic granules red. Note
how the lytic granules are dispersed
throughout the T cell. Panel b depicts a
cytotoxic T cell bound to a (larger)
.Release of lytic granules at site of cell "'lnla.c;t.
target cell. The lytic granules are now
clustered at the site of cell-eell contact
in the bound T cell. The electron
micrograph in panel c shows the release
of granules from a cytotoxic T cell.
Panels a and b courtesy of G. Griffiths.
Panel c courtesy of E.R. Podack.
 . 'protein ·1(1,';t·~ ; '",.,. ;'\> •...
."
. gralJ!!Jes.Qf :,:: ' ~cti.OJlsC!n'~~et cellr
"cYt~tox~cCT c:lIs , , . .
--
Aids in delivering contents
Periorin of granules into the
cytoplasm of target cell

Serine proteases,
Granzymes which activate apoplosis
once in the cytoplasm
of the target cell

Granulysin :I Has antimicrobial actions

and can Induce apoplosis

Fig. 8.37 Cytotoxic effector proteins

released by cytotoxic T cells.

. Engagement .0ttCR, by petltide:MHC complex

ca¥es'dirette9releas~ o'.. ~rfo~n ~rliL ".
", 9ta~rflJle~cQfTlP'exedWit/) se~l~cJD; .

~ virus-infected cell

·Gra~zYme. isdeiivered i~to t1i~ 'cytOSOI' ~f

-
infected celi and iitrge!S BID a,ndpri>-caspase-3

c
BID pro-easpase-3

. Trunca~eCi B!D ($10) dIsrupts ·r!\itoe.hondrial

. out~r iit.embran·e, ana activatedc~spa.§e·3
.cleaves ICAD, releasing caspasNclivated
,;' . DNfise (C,4D). "

Fig. 8.38 Perforin, granzymes and without apparent pore formation, and the
, .. CAD
serglycin are released from cytotoxic introduced granzymes then act on
granules and deliver granzymes into specific intracellular targets such as the
caspase-3
the cytosol of target cells to induce proteins BID and pro-caspase-3. Either
apoptosis. Recognition of its antigen on a directly or indirectly, the granzymes cause
'. , virus-infected cell by a cytotoxic CD8 the cleavage of BID into truncated BID
Releilse of cytochrolpe c i~to e~o$ol . T cell induces the release of the contents (tBID) and the cleavage of pro-caspas e­3
activates: ap6plosis j and:CAD' induces,
. .,. ONA fragmentat!.011 of its cytotoxic granules in a directed to an active caspase (second panel). tBID
fashion. Perforin and granzymes, acts on mitochondria to release
complexed with the proteoglycan cytochrome c into the cytosol, and

cleaved ICAD ~ ..
•• 4
serglycin, are delivered as a complex to activated caspase-3 targets ICAD to
release caspase-activated DNase (CAD)
~
the membrane of the target cell (top
panel). By an unknown mechanism, (third panel). Cytochrome c in the cytosOl
perforin directs the entry of the granule promotes apoptosis, and CAD fragments
contents into the cytosol of the target cell the DNA (bottom panel).
 Activated caspase 3 cleaves !-CAD,
~. Fasllgand (Fasl) binds \Q Fas. lhe inhibitor 01 CAD, ..nlc!lls
~ .... ~surfaces as released 10 enter the nucleus .nd
cleave OIIA

Ag. 6.24 Binding of Fas ligand 10 Fas initiates the process caspase domain that in tum can activate other caspases. The
of apoplosls. When Fas ligand (FasL) blnds Fas this produces ensuing caspase cascade culminates In the activation of the
a signal which results In adaptor proteins binding to the caspase-activatable DNase (CAD), which Is present in all cells
clustered death domains 0'
the Fas trimer. One of these Is the
protein FADD, which in tUm Interacts through a second death
In an' 'Inactive cytoplasmic form bound to an inhibitory protein
called I-CAD. Wheo I-CAD Is broken down by caspases, CAD
domain with the protease caspase a. Clustered caspase a can can enter the nudeus. where h cleaves DNA into the 200-base­
transaetivate, cleaving caspase 8 itself 10 release an active pair fragments that are characteristic of apoptosis,