What is immunological memory?

1
Memory T cells
What is immunological memory?
The capacity of immune cells to remember past
infections
Which immune cells have the capacity to remember?
Specialized cells known as memory B and T
lymphocytes
Why it is important to understand the generation
of memory lymphocytes?
Immunological memory is the basis of vaccination.
The ultimate goal of a vaccine is to develop
long-lived protection, whereby the first
encounter with a pathogen is remembered, which
leads to enhanced memory responses that either
completely prevent infection or greatly reduce
the severity of disease.
2
Memory T cells

• In this lecture, we will focus on memory T cells
  , and in particular on CD8 T cell memory.
• We will examine
• Characteristics of memory T cells
• Models proposed for the generation of memory T
  cells
• Subsets of memory T cells
• Next week we will
• Examine recent data that define the lineage
  relationships between the two subsets of memory
• CD8 T cells, and their ability to persist and to
  confer protective immunity.

Lineage relationship and protective immunity of
memory CD8 T cell subsets Wherry, J.E. et
al. Nature Immunol 4, 225-234 (2003).
3
Characteristics of Memory T cells
What is the essential characteristic of memory T
cells?
Memory T cells respond more rapidly and more
aggressively than naïve T cells.
What is the physiological basis for the faster
response of memory T cells?

• Increased number. Memory T cells are present in
  higher numbers than naïve T cells. The frequency
  of a
• given antigen-specific T cells in an immune
  animal can be 1000X higher than in a naïve animal.

• Gene-expression profile which is reprogrammed by
  changes in chromatine structure. For example,
  mRNA
• for IFN-g and cytotoxic molecules such as
  perforin and granzyme B are not found in naïve T
  cells
• whereas these transcripts are elevated in memory
  CD8 T cells. Therefore, memory CD8 T cells
• have the capacity to produce larger quantities of
  these effector proteins more rapidly than naïve T
  cells.

• Anatomical location. Different pattern of
  expression of cell surface proteins involved in
  cell adhesion and
• chemotaxis that allow them to gain access to
  non-lymphoid tissues, the sites of microbial
  entry..

• Longevity. Memory T cells are maintained for a
  long time due to antigen-independent homeostatic
  
• proliferation.They are able to maintain their
  number by continual low-level proliferation
  (like a stem cells)
• in the absence of Ag. The cytokines IL-2, IL-7
  and IL-15 are involved in the homeostatic
  proliferation of
• memory T cells. The longevity of memory T cells
  explain how they can confer long-term protective
• immunity.

