Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity

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Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment? Complexities of Interpretation due to the Heterogeneity of Memory CD4 T Cells, Including T Follicular Helper Cells Shane Crotty1,2,3

1Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037 2Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), La Jolla, California 92037 3Department of Medicine, Division of Infectious Diseases, University of California, San Diego, La Jolla, California 92093 Correspondence: [email protected]

Plasticity is the ability of a cell type to convert to another cell type. There are multiple effector CD4 T-cell subtypes, including TH1, TH2, TH17, TH1 , CD4 CTL, TH9, and TFH cells. It is commonly thought that a CD4 T cell can readily show full plasticity—full conversion from one differentiated cell—and this propensity to plasticity is possessed by memory CD4 T cells. However, there remains no direct demonstration of in vivo–generated resting memory CD4 T-cell conversion to a different subtype on secondary antigen challenge in vivo in an intact animal at the single-cell level. What has been clearly shown is that CD4 T cells possess extraordinary capacity for phenotypic heterogeneity, but that is a distinct property from plasticity. Heterogeneity is diversity of the resting memory CD4 T-cell population, not conversion of a single differentiated cell into another subtype. Apparently, plasticity at the population level can be accomplished by either mechanism, as heterogeneity of CD4 T-cell subpopulations could affect large shifts in subtype distribution at the overall population level via differential exponential expansion and death.

GREAT DEBATES

What are the most interesting topics likely to come up over dinner or drinks with your colleagues? Or, more importantly, what are the topics that don’t come up because they are a little too controversial? In Immune Memory and Vaccines: Great Debates, Editors Rafi Ahmed and Shane Crotty have put together a collection of articles on such questions, written by thought leaders in these fields, with the freedom to talk about the issues as they see fit. This short, innovative format aims to bring a fresh perspective by encour- aging authors to be opinionated, focus on what is most interesting and current, and avoid restating introductory material covered in many other reviews. The Editors posed 13 interesting questions critical for our understanding of vaccines and immune memory to a broad group of experts in the field. In each case, several different perspectives are provided. Note that while each author knew that there were additional scientists addressing the same question, they did not know who these authors were, which ensured the independence of the opinions and perspectives expressed in each article. Our hope is that readers enjoy these articles and that they trigger many more conversations on these important topics.

Editors: Shane Crotty and Raﬁ Ahmed Additional Perspectives on Immune Memory and Vaccines: Great Debates available at www.cshperspectives.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032102 1 Downloaded from http://cshperspectives.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

