do memory cd4 t cells keep their cell-type programming...

Do Memory CD4 T Cells Keep Their Cell-TypeProgramming: Plasticity versus Fate Commitment?Complexities of Interpretation due to theHeterogeneity of Memory CD4 T Cells,Including T Follicular Helper CellsShane Crotty1,2,3

1Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California 920372Scripps 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 effectorCD4 T-cell subtypes, including TH1, TH2, TH17, TH1�, CD4 CTL, TH9, and TFH cells. It iscommonly thought that a CD4 T cell can readily show full plasticity—full conversion fromone 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 CD4T-cell conversion to a different subtype on secondary antigen challenge in vivo in an intactanimal at the single-cell level. What has been clearly shown is that CD4 T cells possessextraordinary capacity for phenotypic heterogeneity, but that is a distinct property fromplasticity. Heterogeneity is diversity of the resting memory CD4 T-cell population, notconversion of a single differentiated cell into another subtype. Apparently, plasticity at thepopulation level can be accomplished by either mechanism, as heterogeneity of CD4 T-cellsubpopulations could affect large shifts in subtype distribution at the overall population levelvia differential exponential expansion and death.

GREAT DEBATES

What are the most interesting topics likely to come up over dinner or drinks with yourcolleagues? Or, more importantly, what are the topics that don’t come up because theyare a little too controversial? In Immune Memory and Vaccines: Great Debates, EditorsRafi Ahmed and Shane Crotty have put together a collection of articles on such ques-tions, written by thought leaders in these fields, with the freedom to talk about the issuesas 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 avoidrestating introductory material covered in many other reviews.

The Editors posed 13 interesting questions critical for our understanding of vaccinesand immune memory to a broad group of experts in the field. In each case, severaldifferent perspectives are provided. Note that while each author knew that there wereadditional scientists addressing the same question, they did not know who these authorswere, which ensured the independence of the opinions and perspectives expressed ineach article. Our hope is that readers enjoy these articles and that they trigger manymore 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

on August 11, 2019 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

mailto:[email protected]

mailto:[email protected]

http://www.cshperspectives.org



http://www.cshperspectives.org/site/misc/terms.xhtml

http://cshperspectives.cshlp.org/

Here, plasticity is defined as the conversion ofa single cell possessing a well-characterized

CD4 T-cell type into a cell no longer possessingthat phenotype and instead possessing a differ-ent well-characterized CD4 T-cell phenotype.For example, conversion of a memory TH1 cell(T-betþIFN-gþCXCR52Bcl62) into a TFH cell(Bcl6þCXCR5þT-bet2IFN-g2) would be plas-ticity. Separately, heterogeneity within a well-characterized CD4 T-cell population is definedhere as a collection of varied phenotypes(,100% of the cell population) linked by ashared core phenotype. For example, heteroge-neity among TH1 cells can be observed by flowcytometry or mass cytometry by defining TH1cells as T-betþIFN-gþ cells and then observingfractions of the population expressing tumornecrosis factor (TNF), or interleukin (IL)-2,or Blimp1, or IL-10, or Eomes, etc. As anotherexample, heterogeneity among TH2 cells can beobserved by flow cytometry or mass cytometryby defining TH2 cells as GATA3hi cells and thenobserving fractions of the population express-ing IL-5, IL-4, IL-13, CRTH2, CCR4, or IL-10,etc. As another example, heterogeneity amonggerminal center (GC) TFH cells can be observedby flow cytometry or mass cytometry by defin-ing GC TFH cells as Bcl6þCXCR5þ cells andthen observing fractions of the population ex-pressing CXCL13, IL-21, IL-4, or CXCR3, etc.(Crotty 2014; Vinuesa et al. 2016). Heterogene-ity at the whole population level further includesthe range of differentiated CD4 T-cell subtypespresent, including TH1, TH2, TH17, TH1�, CD4CTL, TH9, and TFH cells, and perhaps evensome form of “unbiased” TH0-type cells. Bothplasticity and heterogeneity must be describedbased on analyses at the single-cell level.

