Transcript Tracking antigen specific T cell dynamics in vivo

T cell Activation in Vivo
Carrie Miceli
May 16, 2005
• Assigned reading:
Drew M. Catron, Andrea A. Itano, Kathryn A. Pape, Daniel
L. Mueller, and Marc K. Jenkins Visualizing the First 50
Hr of the Primary Immune Response to a Soluble
Antigen Immunity 2004 21: 341-347.
• Additional Reading:
– Jenkins et al., In Vivo Activation of Antigen Specific CD4 T cells.
Annual Review of Immunology 2001
Reinhardt and Jenkins Whole-body Analysis of T cell Responses
Current Opinion in Immunology 2003 15:366-371
Germain and Jenkins. In vivo antigen presentation. Current
Opinion in Immunology 2003 16: 120-125
Tracking antigen specific T cell dynamics in vivo
• The problem: frequency of an ag specific T cell 1/105-1/106
• The dream solution: TCR transgenic mice with monoclonal
specificity
– Reality sets in, these mice are not normal
– Antigen specific T cells are too abundant, no room for all cells to
expand properly
– Initial expansion followed by immediate crash
– no productive immunity
• The real solution (Marc Jenkins)
– Adoptive transfer of TCR transgenic T cells into wt congenic
– Seed TCR tg T cells at 0.3% (still not normal)
– Availability of anti-TCR clonotypic antibody allows for
identification of TCR tg T cells
– Get expansion, homing, and differentiation of TCR tg T cells in
response to specific antigen
• MHC/peptide tetramers -an alternate solution (mark davis)
OT-II B6.PL (Thy 1.1)
OVA peptide-I-Ab
SM1 B6.PL (Thy 1.1)
Salmonella
FliC peptide-I-Ab
B6 (Thy 1.2)
D3 T cell expansion
20-100X antigen w/ adj
10-20 antigen alone
PM JK
DL KM
KH
BH FC
MO
• Histology allows for in situ detection; flow allows for
quantitation; activation and differentiation markers
• Transferred T cells can be from knockout X TCR transgene
• Recipient can be knockout to determine APC or tissue
requirements
– Can also adoptively transfer APC or specific B cells
• Can take advantage of congenic strains of allelic CD45,
CD8, or thy-1 mice
• Can track number of cell divisions using CFSE
• Can manipulate route of antigen delivery
– Conditions of activation or inactivation (adjuvent or site
and dose)
• CFSE allows determination of number of cell divisions by flow cytometry.
One can stimulate and monitor in vitro; adoptively transfer and monitor in
vivo fate; or transfer and both stimulate and track fate in vivo
Knock-out
Wild-type
Question answered using this approach.
• Where do T cells first encounter antigen?
• What are the molecular requirements for activation,
homing to B cell zones, B cell help?
• Do T cells die after several rounds of division?
• Do they home to the tissues?
• Do they become memory T cells?
• Are those that home to tissues different from those
that stay in lymphoid tissues, die or become
memory Ts?
KS
CA
TEa TCR recognizes Ea45-75 and can be detected with clonotypic ab
Y-Ae ab recognizes surface pEa52-68/Iab. Soluble peptide labeled with RFP
Ea 45-75
RFP
TEa
T cell
pEa 52-68/
I-Ab
Y-Ae
AR
Peptide-MHC II+ cells present
at 4-14 hours are Langerhans
cells that picked up soluble
antigen from lymph.
EaRFP
Y-Ae
B220
Peptide-MHC II+ cells present
at >18 hours are dermal
dendritic cells that picked up
antigen at the injection site.
