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Seminars in Immunology 21 (2009) 53–61

Contents lists available at ScienceDirect

Seminars in Immunology

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D4 memory T cells: What are they and what can they do?

egan K.L. MacLeoda,∗, Eric T. Clambeya, John W. Kapplera,b,c, Philippa Marracka,c,d

Howard Hughes Medical Institute and Integrated Department of Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USAProgram in Biomolecular Structure, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USADepartment of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USADepartment of Biochemistry and Molecular Genetics, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA

r t i c l e i n f o a b s t r a c t

eywords:D4ytokineelpemory

rotection

Immunological memory provides the basis for successful vaccines. It is important to understand theproperties of memory cells. There is much known about the phenotype and functions of memory CD8T cells, less about memory B cells, while CD4 memory T cells have proved difficult to study. Differencesin the types of memory CD4 cells studied and the difficulties of tracking the small number of cells haveled to conflicting and unclear results. Here we discuss the different systems used to study CD4 memorycells and ask whether, and in what circumstances, memory CD4 cells could provide protection against

infections.

. Introduction

CD4 T cells play a central role in the immune system, coordi-ating both adaptive and innate responses. CD4 memory T cells,owever, have a much less well-defined role; in fact their very exis-ence is sometimes called into question [1–3]. Why is the subjectf CD4 memory still so controversial? One of the major reasons forhis is the variety of systems studied, and the different criteria usedo define CD4 memory T cells. Here we discuss the appropriatenessf a number of the systems used to study CD4 memory and, in lightf this, examine how much we really know about the elusive CD4emory T cell. Finally we would like to shift the focus away from

he question of how exactly we define a CD4 memory T cell to themportant, and sometimes over-looked—over-looked, question ofow memory CD4 T cells can provide protection against a varietyf pathogens.

Upon exposure to antigen, for example during an infection orfter vaccination, antigen specific T cells are activated, prolifer-te and differentiate into effector cells. The increased number of

ntigen specific cells with effector functions can act to clear thenfection, but then, however, the vast majority of these cells die. Theurviving cells are memory cells [4,5]. These memory cells can pro-ide protection or an enhanced response upon re-exposure to the

Abbreviations: BCG, Bacillus Calmette-Guérin; DC, dendritic cell; IFN�, interferonamma; IL, interleukin; LCMV, lymphocytic choriomeningitis virus; OVA, ovalbu-in; TB, tuberculosis; TCR Tg, T cell receptor transgenic; TGF�, transforming growth

actor beta; TNF�, tumor necrosis factor alpha; VSV, vesicular stomatitis virus.∗ Corresponding author. Tel.: +1 303 398 1308; fax: +1 303 270 2166.

E-mail address: macleodm@njc.org (M.K.L. MacLeod).

044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2009.02.006

© 2009 Elsevier Ltd. All rights reserved.

same pathogen or antigen. The enhanced response is a consequenceof two major changes following the initial exposure. First, althoughmost of the activated cells die following the first response, theremaining cells are present at higher frequencies than the originalnaïve T cell. This higher frequency of antigen specific cells increasesthe likelihood that any re-infection will be detected quickly, allow-ing the immune response to get underway before the pathogen hastime to spread.

The second difference between naïve and memory cells is thatmemory cells are able to make effector responses more rapidly thanprimary responding cells [6,7]. Depending on the type of infec-tion and the signals relayed from antigen presenting cells, such asdendritic cells (DCs), the responding T cells can make a range ofresponses. The number of known CD4 T cell subsets has grown inrecent years to include not just T helper (Th) 1 and Th2 cells but alsoTh17 cells and regulatory T cells (Treg) [8]. Th1 cells predominatelymake inflammatory effector cytokines such as interferon� (IFN�)and tumor necrosis factor� (TNF�) that are required for clearancesof viruses and intracellular bacteria [9,10]. Th2 cells can make anumber of interleukins (ILs) including IL4, IL5 and IL13 and areimplicated in protection against helminth parasites [9,10] but alsoplay a role in allergic responses [11]. The more recently identifiedTh17 cells are thought to be involved in protection against extra-celluar bacteria but also play a role in autoimmunity [12,13]. Tregcells can either be generated in the thymus (natural Tregs) or canbe generated during immune responses (inducible Tregs) [14]. The

properties and functions of Tregs have been extensively reviewedelsewhere [15,16] and so will not be discussed further in this review.

