Transcript Memories M. Carrie Miceli May 17, 2002

Memories
M. Carrie Miceli
May 21, 2004
• Assigned Reading:Effector and Memory T
cell differentiation: Implications for
Vaccine Development. Nature Reviews
Immunology, April 2002
• Janeway and Travers etc 412-420
Memories of Memory
 430 B.C Greece, in "the plague of Athens" it was noted by
Thucydides that "the same man was never attacked twice".
 The concept of immune memory is born
 18th century natural experiment on the remote Faroe Islands
 1781 measles outbreak
 Islands remain measles free for 65 years with relatively few
people coming or going
 1846 major outbreak affecting 75-95% of the population
 "of the many aged people still living on the Faroes who had
measles in 1781, not one was attacked a second time"
 "all the old people who had not gone through with measles
in earlier life were attacked when they were exposed to
infection” Ludwig Panum (Danish Physician)
 Immune memory is long lived (65 years!)
 Re-exposure to measles virus is unnecessary
Both CD4 and CD8 T cell responses can be
broken down into three distinct phases:
• Activation and expansion
– During the initial phase which typically lasts about a week,
antigen driven expansion of the specific T cells and their
differentiation into effector cells occur.
– In several viral systems between 100 and 5000 fold expansion
of virus specific CD8+ T cells takes place.
– Substantial expansion of CD4+ T cells has also been reported
for several antigens (1200 fold expansion of CD4+ to PCC)
• Death
– A period of death then ensues (days 7-30) during which most of
the activated T cells undergo apoptosis and effector activity
subsides as the amount of antigen declines.
– This contraction of the T cell response is as dramatic as the
expansion and in most cases more than 95% of the antigen
specific cells disappear.
• Memory
– stable pool of memory cells can persist for many years.
– accelerated T cell responses seen upon re-exposure to antigen
due to:
• Increased frequency (5-100 fold increase)
• Qualitative changes in memory T cells that allow them to
respond faster and develop into effector cells more
efficiently than naïve cells.
– Genes for effectors such as g-IFN, perforin and granzyme B
are consitutively expressed. Controlled at the level of
translation. This allows for quicker and higher expression of
effectors.
• Express larger amounts of adhesion and/or accessory
molecules.
• Affinity Maturation (sort of). No somatic hypermutation
but higher “affinity” clones out compete lower affinity
clones during secondary exposures (perhaps this is “boost”
phenomenon).
Figure 1 | Antiviral CD8+
and CD4+ T-cell responses.
The three phases of the T-cell
immune response (expansion,
contraction and memory) are
indicated. Antigen-specific T
cells clonally expand during the
first phase in the presence of
antigen. Soon after the virus is
cleared, the contraction phase
ensues and the number of
antigen-specific T cells
decreases due to apoptosis.
After the contraction phase, the
number of virus-specific T cells
stabilizes and can be
maintained for great lengths of
time (the memory phase). Note
that, typically, the magnitude
of the CD4+ T-cell response is
lower than that of the CD8+ Tcell response, and the
contraction phase can be less
pronounced than that of CD8+
T cells. The number of memory
CD4+ T cells might decline
slowly over time, as reported
recently
How do we know?
• Track antigen specific T cells
– Adoptive transfer of TCR transgenic T cells
into syngeneic hosts at low (but not too low)
concentrations.
– Peptide/MHC tetramers
Tracking antigen specific responses with MHC/tetramers
Kaja Murali-Krishna Rafi Ahmed
Counting Antigen-Specific CD8 T Cells: Immunity1998 8: 177
•
Figure 4. Quantitation and Visualization of Antigen-Specific Memory CD8 T Cells
during LCMV Infection BALB/c (A) and C57BL/6 (B) mice were checked at the
indicated days postinfection for the number of virus-specific CD8 T cells in the spleen
by IFN single-cell assays and MHC class I tetramer staining. Data represent average
values obtained from three to five mice at each time point. The frequencies of peptidespecific cells/total CD8 T cells are indicated for each of the epitopes at selected time
points. On day 3,the frequencies for NP118 were 1 in 1000 and for GP283, 1 in 10,000;
on day 5, the frequencies were 1 in 4 for NP118 and 1 in 30 for GP283. In C57BL/6
mice the day 5 frequencies were 1 in 12 for NP396, 1in 27 for GP33, and 1 in 180 for
GP276. On day 0 postinfection the number of peptide-specific CD8 T cells was below
detection (dotted line).(C) Spleen cells from LCMV-infected BALB/c mice 240 days
postinfection were incubated with peptide NP118–126 for 5 hr, followed by staining for
surface CD8 and intracellular IFN. Antigen-specific cells from the same mouse were
visualized by staining freshly explanted spleen cells with LdNP118–126 tetramer. (D)
C57BL/6 mice 270 days postinfection were incubated in the presence of the indicated
peptides followed by intracellular IFN staining, or freshly explanted spleen cells were
stained with the indicated MHCtetramer–peptide complexes. Numbers represent the
percentage of CD8 T cells that are antigen specific.