S. Crotty

ere, plasticity is defined as the conversion of severely immunocompromised (e.g., T-cell- Ha single cell possessing a well-characterized deficient mice). Such experiments show that CD4 T-cell type into a cell no longer possessing CD4 T-cell plasticity can occur under extreme that phenotype and instead possessing a differ- conditions, but the experiments have no dem- ent well-characterized CD4 T-cell phenotype. onstrated relevance to what CD4 T cells actually For example, conversion of a memory TH1 cell do or experience in an intact animal. In con- þ þ 2 2 (T-bet IFN-g CXCR5 Bcl6 ) into a TFH cell trast, if transferred cells do maintain stability, (Bcl6þCXCR5þT-bet2IFN-g2) would be plas- those results are more credible, because they ticity. Separately, heterogeneity within a well- show stability of cell identity even when exposed characterized CD4 T-cell population is defined to nonphysiological stresses. Apparent plasticity here as a collection of varied phenotypes of differentiated CD4 T cells in vitro is generally (,100% of the cell population) linked by a not convincing, both because the in vitro exper- shared core phenotype. For example, heteroge- iments lack demonstrated in vivo relevance neity among TH1 cells can be observed by flow and because the experiments are performed cytometry or mass cytometry by defining TH1 at the cell population level, masking the impact cells as T-betþIFN-gþ cells and then observing of outgrowth of minor cell populations. The fractions of the population expressing tumor strictest criterion for demonstration of plastic- necrosis factor (TNF), or interleukin (IL)-2, ity is the use of a lineage marker reporter trans- or Blimp1, or IL-10, or Eomes, etc. As another genic mouse, tracking, over time, cells marked example, heterogeneity among TH2 cells can be irreversibly. Such an experiment directly estab- observed by flow cytometry or mass cytometry lishes the transcriptional history of a given cell. hi by defining TH2 cells as GATA3 cells and then Many lineage-tracking experiments have been observing fractions of the population express- performed on nTregs, making use of Foxp3- ing IL-5, IL-4, IL-13, CRTH2, CCR4, or IL-10, IRES-GFP/YFP/RFP-Cre-based designs (Rub- etc. As another example, heterogeneity among tsov et al. 2008, 2010; Zhou et al. 2009; Miyao germinal center (GC) TFH cells can be observed et al. 2012). The central conclusions from the by flow cytometry or mass cytometry by defin- two later studies with more sophisticated mod- þ þ ing GC TFH cells as Bcl6 CXCR5 cells and ified Foxp3 gene reporter constructs was that then observing fractions of the population ex- Foxp3þ nTregs are very stable, with almost no pressing CXCL13, IL-21, IL-4, or CXCR3, etc. plasticity (Rubtsov et al. 2010; Miyao et al. (Crotty 2014; Vinuesa et al. 2016). Heterogene- 2012). In contrast, substantial gene-expression ity at the whole population level further includes heterogeneity could be observed in conditions the range of differentiated CD4 T-cell subtypes of stress and while still maintaining core Foxp3þ present, including TH1, TH2, TH17, TH1 , CD4 nTreg programming. Still, the stability conclu- CTL, TH9, and TFH cells, and perhaps even sions drawn from such studies are not necessar- some form of “unbiased” TH0-type cells. Both ily directly transferrable for antigen-specific plasticity and heterogeneity must be described CD4 T-cell responses and CD4 T-cell memory, based on analyses at the single-cell level. because nTregs develop their initial program- Reports of T-cell program plasticity are un- ming during thymic development. convincing when the data are population-level changes in phenotypes. Such results can easily STABILITY DURING A PRIMARY RESPONSE be the outcome of outgrowth of a minor cell population to become the dominant cell popu- There are no lineage marker reporter mouse lation, or vice versa, particularly given the ex- studies showing plasticity of TH1, TH2, TH17, ponential proliferation that T cells are capable or TFH cells during a primary immune response of. Also unconvincing are the relevance of in an intact animal. Thus, excluding thymic- reports of cell program plasticity for which derived Tregs, there is no definitive evidence the central experiments are cell transfers into of physiologically relevant CD4 T-cell plasticity new hosts, particularly new hosts that are during a primary immune response. Cell-trans-

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Do Memory CD4 T Cells Keep Their Cell-Type Programming?

