TFH memory: More or less TFH?

Eur. J. Immunol. 2012. 42: 1977–1980 HIGHLIGHTSDOI: 10.1002/eji.201242755 1977C

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TFH memory: More or less TFH?

Tom Barr and David Gray

Institute of Immunology and Infection Research, School of Biological Sciences, University ofEdinburgh, Edinburgh, United Kingdom

Follicular T-helper (TFH) cells are a subset of CD4+ T cells that is specialized to helpB cells. Their capacity to give rise to enhanced TFH memory responses has not beendocumented. A study by Weber et al. [Eur. J. Immunol. 2012 42: 1981–1988] in this issueof the European Journal of Immunology addresses this question, and a picture is emergingfrom this and several other recent studies which suggests that the formation of memoryTFH and of central memory (TCM) cells are intimately bound and that the heterogeneityof what we currently call “TFH’’ cells clouds this issue. In this Commentary, we discussthese complexities and ask what memory TFH cells are doing given that the germinalcenters are a feature of primary, but not secondary immune responses.

Keywords: Bcl-6 � Central memory T cells (TCM) � CXCR5 � Follicular T-helper cells (TFH)

See accompanying article by Weber et al.

During their stay in secondary lymphoid tissues (e.g. spleen andlymph nodes), T and B cells segregate into largely separate com-partments, T zones and B zones (the latter also known as follicles).However, to bring about the cooperative interactions required togenerate most antibody responses, B and T cells need to meet.B cells can meet cognate T cells as they migrate through the outerT zone toward the B-cell follicle, and a prelude to a full-blownT-dependent B-cell response is the migration of antigen-specificT and B cells to the border area between the T zone and follicle [1](Fig. 1). This migration and the subsequent entry of some of theseT cells into B-cell follicles are caused by balancing the expres-sion levels of the chemokine receptors CCR7 and CXCR5 on Tcells [2]. The T cells that enter B-cell follicles are specialized tohelp B cells and have become known as follicular T-helper (TFH)cells. TFH are critical for the formation and function of germinalcenters (GCs) to bring about the generation of memory B cells andlong-lived plasma cells (reviewed by Crotty; [3]). Originally TFH

cells were defined as CXCR5+ CD4+ T cells that expressed otheractivation markers, such as ICOS or PD1, and crucially their sur-

Correspondence: Prof. David Graye-mail: [email protected]

vival was dependent on B cells (now known to be via ICOS-ICOSLsignals; [4]). Initially, a full CD4+ T-cell differentiation programwas not assigned to TFH cells; however, the demonstration thatthe transcriptional repressor Bcl-6 acts as a master regulator ofTFH-cell development and function has established TFH cells asa distinct T-cell lineage [5]. The question now arises that if TFH

cells are a lineage with specialized functions, should they not alsoexhibit the property of memory as other subsets (e.g. Th1 andTh2) do? Until now this has been unclear.

In this issue of the European Journal of Immunology, a studyby Weber et al. [6] seeks to address this. They have used two, sim-ilar TCR transgenic systems (OVA peptide-specific OT-II or LCMVpeptide-specific SMARTA mice) to generate and sort antigen-specific TFH and non-TFH effector cells. These effector cells weregenerated following transfer to, and peptide stimulation in, afirst adoptive host (Thy1-distinct). After primary antigen-specificexpansion (day 8) the transgenic T cells were sorted and trans-ferred to a second adoptive host, where they were left for 28 daysin the absence of any antigen before rechallenge. A schematic dia-gram of the experiments is provided in Fig. 2. Analysis at day 7postimmunization showed that the non-TFH cells survived and/orexpanded much better than TFH cells; however, TFH cells werereadily detectable. Phenotypic analysis of the TFH cells during theeffector phase (day 8 in draining lymph nodes in the first adoptive

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1978 Tom Barr and David Gray Eur. J. Immunol. 2012. 42: 1977–1980

Figure 1. Summary of possible memory outcomes for TFH and TFH -related cells, based on the papers discussed herein. All of the cells labeledas TFH (or TFR) express CXCR5. Primary TFH generation is extensively reviewed in [3]. The consensus is that dendritic cells are needed as APCs,while B cells provide survival/programming signals. It is presently unclear whether follicular mantle (non-GC) TFH cells and GC TFH cells aregenerated separately or if one derives from the other. The function of non-GC TFH cells is not known; they may support the extrafollicular plasmacell response [11]. However, we speculate that non-GC TFH cells contain precursors of TCM cells. 1See [4]. 2Speculation based on [4,9]. 3See [6,19].4See [14].