Memory cells can be distinguished from naïve cells based on anumber of cell surface molecules and these alterations are oftenused to define memory cells [4,5]. Whether these changes are

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ufficient to define memory cells is discussed below. Changesn cell surface molecules following T cell activation include thepregulation of the receptor for hyaluronate, CD44. This may allowctivated and memory T cells to enter inflamed peripheral siteshere an infection could be present [17]. In humans, changes in the

soforms of CD45 that are expressed are often used to differentiateaïve and memory cells with naïve cells expressing CD45RA andemory cells expressing CD45RO [5].T cell activation results in the downregulation of CD62L and

CR7, molecules that are required to enter lymph nodes and accesshe T cell area of the lymph node, respectively. While the changesn CD45 expression and the upregulation of CD44 are probably per-

anent changes, some memory cells re-express CD62L and CCR7.his has led to the description of two subsets of memory cells. Firstescribed in the peripheral blood of humans [18], T central mem-ry cells (TCM) cells are similar to naïve T cells in that they expressD62L and CCR7 and make IL2 following re-activation. T effectoremory cells (TEM) cells make effector cytokines (such as IFN� or

L4) and are less likely to traffic through lymph nodes due to theirow expression of CD62L and CCR7. Although there is evidence forhe existence of these subsets, in other reports the distinction is lesslear [19–25]. In mice, protection from re-infection appears to beest provided by TCM CD8 T cells [26]. Whether the same is true forD4 T cells is an area of debate [27].

. Not all memory cells are equal

Before we can understand what role CD4 memory T cells can playn protective secondary responses, it is important to have a clearefinition of what they are. Can the term memory be used loosely toescribe any T cell that does not have a naïve phenotype? Or, at thether end of the spectrum, is it critical to know the specificity of theCR and thereby have some understanding of what the stimulatingntigen and activation environment was?

CD4 memory cells have been difficult to study because, unlikeD8 T cells, they are not usually present in large numbers afterxposure to antigen [28,29]. Therefore CD4 memory cells are oftentudied in the form of “memory phenotype” cells from animals thatave not been intentionally immunized with antigen but whichevertheless bear the surface markers expected of memory cells,.g. high levels of CD44, low levels of CD62L and changes in the iso-orm of CD45 that are expressed (Table 1, [30–32]). These cells havehe advantages that they are available in reasonably large num-ers from unmanipulated individuals and can easily be isolated. Theajor disadvantages of using these cells is that their antigens are

nknown and, indeed, they may not have been created by reactionith antigen at all [33].

The proportion of memory phenotype CD4 and CD8 T cellsncreases with age in both mice and humans [30]. In mice kept inpecific pathogen free conditions the origin of these cells is partic-

able 1odels for studying memory CD4 T cells in mice.

odel Advantages

emory phenotype cells Plentiful and eCR Tg cells: activated in vitro Easily generat

CR Tg cells: activated by antigen + adjuvant in vivo. Easily generatenvironmen

ndogenous antigen-specific cells activated by antigen+adjuvant Genuine memand activation

emory CD4 T cells in mice can be studied using a number of different systems, which h

mmunology 21 (2009) 53–61

ularly unclear. Some of these cells may be a result of proliferation ofthe first T cells that leave the thymus and enter a lymphopenic envi-ronment [34], although proliferation in neonates does not alwayslead to the upregulation of CD44 [35]. Memory phenotype cellsmay also be generated as a result of exposure to environmentalantigens and in this case they may not have received the costimu-latory signals considered necessary to generate bona fide memorycells. Although memory phenotype cells can have properties sim-ilar to those of genuine memory cells [30–32], their unknownhistory makes any data based on them questionable. Humans areexposed to many more antigens, and this is more likely to occurin inflammatory settings. Therefore, human memory phenotypecells are likely to contain many antigen-elicited, genuine memorycells. Indeed, these cells do act like memory cells in many regards[36–39].

To circumvent the problem of the unknown specificity of mem-ory phenotype cells, T cells expressing a transgenic T cell receptor(TCR Tg) can be used (Table 1). Memory cells can be generated by thetransfer of small numbers of TCR Tg cells to wild-type mice that aresubsequently immunized or infected. However, if small numbersof TCR Tg cells are transferred, this does not solve the problem ofhow to detect and isolate the memory cells. To surmount this prob-lem, cells have either been transferred at very large frequencies,transferred into lymphopenic hosts, or transferred after activationin vitro [7,19,20,40,41]. A number of artifacts have recently beendescribed by several groups following the transfer of large num-ber of TCR Tg cells [42–45] demonstrating that this is not the mostuseful way to study memory T cells. The transfer of TCR Tg cellsto lymphopenic hosts provides a straight-forward way in whichto generate large numbers of memory cells that can easily be re-isolated. However, these cells are generated (regardless of whetherthey were activated in vivo or in vitro prior to transfer) and main-tained in very artificial environments.