4
Stages of a T cell response
The kinetics of a first T cell response to a
pathogen can be divided into three distinct
phases. The first is expansion, in which
antigen-specific lymphocytes are activated to
divide. The number of lymphocytes rapidly
becomes enormous, and the lymphoid organs (such
as the spleen and lymph nodes) enlarge to
accommodate them. The activated T cells then
begin to take on 'effector' functions they
secrete cytokines and kill infected target
cells. The second phase is contraction and
occurs soon after the pathogen is cleared. Over
95 of the antigen-specific T cells then die.
Finally comes the memory phase, in which those
T cells that have been spared by the contraction
phase survive for long periods, forming a stable
pool of 'memory' cells.
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.
5
Models of memory T cell differentiation
The divergent model proposes that a naïve T cell
can give rise to daughter cells that develop into
either effector or memory T cells. Accordingly,
naïve T cells can bypass an effector-cell stage
and develop directly into memory T cells.
6
Earlier discovery a green fluorescent protein
reporter gene (T-GFP) is expressed in naive and
short-term activated T cells but is silent in
terminally differentiated effector cells. In
T-GFP transgenic mice, GFP expression can be
conveniently used to monitor T cell
differentiation. Manjunath et al. have
generated naive T cells from T-GFP mice carrying
a class Irestricted T cell receptor (TCR) that
recognizes a specific viral peptide. They
stimulated these cells in vitro with antigen for
2 days and expanded them in the presence of
different cytokines. When cultured in high doses
of IL-2 (CD8IL-2), the cells become large blasts,
express high levels of activation markers, lose
expression of GFP and acquire the capacity to
produce IFNg- and to kill target cells. The same
cells cultured in the presence of low doses of
IL-2 or with IL-15 (CD8IL-15), become small,
retain GFP, and fail to acquire cytotoxic
function, although they acquire IFNgproducing
capacity. Importantly, after adoptive transfer,
CD8IL-15 cells survive for several weeks and,
upon antigen rechallenge, mount a secondary
response that is comparable to that mediated by
endogenously generated memory cells.
Effector differentiation is not prerequisite for
generation of memory cytotoxic T lymphocytes N.
Manjunath et al. J. Clin. Invest. 108, 871-878
(2001)
7
Models of memory T cell differentiation
According to the linear differentiation model,
memory T cells are the progeny of effector T
cells. There are many in vitro and in vivo
studies that have established that long-term
memory results when naïve T cells are induced to
undergo several rounds of cell division in
response to antigen in vitro and are then
adoptively transferred in vivo in the absence of
antigen. For example, Opferman et al.
demonstrated that only effector T cells that
have divided more than five times in vitro can
generate memory T cells.
Linear differentiation of cytotoxic effectors
into memory T lymphocytes Joseph T. Opferman,
Bertram T. Ober, Philip G. Ashton-Rickardt Science
283, 1745- 1748 (1999)
8
Models of memory T cell differentiation
The decreasing potential hypothesis. The previous
model does not definitely resolve the issue of
whether memory cells arise from fully
differentiated effectors. It is notable that
memory fails to occur when T cells undergo
exhaustive' proliferation to high doses of
viruses in this situation, effector T cells are
generated in enormous numbers but then die en
masse, presumably because the cells are all
driven to a terminal stage of differentiation
where they can not escape from
activation-induced cell death' (AICD). This
phenomenon is known as clonal exhaustion. Thus,
cumulative encounter with Ag increases
susceptibility of effector T cells to apoptosis
and reduced formation of memory T cells.
9
Thus, increasing cell stimulation and division
are associated with progress to terminal
differentiation and a reduction in the memory
potential. In other words, cells that divide many
times are more likely to die than to survive as
memory cells. Accordingly, the decreasing
potential hypothesis proposes that memory cells
may normally arise from a subset of cells that
express a full range of effector functions but,
perhaps because of lack of prolonged contact
with antigen, do not initiate AICD. Hence memory
cells might originate from a population of
effector cells that only arrive during the later
stages of the immune response, when the Ag is
removed or greatly decreased in concentration.
10
Thus, the mechanisms involved in the generation
of memory T cells remain poorly
understood despite that the practice of
variolation (inoculation of virus taken from
pustules of smallpox victims) was used for
protection against smallpox well before
1796. Part of this deficiency may arise from the
fact that memory T cells are heterogeneous.
11
Subsets of Memory T cells
Based on the expression of CCR7 two subsets of
memory T cells have been recently identified.
Figure 1 CCR7 and CD62L are co-expressed on a
subset of peripheral blood memory CD4 and CD8
T cells. CD4 (a, b) and CD8 (c, d) lymphocytes
were stained with monoclonal antibodies to
CD45RA and CCR7, which identified three and four
subsets, respectively. These subsets were sorted
and analysed for the expression of CD62L, and
the percentage of bright cells is indicated (b,
d). Upon serial analysis, the proportion of
cells in the different compartments was rather
stable in the same individual, but more variable