Reports of T-cell program plasticity are un-convincing when the data are population-levelchanges in phenotypes. Such results can easilybe the outcome of outgrowth of a minor cellpopulation to become the dominant cell popu-lation, or vice versa, particularly given the ex-ponential proliferation that T cells are capableof. Also unconvincing are the relevance ofreports of cell program plasticity for whichthe central experiments are cell transfers intonew hosts, particularly new hosts that are

severely immunocompromised (e.g., T-cell-deficient mice). Such experiments show thatCD4 T-cell plasticity can occur under extremeconditions, but the experiments have no dem-onstrated relevance to what CD4 T cells actuallydo or experience in an intact animal. In con-trast, if transferred cells do maintain stability,those results are more credible, because theyshow stability of cell identity even when exposedto nonphysiological stresses. Apparent plasticityof differentiated CD4 T cells in vitro is generallynot convincing, both because the in vitro exper-iments lack demonstrated in vivo relevanceand because the experiments are performedat the cell population level, masking the impactof outgrowth of minor cell populations. Thestrictest criterion for demonstration of plastic-ity is the use of a lineage marker reporter trans-genic mouse, tracking, over time, cells markedirreversibly. Such an experiment directly estab-lishes the transcriptional history of a given cell.Many lineage-tracking experiments have beenperformed on nTregs, making use of Foxp3-IRES-GFP/YFP/RFP-Cre-based designs (Rub-tsov et al. 2008, 2010; Zhou et al. 2009; Miyaoet al. 2012). The central conclusions from thetwo later studies with more sophisticated mod-ified Foxp3 gene reporter constructs was thatFoxp3þ nTregs are very stable, with almost noplasticity (Rubtsov et al. 2010; Miyao et al.2012). In contrast, substantial gene-expressionheterogeneity could be observed in conditionsof stress and while still maintaining core Foxp3þ

nTreg programming. Still, the stability conclu-sions drawn from such studies are not necessar-ily directly transferrable for antigen-specificCD4 T-cell responses and CD4 T-cell memory,because nTregs develop their initial program-ming during thymic development.

STABILITY DURING A PRIMARY RESPONSE

There are no lineage marker reporter mousestudies showing plasticity of TH1, TH2, TH17,or TFH cells during a primary immune responsein an intact animal. Thus, excluding thymic-derived Tregs, there is no definitive evidenceof physiologically relevant CD4 T-cell plasticityduring a primary immune response. Cell-trans-

S. Crotty

2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032102



fer experiments have attempted to addressstability or plasticity of antigen-specific CD4 Tcells during a primary immune response. Weobserved that TFH and TH1 cells during a viralinfection establish largely irreversible cell fatesby 72 h postinfection, based on cell transfersof virus-specific TH1 or TFH cells from virallyinfected mice into time-matched virally infect-ed mice (Choi et al. 2013). Similar pronouncedcell-fate commitment results were indepen-dently reported using a protein immunizationand an RFP-Bcl6 reporter mouse strain whentransferring CXCR52Bcl62 or CXCR5þBcl6þ

cells at day 7 postinfection (Liu et al. 2012).Plasticity of TH1 and TH2 cells to become TFH

cells has been reported; however, those experi-ments used in vitro–generated TH1 and TH2cells transferred into mice (Liu et al. 2012) orin vitro polarized cells then repolarized underdifferent in vitro conditions (Lu et al. 2011). It isalmost certainly the case that there is a windowof time early during effector CD4 T-cell differ-entiation in a primary immune response when agiven CD4 T cell possesses pluripotency, simul-taneously expresses lineage-defining transcrip-tion factors (e.g., Bcl6 and T-bet and RORgt)(Nakayamada et al. 2011; Oestreich et al. 2012),and maintains the capacity to respond to differ-ent extrinsic signals and subsequently committo one differentiated cell type (e.g., TFH or TH1or TH17) (DuPage and Bluestone 2016). Thus,simple questions regarding durable stabilityversus plasticity must be assessed after thatpoint, which is nontrivial to accomplish.

