Anatomy of Immune Responses

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Anatomy of Immune Responses

• Micro 204 Molecular and Cellular Immunology
• 2009
• Lecturer Jason Cyster

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(No Transcript)
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Lymphatics
Lymph is filtered by Lymph Nodes before returning
to circulation (liters per day) Lymph contains T
and B cells and dendritic cells
(thin walled)
One-way valves
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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?

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Secondary Lymphoid Organs
(Goodnow Cyster, 1997, Current Biol. 7, R219)
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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

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Follicle (B zone)
Anatomy of the Spleen
Splenic (red pulp) cord
Splenic (venous) sinus
PALS or T zone (periarterial lymphoid sheath)
Pulp vein
Capsule
Trabecular vein
Trabecular artery
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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
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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
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Lymphoid organ chemokines
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Lymphoid organ chemokine expression in murine
Lymph Node
CXCL13 (BLC)
CCL21 (SLC)
CCL19 (ELC)
-gt CXCR5
-gt CCR7
-gt CCR7
from Cyster, 1999 Science 286, 2098
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CXCR5 is required for B cell migration into
follicles
CXCR5-/- B cells -gt WT
WT B cells -gt WT
red transferred B cells brown endogenous B
cells
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Lymphocyte migration within lymphoid tissue

• Two-photon microscopy of intact lymph node
• High speed imaging at depths up to 500 µM
• 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

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Lymphocytes migrate along stromal processes
Bajénoff et al., 2006 Immunity 25, 989
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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
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Summary 1

• Secondary lymphoid organs
• lymph nodes, Spleen, Peyers patches
• function to filter antigen from body fluids
• bring together antigen, antigen-presenting cells
  and antigen-specific lymphocytes
• support lymphocyte activation and differentiation
  events

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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

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DC precursors travel via blood to tissues
-gt Some tissue DC derive from specialized
precursors, others may differentiate from blood
monocytes -gt Some DC are maintained locally (by
proliferation e.g. Langerhans cells in the
skin), others are continually replenished by bone
marrow derived precursors
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Immature (sentinel) DCs in peripheral tissue
longitudinal section
rat tracheal epithelium
tangential section
Schon-Hegrad et al., (1991) J. Exp. Med. 173,
1345
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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
Langerhans Cells
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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
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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

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3. How do antigen-bearing DCs and
antigen-specific T cells find each other?
BLC
SLC ELC
T zone stromal cell (producing CCL21)
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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

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B cell antigen encounter
antigen
lymph fluid
Sinus Macrophage
FDC
hev

• B cells bind intact antigen through their
  surface Ig / B cell receptor (BCR)
• Antigen that enters via blood or lymph reaches
  the follicle and can be captured directly by B
  cells
• Follicular dendritic cells (FDC) can display
  antigen on their surface in an intact form for
  long periods

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Antigen-capturing Subcapsular Sinus macrophages
Subcapsular sinus macrophages capture and display
(opsonized) antigens Medullary macrophages
phagocytose and degrade antigens
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Deposition of Immune Complexes occurs in distinct
phases
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
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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
Follicle
Complement Receptor
Non-specific B cell
FDC
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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
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5. How do B cells find helper T cells specific
for the same antigen?
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Changes in lymphocyte homing during
T-dependent antibody responses
F
B
F
F
M
G
C
B
T
B
T
T
T
T
B
T
T
T
T
T
T
T
T
T
T
D
C
T
T
P
P
P
P
P
Plasma Cell and Germinal Center formation
T/B collaboration near the
Antigen encounter
follicle/T zone boundary
Antigen-specific B cell
Antigen-specific Plasma cell
P
Antigen-specific T cell
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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
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BCR engagement increases CCR7 surface levels
HEL-specific Ig-transgenic
Non-transgenic
CCR7
CXCR5
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B cells deficient in T zone chemokine receptor
fail to migrate to follicle / T zone boundary
CCR7 deficient Ig-tg B cells
Wildtype Ig-tg B cells
F
F
T
T
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Activated B-cell localization in outer T zone
determined by balanced responsiveness to T and B
zone chemokines
T zone
B zone (follicle)
CXCL13
vessel
T zone stromal cell
B
CCL21
Ag
CXCR5
B zone stromal cell
CCR7
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Migration of activated T cells to B cell area is
CXCR5 dependent

• 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 follicle)
• upregulate costimulatory molecules (e.g. ICOS)
  and cytokines (e.g. IL21) that facilitate B cell
  responses

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Changes in lymphocyte homing during
T-dependent antibody responses
F
B
F
F
M
G
C
B
T
B
T
T
T
T
B
T
T
T
T
T
T
T
T
T
T
D
C
T
T
P
P
P
P
P
Plasma Cell and Germinal Center formation
T/B collaboration near the
Antigen encounter
follicle/T zone boundary
Antigen-specific B cell
Antigen-specific Plasma cell
P
Antigen-specific T cell
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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 gt10 min,
  some for more than 1 hr
• Antigen non-specific conjugates persist lt10 min

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Effector T cell Trafficking

• Activated T cells exit lymphoid tissue -gt
  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)

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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

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Recommended Reading
Henrickson SE, Mempel TR, Mazo IB, Liu B,
Artyomov MN, Zheng H, Peixoto A, Flynn MP, Senman
B, Junt T, Wong HC, Chakraborty AK, von Andrian
UH. 2008. T cell sensing of antigen dose governs
interactive behavior with dendritic cells and
sets a threshold for T cell activation. Nat
Immunol. 9, 282-91. 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.
200847-56 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.
21279-88. Mempel TR, Henrickson SE, Von Andrian
UH. (2004) T-cell priming by dendritic cells in
lymph nodes occurs in three distinct phases.
Nature 427154-9. Okada, T., Miller, M.J.,
Parker, I., Krummel, M.F., Neighbors, M.,
Hartley, S.B., OGarra, 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 Batista FD,
Harwood NE. 2009. The who, how and where of
antigen presentation to B cells. Nat Rev Immunol.
9, 15-27. Phan TG, Gray EE, Cyster JG. 2009. The
microanatomy of B cell activation. Curr. Opin.
Immunol 21, 258-65. Alvarez D, Vollmann EH, von
Andrian UH. 2008. Mechanisms and consequences of
dendritic cell migration. Immunity 19,
325-42. 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. 4733-9 Jenkins M.K. et al.,
(2001) In vivo activation of antigen-specific
CD4 T cells. Annu. Rev. Immunol. 19 23