Anatomy of Immune Responses
Micro 204: Molecular and Cellular Immunology
2008
Lecturer: Jason Cyster
Lymphatics
Lymph is filtered by
Lymph Nodes before
returning to circulation
(thin walled) (liters per day)
One-way valves
Lymph contains T and
B cells and dendritic
cells
Qu i ckTi me ™ a nd a
TIFF (LZW) de co mp res so r
a re ne ed ed to se e th is pi ctu re.
Lecture Outline
1. What are Secondary Lymphoid Organs and how
do they function?
2. Why are Dendritic Cells so effective at initiating
adaptive immune responses?
3. How do antigen-bearing DCs and antigen-
specific T cells find each other?
4. How do B cells come in contact with intact
antigen?
5. How do B cells find helper T cells specific for the
same antigen?
Secondary Lymphoid Organs
lymph fluid intestinal contents
ca hev hev
(Goodnow & Cyster, 1997, Current Biol. 7, R219)
blood
Spleen - A filter of the blood
• Two main functions of the spleen carried out in two major
regions
1) White-pulp is where immune responses against blood-borne antigens
occur
2) Red-pulp is responsible for monitoring and removing old or damaged
RBC
• Red-pulp consists of thin walled splenic (or venous) sinuses
and dense collections of blood cells (including numerous
macrophages) that form red-pulp cords (or cords of Billroth)
• Blood supply: branches of central arteries open directly into
red-pulp cords, adjacent to the splenic sinuses (open
circulation)
– Released RBC must cross the sinus walls; interendothelial slits are a
major mechanical barrier and only the most supple mechanically
resilient RBC survive; old and damaged cells are removed by
macrophages
Follicle Splenic (red pulp)
Anatomy of (B zone) cord Splenic (venous)
the Spleen sinus
PALS or T zone
(periarterial lymphoid
sheath)
Pulp
vein
Capsule
Trabecular vein
Trabecular artery
Lymphocytes traverse HEVs to enter lymph nodes and
then compartmentalize in B cell follicles and T cell zones
Follicle or B zone
- B cells
- FDCs
HEV
T cell area (paracortex)
- T cells
- DCs
LN section stained with:
B220
PNAd
The ‘infrastructure’ of the lymph node
Follicle
T zone
Scanning EM of collagen fiber network in rat LN after removal of cells
from Gretz et al., 1997, Imm. Rev. 156, 11
Lymphoid organ chemokines
BLC (CXCL13) B cells
% input cells
migrated
CXCR5
T cells
granulocytes
[BLC]
ELC (CCL19)
T cells
SLC (CCL21) DCs
% input cells
migrated
CCR7 B cells
granulocytes
[ELC] or [SLC]
Lymphoid organ chemokine expression in
murine Lymph Node
CXCL13 (BLC) CCL21 (SLC) CCL19 (ELC)
-> CXCR5 -> CCR7 -> CCR7
from Cyster, 1999
Science 286, 2098
CXCR5 is required for B cell migration
into follicles
WT B cells -> WT CXCR5-/- B cells -> WT
red = transferred B cells
brown = endogenous B cells
Lymphocyte migration within
lymphoid tissue
• Two-photon microscopy of intact lymph node
• High speed imaging at depths up to 500 µM
QuickTime™ and a
TIFF (LZW) deco mpressor
are neede d to se e this picture.
• Demonstrate that naïve B and T lymphocytes undergo
extensive ‘random’ migration behavior
• 5-6 µM / min for B cells
• 10-12 µM / min for T cells
Schematic view of a lymph node
BLC
SLC
ELC
In mice lacking BLC (CXCL13) or CXCR5, B cells fail to home to B zones (follicles)
In mice lacking SLC (CCL21) and ELC (CCL19) or CCR7, T cells and DCs fail to home to
T zones
Summary 1
Secondary lymphoid organs:
• lymph nodes, Spleen, Peyer’s patches
• function to filter antigen from body fluids
• bring together antigen, antigen-presenting
cells and antigen-specific lymphocytes
• support lymphocyte activation and
differentiation events
2. Why are Dendritic Cells (DC) so effective at
initiating adaptive immune responses?
