Cell-mediated Cytotoxicity Dr. Diebel 5/14/14

Question 1

Mediators of cell mediated immunity

Answer 1

Antigen non-specific effector cells
-NK cells. Produce cytotoxins (performs and granzymes), IFN-gamma, TNF, Fas ligand (FASL). Mechanism of killing is cytotoxic granule release and FASL-FAS interactions. NK cells bind NK receptor and tonic activating ligand and recognize lack of MHC I or NK receptor/tonic activating ligand and stress-induced activating ligand.

Antigen specific effector cells
CD8+ cells (cytotoxic T cells, T cells, CTLs, TC cells). Produce cytotoxins (performs and granzymes), IFN-gamma, TNF, Fas ligand (FASL). Mechanism of killing is cytotoxic granule release and FASL-FAS interactions. CTL TCR and MHC I bind along w/ CD8 mediating cytotoxicity.

Question 2

CD8+ T cells (CTLs)

Answer 2

Naive T cells are called CTL precursors (CTL-P) to indicate their immature state.

A CTL-P matures after being activated by an interaction w/ a Th1 CD4+ T cell

Maturation required three sequential signals

1. Antigen-specific signal transmitted by TCR upon recognition of the proper peptide:MHC I complex present on an APC
2. Co-stimulatory signal is transmitted by CD28:B7 interaction between CTL-P cell and licensed APC
3. Signal induced by the IL-2 (needed for differentiation) secreted by a Th1 CD4+ T cell results in the proliferation and differentiation of the antigen-activated CTL-P cell to a fully active CTL

Question 3

Naive vs. Mature CTL

Answer 3

Naive
Does not express IL-2 or IL-2R only occurs after activation
Expresses CD45RA
Express low levels of cell adhesion molecules CD2 and LFA-1
No Cytotoxic activity

Mature
Expresses high affinity IL-2R and requires high level of IL-2 to proliferate
Synthesizes low levels of IL-2
Expresses CD45RO
Expresses High levels of the adhesion molecules CD2 and LFA-1
Exhibits cytotoxicity

Memory
May not require CD4 help to reactivate. Requires only low levels of IL-2 which can be produced by the activated CTLs)

Question 4

CTL killing: Binding of the target cell

Answer 4

TCR-CD3 complex on the CTL recognizes the peptide:MHC class I complex on the target cell

LFA-1 on the CTL binds to ICAMs on the target cell

Antigen activation converts the LFA-1 from a low affinity state to a high affinity state for better binding. After about 5-10 minutes the LFA-1 returns to a low-affinity state, resulting in the dissociation of the CTL from the target cell.

Question 5

CTL killing mechanisms

Answer 5

1. Perforin and granzyme secretions
   - perforin forms a pore on target cell membranes
   - granzyme molecules activate apoptosis by cleavage of caspases
2. Fas ligand protein on the cell membrane surface
   - Membrane bound FasL binds to Fas on the membrane of the target cells and initiates killing
   - Activates apoptosis by cleavage of caspases
3. CTLs can also kill by TNF production and secretion

Question 6

CTL Killing: Pathways to apoptosis

Answer 6

Granzyme is a serene protease that is specific to apoptosi.

Granzyme B can go to caspase 3 OR Bid–>mitochondria–>cytochrome C.

FASL–>FASL–>FDD–>(cleaved) caspase 8–>Bid–> mitchondria–>cytochrome C

Question 7

NK cells

Answer 7

Play a major role in killing virus-infected cells, intracellular pathogen-infected cells, and tumor cells.

NK cells produce a number of important cytokines including IFN-gamma.

• IFN-gamma tilts the immune response toward Th1 cells by inhibiting Th2 cells and inducing IL-12 production by macrophages and DC.
• IFN-gamma can activate macrophages (M1) and NK Cells

NK cell activity is stimulated by IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and IL-25

Question 8

NK cells and early defense against viruses

Answer 8

Type 1 interferon through TLR3–>IRF3(NF-kB)–>IFN-beta

TLR7/8–>IFN-alpha–IRF7(NF-kB)–>IFN-alpha

Type 1 interferon will draw in NK cells to hold down the fort and supply signals to get CD8 into the area.

Question 9

NK cells versus CTLs

Answer 9

NK cells express CD16 and FcRIII
NK cells do not need to be educated in the thymus
NK cells do not undergo gene rearrangement of receptor genes (don’t make TCR or CD3)
NK cell killing is not MHC restricted

NK cell killing is similar to CTL killing

• FasL expressed on surface can kill cells expressing Fas
• Perforin and granzyme released from granules
• TNF expressed on the surface and excreted

Question 10

NK cell receptors

Answer 10

NK cells have both activation and inhibitory (higher affinity than activating) receptors.

NK cell receptors fall into two categories.

1. Lectin-like receptors
   - bind proteins rather than polysaccharides
2. Immunoglobulin-like receptors (KIR=killer cell immunoglobulin-like receptor)
   - bind to most MHC I molecules

Question 11

Lectin-like receptors of NK cells

Answer 11

non-inhibitory receptor
-CD94 and NKG2C. When they bind nothing is stopping them.

Inhibitory receptor
-CD94 and NKG2A. ITIM which inhibits down stream activation.

ITAM (CD3, CD79a/b) is a class of activating domains

Question 12

Killer immunoglobulin-like receptors

Answer 12

non-inhibitory receptors

• short
• KIR-2D
• KIR-3D

Inhibitory receptors

• Long
• KIR-2D
• KIR-3D
• ITIM

Question 13

NK Cell receptors and ligands

Answer 13

KIR family (MHC I)

Receptor Ligand
KIR2DL1 HLA-C2

KIR2DS1 HLA-C2 (activating)

KIR3DL1 HLA-Bw4

KIR3DL2 HLA-A3 and A11

KIR2DL2/3 HLA-C1

C-type lectin-like family

CD94-NKG2A HLA-E

Certain NK receptors predispose to certain diseases (e.g. HLA-Bw4 and HIV is not as severe)

Question 14

HLA-E

Answer 14

In the ER sometimes a leader sequence interacts w/ HLA-E which goes onto the surface. Target is NKG2A.

Advantage would be in a cell that is having trouble getting MHC I out.

Question 15

TNF released by?

Answer 15

NK cells, monocytes, and macrophages

Question 16

Perforin released by?

Answer 16

NK cells and esosinophils

Question 17

Granzyme released by?

Answer 17

NK cells

Question 18

Four categories of Ab fnx?

Answer 18

1. Virus and toxin neutralization
2. Opsonizatoin
3. Complement fixation
4. Antibody-dependent cell-mediated cytotoxicity