.
Figure 4
•increased frequency
•increased potency on
a per cell basis (not
shown)
Lik e antibodi es T CRs migh t und ergo“affini ty
maturation”, (but not so matic mutation , at least no t
often).
A Kinetic Basis ForT Cell Receptor Reperto ire Sele ctio n
During a n Immune Response
Peter Savage1… Mark Davis, Immunity, Vol. 10, 485-492, April, 1999,
The ba sis for T cell antigen recep tor (TCR) repertoire selection upon
repea ted antigen ic cha llenge is unc lear. We eva luated the avidity and
dissociation k inetics of pep tide/major histocompatibilit y co mplex (MHC )
tetramer binding to antigen -specific T lymphocy tes isolated following
primary or s econda ry immunization. The data revea l a narrowing of the
secondary reperto ire relative to the primary repertoire, large ly
resulting from the loss of cells expressing TCRs with the fastest
dissociation rates for peptide/MHC binding. In add ition, T cells in the
seconda ry response exp ress TCRs of high er ave rage affinity for
pep tide/MHC than cells in the p rimary respon se. These res ults provide a
link between the kinetics and affinity o f TCR-peptide/MHC interac tions
and TCR sequence selection during the course of a n immune response.
Look at tetramer dissociation as a
measure of affinity (?) really avidity
6 weeks
>14 weeks
Figure 3. Tetramer Staining
Decay Kinetics for Primary or
Secondary Populations Stained
with MCC/I-Ek Tetramer
Staining decay plot overlays for
20 primary and 19 secondary
samples. The natural logarithm
of the normalized fluorescence
is plotted versus time after
14.4.4 addition. Tetramer
staining was evaluated at 0, 20,
40, 60, 90, 120, and 180 min
after 14.4.4 addition. Mean
decay rates for primary and
secondary populations were
comparable over the 120-180
min interval (data not shown).
The upper and lower dashed
lines represent MCC/I-Ek
tetramer dissociation and
MCC(102S)/I-Ek tetramer
dissociation from 2B4 cells,
respectively.
How Do We Make Memories?
Figure 5 | Models of memory T-cell differentiation.
a | Model 1 represents a divergent pathway, whereby a
naive T cell can give rise to daughter cells that develop
into either effector or memory T cells, a decision that
could be passive or instructive. In this model, naive T
cells can bypass an effector-cell stage and develop
directly into memory T cells. b | Model 2 represents a
linear-differentiation pathway, whereby memory T cells
are direct descendants of effector cells. This model
indicates that memory T-cell development does not
occur until antigen (Ag) is removed or greatly decreased
in concentration. c | In model 3, which is a variation of
model 2, a short duration of antigenic stimulation
favours the development of central memory T cells,
whereas a longer duration of stimulation favours the
differentiation of effector memory T cells. d | Model 4
represents the decreasing-potential hypothesis, which
suggests that effector T-cell functions steadily decrease
as a consequence of persisting antigen (as observed in
chronic infections). In addition, accumulative encounters
with antigen lead to increased susceptibility of effector
cells to apoptosis, and reduced numbers of memory T
cells are formed. As suggested in model 2, the
development of memory T cells occurs following antigen
clearance. It is not known whether dysfunctional effector
T cells can give rise to functional memory T cells, but
this model suggests that T cells might regain function
over time following the removal of antigen. CCR7, CCchemokine receptor 7.
Are memory T cells derived from
effector pool or from distinct precursors?
• Evidence for each
• Linear development -Jacob and Baltimore
• Distinct effectors
– Lauvau, G. et al. Priming of memory but not effector CD8 T cells
by a killed bacterial vaccine. Science 294, 1735-1739
– It has been proposed that central memory T cells do not adopt
effector-cell properties during the primary T-cell response, but they
persist and form a protective reservoir that can give rise to
secondary effector T cells if antigen is re-encountered
– Look at knockouts
• Need HSA for memory, but not effectors
• Need CD28 for both
• Do different requirements imply different lineages?
Figure 1 Strategy used by Jacob
and Baltimore2 to label
activated T cells in vivo. Mice
have two transgene constructs.
In the first, the granzyme B
promoter drives expression of
the Cre recombinase. Granzyme
B is an enzyme used by
activated T cells to kill infected
cells, and its promoter is active
only in activated T cells. The
second, reporter transgene
contains a T-cell promoter
(CD2), a stop codon
flanked by substrates for Cremediated re-combination (loxP),
and a reporter gene (PLAP). a,
In naive T cells the granzyme B
promoter is silent so cells do not
transcribe the Cre recombinase.
