MemoriesM. 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 Memory430 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+ 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

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 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 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)

Like antibodies TCRs might undergo“affinity

maturation”, (but not somatic mutation, at least not

often).

A Kinetic Basis For T Cell Receptor Repertoire Selection

During an Immune Response

Peter Savage1…Mark Davis, Immunity, Vol. 10, 485-492, April, 1999,

The basis for T cell antigen receptor (TCR) repertoire selection uponrepeated antigenic challenge is unclear. We evaluated the avidity and

dissociation kinetics of peptide/major histocompatibility complex (MHC)

tetramer binding to antigen-specific T lymphocytes isolated following

primary or secondary immunization. The data reveal a narrowing of the

secondary repertoire relative to the primary repertoire, largely

resulting from the loss of cells expressing TCRs with the fastestdissociation rates for peptide/MHC binding. In addition, T cells in the

secondary response express TCRs of higher average affinity for

peptide/MHC than cells in the primary response. These results provide a

link between the kinetics and affinity of TCR-peptide/MHC interactions

and TCR sequence selection during the course of an immune response.

Figure 3. Tetramer Staining Decay Kinetics for Primary or Secondary Populations Stained with MCC/I-Ek TetramerStaining 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 >14 weeks

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.

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 codonflanked 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.

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 CD2–STOP–PLAP granzyme-B–Cre mice. Mice were immunized with asingle intraperitoneal injection of 5 104 PFU of the Armstrong strain of LCMV. Splenocytes of uninfected (a) and infected doublytransgenic 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 PLAP- CD8+ 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+ PLAP- were sorted by FACS from LCMV-immune (infected >1 month earlier) doubly transgenic mice. 2 105 sorted cells were adoptively transferred into naive recipients and challengedthe 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.

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).

• 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.

Human PBL separated on the basis of CD45RA+(naïve) and RA- (memory)

CD45 RA expressed by human naïve T cells

CD45R0, not CD45RA, is expressed in memory T cells

Figure 2 CCR7+ and CCR7- memory T cells display different effector functions. a, b, The three subsets of CD4+ T cells were sortedaccording 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+

Figure 4 CCR7+ memory cells show enhanced responsiveness to T-cell receptor triggering and potently activate dendritic cells toproduce IL-12. a, Proliferative response of naive T cells (squares), CCR7 + (triangles) and CCR7- (circles) memory T cells to differentconcentrations of plastic-bound anti-CD3 monoclonal antibody in the absence (empty symbols) or in the presence (filled symbols) ofanti-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).