The advent of both human and mouse MHC (major histocompat-ibility complex) class I and II tetramers have enabled the countingand phenotypic analysis of endogenous memory cells ex vivo. Firstgenerated by Altman and co-workers, MHC tetramers have revolu-tionized T cell immunology [46]. Class I tetramers are now widelyused; class II tetramers, on the other hand, have proved to be morechallenging both to produce, and to use to track the smaller numberof responding CD4 T cells. Several groups (including our own) havenow successfully produced and used human and mouse MHC classII tetramers to examine CD4 T cells and their responses [47–54].There are two major drawbacks to using MHC tetramers to trackT cell responses (Table 1). The first is that it is essential to know

the T cell epitope(s) from the protein(s) of interest, although thisis becoming easier as our knowledge of the binding patterns ofpeptides to the MHC improves [55,56]. The second hurdle is thatonce one epitope has been determined and MHC tetramers pro-duced, examining the T cell response to this one epitope may not

Disadvantages

asily identified. No known history of activation.ed. Not exposed to the normal cells and

signals during activation.ed, known antigen and activationt.

1. Transfer to wild-type mice: Rejectionby wild-type host, unusual phenotypeif used in large numbers, difficult todetect if used in small numbers.2. If transferred to lymphopenic mice:Survive in the absence of competition.

ory cells. Knowledge of antigenenvironment.

Difficult to detect by class II tetramersor by cytokine production. Need toknow major epitopes.

ave a variety of advantages and disadvantages.

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rovide a complete picture of a response, especially if a complexicroorganism is studied.In summary, we believe that there are certain criteria that must

e met for a memory cell to be considered a genuine memory cell.ost important is the knowledge of the specificity of the T cell and

he environment in which the T cell was activated. In experimentalnimal models, this means that the animal has been intentionallyxposed to an antigen or infection. That the T cell is activated in vivon an animal with a full lymphoid compartment is also key, ensuringhat the activation and subsequent generation of the memory cellccurs normally. In humans, genuine memory cells can be identi-ed with MHC tetramers that recognize antigen-elicited T cells, forxample from pathogens or vaccines to which the individual haseen exposed. In our eyes, there is little reason to continue to studyemory phenotype cells when it is clearly feasible to study genuineemory cells [47–54].

. The controversy

CD4 T cell memory has always been a controversial issue. Theontroversies cover a wide range of problems including: how mem-ry cells are generated (stochastic vs selected); how and to whatxtent (if any) they are maintained over time; how many subtypesxist; and what role (if any) they play in protecting the host frome-infection?

Zinkernagel has long argued that the presence of long-lived anti-en specific cells does not test the presence of protective memory,hat the only way to test for memory is with the use of survivalssays [1,2]. Zinkernagel and Hengartner propose that protections provided either by pre-existing neutralizing antibodies or by

cells that are “pre-activated,” a characteristic that requires theresence of persistent antigen [2]. Whether cells that are continu-usly exposed to antigen can be considered memory cells is morehan a question of semantics. Certainly cells that actively “see” theirntigen will have a different phenotype than cells not exposed tontigen. This persistent antigen may not necessarily be a good things CD4 and CD8 T cells exposed to antigen continuously can becomexhausted and/or anergic [57–59]. However, in some settings per-istent antigen may be important in the continual generation ofemory cells [59], or in the maintenance of certain memory cell

henotypes [60].Bell and Westermann have recently argued that the CD4 T cells

hat survive following an immune response cannot be consideredmemory” cells as they are not permanently altered by the activa-ion process either in terms of phenotype or function [3]. Rather,hey suggest that the “memory” response is just a function of thencrease in the precursor frequency of antigen specific cells aftern immune response and that these cells reside in the “naïve” Tell compartment. We and others have found the opposite to berue: long-lived antigen specific cells, identified by MHC class IIetramers, are CD44hi [47,48,61–63]. Bell and Westerman make thentriguing suggestion that re-expression of the heavily glycosylatedaïve isoform of CD45 prevents MHC tetramers from binding tond identifying “memory” cells with a naïve phenotype [3]. Thiseems unlikely as naïve T cells can be stained with MHC tetramers47,52,64]. Certainly antigen specific CD4 T cells can be identifiedor some time following an immune response but how well theyre maintained is an issue that has not yet been resolved.