host) or memory phase (day 14 in spleens in the second adoptivehost; Fig. 2) revealed that in the memory phase, the TFH cells lostTFH markers, downregulating the transcription factor Bcl-6, alongwith key molecules PD1 and CXCR5. Upon rechallenge (in thesecond host) most of the transferred TFH cells rapidly reexpressedthese molecules as well as ICOS, IL-21, and CD40L. Interestingly,most of these molecules were also upregulated by the transferrednon-TFH cells upon rechallenge, the exception being CXCR5. As awhole, this suggests that once antigen is no longer available TFH

cells undergo significant contraction and that some survive to entera quiescent phase during which they lose expression of moleculesthat normally characterize them, in particular Bcl-6 and CXCR5.Upon antigen-specific reactivation, they regain the exact same TFH

phenotype, although the expression levels of many molecules dif-fer compared with those of primary TFH cells (e.g. CXCR5, Bcl-6,PD1, IL-21, CD40L are higher, ICOS is lower). These results sug-gest that TFH cells enter an antigen-independent memory phaseand can be reactivated by antigen (Fig. 1).

Are there caveats to this conclusion? As ever, yes there are.To enable detection of antigen-specific cells the authors haveused TCR transgenic T cells and transferred numbers that rep-resent at least tenfold higher frequencies than endogenous Ag-specific CD4+ T cells. Over a 28-day period without antigen in thefirst host, they observe a precipitous drop in donor cell numbers;

however, although we do not know the relative survival of TFH andnon-TFH cells, we do know that the number of TFH cells comparedwith that of non-TFH cells after immunization is almost tenfoldlower [6]. This might mean that TFH cells survive less well thannon-TFH cells and if the authors had waited longer than 28 days (arather short time in the life of a memory cell), the TFH cells mighthave disappeared completely. This would leave us with the oppo-site conclusion: TFH cells do not survive as memory cells. Whilethis study may not be entirely conclusive, it does show that TFH

cells survive (antigen free) longer than we previously believed andthat during this period, and upon meeting antigen, they behaveexactly like memory cells.

How does this fit with other work in this area? In humans, aCXCR5+ T-cell subset has been proposed to be a TFH-like mem-ory population [7,8]. In mice, MacLeod et al. [9], using I-Ab/GP66MHC tetramers to identify antigen-specific memory T cells 8 weeksafter LCMV infection, found that the memory population exhibitedincreased levels of TFH markers (CXCR5, Bcl-6, PD1, and ICOS).They felt, however, that this did not represent a TFH-cell mem-ory subset as a discrete population expressing high levels of 2or 3 of the TFH markers could not be identified [9]. The datapresented by Weber et al. [6] suggest that in the memory phasethe TFH cells largely lose their TFH markers. MacLeod et al. [9]went on to sort and transfer the CXCR5hi and CXCR5lo memory


Eur. J. Immunol. 2012. 42: 1977–1980 HIGHLIGHTS 1979

Figure 2. Schematic diagram of the experiments described by Weber et al. [6].

populations to adoptive hosts, followed by GP66-peptide immu-nization. They found that the CXCR5hi cells (compared withCXCR5lo cells) showed enhanced B-cell helper activity (measuredby antibody responses) and concluded that CXCR5hi T cells morereadily migrate to B-cell follicles and interact with B cells [9].There are clearly differences in methodology and execution ofthese two studies, but the outcomes could be reconciled by thefact that antigen supply might have differed during the memoryphase in each model; for instance, memory cells in LCMV-infectedmice [9] may be exposed for longer to low levels of antigen, thussustaining many of the activation markers.

The CXCR5hi memory cells described by MacLeod et al. [9]also expressed CCR7, and this comprises a population that hasbeen proposed to be central memory T (TCM) cells or precur-sors to TCM cells [4]. Thus, in an antigen-specific (MHC class IItetramer) Listeria infection model, Pepper et al. [4] show thatthe CXCR5+CCR7+TbetloPD1− cells are TCM memory precursors(giving rise on rechallenge to Th1 effector cells, more TCM cellsand some TFH cells). These cells, despite expressing CXCR5, arelocated in T zones, presumably as a result of CCR7 expression [4].They did not test in this study whether bona fide TFH cells(CXCR5+PD1+) had any capacity as memory cells following adop-tive transfer [4]. All of this leads us to consider a very importantmatter: the heterogeneity of TFH cells. Originally, the term “TFH