fer experiments have attempted to address clone can differentiate into multiple different stability or plasticity of antigen-specific CD4 T CD4 T-cell types (e.g., TFH and TH1) as they cells during a primary immune response. We divide during a primary immune response observed that TFH and TH1 cells during a viral (Tubo et al. 2013). Furthermore, those effector infection establish largely irreversible cell fates cells can then develop into memory TFH and TH1 by 72 h postinfection, based on cell transfers cells in frequencies comparable with the fre- of virus-specific TH1orTFH cells from virally quencies of TFH and TH1 cells generated by infected mice into time-matched virally infect- that clone during the effector phase of the CD4 ed mice (Choi et al. 2013). Similar pronounced T-cell response (Tubo et al. 2016). Human T-cell cell-fate commitment results were indepen- receptor (TCR) sequencing clonotype analysis of dently reported using a protein immunization antigen-specific human memory CD4 T cells has and an RFP-Bcl6 reporter mouse strain when shown that a given TCR sequence can be found 2 2 þ þ transferring CXCR5 Bcl6 or CXCR5 Bcl6 in TH1, TH2, and TH17 antigen-specific central cells at day 7 postinfection (Liu et al. 2012). memory cells (Becattini et al. 2015), consistent Plasticity of TH1 and TH2 cells to become TFH with the mouse model observation. cells has been reported; however, those experi- During a primary immune response, it ments used in vitro–generated TH1 and TH2 has been observed that TFH cells can have gene cells transferred into mice (Liu et al. 2012) or expression of other T-cell differentiation pro- in vitro polarized cells then repolarized under grams. In the context of a mouse with an acute different in vitro conditions (Lu et al. 2011). It is lymphocytic choriomeningitis virus (LCMV) almost certainly the case that there is a window infection, the mantle TFH cells (mTFH, outside of time early during effector CD4 T-cell differ- of GCs) and GC TFH cells express T-bet and entiation in a primary immune response when a interferon g (IFN-g) at substantially higher given CD4 T cell possesses pluripotency, simul- amounts than naı¨ve CD4 T cells (Johnston taneously expresses lineage-defining transcrip- et al. 2009; Yusuf et al. 2010; Ray et al. 2015). tion factors (e.g., Bcl6 and T-bet and RORgt) In our first paper on TFH cells, we stated “it is (Nakayamada et al. 2011; Oestreich et al. 2012), notable that T-bet and IFN-g were still ex- and maintains the capacity to respond to differ- pressed in the TFH in vivo, although at lower ent extrinsic signals and subsequently commit levels than in TH1/non-TFH LCMV-specific to one differentiated cell type (e.g., TFH or TH1 CD4 T cells. These observations are consistent or TH17) (DuPage and Bluestone 2016). Thus, with a model in which TFH cells follow their own simple questions regarding durable stability differentiation pathway but are not an isolated versus plasticity must be assessed after that lineage and can show partial characteristics of point, which is nontrivial to accomplish. TH1/TH2 polarization depending on environmental conditions.” Similar observations have been made for simian immunodeficiency virus STABILITY DURING TRANSITION FROM (SIV) infection of rhesus macaques (Iyer et al. EFFECTOR CELL TO MEMORY CELL 2015). Given that both LCMV and SIV infec- The transition from an effector CD4 T cell to a tions are extreme TH1-biased immune respons- central memory CD4 T cell appears to also be es, the presence of TH1 gene expression by TFH a transition from a cell with a highly polarized cells in LCMV, and SIV immune response may gene-expression program to a cell with a less represent uncommon exceptions. In support polarized gene-expression program. This may of that concept, human tonsillar GC TFH cells be key to understanding the apparent plasticity expressing TH1, TH2, or TH17 cytokines are of memory CD4 T cells, discussed below. rarely observed (Ma et al. 2009; Yu et al. 2009; Based on single-cell transfer studies in Dan et al. 2016; Havenar-Daughton et al. 2016). mouse model systems, most CD4 T-cell clones For example, ,1% of GC TFH cells produce IL- are capable of generating memory cells (Tubo 17 (Yu et al. 2009; Wong et al. 2015; Dan et al. et al. 2016), and a given individual CD4 T-cell 2016). Mouse GC TFH cells rarely produce IL-

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13, even under very strong TH2 polarizing hel- then become derepressed in some cells in the minth infection conditions (Liang et al. 2012), absence of Bcl6 protein, resulting in the central or house dust mite sensitization (Ballesteros- memory TFH cell acquiring a partially mixed Tato et al. 2016) (TFH cells normally express TFH/TH1 phenotype (Fig. 1, model 1). Such IL-4 as part of canonical TFH programming, an event may be considered “imprinted partial distinct and independent of TH2 programming, plasticity” because the phenotype of the cell and thus TFH expression of IL-4 is not an indi- would be dependent on the initial signals it re- cation of any TH2 gene programming [Crotty ceived during T-cell priming, even if the gene- 2011]). Furthermore, Bcl6 represses many TH1, expression program was kept silent for long TH2, and TH17 genes and can prevent TH1 dif- periods of time. If the cell were to subsequently ferentiation (Johnston et al. 2009; Oestreich become a TH1 cell and lose TFH characteristics, et al. 2012; Hatzi et al. 2015). A counterargu- that would be “imprinted full plasticity.” This ment can now be made that cytokine expression concept has not been directly tested. by intracellular cytokine staining is insufficient- A second possibility (model 2) is that a GC ly sensitive to determine whether a GC TFH cell TFH cell that does not have any TH1, TH2, or may possess TH1, or TH2, or TH17 gene expres- TH17 gene expression or imprinting may be in- sion, because GC TFH cells are intrinsically duced to activate a partial TH1, TH2, or TH17 stingy cytokine producers, and intracellular gene-expression program if there are TH1, TH2, cytokine staining missed 98% of human or or TH17 differentiation inductive signals still macaque antigen-specific GC TFH cells (Dan present in the environment when the GC TFH et al. 2016; Havenar-Daughton et al. 2016). cell is transitioning to become a memory cell Thus, single-cell RNAseq of GC TFH cells may and losing Bcl6 protein expression (Fig. 1). be required to better understand whether GC Such an event would be CD4 T-cell plasticity, TFH cells with partial TH1, TH2, or TH17 het- termed “de novo partial plasticity” here, to dis- erogeneity characteristics are common or rare. tinguish it from imprinted plasticity. Considering the process from the opposite Alternatively, a substantial proportion of direction, it is clear that human memory TFH memory TFH cells may be generated very early cells can show certain phenotypic markers com- during an immune response derived from man- monly associated with TH1, TH2, or TH17 cells tle TFH cells (mTFH) without going through a (discussed more in the next section). What is GC TFH cell stage. Evidence for such a pathway the ontogeny of those cells? One possibility is comes from studies of Sh2d1a2/2 mice and that TFH cells can be imprinted with a fractional humans, which have CD4 T cells that can differ- amount of TH1 gene programming by an anti- entiate into mTFH cells but not GC TFH cells and gen-presenting cell during the early stages of have evidence of TFH cell memory (He et al. T-cell priming in response to a TH1 pathogen, 2013). If memory TFH cells are generated via and, although that gene-expression program such a pathway in immunocompetent mice is efficiently squelched by Bcl6 in the effector and humans, it is plausible that memory TFH mTFH and GC TFH progeny of that cell, a partial cells derived from mTFH cells may be less polar- TH1 program remains imprinted (Fig. 1). As a ized than memory TFH cells derived from GC GC TFH cell transitions into a memory cell, it TFH cells, with less fixed-fate programming, and loses expression of Bcl6 protein (Kitano et al. therefore may be more likely to have mixed at- 2011; Liu et al. 2012; Choi et al. 2013; Hale et al. tributes of TFH and TH1 or other programs 2013; Locci et al. 2013; Ise et al. 2014), thus (model 3). As is the case for models 1 and 2, derepressing a range of genes, including Ccr7 this concept has also not been directly tested. and Il7ra (Fig. 1) (Kitano et al. 2011; Choi Given the observations and models de- et al. 2013). One possibility is that the loss of scribed above, the presence of CXCR3þ þ Bcl6 protein as the GC TFH cell transitions into a CXCR5 memory CD4 T cells in human memory TFH cell can allow a partial TH1pro- peripheral blood is consistent with differentia- gram imprinted at the time of T-cell priming to tion models based on heterogeneity, imprinted