AA
SS
In Situ Interactions Between Antigen-Specific
CD4 T Cells and Peptide-MHC II+ APC
T cells
CD69
Y-Ae
TEa
B6
50
40
4 hours
T cells
Y-Ae
24 hours
% T Cell/Y-Ae+ Cell
Interactions
30
20
10
0
TEa
20
C57Bl/6
*
15
10
5
0
TEa
JC
C57Bl/6
DC Movie/subcuteneous injection
• T cell in paracortex/T cell zone first encounters antigen (4
hrs post injection) on a LN resident Langerhans cell
(migrated there earlier)…both antigen specific and non
specific give it a try
• T cell activated to express CD69 and lymphokines (and
perhaps chemokine receptor responsible for interaction
with interstitial dendritic cells)
• Second wave of antigen presentation at 18 hrs by newly
immigrated tissue/interstitial dendritic cells
• T cells activated in the first wave specifically migrate to
and interact with these immigrated interstitial dendritic
cells and are stimulated to divide
• Some leave to tissues, some migrate to B cell follicle to
help
In Situ Detection of IL-2 Production
by Antigen-Specific CD4 T Cells
No Antigen
OVA/LPS, 12 h
CJ
In Vivo IL-2 Production by Naive CD4 T Cells
Is Dependent on CD28
35
100
30
%CD86+ DC
%IL-2+ OT-II Cells
TLR4 Expression is Required for the
Adjuvant Effects of LPS
25
20
15
10
5
80
60
40
20
0
0
0
2
4
6
8
0
Hours After Injection
B10 OVAp i.v.
B10 OVAp/LPS i.v.
TLR4-/- OVAp i.v.
TLR4-/- OVAp/LPS i.v.
2
4
6
8
In Vivo Clonal Expansion of Antigen-Specific
CD4 T Cells
Death or exodus?
MMW MD
RA
EP
HvB
RZ
OT-II Recipient, 5 Days
After OVAp/IFA Injection in Tail
LL
OT-II
DAPI
Lymphokine Production Correlates with
Cell Division History
Starting
CFSE
Level
Whole mouse movie
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Spleen entry chemokine dependent, CD62L-independent
Lymph node entry CCR7 dependent; CD62L dependent
Free antigen flows through afferent lymph into T Cell area, taken up by
resident Langerhans cells
Dermal DC take up antigen at the injection site, migrate to T cell area in
response to IL-1, TNF
Naïve T cell activated by resident langerhans cell to express CD69,
produce IL-2, more if B7 induced on APC
Activated T cell seeks out migrating dermal dendritic cells, is reactivated
T cells divide; some many times some few times
T cells leave lymph node to efferent lymph
Highly divided T cells lose CD62L, gain fPSGL-1
Highly dividing tissue homing (lungs and other tissues as well as
specifically to site of injection). After APC death many T cells die there.
Nonlymphoid seeking and effector lymphokine producing
less dividing lymph node seeking; retain CD62L, produce IL-2
A productive primary CD4 T cell response to antigen in the presence of adjuvant-induced
inflammation Jenkins Annual Rev of Immunol..2001
Response in the lymph nodes after subcutaneous injection of antigen plus adjuvant. This is the type of response
that generates effector lymphokine-producing memory cells and is induced by microbes because they contain
foreign proteins and molecules with adjuvant properties. Adjuvant molecules are recognized by pattern
recognition receptors on cells of the innate immune system at the antigen injection site, causing the release of
TNF- and IL-1. These cytokines signal the local tissue dendritic cells or monocytes to leave the tissue and
migrate via an afferent lymphatic vessel to the draining lymph node after first ingesting antigen. During the
migration process, these cells mature to produce peptide-MHC complexes from the ingested antigen and deliver
these to the cell surface along with newly synthesized B7 molecules. After arriving at the lymph node, the
dendritic cells crawl through the floor of the subcapsular sinus and present peptide-MHC complexes to naïve
antigen-specific CD4 T cells in the T cell area. The T cells produce high levels of IL-2 and an unknown T cell
growth factor, and they proliferate. This proliferation occurs in an IL-12-rich environment due to IL-12
produced by dendritic cells in response to the adjuvant. Those that divided the most and experienced the highest
concentration of IL-12 lose CCR7, gain P-selectin ligand, and acquire the capacity for rapid IFN- production.