By examining memory phenotype cells or memory TCR Tg cells,any characteristics of memory cells have been described. For

xample, it is clear that memory cells are activated more rapidly

han naïve cells, that they are more likely to make effector responsesnd require lower doses of antigen and costimulatory signals thanrimary responding cells, and they are more likely to be found

n tertiary organs [7,19,65–67]. However, it is important to makehe distinction between intrinsic differences between memory and

mmunology 21 (2009) 53–61 55

naïve cells and the consequences of just having more cells specificfor a particular antigen. When using TCR Tg cells this problem canbe easily resolved as equal numbers of the naïve and memory cellscan be examined. However, as mentioned above, there are majorcaveats to experiments involving the transfer of large numbers ofTCR Tg cells [42–45].

Numerous convincing studies have demonstrated that memorycells are intrinsically different from naïve cells. For example, Chan-dok et al. showed that the levels of the molecules required for TCRsignaling are increased in memory CD4 T cells as compared to naïvecells, enabling the memory cells to respond more quickly to antigen[65]. Using cytokine reporter mice, Mohrs et al. showed that, follow-ing antigen-exposure, memory CD4 T cells expressed mRNA for theeffector cytokine, IL4, and upon rechallenge, these cells made IL4protein more quickly than primary responding cells [68]. These twostudies demonstrate two of the classic features of memory cells:they respond more rapidly and their response is dominated by pro-duction of effector cytokines. The third classic feature of memoryresponses is that they are larger than primary responses. We haverecently made the somewhat surprising observation that memoryCD4 T cells do not proliferate for as long a time as naïve T cellsas a consequence of changes in cytokine production [47], therebyexplaining the earlier peak response of memory cells. This may alsobe due to direct inhibition of memory responses as a consequence ofpresentation of peptide-MHC complexes by the responding mem-ory CD4 T cells to each other [69]. Therefore, the larger size ofmemory responses is dependent on their increased number, nottheir ability to produce more progeny. This means that, if memoryCD4 T cells decline over-time, the size of the recall response will beproportionally reduced.

4. The controversy continues: antigen and memory cellsurvival

The long-term survival (or absence thereof) of CD4 memory cellsis one of the memory CD4 field’s most problematic subjects. Forexample, whether antigen is required to maintain memory cellshas been a long-standing controversy. Initially, Gray and Matzingerfound that continued exposure to antigen was required for rat anti-gen specific memory CD4 cells to maintain their ability to help Bcells [70]. However, the finding that in vitro activated CD4 TCR Tgcells could survive following transfer to MHC class II deficient ani-mals, suggested that memory cells could survive in the absenceof TCR signals [71]. There are some major caveats to this latterstudy. Significantly, there are no other CD4 T cells in an MHC classII deficient host, so the transferred memory cells survived in anartificial environment in which there was no competition for sur-vival with other CD4 T cells, for example for the important cellsurvival cytokine, IL7 [31,72,73]. Moreover, it has now been shownthat although transferred CD4 memory cells can survive in MHCclass II deficient hosts, they are functionally impaired in the absenceof the tonic TCR signals provided by interactions with MHC II [74].Thus, in such experiments it is important to evaluate not just thepresence of memory cells but also their function.

Importantly, when examined in non-lymphopenic mice, antigenspecific CD4 memory T cells have been found to decline [47–49,63]and the numbers of specific CD4 T cells in humans decline fasterthan specific CD8 T cells or B cells [75,76]. These studies suggestthat, even if there is a specific survival signal for memory CD4 Tcells, the cells are unable to achieve a long-term survival advan-tage. A recent study by Hataye et al. made the intriguing suggestion

that memory cells not only require contact with MHC class II forsurvival but also compete for signals derived from this contact [43].This conclusion was reached from the finding that both naïve andmemory TCR Tg cells decline in wild-type hosts at much greaterrates following the transfer of large numbers of T cells compared

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ig. 1. Memory CD4 T cells could protect the host in a number of possible ways. Most by either making early effector cytokines to direct other cells; provide help to

o the transfer of very few cells. Duffy et al. reported the samehenomenon but in their case the faster decline of the same TCRg cells was caused by rejection by the wild-type host, with theransfer of more cells inducing an enhanced response against theransferred TCR Tg cells [77]. The lesson from these studies is thatata based solely on TCR Tg cells may contain pitfalls and any resultshould be confirmed using antigen specific endogenous memoryD4 T cells.