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Do Memory CD4 T Cells Keep Their Cell-Type Programming?

Model 1. Imprinted partial plasticity

CXCR5+PD-1+Bcl6hi CXCR5hiPD-1hiBcl6hi CXCR5+PD-1+Bcl6lo CXCR5+PD-1–/loBcl6lo CXCR5+PD-1+Bcl6hi IFN-γloCXCR3–T-betlo IFN-γ–CXCR3–T-betlo IFN-γ–CXCR3–T-betlo IFN-γ–CXCR3–T-betlo IFN-γ–CXCR3–T-betlo

DC + Ag

mT FH GC TFH Transitioning Memory 2° TFH TFH TFH

CXCR5+PD-1+Bcl6hi CXCR5hiPD-1hiBcl6hi CXCR5+PD-1+Bcl6lo CXCR5+PD-1–/loBcl6lo DC + T 1 + + + – – – + + + + + H IFN-γ CXCR3 T-bet IFN-γ CXCR3 T-bet IFN-γ CXCR3 T-bet IFN-γ CXCR3 T-betlo 2° T FH+ + stimuli ? CXCR5 Bcl6 IFN-γ–CXCR3–T-bet– + Ag 2° TFH1 CXCR5+Bcl6+ + + + mTFH1 GC TFH Transitioning Memory IFN-γ CXCR3 T-bet TFH1 TFH1 ? 2° T 1 = TH1 bias H CXCR5–Bcl6– IFN-γ+CXCR3+T-bethi

Model 2. De novo partial plasticity

Transitioning Memory T T mTFH GC TFH FH FH 2° TFH

+ Ag CXCR5+Bcl6+ IFN-γ–CXCR3–T-bet–

CXCR5+PD-1+Bcl6lo γ+ + + IFN- CXCR3 T-bet 2° TFH TH1 + + ? CXCR5 Bcl6 inflammatory IFN-γ–CXCR3–T-bet– environment + Ag 2° TFH1 CXCR5+Bcl6+ Transitioning Memory IFN-γ+CXCR3+T-bet+ ? TFH1 TFH1 2° TH1 CXCR5–Bcl6– IFN-γ+CXCR3+T-bethi

Model 3. Early generated memory cells have greater plasticity

Transitioning Memory

mTFH GC TFH TFH TFH 2° TFH + Ag

? 2° TFH + Ag 2° T 1 Transitioning Memory ? FH TFH TFH

2° TH1

Figure 1. Three models of the development of heterogeneous or plastic CD4 T-cell memory. Each model is discussed in the main text.