STABILITY DURING TRANSITION FROMEFFECTOR CELL TO MEMORY CELL

The transition from an effector CD4 T cell to acentral memory CD4 T cell appears to also bea transition from a cell with a highly polarizedgene-expression program to a cell with a lesspolarized gene-expression program. This maybe key to understanding the apparent plasticityof memory CD4 T cells, discussed below.

Based on single-cell transfer studies inmouse model systems, most CD4 T-cell clonesare capable of generating memory cells (Tuboet al. 2016), and a given individual CD4 T-cell

clone can differentiate into multiple differentCD4 T-cell types (e.g., TFH and TH1) as theydivide during a primary immune response(Tubo et al. 2013). Furthermore, those effectorcells can then develop into memory TFH and TH1cells in frequencies comparable with the fre-quencies of TFH and TH1 cells generated bythat clone during the effector phase of the CD4T-cell response (Tubo et al. 2016). Human T-cellreceptor (TCR) sequencing clonotype analysis ofantigen-specific human memory CD4 T cells hasshown that a given TCR sequence can be foundin TH1, TH2, and TH17 antigen-specific centralmemory cells (Becattini et al. 2015), consistentwith the mouse model observation.

During a primary immune response, ithas been observed that TFH cells can have geneexpression of other T-cell differentiation pro-grams. In the context of a mouse with an acutelymphocytic choriomeningitis virus (LCMV)infection, the mantle TFH cells (mTFH, outsideof GCs) and GC TFH cells express T-bet andinterferon g (IFN-g) at substantially higheramounts than naıve CD4 T cells (Johnstonet al. 2009; Yusuf et al. 2010; Ray et al. 2015).In our first paper on TFH cells, we stated “it isnotable that T-bet and IFN-g were still ex-pressed in the TFH in vivo, although at lowerlevels than in TH1/non-TFH LCMV-specificCD4 T cells. These observations are consistentwith a model in which TFH cells follow their owndifferentiation pathway but are not an isolatedlineage and can show partial characteristics ofTH1/TH2 polarization depending on environ-mental conditions.” Similar observations havebeen made for simian immunodeficiency virus(SIV) infection of rhesus macaques (Iyer et al.2015). Given that both LCMV and SIV infec-tions are extreme TH1-biased immune respons-es, the presence of TH1 gene expression by TFH

cells in LCMV, and SIV immune response mayrepresent uncommon exceptions. In supportof that concept, human tonsillar GC TFH cellsexpressing TH1, TH2, or TH17 cytokines arerarely observed (Ma et al. 2009; Yu et al. 2009;Dan et al. 2016; Havenar-Daughton et al. 2016).For example, ,1% of GC TFH cells produce IL-17 (Yu et al. 2009; Wong et al. 2015; Dan et al.2016). Mouse GC TFH cells rarely produce IL-

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

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032102 3



13, even under very strong TH2 polarizing hel-minth infection conditions (Liang et al. 2012),or house dust mite sensitization (Ballesteros-Tato et al. 2016) (TFH cells normally expressIL-4 as part of canonical TFH programming,distinct and independent of TH2 programming,and thus TFH expression of IL-4 is not an indi-cation of any TH2 gene programming [Crotty2011]). Furthermore, Bcl6 represses many TH1,TH2, and TH17 genes and can prevent TH1 dif-ferentiation (Johnston et al. 2009; Oestreichet al. 2012; Hatzi et al. 2015). A counterargu-ment can now be made that cytokine expressionby intracellular cytokine staining is insufficient-ly sensitive to determine whether a GC TFH cellmay possess TH1, or TH2, or TH17 gene expres-sion, because GC TFH cells are intrinsicallystingy cytokine producers, and intracellularcytokine staining missed �98% of human ormacaque antigen-specific GC TFH cells (Danet al. 2016; Havenar-Daughton et al. 2016).Thus, single-cell RNAseq of GC TFH cells maybe required to better understand whether GCTFH cells with partial TH1, TH2, or TH17 het-erogeneity characteristics are common or rare.

