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Ag Processing and Presentation
Ag pulsed onto macrophages is much more immunogenic than Ag alone
Response to L. monocytogenes studied in mice:
Ag
0 ug/ml Ab
7 days
MP
100 ug/ml Ab
Radiolabled amino acids fed to L. monocytogenes, added to macrophages
T cells
(immune to L.m.)
% Complete
L.m.
Mø monolayer
100
1
2
3
50
4
20
40
60
80
100
1. Binding complete by 20 min
2. Internalization complete by 40 min
3. Degradation of Lm gradual, complete by 90 min (acid sol vs precipitatable counts
4. T cells begin binding Mf after 40 -90 minutes
T cells do not recognize native Ag, it must be processed
% complete binding
Glutamate-Fixed Macrophages
Do not present Ag
Blocked at binding and internalization steps
If fix Macrophages at various times after adding Ag:
100
T cell
binding to
monolayer
50
10 20 30 40 50 60 70
minutes with Ag before fixation
Internalization of Ag required for T cell binding
What is “Processing”?
OVA: native
denatured
1
denatured and digested
Th (OVA)
2
Fixed/normal Mø
24 hr
measure IL-2 production by T cells
Form of
APC:
native
normal
fixed
640
0
urea/2ME
640
0
Ag :
Trypsin
CNBr
640
640
640 (
640
pg/ml)
Fixed APC can only present pre-digested Ag
Processing is internalization, digestion, and presentation of Ag on surface
Processing occurs in lysosomes, can eliminate by neutralizing pH
B cells can present Ag
Ag-specific B cells
Present Ag efficiently
Absorb
Mø
B-cells
+
Mø
Non-Adherents
a-thy-1
+C'
Ag binds surface Ig
Is internalized and
processed
TNP-BSA
(sev eral injections)
discard non-adherents
TNP-gelatin
melt gelatin
Recov er a-TNP B-cells
1
2
Mø + BSA
Mø + TNP-BSA
B cells + BSA
B cells + TNP-BSA
TNP B cells + BSA
TNP B-cells
+TNP-BSA
prolif of
a-BSA
T-cell clone
.01
.10
1.0
10.0
µg Ag added to culture
100
Any epitope on the Ag
Can be presented, not
Just the epitope binding
The Ig
T helper cell can be
Specific for any epitope
On Ag
II
BRACIALE'S EXPT.
CD4+
FLU
INFECTED
12 days
Infect with influenza
B CELL
SPLEEN
CELLS
OR
MØ
CD8+
I
+ CHLOROQUINE
CD4+ RESPONSE-SHUT DOWN
CD8+ RESPONSE-STILL OK
+ IRRADIATED VIRUS
CD4+ RESPONSE-STILL OK
CD8+ RESPONSE-SHUT DOWN
Chloroquine stops lysosome function
Treatment of APC with chlorquine stops CD4 reponse, not CD8
Irradiated virus enters cells, will not infect
Irradiated virus detected by CD4 cells, not CD8 cells
Class I and II pathways are different. Class II uses lysosomes, Class I does not
Class I pathway samples proteins
from inside the cell
Proteins degraded in cytoplasm by proteosome complex
Transported into the ER by ATP-driven transporter
proteins TAP-1. TAP-2
Peptides lodge into MHC I molecules held
by chaperones in ER
Chaperones dislodge, complex transported
from ER-to golgi-to cell membrane
Class II pathway samples proteins
from outside the cell
Class II molecules reside in ER,
but Ag-binding cleft is blocked
A molecule “invariant chain” is lodged in cleft,
preventing Ag binding
This complex is transported
through golgi to endocytic vessicles,
Tail of invarient chain helps target complex
To vessicles
Ag is taken into the cell via phagocytosis,
the phagosome fuses with endocytic vessicle
containing MHC II
Plug in Ag cleft (CLIP) is removed by HLA-DM
peptide loads into MHC II, moves to cell surface
Cross Presentation by APC to MHC I
Exogenous Ag enters into phagosome
Proteins exported from phagosome to cytoplasm
Degraded by protesome
Phagosome at some point fuses with ER, obtains MHC I and associated machinery
Peptides transported into phagosome/ER fusion organelle by TAP
Loaded into Class I, presented at cell surface
Net result is that
Exogenous Ag can
Be presented on
Class I molecules
To CD8 T cells
Each MHC molecule will pick up only a limited subset of peptides
These are determined by “anchor residues” which interact with
Amino acids in the Ag-binding cleft
HLA-A2 H2N_ Leu _ _ _ _ _ _ Val COOH
H-2Kb H2N _ _ _ _ Tyr/Phe _ _ Leu COOH
Varying the constellation of MHC molecules means more peptides
Can be presented
MHC molecules must have a peptide occupying cleft to be
Cell surface associated