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partial plasticity, or de novo partial plasticity. MEMORY CD4 T-CELL PHENOTYPE CXCR3 is expressed by TH1 cells and is a direct HETEROGENEITY AND STABILITY DURING target of T-bet, and the CXCR3þ CXCR5þ RESTING MEMORY memory CD4 T cells in human peripheral blood cells are almost all capable of producing Resting memory CD4 T cells appear to be largely IFN-g on stimulation (Morita et al. 2011; stable over time by major lineage-defining phe- Bentebibel et al. 2013; Locci et al. 2013; notypic markers, as shown for antigen-specific þ Obeng-Adjei et al. 2015). Thus, CXCR3 TFH,TH1, and TH2 cells in mouse models CXCR5þ memory CD4 T cells could have (Harrington et al. 2008; Hale et al. 2013; þ potentially derived from CXCR3 GC TFH cells Hondowicz et al. 2016; Tubo et al. 2016). Human 2 or mTFH cells (Iyer et al. 2015), or CXCR3 GC data support the same conclusion but the anti- TFH cells or mTFH cells that were exposed to gen-specific data are limited (Locci et al. 2013; aTH1 environment while transitioning to Bancroft et al. 2016; Da Silva Antunes et al. memory TFH cells. 2017). No longitudinal data on antigen-specific CD4 T-cell biology usually does not fit tidy resting memory CD4 T-cell phenotypes from in- single pathway models; heterogeneity of pheno- dividual human donors are available at single- types and differentiation patterns are common, cell flow-cytometric resolution, which is much as this is likely important to confound pathogen needed to demonstrate memory CD4 T-cell sub- immune evasion strategies (Crotty 2012). Thus, set stability. a new report is surprising, but perhaps should TH17 cells may be an exception to memory. þ not have been. Development of TH2 cells (IL-5 There remains little evidence showing clear þ 2 IL-13 CXCR5 ) in the lungs in response to a demonstration of in vivo–generated TH17 second exposure to house dust mite antigens memory cells in mice. TH17 memory was absent was observed to be dependent on effector TFH in intact mice in one longitudinal antigen-spe- cells, with multiple lines of evidence pointing to cific CD4 T-cell study (Pepper et al. 2010). A þ hi differentiation of GC TFH cells (CXCR5 PD-1 later paper reported TH17 memory, but it de- þ IL-21 ) into TH2 cells (Ballesteros-Tato et al. pended on transfer of in vitro–generated TH17 2016). These appeared to be fully differentiated cells (Muranski et al. 2011). Although an Il17a active GC TFH cells, to the best of the ability of lineage marking reporter mouse has been the authors to sort a pure cell population, with available for many years (Hirota et al. 2011), the previously stated caveats. In contrast, a there is a lack of publications on in vivo–gen- different group, using a different IL-21 reporter erated TH17 memory. In humans, presence of mouse, did not observe TFH cells to be precur- antigen-experienced TH17 cells to Candida al- þ þ þ sors to TH2 cells (IL-33R IL-5 IL-13 )ina bicans has been clearly demonstrated (Zielinski similar house dust mite model (Coquet et al. et al. 2012); however, recurrent or continual 2015), but they did not gate on CXCR5þ cells exposure was not excluded, and a resting mem- 2 for the cell sorts. The two groups also trans- ory TH17 phenotype (e.g., Ki67 ) was not ferred cells at different times after the primary shown for C. albicans–specific cells. Thus, antigen exposure, which may result in tracking although the existence of resting stable memory cells at different points of cell-fate commitment. TH17 cells seems biologically reasonable and When TFH!TH2 plasticity occurred, the TFH there is indirectly supportive literature (Linden- cells were taken 6 d after the primary immuni- strøm et al. 2012; McGeachy 2013), data show- zations (Ballesteros-Tato et al. 2016). In neither ing antigen-specific resting memory TH17 cells study were resting memory CD4 T cells used with single-cell analysis are currently quite (cells without activation marker expression limited. taken at .30 d after the last antigen exposure). Although there has been evidence of hetero- Lineage-tracking models that do not depend on geneity within CD4 T-cell subsets, going back to cell transfers are likely to be the only means of early descriptions of TH1 and TH2 cells (e.g., resolving such disparate observations. TH2 cells producing some or all of IL-4, IL-5,

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Do Memory CD4 T Cells Keep Their Cell-Type Programming?