• immature ‘sentinel’ DCs are present in most tissues,
continually sampling their microenvironment for antigen
– by pinocytosis, phagocytosis and engulfment of dying (apoptotic)
cells
• detection of ‘danger signals’ (e.g. LPS, dsRNA, bacterial
DNA, necrotic cells, TNF, IL-1, CD40L) causes the cells
to mature
– decrease adhesion to local tissue cells (e.g. keratinocytes)
– increase expression of receptors (CCR7) for chemokines made
by lymphatic endothelial cells and lymphoid organ T zones
– process internalized Ag, upregulate MHC and costimulatory
molecules
• migrate into lymphoid T zone
• present antigen to T cells
DC precursors travel via blood to tissues
-> Some tissue DC derive from specialized precursors, others may
differentiate from blood monocytes
-> Some DC are maintained locally (by proliferation e.g. Langerhan’s
cells in the skin), others are continually replenished by bone marrow
derived precursors
Immature (sentinel) DCs in peripheral tissue
longitudinal
section
tangential
section
Schon-Hegrad et al., (1991)
J. Exp. Med. 173, 1345
Rapid emigration of sentinel DC from peripheral tissue in response to LPS
Roake et al., (1995)
J. Exp. Med. 181, 2237
DCs migrate from periphery to lymphoid organ T zone
bearing antigen Skin draining Lymph Node (day 1)
contact
sensitizer
(FITC) B zone
T zone
DC
Note: immature DC of skin are known as Langerhan’s Cells
Chemokine CCL21 (SLC) expression by lymphatic endothelium
in situ hybridization (ISH) to detect CCL21 mRNA expression
Liver Small Intestine
gut lumen
bright field dark field
under bright field illumination, deposited silver grains appear black;
under dark field (Nomarski) optics they appear silver
from Gunn et al.,
1999, PNAS 95, 258
Dendritic Cell Subsets QuickTime™ an d a
CD11c ->
TIFF (LZW) decomp resso r
are need ed to see this picture.
CD4 ->
• Different lineages of DC exist
– All are CD11c+ CD45 -> CD8 ->
– CD4+ and CD11b+ ‘myeloid’ DC
– CD8+ DEC205+ ‘lymphoid’ DC, involved in cross-presentation
– B220+ plasmacytoid DC, involved in IFNa/b production
• Different stimuli provoke immature DC to undergo
distinct maturation pathways, possibly secreting different
cytokines (e.g. becoming IL12+ or IL12-)
• DC from different tissues demonstrate an ability to
imprint T cells with a homing receptor profile that favors
homing to the tissue of origin of the DC
DC from gut imprint T cells for gut homing
Summary 2
DC are effective at initiating immune responses
because:
• The immature cells are located in sentinel positions
• They are highly efficient at processing and presenting
antigen
• They migrate rapidly to lymphoid T zones
• They express high levels of costimulatory molecules
for provoking activation of T cells
• DC influence the differentiation pathway of the T cell in
terms of cytokine induction and homing receptor profile
3. How do antigen-bearing DCs and antigen-
specific T cells find each other?
BLC
SLC
ELC
T zone
stromal cell
(producing
CCL21)
Summary 3
Antigen-bearing DCs and T cells find each
other by:
• migrating to a common microenvironment within
lymphoid organs
• DCs stop in T zone while T cells migrate rapidly
through the zone surveying the DCs for MHC-
peptide complexes
Mechanisms of B cell exposure to antigen
Splenic White Pulp Cord Lymph Node Peyer’s Patch
Sinus Macrophage
FDC
• B cells bind intact antigen through their surface Ig / B cell receptor (BCR)
• Antigen that enters via blood or lymph can be captured by sinus lining macrophages
and displayed to B cells
• Follicular dendritic cells (FDC) can display antigen on their surface in an intact form
for long periods
Follicular Dendritic Cells (FDCs)
• Resident in lymphoid follicles
– highly extended processes, can contact many migrating B cells
– not of hematopoietic origin and thus not related to DCs of T zone (possibly of
mesenchymal ‘fibroblastic’ origin)
– produce CXCL13
• Development dependent on LTa1b2 and TNF
• Express receptors that bind antigen coated in complement C3d
(CRs) and antibody (FcRs)
• Play a role in the Germinal Center reaction
Scanning EM of
isolated FDC
Skazal et al. 1985
JI 134, 1349
Antigen-capturing Subcapsular Sinus macrophages
Subcapsular sinus macrophages
capture and display (opsonized)
antigens
Medullary macrophages phagocytose
and degrade antigens
QuickTime™ and a
TIFF (Uncompressed) decompressor
are neede d to see this picture.
Deposition of Immune Complexes occurs in distinct phases
15 min 2h 8h
Capsule
Follicle
FDC
PE Immune Complex
Complement Receptor-1 (CD35)
B cells (B220)
Immune Complexes are made up of Antigen, Antibody (IgM or IgG) and (typically)
Complement (C3b). They are a form of opsonized antigen. They will usually be
multivalent (contain multiple units of the antigen). Antigens coated by C3b alone are
also termed opsonized and are handled in a similar way
B cells capture opsonized antigen from SCS
macrophages and transport to FDC
Afferent Lymphatic
Lymph Flow
Immune Complex
Lymph Node Capsule
Sinus Macrophage
Subcapsular
Sinus
B
Migration
Follicle
Complement
Receptor B
Non-specific B cell FDC
BCR
B B
Antigen-specific B cell
B cell Antigen Encounter
Afferent Lymphatic
Lymph Flow
Immune Complex
Lymph Node Capsule
Sinus Macrophage
Subcapsular
Sinus
Follicle
B
B
B B
Migration
BCR FDC
B
Seek T cell help Complement Receptor
Antigen-specific B cell
5. How do B cells find helper T cells
specific for the same antigen?