among individuals, the variability being more
pronounced in the CD8 than in the CD4
compartment. Comparable results were obtained
using two anti-CCR7 antibodies (clones 3D12 and
10H5).
Two subsets of memory T lymphocytes with distinct
homing potentials and effector functions
FEDERICA SALLUSTO, DANIELLE LENIG,
REINHOLD FÖRSTER, MARTIN LIPP
ANTONIO LANZAVECCHIA Nature 401, 708 - 712
(1999)
12
Figure 2 CCR7 and CCR7- memory T cells display
different effector functions. 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) (ref. 26). Comparable results
were obtained in 12 healthy donors.
Memory CCR7
Naive
Memory CCR7-
Effectors
Two subsets of memory T lymphocytes with distinct
homing potentials and effector functions
FEDERICA SALLUSTO, DANIELLE LENIG,
REINHOLD FÖRSTER, MARTIN LIPP
ANTONIO LANZAVECCHIA Nature 401, 708 - 712
(1999)
13
Two subsets of memory T cells CCR7 CD62Lhigh
? Central memory T cells (CM) CCR7- CD62Llow ?
Effector memory T cells (EM) With functional
differences TCM ? IL-2, little IFN-g, no
perforin TEM ? little IL-2 but high IFN-g and
perforin And different homing potential CD62L
interacts with PNAd on HEV, which mediates
attachment and rolling. CCR7 binds to chemokines
CCL19 and CCL21 that are presented on the luminal
surface of endothelial cells in lymph nodes
which causes firm arrest and the initiation of
extravasation. Studies have shown that CCR7
CD62Lhigh T cells migrate efficiently to
peripheral lymph nodes, whereas T cells lacking
these two molecules do not. Rather, CCR7-
CD62Llow T cells can be found in other sites,
such as the liver and lungs.
14
Masopust et al. tracked the migration of CD8
memory T cells with tetramers composed of major
histocompatibility complex (MHC) molecules bound
to an antigenic peptide. Their tetramer was
composed of mouse MHC class I molecules and a
peptide derived from vesicular stomatitis virus
(VSV). This tetramer identified VSV-specific CD8
T cells in mice that had been infected with this
virus. The dynamics and distribution of
VSV-specific CD8 T cells were revealed by
analyzing which T cells bound to the tetramer.
Remarkably, 9 days after infection, nonlymphoid
tissues including kidney, liver, and peritoneum
contained extremely high numbers of VSV-specific
CD8 T cells (which constituted up to 40 of the
total CD8 population). Even after 296 days,
some tissues still retained VSV-specific T memory
cells that constituted as much as 4 of the
total CD8 population. Despite the high
proportion of VSV-specific CD8 cells in
nonlymphoid tissues, this T cell subpopulation
had almost totally disappeared from lymphoid
tissues. Thus, the clonally expanded effector and
memory T cells had become redistributed to the
body's nonlymphoid tissues, the very places where
protection against pathogens is needed the most.
Preferential localization of effector memory
cells in nonlymphoid tissue David Masopust,
Vaiva Vezys, Amanda L. Marzo, Leo
Lefrancois Science 291, 2413- 2417 (2001)
15
Figure 1. Infection with VSV leads to the
appearance of virus-specific CD8 T cells in
lymphoid and nonlymphoid tissues. C57Bl/6J mice
were infected intravenously with 106
plaque-forming units (PFU) of VSV-Indiana, and
8 days (A) or 81 days (B) later, mice were
perfused and lymphocytes were isolated from the
indicated tissues. The percentage of
antigen-specific CD8 T cells was assessed by
staining with N52-59/Kb tetramer and antibodies
to CD8 and CD11a, followed by fluorescence flow
cytometry. Plots shown are gated on CD8
lymphocytes values are mean percentages of
tetramer cells within the CD8 T cell population
derived from at least four mice. Control
staining with a Kb tetramer containing SIINFEKL
was negligible. PLN, peripheral lymph nodes
MLN, mesenteric lymph nodes PBL, peripheral
blood lymphocytes LP, small intestine lamina
propria IEL, small intestine intraepithelial
lymphocytes BM, bone marrow Perit, peritoneal
cavity lymphocytes.
16
They also demonstated that the T cells
redistributing to nonlymphoid tissues have an
activation profile characteristic of Sallusto's
effector-memory T cells. In contrast, lymphoid
memory T cells, that did not have Immediate
effector ability resembles Sallusto's
central-memory T cells.
Fig. 3. Virus-specific CD8 memory T cells in
peripheral but not lymphoid tissues are
constitutively cytolytic. (B) Twenty days after
VSV infection, lymphocytes were incubated for
4 to 5 hours with 51Cr-labeled untreated EL4
target cells (27) or target cells pulsed with
N52-59 peptide. ET ratio was 2001 for all
tissues. ET values shown in plots are corrected
for the number of tetramer cells in each
population. (C) As described in (B), except
lymphocytes were isolated from mice primed with
106 PFU of VSV-New Jersey, rested gt7 months,
then infected with VSV-Indiana and rested an
additional 224 days. ET ratio was 3001 for all
tissues. ET values shown in plots arecorrected
for the number of tetramer cells
Preferential localization of effector memory
cells in nonlymphoid tissue David Masopust,
Vaiva Vezys, Amanda L. Marzo, Leo
Lefrancois Science 291, 2413- 2417 (2001)
17
Reinhardt et al.reached a similar conclusion
although they tracked the redistribution of CD4
T cells using a totally different approach.
They followed migrating antigen-specific CD4 T
cells by transferring naïve T cells with a
defined antigenic specificity (derived from a T
cell receptor transgenic mouse) into recipient
mice and using Thy-1 as a marker for the
transferred cells. The authors painstakingly
determined the presence of antigen-reactive CD4