IL-13, and IL-10), the vastness of the dimen- defined. Given that TFH cells produce IL-4 in a sional space of memory T-cell phenotypic het- TFH-specific manner (independent of the TH2 erogeneity was first made evident by the human program) (Crotty 2011), identification of a memory CD8 T-cell mass spectrometry study of TFH2 cell requires demonstration of a resting Newell and Davis (Newell et al. 2012). There was memory CXCR5þ CD4 T cell capable of such phenotypic variety of memory CD8 T cells producing IL-5 and/or IL-13 at the single-cell to influenza and cytomegalovirus (CMV) that level—a criterion not reached in the current the authors did not even attempt to put a num- literature. Such cells may exist, but they have ber on the total range of memory CD8 T-cell not been shown directly, and they may be a phenotypes observed; instead, the diversity was minor fraction of the CXCR32CCR62 memory best calculated as large spaces of phenotypic TFH cells. There is clearly vast phenotypic het- variation in three-dimensional principal com- erogeneity among resting memory CD4 T cells, ponent analysis (PCA) plots (Newell et al. including resting memory TFH cells. However, 2012). Subsequent mass spectrometry analysis at the level of cytokine production, it is still of human CD4 T cells has shown even more unknown how rare memory TFH cells with cy- heterogeneity (Wong et al. 2015), consistent tokine production attributes of other CD4 T- with the diversity of TH1, TH2, TH17, TFH, cell subsets are, except for overlap between TH1 TH9, TH1 , CD4 CTL, and iTreg biology. Impor- and TFH programs in memory CD4 T cells, tantly, phenotypic diversity in human memory which has been clearly shown by multiple CD4 T cells is seen even at the level of individual groups. TCR clonotypes (Becattini et al. 2015). One example of heterogeneity is the presence or ab- PLASTICITY WHEN MEMORY CD4 T CELLS sence of PD-1 expression by resting memory ARE RECALLED? TFH cells (Locci et al. 2013). Heterogeneity is clearly present among chemokine receptor ex- A first study using an IL-21-GFP reporter pression by memory CD4 T cells. A population mouse observed extensive plasticity of IL-21- þ þ þ þ 2 þ of CCR6 CXCR5 resting memory CD4 T cells GFP CXCR5 TFH or IL-21-GFP CXCR5 is present in human peripheral blood, and it has cells after transfer into new hosts, with ,50% been suggested those cells represent “TFH17” of the memory cells maintaining CXCR5 ex- memory cells (Morita et al. 2011). However, pression, and a majority of the cells observed CCR6 expression is not specific to TH17 cells after recall by influenza infection were 2 and does not correlate well with TH17 program- CXCR5 (Lu¨thje et al. 2012). A newer IL-21 ming in many cases. One report found no IL- fluorescent protein reporter mouse model ob- þ þ + + 17a expression by stimulated CCR6 CXCR5 served robust stability of IL-21 CXCR5 TFH memory CD4 cells in single-cell analysis (Wong CD4 T cells after transfers into new hosts, but et al. 2015). Therefore, most CCR6 expression did not test memory time points (Weinstein by memory TFH cells may be unrelated to TH17 et al. 2016). In the context of an acute LCMV biology and simply reflective of preferential infection, memory CD4 T cells appear to largely chemotaxis needs. A similar situation exists maintain their TH1orTFH programming upon for “TFH2” memory cells. “TFH2” memory 2˚ response. TCR transgenic memory CD4 T CD4 T cells in human peripheral blood are cells with a resting TH1 phenotype all became hi lo 2 commonly defined only on the basis of negative effector TH1 cells (T-bet Bcl6 CXCR5 markers (CXCR32CCR62) (Morita et al. 2011; Granzyme Bþ) when transferred into a new Jacquemin et al. 2015), based on the assump- host that was then infected with LCMV. In the tion that all TFH memory cells must have a TH1, same study, the major of memory CD4 T cells TH2, or TH17 bias (which has certainly not with a resting TFH phenotype became effector been demonstrated at the single-cell level for TFH and GC TFH cells on rechallenge (T- lo þ þ 2 GC TFH cells or resting memory TFH cells); bet Bcl6 CXCR5 Granzyme B ). A fraction thus, the concept of TFH2 cells remains poorly of the memory TFH cells did lose CXCR5 in