Changes in lymphocyte homing during
T-dependent antibody responses
B
F T B
F FM
B T
B T GC
T T T
B
T
T
T T
T
DC
T
T
T T T T
T
P PP PP
T/B collaboration near the Plasma Cell and
Antigen encounter Germinal Center
follicle/T zone boundary
formation
B Antigen-specific B cell P Antigen-specific Plasma cell
T Antigen-specific T cell
B cell antigen receptor engagement induces
B cell movement to outer T zone
i.v. antigen
6-8 hr
Spleen Spleen
brown = all endogenous B cells
red = antigen specific B cells
BCR engagement increases CCR7 surface levels
HEL-specific Ig-transgenic Non-transgenic
CCR7
CXCR5
B cells deficient in T zone chemokine receptor fail to
migrate to follicle / T zone boundary
Wildtype Ig-tg B cells CCR7 deficient Ig-tg B cells
F F
T
T
Activated B-cell localization in outer T zone determined by
balanced responsiveness to T and B zone chemokines
T zone B zone (follicle)
vessel
T zone B
stromal cell
+Ag
CXCR5
B zone
stromal cell
CCR7
Migration of activated T cells to B cell area is
CXCR5 dependent
QuickTime™ and a
TIFF (LZW) decompressor
are neede d to see this picture.
-some activated CD4 T cells become ‘Follicular Helper T cells (TFH)’
-they upregulate CXCR5 and downregulate CCR7 and S1P1 (causing them to
stay in the responding lymphoid organ and move into the follicle)
-upregulate costimulatory molecules (e.g. ICOS) and cytokines (e.g. IL21) that
facilitate B cell responses
Changes in lymphocyte homing during
T-dependent antibody responses
B
F T B
F FM
B T
B T GC
T T T
B
T
T
T T
T
DC
T
T
T T T T
T
P PP PP
T/B collaboration near the Plasma Cell and
Antigen encounter Germinal Center
follicle/T zone boundary
formation
B Antigen-specific B cell P Antigen-specific Plasma cell
T Antigen-specific T cell
Summary 5
Antigen specific B cell - CD4 T encounter:
• cells move to a common location in lymphoid tissue
• B-T conjugate pairs are highly motile
• Antigen specific conjugates persist for >10 min,
some for more than 1 hr
• Antigen non-specific conjugates persist circulation
– ability to re-enter lymphoid tissue is reduced (decrease in
CCR7, L-selectin)
• Increased ability to enter inflammed tissue due to
increased expression of:
– ligands for E- and P- selectins
– receptors for inflammatory chemokines (e.g. CXCR3)
– adhesion molecules (e.g. integrin a4b7)
Effector T cells in non-lymphoid tissue
• Effector T cells attracted to site in response to
chemokines
– produced by tissue cells exposed to microbial products
(e.g. epithelial cells, keratinocytes, mast cells,
macrophages)
• Macrophages and DCs in tissue present Ag to CD4
T cells
– CD4 T cells release cytokines that activate macrophages
to promote killing of ingested organisms
• All cells (except RBC) express MHC class I and can
be recognized (and killed) by effector CD8 T cells
Recommended Reading
Required Reading:
Phan TG, Grigorova I, Okada T, Cyster JG. 2007. Subcapsular encounter and complement-dependent transport of immune
complexes by lymph node B cells. Nat Immunol. 8: 992-1000.
M. Bajénoff, J. Egen, L. Koo, J. Laugier, F. Brau, N. Glaichenhaus, R. Germain. 2006 Stromal Cell Networks Regulate Lymphocyte
Entry, Migration, and Territoriality in Lymph Nodes. Immunity25, 989-1001
Miller MJ, Safrina O, Parker I, Cahalan MD. (2004) Imaging the Single Cell Dynamics of CD4+ T Cell Activation by Dendritic Cells in
Lymph Nodes. J Exp Med. 200:847-56
Other primary papers:
Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, Blankenstein T, Henning G, Forster R. (2004) CCR7 governs skin
dendritic cell migration under inflammatory and steady-state conditions. Immunity. 21:279-88.
Mempel TR, Henrickson SE, Von Andrian UH. (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.
Nature 427:154-9.
Okada, T., Miller, M.J., Parker, I., Krummel, M.F., Neighbors, M., Hartley, S.B., O’Garra, A., Cahalan, M.D. and Cyster, J.G. 2005.
Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biology 3:
e150.
Useful Reviews:
Cyster JG. (2005) Chemokines, Sphingosine-1-Phosphate, and Cell Migration in Secondary Lymphoid Organs.
Annu Rev Immunol. 23. 127-159
Itano AA, Jenkins MK. (2003) Antigen presentation to naive CD4 T cells in the lymph node.
Nat Immunol. 4:733-9
Jenkins M.K. et al., (2001) In vivo activation of antigen-specific CD4 T cells. Annu. Rev. Immunol. 19; 23
Cyster, J.G. (1999) Chemokines and cell migration in secondary lymphoid organs. 286; 2098
Gretz, J.E., Anderson, A.o. and Shaw, S. (1997) Cords, channels, corridors and conduits: critical architectural elements facilitating
cell interactions in the lymph node cortex. Immunological Reviews 156; 11