Although overt antigen may not be absolutely required for CD4emory cell survival, it may be important for the phenotype and

ocation of the cells. Cells in peripheral organs such as the lungr the peritoneal cavity have a different phenotype from cells in

ymphoid organs [63,66]. However, the migration of the memoryells to these peripheral sites, rather than persistent antigen, maye what causes the activated phenotype [66]. As Zinkernagel andengartner argue, antigen may be required to maintain certain Tell functions [2]. For example, the presence of persistent antigenn the draining lymph node caused T cells to maintain high levelsf ICOS and CXCR5, indicative of the T follicular helper subset [60].herefore, persistent antigen may have an important role to play inaintaining the helper activity of some T cells.In some cases CD4 cells are potentially constantly exposed to

ntigen, for example in chronic infections. Although the repeatedxposure to antigen can diminish the effector function of CD4 T cells59], IFN� producing CD4 cells have been shown to play an impor-ant, although as yet unclear, role in preventing re-activation of theerpes virus, �-herpes virus [78]. Whether these T cells, which arectively involved in an on-going immune response, can be calledemory cells rather than effector cells, is a debatable question.

. Can CD4 memory T cells provide protective responses?

Rather than define memory based on cell surface phenotype,erhaps a more useful way is by an alteration in the response fol-

owing a second exposure to the antigen; demonstrating function,ather than just counting numbers of cells. This leads to the moremportant question of whether memory CD4 T cells can provide arotective response. It is clear that the humoral response and CD8cell responses can be protective in both human and mouse infec-

CD4 T cells, re-activated by antigen exposure, expand and could act to protect thece the B cell or CD8 T cells responses; or possibly kill infected cells directly.

tion models. For example, protective antibody can be measuredmany years after infection or vaccination [75,76,79]. Similarly, CD8memory responses can often be examined by measuring ex vivocytotoxicity, or in the mouse by challenge infections [80,81].

There are several ways in which memory CD4 T cells could beprotective: (1) by making effector cytokines early in the responseand in large quantities, (2) by enhancing B cell responses, (3) byenhancing CD8 T cell responses and (4) by directly killing infectedcells (Fig. 1).

5.1. Early effector cytokines

Mycobacterium tuberculosis (TB) latently infects one third of theworld’s population and causes the death of around 2 million peopleevery year [82,83]. The current vaccine, BCG (Bacillus Calmette-Guérin), provides varying levels of protection [84] but is thoughtto act by generating IFN� producing CD4 T cells that activateinfected macrophages in the lung, enabling them to kill the bac-teria [85,86]. The presence of long-lived TB specific CD4 memorycells can be demonstrated by the tuberculin skin test that containssmall amounts of TB proteins and causes a type IV hypersensitivityreaction in vaccinated or infected individuals.

In mouse models of TB, CD4 memory cells provide a small levelof protection in mice either immunized with BCG or infected, thendrug-cured, with TB itself [87,88]. Although a difference in bacterialgrowth between normal and vaccinated mice is observed only afterday 15 of infection, protection correlates with an early increase intype 1 cytokine production (including TNF� and the chemokines IP-10 and MCP-1) and the presence of IFN� producing CD4 cells in thelungs [87]. Interestingly, this is not accompanied by a large increasein the number of IFN� producing cells in the lung, suggesting thatlarge numbers of antigen specific cells may not be needed for theprotective effect. It is perhaps not surprising, therefore, that a largenumber of in vitro activated TCR Tg cells were able to provide only

limited protection following transfer to mice subsequently infectedwith TB [89]. Goldsack and Kirman argue that IFN� production byCD4 cells is not sufficient to provide protection from TB and sug-gest that it is important to consider other protective mediators [83].For example, Khasder et al. have reported a critical role for IL17

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roducing CD4 cells in the amplification of the T cell response to TBn the lung [90].

A clearer role for CD4 produced IFN� has been shown in otheracterial models. IFN� producing CD4 cells generated by vaccina-ion with specific antigen and adjuvant provide protection fromither Chlamydia muridarum or Bacillus anthracis [91,92]. Althoughromising, in both studies the CD4 cells examined may well beffector rather than memory cells either because of the timeetween the last vaccination and the challenge [91] or because thexperimenters used an adjuvant that induces a depot of antigen92].