The CD2 promoter is active but
the stop codon prevents
expression of PLAP. b,
In activated T cells, the
granzyme B promoter is active
so cells express the Cre
recombinase. This enzyme
removes the stop codon from the
reporter transgene so PLAP is
expressed. Once this stop codon
has been removed, PLAP is
permanently expressed. c,
Memory cells contain PLAP, but
they no longer express the Cre
recombinase.
Nature 399, 593 - 597 (1999) Modeling T-cell memory by
genetic marking of memory T cells in vivo
JOSHY JACOB* AND DAVID BALTIMORE
Figure 5 CD8+ Tcell response to
LCMV infection in
CD2–STOP–PLAP
granzyme-B–Cre
mice. Mice were
immunized with a
single intraperitoneal
injection of 5 104
PFU of the
Armstrong strain of
LCMV. Splenocytes
of uninfected (a) and
infected doubly
transgenic mice were
analysed by flow
cytometry at 8 (b),
30 (c) and 90 (d)
days post infection.
fig 6 While both PLAP+ and PLAP- T
cells from a primary response can kill;
during recall response PLAP+ better
• Figure 6 PLAP+ CD8+ T cells are LCMV-specific. a, Direct ex
vivo killing of LCMV-infected target cells by PLAP+ and PLAPCD8+ T cells from mice infected eight days earlier with LCMV.
PLAP+ and PLAP- CD8+ T cells from CD2–STOP–PLAP times
granzyme-B–Cre doubly transgenic mice were isolated by FACS
and assayed for direct ex vivo cytotoxicity in a 6 h 51Cr-release
assay. Shown is a representative direct ex vivo cytotoxicity assay
of sorted PLAP+ and PLAP- CD8+ T cells (H-2b/q) against
uninfected or LCMV-infected MC57G (H-2b) target cells. The
percent specific lysis is plotted against effector:target (E/T) ratios
used. b, Secondary bulk CTL assay. CD8+ PLAP+ and CD8+
PLAP- T cells were isolated by FACS from mice infected eight
days earlier with LCMV. The sorted CD8+ PLAP+ and CD8+
PLAP- T cells (H-2b) were stimulated in vitro with irradiated
LCMV-infected peritoneal exudate cells (H-2b), irradiated
syngeneic splenocytes (H-2b) and interleukin-2 (IL-2). After six
days of incubation at 37 °C the cells were assayed for cytotoxicity
against uninfected or LCMV-infected MC57G (H-2b) target cells
in a 6 h 51Cr-release assay.
• Supports linear model; plap + effectors turn
into memory T cells
• Indicates not all effectors can become
memory? Implicates granzyme b + cells as
specialize subset that does become memory
T (no, actually an artifact)
Figure 7 Adoptive transfer of CD8+ T cell
memory. CD8+ PLAP+ and CD8+ PLAPwere sorted by FACS from LCMVimmune (infected >1 month earlier)
doubly transgenic mice. 2 105 sorted cells
were adoptively transferred into naive
recipients and challenged
the next day with 2 106 PFU clone-13
LCMV, intravenously. Eight days later
their spleens were removed and LCMV
titres were determined. The mean LCMV
titres (log10) per spleen of mice that
received CD8+ PLAP+ and CD8+ PLAPT cells are shown.
Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A.
Two subsets of memory T lymphocytes with distinct homing
potentials and effector functions. Nature 401, 708-712 (1999).
Human PBL separated on the basis of CD45RA+(naïve) and RA- (memory)
• Expression of CCR7, a chemokine receptor that controls homing to
secondary lymphoid organs, divides human memory T cells into two
functionally distinct subsets.
• CCR7- memory cells express receptors for migration to inflamed
tissues and display immediate effector function.
• In contrast, CCR7+ memory cells express lymph-node homing
receptors and lack immediate effector function, but efficiently
stimulate dendritic cells and differentiate into CCR7- effector cells
upon secondary stimulation. The CCR7+ and CCR7 - T cells, which
we have named central memory (TCM) and effector memory (TEM),
differentiate in a step-wise fashion from naive T cells, persist for years
after immunization and allow a division of labour in the memory
response.
