Memories M. Carrie Miceli May 21, 2004

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MemoriesM. Carrie MiceliMay 21, 2004

• Assigned ReadingEffector and Memory T cell
  differentiation Implications for Vaccine
  Development. Nature Reviews Immunology, April
  2002
• Janeway and Travers etc 412-420

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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

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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.
  

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• 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).

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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 T-cell 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
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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

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Tracking antigen specific responses with
MHC/tetramers
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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 peptide-specific
  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 NP118126 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 LdNP118126 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
  MHCtetramerpeptide complexes. Numbers represent
  the percentage of CD8 T cells that are antigen
  specific.

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• Figure 4
• increased frequency
• increased potency on
• a per cell basis (not shown)

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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.
Look at tetramer dissociation as a measure of
affinity (?) really avidity
6 weeks
gt14 weeks
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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,
CC-chemokine receptor 7.
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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?

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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 Cre-mediated 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.
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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 T-cell response to LCMV infection
in CD2STOPPLAP granzyme-BCre 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.
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fig 6 While both PLAP and PLAP- T cells from a
primary response can kill during recall response
PLAP better
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• Figure 6 PLAP CD8 T cells are LCMV-specific. a,
  Direct ex vivo killing of LCMV-infected target
  cells by PLAP and PLAP- CD8 T cells from mice
  infected eight days earlier with LCMV. PLAP and
  PLAP- CD8 T cells from CD2STOPPLAP times
  granzyme-BCre 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 effectortarget (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.

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• 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)

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Figure 7 Adoptive transfer of CD8 T cell memory.
CD8 PLAP and CD8 PLAP- were sorted by FACS
from LCMV-immune (infected gt1 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
PLAP- T cells are shown.
21
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.

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CD45 RA expressed by human naïve T cells CD45R0,
not CD45RA, is expressed in memory T cells
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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)
naive
CCR7 CD45A-
CCR7- CD45A-
CCR7- CD45RA
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Figure 4 CCR7 memory cells show enhanced
responsiveness to T-cell 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.
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• 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.

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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?)

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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?

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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.
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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).