Antigen-activated T cells that do not achieve this threshold number of cell divisions, or IL-12 concentration,
remain CCR7+ and acquire rapid IL-2 production pot ential, but not the capacity for IFN- production. After
leaving the lymphoid tissues during the primary response, the CCR7+ cells recirculate through lymphoid tissues
like naïve T cells. In contrast, the CCR7- cells are excluded from lymph nodes and remain in the blood or enter
nonlymphoid tissues that express P-selectin. As long as residual antigen is present to drive the survival of CCR7cells, then a second exposure to antigen will result in rapid production of effector lymphokines at the site of
antigen entry either by CCR7- cells that happen to reside in that tissue or by CCR7- cells that are rapidly
recruited from blood. As antigen is cleared from the body, CCR7- cells die or revert to the CCR7+ phenotype; in
either case only CCR7+ memory cells remain. If antigen enters the body during this phase, then CCR7+ cells
will be activated in the lymphoid organs and rapidly differentiate into CCR7-, effector lymphokine-producing
cells capable of migrating to the site of antigen deposition. This process would be more efficient than the
primary response because CCR7+ memory cells could achieve effector lymphokine production faster than naïve
T cells, and extrinsic factors such as antibodies from the primary response would facilitate antigen presentation.
Diagrammatic representation
of an unproductive CD4 T
cell response to antigen in the
absence of inflammation.
Presentation of an injected antigen is relatively
inefficient because it depends on the low level of
dendritic cell migration that occurs under
noninflammatory conditions, or the small amount
of antigen that leaks across the subcapsular barrier
to be taken up by lymphoid–tissue resident
dendritic cells. In either case, antigen presentation
is carried out by dendritic cells that don’t express
high levels of co-stimulatory ligands. Lack of the
anti-apoptotic effects of inflammatory cytokines
cause most of the T cells to die. The minimal
signaling through CD28, IL-1 receptor, and
receptors for differentiating cytokines such as IL12 and IL-4, experienced in the primary response
would limit IL-2 and effector lymphokine
production potential in any memory cells that
survived. The combination of death of most of the
expanded antigen-specific T cell
Population and the functional defects in the
survivors could explain the induction of peripheral
tolerance by antigen administration in the absence
of inflammation.
Tracking antigen specific responses with MHC/tetramers
Flow cytometric detection of
antigen-specific T cells using
fluorochrome labeled peptideMHC complexes.
Refold soluble empty class I MHC
antigens with a single antigenic
peptide.
Peptide/MHC complex is
then biotinylated and mixed with
streptavidin fluorochrome to
produce a tetramer
For class II MHC, need to
covalently attach peptide
Science 1996 Oct Phenotypic analysis of antigen-specific T
lymphocytes Altman JD…... Davis MM.
Figure 2. Correlation of antigen-specific staining in three of
four patients with peptide-specific killing activity in CTL
bulk cultures. Peripheral blood mononuclear cells from four
healthy HIV-infected donors were separated as described (5).
The CD4 counts for each patient at the time of analysis were
as follows: patient 065, 410; patient 868, 330; patient 077,
270; and patient 606, 510. Two million peripheral blood cells
from each patient were stained with anti-CD8a-CyChrome
and phycoerythrin-labeled HLA-A2-Gag (solid line) or
HLA-A2-Pol (dotted line) tetramers as indicated (A through
D) (18). Software gates were set to display only CD8+ small
lymphocytes. The percentages of CD8+ cells that were
positive for either A2-Pol or A2-Gag within gates as
displayed are included in each panel. The reproducibility of
the A2-Pol+ and A2-Gag+ populations in patient 065 (A)
was tested through analysis of five separate stains with each
reagent; the standard deviations are reported. Bulk cultures
were assayed for CTL activity (E
through H) (19) on days 14 through 16 at an effector:target
ratio of 50:1. Black bars, lysis of Gag-loaded targets; white
bars, lysis of Pol-loaded targets.