CD45 RA
expressed by
human naïve T
cells
CD45R0, not
CD45RA, is
expressed in
memory T cells
naive
CCR7+
CD45A-
CCR7CD45ACCR7CD45RA+
Figure 2 CCR7+ and
CCR7- memory T cells
display different effector
functions. a, b, The three
subsets of CD4+ T cells
were sorted
according to the expression
of CCR7 and CD45RA as in
Fig. 1 and tested for their
capacity to produce IL-2,
IFN-, IL-4 and IL-5 (a)
and for the kinetics of
surface CD40L
upregulation (b) following
polyclonal stimulation. c, d,
The four subsets of CD8+ T
cells were sorted according
to the expression of CCR7
and CD45RA as in Fig. 1
and tested for their capacity
to produce IL-2 or IFN- (c)
or were immediately stained
with anti-perforin antibody
(green) and counterstained
with propidium iodide (red)
(d). In the CD8+
CD45RA+ compartment,
CCR7 expression allows us
to discriminate naive cells
(1) from effector cells (4)
Figure 4 CCR7+ memory cells
show enhanced responsiveness to Tcell receptor triggering and potently
activate dendritic cells to
produce IL-12. a, Proliferative
response of naive T cells (squares),
CCR7 + (triangles) and CCR7(circles) memory T cells to different
concentrations of plastic-bound
anti-CD3 monoclonal antibody in
the absence (empty symbols) or in
the presence (filled symbols) of
anti-CD28. b, IL-12 p70 production
by dendritic cells cultured with
naive T cells (squares) or CCR7+
memory T cells (triangles).
Dendritic cells were pulsed with
toxic shock syndrome toxin (TSST)
at 100 ng ml -1 (empty symbols) or
1 ng ml-1 (filled symbols).
Both T-cell populations contained
similar proportions of V2 + cells.
•
A model was proposed in which the tissue-homing effector
memory T cells (TEM; CD62Llo, CCR7lo), which are capable of
immediate effector functions, could rapidly control invading
pathogens. The lymph-node-homing central memory T cells
would be available in secondary lymphoid organs ready to
stimulate dendritic cells, provide B-cell help and/or generate a
second wave of T-cell effectors (CEM; CD62Lhi,CCR7hi).
• Additional data suggest that TEM and CEM may be equally
good at effector cytokines, but only CEM can make IL-2 and
have greater proliferative capacity, thus they are better at
expanding after restimulation.
• Though initial data suggested to be distinct lineages, some
data supports the idea that after infection tissue CD62Llo,
CCR7lo cells differentiate into TEM cells, but ultimately revert
to CEM cells with the aquired capacity to participate in
homeostatic turnover.
How do you keep the memories alive?
• Persistent of antigen? Is re-exposure to antigen
necessary for maintenance of memory (does some
antigen hide out in vivo). Adoptive transfer of memory
Ts into antigen free mice says no.
• Do cross-reactive antigens function to re-stimulate
memory? (probably not)
• Engagement of accessory molecules on the surface of
memory cells
• Response to lymphokines as “bystanders” ..yes
– IL-15 plays a role in homeostatic proliferation
– IL-7 plays a role in survival (by upregulation of BCL-2)
• TCR engagement by peptide/MHC. TCR tickling?
Remember positive selection? Not for survival or
homeostatic proliferation (but perhaps for quality
memories yes?)
Do memory T cells “turn over”?
 Analysis of LCMV –specific memory CTLs have shown
that a small fraction (5-10%) of memory CTLs are in cycle
at a given time.
 It is not known whether the resting and the cycling cells
represent distinct populations or whether over the lifetime
of the mouse all the memory cells will divide (probably the
latter).
 Is turnover a specific mechanism for maintaining specific
memory or is it a general mechanism for maintaining the
total number of peripheral cells?
Fig. 10.25 The affinity as well as the amount of
antibody increases with repeated immunization.
The upper panel shows the increase in the level
of antibody with time after primary, followed by
secondary and tertiary, immunization; the lower
panel shows the increase in the affinity of the
antibodies. The increase in affinity (affinity
maturation) is seen largely in IgG antibody (as
well as inIgA and IgE, which are not shown)
coming from mature B cells that have undergone
isotype switching and somatic hypermutation to
yield higher-affinity antibodies. Although some
affinity maturation occurs in the primary
antibody response, most arises in later responses
to repeated antigen injections. Note that these
graphs are on a logarithmic scale.
Fig. 10.26 The mechanism of affinity
maturation in an antibody response.
At the beginning of a primary
response, B cells with receptors of a
wide variety of affinities (KA),most of
which will bind antigen with low
affinity, take up antigen, present it to
helper T cells, and become activated
to produce antibody of varying and
relatively low affinity (top panel).
These antibodies then bind and clear
antigen, so that only those B cells with
receptors of the highest affinity can
continue to capture antigen and
interact effectively with helper T cells.
Such B cells will therefore be selected
to undergo further expansion and
clonal differentiation and the
antibodies they produce will dominate
a secondary response, (middle panel).
These higher affinity antibodies will
in turn compete for antigen and select
for the activation of B cells bearing
receptors of still higher affinity in the
tertiary response (bottom panel).