Th2 memory cells can also provide protection, for example inice first infected and then drug-cleared with the Helminth par-

site Heligmosomoides polygyrus [93]. In this case, the rapid IL4esponse by the memory CD4 T cells results in an increase of alter-atively activated macrophages in the gut that act as effector cells inarasite clearance. The rapid production of IL4 in the recall responseo H. polygyrus was elegantly demonstrated by Mohrs et al. whosed IL4 reporter mice to show that IL4 protein is made in the laminaropria just a few days after re-infection [68].

The protective role of cytokine producing memory CD4 T cellsn humans is much more difficult to demonstrate. IFN� and TNF�roducing vaccinia specific and IFN� producing measles specificD4 T cells can be found in individuals many years after immu-ization with the viruses [75,76]. However, protection from theseiruses is probably provided by antibody (reviewed in [94,95]).ore relevantly, a correlation was found between the presence

f IFN� producing CD4 T cells specific for a malaria protein and aeduced incidence of malaria, suggesting that these cells providedome protection to re-infection [96]. Th2 cells may be important inumans for protection against parasitic worms that reside in the gut97] and, although protection following drug-treatment from theelminth, Schistosoma mansoni, is correlated with Th2 responses98], natural protection appears to involve a mixed Th1/2 response99].

It has recently become clear that protective T cell responsesay depend more on the quality of the cytokine producing cell

ather than just on the presence of cells that can make the appro-riate cytokine response. Several recent studies have described theresence of “multipotent” cytokine producing memory CD4 T cells22,27,100,101]. These cells make a range of cytokines, such as IFN�,NF� and IL2 and often make higher levels of cytokine than single-ytokine producing cells. Importantly, the presence of multipotentytokine producing cells correlates with protection in both Leish-ania [100] and vaccinia [101] infection models in mice.These studies suggest that the critical factor for generating

rotective CD4 memory T cells would be to induce effector mem-ry cells making the appropriate cytokine response, IFN� for Th1esponses such as influenza or tuberculosis, IL4 for Th2 responsesuch as helminth infections. There had been some evidence thatytokine producing cells in the primary response could not developnto memory cells [40], suggesting that vaccines should aim to gen-rate TCM cells rather than TEM cells. However, two recent studiesave convincingly shown that cells making IFN� in the primaryesponse can survive into the memory pool [102,103] and cells thatave activated their IL4 locus during priming can also become mem-ry cells [104]. Therefore, it seems likely that vaccines should aimo induce T cells that can make the appropriate cytokine responseor the infection they are designed to protect against.

.2. Memory CD4 help for B cells

Most vaccines act by producing long-lived antibody producingells and the same may be true for the long-lived protection that isfforded by natural infections. It is clear that CD4 T cells are crit-cal for the majority of primary B cell responses, particularly to

mmunology 21 (2009) 53–61 57

protein antigens. By providing help via cytokine production (suchas the antibody isotype switch factors IL4 and IFN�) and cell sur-face molecules (e.g. CD40L and ICOS), CD4 T cells enable B cells toform germinal centers, class switch and undergo affinity matura-tion [105]. What is less clear is whether CD4 T cells (and in particularantigen specific memory CD4 T cells) are required for memory B cellresponses.

Various experiments and observations have demonstrated thatCD4 T cells are not required for the long-term maintenance of mem-ory B cells/and or antibody release by plasma cells. There is clearevidence that humoral immunity steadily persists for many yearswhile CD4 T cell memory declines [75,76,79]. Moreover, in HIVinfected individuals, humoral immunity to infections and vaccinesencountered prior to HIV infection, is maintained despite a declinein CD4 T cells [106,107].

To answer this question more directly, Vieira and Rajewskydepleted CD4 cells in previously immunized mice [108]. Althoughthe memory B cells persisted in the absence of T cells, they wereunable to make a secondary antibody response. These results sug-gested that CD4 T cells were required to help memory B cellsrespond to antigen.