T cells in all tissues of the recipient animals.
They did this by immunohistochemical analysis of
whole-body sections using an antibody against
Thy-1.1 that only bound to donor-derived T cells.
With this protocol, they first tracked the
migration of transferred naïve T cells, and then
monitored changes in their migration after
injection of the specific antigen. As expected,
naïve T cells initially became localized only
within lymphoid tissues. However, after injection
of antigen and a primary immune response, there
was a striking redistribution of antigen-reactive
T cells to nonlymphoid tissues including the
liver, lungs, and intestinal lamina propria. This
study also demonstrated that memory T cells
migrating to nonlymphoid tissues can rapidly
become effector cells.
Visualizing the generation of memory CD4 T cells
in the whole body R. LEE REINHARDT,
ALEXANDER KHORUTS, REBECCA MERICA, TRACI ZELL
MARC K. JENKINS Nature 410, 101 - 105 (2001)
18
Although, these studies did not address the
phenotype of tissue-derived memory T cells with
respect to CD62L and CCR7, they have confirmed
the presence of antigen-specific memory T cells
in non-lymphoid compartments long after priming,
which supports the notion of an effector memory
subset of T cells.
What is the wider implication of these
studies? First, a clear distinction can be made
between the migration pathways of naïve T cells
and those of tissue memory T cells. Second,
rapid memory responses occur because
antigen-reactive cells are greatly expanded in
number and have redistributed to numerous
tissues to provide "frontline" immune protection.
But there is more to immunological memory than
simply an increase in the number of
antigen-reactive cells. These studies
demonstrated that memory T cells migrating to
nonlymphoid tissues can rapidly become effector
cells. A model was proposed in which the
tissue-homing effector memory T cells, which are
capable of immediate effector functions, could
rapidly control invading pathogens. TEM first
line of defense in tissues 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. TCM
reserve of defenses
19
The major question resulting from these findings
is how these two subsets of memory T cells are
generated. Three models of differentiation have
been proposed
Two subsets of memory T lymphocytes with distinct
homing potentials and effector functions
FEDERICA SALLUSTO, DANIELLE LENIG,
REINHOLD FÖRSTER, MARTIN LIPP
ANTONIO LANZAVECCHIA Nature 401, 708 - 712
(1999)
In vitro stimulation of naive T cells resulted in
the generation of both TCM and TEM cells, whereas
stimulation of TCM cells resulted in their
efficient differentiation to TEM cells. These
data were consistent with a linear
differentiation model in which naive T cells
differentiate first to TCM and then to TEM cells,
which were considered end-stage cells.
Naive? effectors?TCM? TEM
20
Migratory properties of naive, effector, and
memory CD8() T cells Weninger W, Crowley MA,
Manjunath N, von Andrian UH. J. Exp. Med. 194,
953-966 (2001)
According to Manjunath et al., the duration of
antigenic stimulation and the type and amount of
cytokines present during priming lead either to
fully differentiated effector cells that home to
peripheral tissues (blue) or to cells that are
devoid of effector function and home to lymph
nodes (green). In the system used by Manjunath
et al., these two cell types can be identified
according to the differential expression of the
T-GFP marker transgene and the lymph nodehoming
receptor CCR7. Both cell types are maintained in
the memory pool (dotted arrows) and, upon
secondary challenge, mediate immediate protection
in nonlymphoid tissues or secondary responses in
lymph nodes.
The repertoires of circulating human CD8 central
and effector memory T cell subsets are largely
distinct. Baron V., Bouneaud C., Cumano A.,
LimA., Arstila T.P., KourilskyP., FerradiniL.,and
Pannetier C. Immunity 18, 193-204 (2003)
Baron et al. analyzed the composition and
dynamics of the CD8 T cell repertoire of these
subsets within the peripheral blood of four
healthy individuals. Both subsets had largely
distinct and autonomous TCRV? repertoires. Their
composition remained stable over a 9 month
period, during which no cell passage between
these subsets was detected despite important
size variation of several clones. In one donor,
four out of six TCRV? clonotypes specific for
the influenza A virus were detected in the
central subset only, while the two others were
shared. Altogether, these observations suggest
that most effector memory T cells may not have
derived from the central memory subset.
21
Lineage relationship and protective immunity of
memory CD8 T cell subsets Wherry, J.E. et
all. Nature Immunol 4, 225-234 (2003)

• We will examine in details a recent publication
  which addresses the following points
• The lineage relationships between TCM and TEM
• Which memory T cell subsets has the greater
  capacity to persist long-term in vivo and undergo
• homeostatic proliferation
• Because TEM are located in non-lymphoid
  tissues, it has been proposed that they may
• represent a more effective population for
  protection from reinfection. They will directly
  compare
• in vivo the protective capacity of the two
  subsets of memory T cells.

Their tools Tetramers that allow the detection
of T cells specific for a peptide of gp30/LCMV
presented by Db. For some experiments they will
also use TCR transgenic T cells that are specific
for the same peptide of gp30/LCMV presented by
Db. These TCR transgenic T cells will be
transferred into normal mice.