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S. Crotty

the 2˚ response, but because those experiments CONCLUDING REMARKS depended on cell transfers, it is unknown whether all of the memory TFH cells would There remains no direct demonstration of in have maintained their TFH program on 2˚ chal- vivo–generated resting memory CD4 T-cell lenge under physiological conditions in an in- conversion to a different subtype on secondary tact animal where they were allowed to maintain antigen challenge in vivo in an intact animal at their normal localization. It is, of course, also the single-cell level. Lineage tracing experiments formally possible that the memory cells would are needed to directly test whether plasticity have showed even more plasticity if studying in occurs and, more importantly, how common an intact host. Another longstanding challenge or rare the process is under physiological con- with sorted cells that have high proliferative ca- ditions. Unfortunately, proper lineage-tracking pacity such as lymphocytes is that even a 1% genetic models are difficult to generate for TFH, 2 CXCR5 contamination of sorted cells could TH17, and TH2 cells. The lineage-defining subsequently expand extensively during the ex- transcription factors for TFH,TH17, and TH2 ponential proliferation following 2˚ challenge, cells are Bcl6, RORgT, and GATA3. GATA3 is thus confounding cell-fate interpretations of constitutively expressed by all CD4 T cells; TH2 cell-transfer experiments. cells are defined by high GATA3 expression. Resting central memory CD4 T cells show Thus, a conventional GATA3 lineage reporter less polarized features than actively responding would mark all T cells. Therefore, development effector cells. Resting central memory TH1or of a high-fidelity TH2 lineage genetic marker is TFH cells have less active transcription and a difficult challenge. A successful TH2 lineage protein expression of many signature features genetic marker would probably need to de- of activated TH1 cells or activated GC TFH cells. pend on transcription from a gene or locus For example, central memory TH1 cells express other than GATA3. Bcl6 is highly expressed low levels of T-bet compared with effector cells, by thymocytes, and thus a Bcl6 lineage report- and resting memory TFH cells express Bcl6 but er mouse constructed based on standard de- at levels indistinguishable from other central signs would be expected to mark most T cells. memory cells. Intuitively, one expects such RORgt is also expressed by thymocytes and, cells to be prone to plasticity. Programmed thus, an RORgt lineage reporter mouse based chromatin modifications may prevent plasticity. on standard designs would be expected to Memory TFH cells rapidly up-regulate Bcl6 in mark most T cells. Therefore, a successful vivo on restimulation (Ise et al. 2014). It is un- TFH or TH17 lineage genetic marker would known whether memory TFH cells with mixed probably need to depend on transcription þ þ TFH –TH1 phenotypes (CXCR3 CXCR5 ) from a gene or locus other than GATA3; IL- 2 þ þ differentiate into TFH (CXCR3 CXCR5 Bcl6 17a is one candidate for TH17 cells (Hirota 2 þ þ þ þ T-bet ), TFH1 (CXCR3 CXCR5 Bcl6 T-bet ), et al. 2011). CXCR5 may be a good candidate þ and/or conventional TH1 cells (CXCR3 for TFH cells. As for TH1 cells, a T-bet lineage CXCR52Bcl62T-betþ) in vivo in an intact reporter should be useful, but a key caveat is mouse or human (Fig. 1). Evidence of plasticity that transient expression of T-bet early on by a was not observed in humans at the level of the naı¨ve CD4 T cell after activation is common overall CD4 T-cell response to pertussis, where- and may have no influence on the future his- in the whole-cell pertussis vaccine and the acel- tory of the cell. The same caveat applies for all lular pertussis vaccine elicit predominantly TH1 constitutively active lineage marker systems, and TH2 polarizing responses, respectively, and and contributed to controversy over the ontog- reimmunization with the acellular pertussis eny of “exTregs.” Transient expression of Foxp3 vaccine elicits a CD4 T-cell recall response by some cells may result in erroneous conclu- with the same TH1orTH2 characteristics for sions on the basis of very transient Cre expres- whichever vaccine the individual was initially sion (Rubtsov et al. 2008; Zhou et al. 2009; immunized (Bancroft et al. 2016). Miyao et al. 2012). This concern may be

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Do Memory CD4 T Cells Keep Their Cell-Type Programming?