cells” was coined to describe the CD4+ T cells that are found inB-cell follicles. Subsequently, it became clear that there was aphenotypic and functional distinction between TFH cells in GCsand those in the follicular mantle [3]. Even more recently it has

become clear that GC TFH cells can be differentiated into TFH effec-tor and follicular regulatory T (TFR) cells [10], and also that cellswith the TFH phenotype are required to support the primary extra-follicular plasma cell response [11] (Fig. 1). Thus, a reevaluationof which cells are called TFH cells is sorely needed. Importantly,all of these populations are CXCR5+ and are Bcl-6 dependent [3].While the activities of TFH cells in the GC are broadly known, wehave barely any data to tell us what the TFH cells in the follicularmantle are doing there. These “TFH” cells may not be in the folliclesimply to help B cells, but may migrate there to prolong antigenpresentation and programming/survival events that are necessaryfor their entry into the memory pool (Fig. 1). We [12,13] andothers [14,15] have shown the dependency of CD4+ T-cell mem-ory on B cells. The recent work of Pepper et al. [4] takes thisfurther by showing that the generation of CXCR5+ TCM cells isB-cell, Bcl-6, and ICOS-dependent.

Although Pepper et al. [4] found that TCM cells did give rise toTFH cells , TCM cells were not as efficient in this respect as naıveT cells. The reason for this may be that the CXCR5+CCR7+PD1−

TCM cells, make mostly IL-2 upon reactivation [4] and this seemsto inhibit the differentiation of TFH cells [16]. It is also worthremembering that the GC reaction during the secondary responseis of a much lower magnitude [17,18] and it has been supposedthat memory B cells do not reenter GCs following reactivation.Thus, there may be little need for an enhanced memory (GC) TFH

response. In this issue, Weber et al. [6] do not test directly thefunction of their memory TFH cells, although these cells seem tomake more IL-4 and IL-21 (interestingly non-TFH memory cells


1980 Tom Barr and David Gray Eur. J. Immunol. 2012. 42: 1977–1980

also expressed more IL-21). In the last month, a paper from Nuttand colleagues [19] has added weight to the notion that TFH cellscan generate functional memory. In their study, Luthje et al. [19]use an IL-21-GFP reporter mouse and influenza infection to lookat the memory response after transfer of IL-21 (GFP) negative andpositive TFH cells. They found that both types of TFH cells gave riseto enhanced secondary TFH-cell populations, but that most of theIL-21 (GFP)+ cells arose from transferred IL-21(GFP)+ cells. As inthe primary response, the memory TFH cells responded with strongIFN-γ/IL-2 production, but importantly were also a major sourceof “conventional” (non-TFH) Th1 effector T cells. In these respectsthe TFH cells generate memory populations in much the same wayas any other T-cell subset, giving rise to both more of the same typeof effectors (in this case TFH cells), but also other types of effectorcells (e.g. Th1). In parallel with the data of Pepper et al. [4], theTFH memory cells also had a propensity to make IL-2 [19], possiblyindicating a reduced capacity to aid GC development.

Together, the results of Weber et al. [6] and Luthje et al. [19]both point clearly to the development of memory cells from theTFH subset. These TFH memory cells consist of populations thatreiterate the TFH program, but also ones that form “conventional”effector T-cell populations, indicating a degree of plasticity andpoly-functionality similar to that of TCM cells. The picture is notas clear as we might wish because, as Jenkins et al. show [4],the central memory T-cell population bears certain similaritieswith TFH cells (CXCR5, Bcl-6) and we need to be aware of theheterogeneity in what we call TFH cells and consequently, how wesort them. Finally, we need to know the function of the reactivatedTFH memory cells, if it is not to drive enhanced GCs.

Conflicts of interest: The authors declare no financial or com-mercial conflict of interest.

References

1 Garside, P., Ingulli, E., Merica, R. R., Johnson, J. G., Noelle, R. J. and Jenk-

ins, M. K., Visualization of specific B and T lymphocyte interactions in

the lymph node. Science 1998. 281: 96–99.

2 Haynes, N. M., Allen, C. D., Lesley, R., Ansel, K. M., Killeen, N. and Cyster,

J. G., Role of CXCR5 and CCR7 in follicular Th cell positioning and appear-

ance of a programmed cell death gene-1high germinal center-associated

subpopulation. J. Immunol. 2007. 179: 5099–5108.

3 Crotty, S., Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 2011.

29: 621–663.

4 Pepper, M., Pagan, A. J., Igyarto, B. Z., Taylor, J. J. and Jenkins, M. K.,

Opposing signals from the Bcl6 transcription factor and the interleukin-2

receptor generate T helper 1 central and effector memory cells. Immunity

2011. 35: 583–595.

