Transcript Two subsets of memory T cells

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.
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
•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.
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 CD8+ T cells. The longevity of memory T cells explain how they can confer long-term protective
immunity.
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.
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.
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 I–
restricted 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 (CD8IL15), become small, retain GFP, and fail to acquire cytotoxic function, although they
acquire IFNg–producing 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)
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)
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 susceptibilityof effector T cells to apoptosis and reduced
formation of memory T cells.
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.
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.
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)
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+
Memory
CCR7-
Naive
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)
Two subsets of memory T cells:
CCR7+ CD62Lhigh  Central memory T cells (CM)
CCR7- CD62Llow  Effector memory T cells (EM)
With functional differences:
TCM
TEM
 IL-2, little IFN-g, no perforin
 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.
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)
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.
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. E:T ratio was 200:1 for all tissues.
E:T 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 10 6 PFU of VSV-New
Jersey, rested >7 months, then infected with VSV-Indiana and rested an additional 224 days.
E:T ratio was 300:1 for all tissues. E:T 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)
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)
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
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
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 node–homing 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.
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.