avoided by using estrogen-responsive Cre pro- memory CD4þ T cell clones primed by pathogens or tein (Rubtsov et al. 2010). vaccines. Science 347: 400–406. Bentebibel SE, Lopez S, Obermoser G, Schmitt N, Mueller As an alternative to lineage-tracking genetic C, Harrod C, Flanõ E, Mejias A, Albrecht RA, Blanken- markers, single-cell transfers into infection- ship D, et al. 2013. Induction of ICOSþCXCR3þCXCR5þ matched hosts may be the best test of stability TH cells correlates with antibody responses to influenza versus plasticity in a recall response. Such ex- vaccination. Sci Transl Med 5: 176ra32. Choi YS, YangJA, Yusuf I, Johnston RJ, Greenbaum J, Peters periments would need to show that transferred B, Crotty S. 2013. Bcl6 expressing follicular helper CD4 cells (not using single-cell transfers) would lo- T cells are fate committed early and have the capacity to calize in the new host to the same regions of form memory. J Immunol 190: 4014–4026. lymph node (LN) and spleen as untransferred Coquet JM, Schuijs MJ, Smyth MJ, Deswarte K, Beyaert R, Braun H, Boon L, Karlsson Hedestam GB, Nutt SL, Ham- antigen-specific resting memory CD4 T cells of mad H, et al. 2015. Interleukin-21-producing CD4þ that subtype (e.g., proper microanatomical lo- T cells promote type 2 immunity to house dust mites. Immunity 43: 318–330. calization of memory TFH cells posttransfer). Crotty S. 2011. Follicular helper CD4 T cells (TFH). Annu In the end, it is unknown whether the ap- Rev Immunol 29: 621–663. pearance of plasticity by memory CD4 T cells at Crotty S. 2012. The 1-1-1 fallacy. Immunol Rev 247: 133– the population level is predominantly because of 142. heterogeneity and outgrowth of subpopulations Crotty S. 2014. T follicular helper cell differentiation, func- or predominantly attributable to plasticity. tion, and roles in disease. Immunity 41: 529–542. Dan JM, Lindestam Arlehamn CS, Weiskopf D, dda Silva It remains possible that true memory CD4 Antunes R, Havenar-Daughton C, Reiss SM, Brigger M, T-cell plasticity may be primarily of interest for Bothwell M, Sette A, Crotty, S. et al.2016. A cytokine- purely academic reasons, as a memory recall independent approach to identify antigen-specific human germinal center T follicular helper cells and rare response with apparent plasticity at the whole- antigen-specific CD4þ T cells in blood. J Immunol 197: cell population level could presumably be ac- 983–993. complished via rapid outgrown of a very minor Da Silva Antunes R, Paul S, Sidney J, Weiskopf D, Dan JM, population of memory cells. Because essentially Phillips E, Mallal S, Crotty S, Sette A, Arlehamn CSL. 2017. 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Do Memory CD4 T Cells Keep Their Cell-Type Programming?

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Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment?: Complexities of Interpretation due to the Heterogeneity of Memory CD4 T Cells, Including T Follicular Helper Cells

Shane Crotty

Cold Spring Harb Perspect Biol published online April 21, 2017

Subject Collection Immune Memory and Vaccines: Great Debates

Is There Natural Killer Cell Memory and Can It Be Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination?: Can Natural Killer Harnessed by Vaccination?: NK Cell Memory and and CD8 T Cells Switch Jobs? Immunization Strategies against Infectious Christine A. Biron and Marcus Altfeld Diseases and Cancer Joseph C. Sun and Lewis L. Lanier Is There Natural Killer Cell Memory and Can It Be Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination?: Vaccination Harnessed by Vaccination?: Natural Killer Cells in Strategies Based on NK Cell and ILC Memory Vaccination Megan A. Cooper, Todd A. Fehniger and Marco Harold R. Neely, Irina B. Mazo, Carmen Gerlach, et Colonna al. Is It Possible to Develop Cancer Vaccines to Is It Possible to Develop Cancer Vaccines to Neoantigens, What Are the Major Challenges, and Neoantigens, What Are the Major Challenges, and How Can These Be Overcome?: Neoantigens as How Can These Be Overcome?: Targeting the Vaccine Targets for Cancer Right Antigens in the Right Patients Haydn T. Kissick Stephen P. Schoenberger Is It Possible to Develop Cancer Vaccines to Which Dengue Vaccine Approach Is the Most Neoantigens, What Are the Major Challenges, and Promising, and Should We Be Concerned about How Can These Be Overcome?: Neoantigens: Enhanced Disease after Vaccination?: There Is Nothing New in Spite of the Name Only One True Winner Olivera J. Finn and Hans-Georg Rammensee Scott B. Halstead

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