In contrast to this finding, Hebeis et al. found that, following thetransfer of memory B cells to Rag knockout mice, B cells respondedequally to re-activation regardless of the presence of transferredimmune CD4 cells [109]. The explanation for this difference prob-ably lies in the different forms of antigen used in the two studies.While Vieira and Rajewsky used soluble antigen, Hebeis et al. usedparticulate antigen. In support of this, while Duffy et al. found thatmemory B cells needed CD4 T cell help to produce antibody inresponse to soluble ovalbumin (OVA) [110], in a separate study,memory B cells activated with OVA and alum (forming a partic-ulate antigen), did not require T cell help [111]. Surprisingly, Duffyet al. found that help could be provided by the transfer of the samenumber of naïve and memory TCR Tg cells, suggesting that memoryCD4 cells are no better than primary responding cells at helpingB cells [110]. However, this may be a consequence of the way inwhich the CD4 memory cells were generated in this study. Mem-ory cells were generated following the transfer of naïve cells intoSCID recipients that were subsequently immunized. Perhaps in thislymphopenic environment, the memory cells lose some of theirhelper activity, indeed, there is some evidence that memory CD4T cells require the presence of B cells in order to survive long term[112].

Although these studies examined the cellular requirements formemory B cell responses, the more relevant question is whethermemory CD4 T cells can provide help to B cells to produce neutral-izing antibodies during infection. Gupta et al. asked this questionby examining the antibody response in mice primed with oneserotype of vesicular stomatitis virus (VSV) and then challengedwith a second serotype [113]. This is an example of heterosubtypicimmunity in which the two serotypes shared common T cell epi-topes but required different antibody responses to neutralize them.Although the VSV specific CD4 memory T cells could enhance pri-mary antibody responses to a hapten that had been conjugated tothe virus, they were unable to enhance primary neutralizing anti-body responses. This result suggests that while the CD4 T cellswere capable of enhancing some antibody response, they wereunable to improve a useful antibody response to the neutralizingepitope.

The ability of CD4 T cells to mediate heterosubtypic immu-nity is a salient issue with the threat of a pandemic influenza

infection looming [114]. Protection from influenza is thoughtto be largely mediated by neutralizing antibodies to the twomain surface proteins, haemagglutinin and neuramindase [114].However, as a consequence of poor proof-reading by the viral poly-merases (antigenic drift) and the reassortment of viral genes from

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ifferent serotypes (antigenic shift), these molecules frequentlyhange, rendering the neutralizing antibodies potentially useless.eterosubtypic immunity has been demonstrated in mouse mod-ls of influenza infection such that mice previously exposed to oneubtype of virus have some level of protection from infection withdifferent subtype [115,116].

Although heterosubtypic immunity has not been convincinglyemonstrated in humans, there is some evidence that adultsxposed to previous influenza infections are less likely to fall sickith new influenza variants [117]. A number of CD4 epitopes have

een found in common between different subtypes of influenza,ven between recent variants and H5N1, the subtype that causesird ‘flu and has been predicted to be a potential cause of an

nfluenza pandemic [55,118,119]. However, the significance of iden-ifying these epitopes can be called into question if CD4 memorycells generated from a previous influenza infection are unable torovide protection from the new infection.

From early studies, heterosubtypic immunity to influenza washought to be mediated by both CD4 and CD8 T cells, although thisrotective effect was found to wane over time [115]. A more recenttudy by Rangel-Moreno et al. demonstrated that, in mice, hetero-ubtypic immunity relies on a number of mechanisms involvingD8 T cell and B cell responses [116]. Although not directly tested

n this study, the authors did not observe an enhanced primary B cellesponse to influenza in the presence of potentially cross-reactiveD4 memory T cells, suggesting that CD4 T cells may play, at most,minor role in heterosubtypic immunity to influenza.

It is theoretically possible for CD4 T cells to provide protectionrom influenza. Brown et al. have examined the ability of in vitroctivated CD4 TCR Tg cells to provide protection from influenzahallenge in mice [120]. The transfer of large numbers of the acti-ated cells could protect mice and the authors showed that thisas partly as a result of enhanced B cell responses as the abilityf the T cells to provide protection was reduced following transfero B cell deficient mice. It still remains to be demonstrated, how-ver, whether small numbers of antigen specific memory CD4 Tells could achieve the same level of protection.

A further concern about studies that use in vitro activated cellss that the range of T cell phenotypes generated in vivo may note reflected in this population. Although influenza virus induces aype 1 immune response, it may be more appropriate to generatefollicular helper memory cells if the aim is to enhance the B cell

esponse [121]. Moreover, TGF� producing memory T cells, ratherhan Th1 cells, may be required to enhance a protective IgA responseo influenza [122,123]. Therefore, any vaccine designed to generate

emory CD4 T cells with the intention of having them help B cells,ust consider what type of B cell is required to protect the host

rom the infection.