5 Johnston, R. J., Poholek, A. C., DiToro, D., Yusuf, I., Eto, D., Barnett, B.,

Dent, A. L. et al., Bcl6 and Blimp-1 are reciprocal and antagonistic regula-

tors of T follicular helper cell differentiation. Science 2009. 325: 1006–1010.

6 Weber, J. P., Fuhrmann, F. and Hutloff, A., T follicular helper cells survive

as long-term memory cells. Eur. J. Immunol. 2012. 42: 1981–1988.

7 Chevalier, N., Jarrossay, D., Ho, E., Avery, D. T., Ma, C.S., Yu, D., Sallusto,

F. et al., CXCR5 expressing human central memory CD4 T cells and their

relevance for humoral immune responses. J. Immunol. 2011. 186: 5556–

5568.

8 Morita, R., Schmitt, N., Bentebibel, S. E., Ranganathan, R., Bour-

dery, L., Zurawski, G., Foucat, E., Dullaers, M. et al., Human blood

CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain

specific subsets that differentially support antibody secretion. Immunity

2011. 34: 108–121.

9 MacLeod, M. K., David, A., McKee, A. S., Crawford, F., Kappler, J. W. and

Marrack, P., Memory CD4 T cells that express CXCR5 provide accelerated

help to B cells. J. Immunol. 2011. 186: 2889–2896.

10 Linterman, M.A., Pierson, W., Lee, S.K., Kallies, A., Kawamoto, S.,

Rayner, T.F., Srivastava, M. et al., Foxp3+ follicular regulatory T cells

control the germinal center response. Nat Med. 2011. 17: 975–982.

11 Lee, S. K., Rigby, R. J., Zotos, D., Tsai, L. M., Kawamoto, S., Marshall, J. L.,

Ramiscal, R. R. et al., B cell priming for extrafollicular antibody responses

requires Bcl-6 expression by T cells. J. Exp. Med. 2011. 208: 1377–1388.

12 Barr, T.A., Brown, S., Mastroeni, P., and Gray, D., TLR and B cell recep-

tor signals to B cells differentially program primary and memory Th1

responses to Salmonella enterica. J Immunol. 2010. 185: 2783–2789.

13 van Essen, D., Dullforce, P., Brocker, T. and Gray, D., Cellular interactions

involved in Th cell memory. J. Immunol. 2000. 165: 3640–3646.

14 Linton, P. J., Harbertson, J. and Bradley, L. M., A critical role for B cells in

the development of memory CD4 cells. J. Immunol. 2000. 165: 5558–5565.

15 Whitmire, J.K., Asano, M.S., Kaech, S.M., Sarkar, S., Hannum, L.G.,

Shlomchik, M.J. and Ahmed, R., Requirement of B cells for generating

CD4+ T cell memory. J Immunol. 2009. 182: 1868–1876.

16 Ballesteros-Tato, A., Leon, B., Graf, B.A., Moquin, A., Adams, P.S., Lund,

F.E. and Randall, T.D., Interleukin-2 inhibits germinal center formation

by limiting T follicular helper cell differentiation. Immunity 2012. 36: 847–

856.

17 Hollowood, K. and Macartney, J., Cell kinetics of the germinal center

reaction–a stathmokinetic study. Eur. J. Immunol. 1992. 22: 261–266.

18 Liu, Y. J., Zhang, J., Lane, P. J., Chan, E. Y. and MacLennan, I. C., Sites of

specific B cell activation in primary and secondary responses to T cell-

dependent and T cell-independent antigens. Eur. J. Immunol. 1991. 21:

2951–2962.

19 Luthje, K., Kallies, A., Shimohakamada, Y., Belz, G., Light, A., Tarlinton,

D. M. and Nutt, S. L., The development and fate of follicular helper T

cells defined by an IL-21 reporter mouse. Nat. Immunol. 2012. 13: 491–498.

Abbreviations: GC: germinal center · TFH cell: follicular T-helper cell ·TCM cell: central memory T cell

Full correspondence: Prof. David Gray, Institute of Immunology andInfection Research, School of Biological Sciences, University ofEdinburgh, Edinburgh, United KingdomFax: +44-131-650-7322e-mail: [email protected]

See accompanying article:http://dx.doi.org/10.1002/eji.201242540

Received: 19/6/2012Revised: 26/6/2012Accepted: 26/6/2012


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TFH memory: More or less TFH?