.3. Memory CD4 help for CD8 T cells

CD4 T cell help is not exclusively for B cells. Many studies havehown that CD4 T cell help improves the production and/or func-ion of CD8 memory T cells (reviewed in [124]). However, there doesot seem to be a requirement for antigen specific memory CD4 Tells [125]. A question that has been less well studied is whetheremory CD4 T cells can enhance a primary CD8 T cell response.

rawczyk et al. examined this question by first priming mice withCs loaded with the immunodominant CD4 T cell epitope from Lis-

eria monocytogenes, then challenging the mice with a recombinant. monocytogenes expressing OVA [126]. The presence of the CD4

emory cells resulted in an increase in the number of OVA-specific

D8 T cells and also significantly reduced the number of bacteriaound in the spleen following challenge. This study suggests thathe role CD4 memory T cells may play in protecting the host couldxtend beyond the obvious enhancement of antibody responses.

mmunology 21 (2009) 53–61

5.4. Killer CD4 T cells

Finally, CD4 T cells can act to reduce a secondary infection bydirectly killing infected cells. There are a number of mechanismsby which T cells can kill, including Fas-FasL interactions or via therelease of granzyme B and perforin [127]. Perforin-mediated killingis more generally thought to be a function of CD8 T cells, however,there are a number of studies that show that CD4 T cells can also killin this way. Early studies demonstrated killing by CD4 cells usingcells cloned from infected mice [128] or from humans [129,130].Although there is some concern that this could be an in vitro artifact[131], a study by Erb et al. demonstrated that cytotoxic CD4 T cellscould be found ex vivo [132].

More recent studies have examined cells directly ex vivo or invivo. Human memory CD4 T cells were found to express perforin inhealthy individuals and such cells were increased in HIV infectedindividuals [133]. These cells expressed surface markers indicativeof end-stage differentiation, suggesting that “killer” CD4 memorycells may be generated by a strong activation stimulus. However,there is no indication whether these cells were involved, let alonerequired, for protective immune responses. One indication thatthese cells could be protective comes from Adhikary et al., whoshowed that cytotoxic human Epstein Barr Virus specific CD4 clonescould control the virus in vitro [134].

In mice, the in vivo presence of cytotoxic CD4 T cells has beendemonstrated following infection with lymphocytic choriomenin-gitis virus (LCMV) [127]. These T cells could kill transferred cellsthat had been pre-pulsed with a CD4 LCMV epitope either via Fas-FasL interactions or by perforin. It is still unclear, however, whetherthese killer CD4 cells were required for, or play a role in, protection.Certainly LCMV specific memory CD8 T cells can clear the infec-tion rapidly in the absence of a memory CD4 T cell response [135].Perforin producing CD4 T cells have been shown to be partly respon-sible for the protection offered by the transfer of large numbers ofactivated CD4 T cells to wild-type hosts subsequently infected withinfluenza [120]. However, this effect was lost if fewer cells weretransferred. It remains to be determined, therefore, whether mem-ory CD4 T cells present at normal frequencies could act in this way.

6. Conclusions

That CD4 memory T cells exist seems clear. Present in an alteredstate and at higher frequencies following an immune response,these cells have the hallmarks of memory. Whether, as a popula-tion, they are long-lived is less clear, prompting the question of whywould the individual not want to maintain the CD4 memory T cellsthat have been generated following a successful immune response?Perhaps the simple answer to this question is that there has beenno selective pressure to preserve memory CD4 T cells, they are sim-ply not needed once long-lived protective antibody and CD8 T cellshave been generated. Certainly, if CD4 memory T cells can only pro-vide protective responses when present at high frequencies, theirpoor long-term survival is an area of important concern.

What is also not clear is what role CD4 memory T cells can play inprotective responses. CD4 T cells are central to all adaptive immuneresponses. It seems unlikely to us then, that memory CD4 T cells donot have an important role to play in at least some infections. Thelack of reliable and successful vaccines against some of the world’sdeadliest infections prompts a re-examination of how memory Tand B cells can offer protection to the host. Since CD4 T cells candirect so many other immune cells, they seem likely to be critical

mediators in protective immune responses. The big question for thememory CD4 T cell field now is not whether memory CD4 T cellsexist, but what CD4 memory T cells can do to protect infected orvaccinated individuals and, perhaps more importantly, to ask howmemory cells capable of protection can be effectively generated.

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M.K.L. MacLeod et al. / Semina

cknowledgements

We thank Frances Crawford for critical reading of theanuscript. This work was supported by NIH grants NIH AI-18785,I-22295, AI-52225.

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