Considering the process from the oppositedirection, it is clear that human memory TFH

cells can show certain phenotypic markers com-monly associated with TH1, TH2, or TH17 cells(discussed more in the next section). What isthe ontogeny of those cells? One possibility isthat TFH cells can be imprinted with a fractionalamount of TH1 gene programming by an anti-gen-presenting cell during the early stages ofT-cell priming in response to a TH1 pathogen,and, although that gene-expression programis efficiently squelched by Bcl6 in the effectormTFH and GC TFH progeny of that cell, a partialTH1 program remains imprinted (Fig. 1). As aGC TFH cell transitions into a memory cell, itloses expression of Bcl6 protein (Kitano et al.2011; Liu et al. 2012; Choi et al. 2013; Hale et al.2013; Locci et al. 2013; Ise et al. 2014), thusderepressing a range of genes, including Ccr7and Il7ra (Fig. 1) (Kitano et al. 2011; Choiet al. 2013). One possibility is that the loss ofBcl6 protein as the GC TFH cell transitions into amemory TFH cell can allow a partial TH1 pro-gram imprinted at the time of T-cell priming to

then become derepressed in some cells in theabsence of Bcl6 protein, resulting in the centralmemory TFH cell acquiring a partially mixedTFH/TH1 phenotype (Fig. 1, model 1). Suchan event may be considered “imprinted partialplasticity” because the phenotype of the cellwould be dependent on the initial signals it re-ceived during T-cell priming, even if the gene-expression program was kept silent for longperiods of time. If the cell were to subsequentlybecome a TH1 cell and lose TFH characteristics,that would be “imprinted full plasticity.” Thisconcept has not been directly tested.

A second possibility (model 2) is that a GCTFH cell that does not have any TH1, TH2, orTH17 gene expression or imprinting may be in-duced to activate a partial TH1, TH2, or TH17gene-expression program if there are TH1, TH2,or TH17 differentiation inductive signals stillpresent in the environment when the GC TFH

cell is transitioning to become a memory celland losing Bcl6 protein expression (Fig. 1).Such an event would be CD4 T-cell plasticity,termed “de novo partial plasticity” here, to dis-tinguish it from imprinted plasticity.

Alternatively, a substantial proportion ofmemory TFH cells may be generated very earlyduring an immune response derived from man-tle TFH cells (mTFH) without going through aGC TFH cell stage. Evidence for such a pathwaycomes from studies of Sh2d1a2/2 mice andhumans, which have CD4 T cells that can differ-entiate into mTFH cells but not GC TFH cells andhave evidence of TFH cell memory (He et al.2013). If memory TFH cells are generated viasuch a pathway in immunocompetent miceand humans, it is plausible that memory TFH

cells derived from mTFH cells may be less polar-ized than memory TFH cells derived from GCTFH cells, with less fixed-fate programming, andtherefore may be more likely to have mixed at-tributes of TFH and TH1 or other programs(model 3). As is the case for models 1 and 2,this concept has also not been directly tested.

Given the observations and models de-scribed above, the presence of CXCR3þ

CXCR5þ memory CD4 T cells in humanperipheral blood is consistent with differentia-tion models based on heterogeneity, imprinted

S. Crotty




Model 2. De novo partial plasticity

= TH1 bias

DC

DC + TH1stimuli

mTFH GC TFH TransitioningTFH

MemoryTFH

Model 1. Imprinted partial plasticity

2° TFH

+ Ag

CXCR5+Bcl6+

IFN-γ+CXCR3+T-bet+mTFH1 GC TFH TransitioningTFH1

MemoryTFH1

2° TFH

+ Ag2° TFH1

2° TH1

CXCR5+PD-1+Bcl6hi

IFN-γ loCXCR3–T-betloCXCR5hiPD-1hiBcl6hi

IFN-γ–CXCR3–T-betloCXCR5+PD-1+Bcl6lo

IFN-γ–CXCR3–T-betloCXCR5+PD-1–/loBcl6lo

IFN-γ–CXCR3–T-betloCXCR5+PD-1+Bcl6hi

IFN-γ–CXCR3–T-betlo

CXCR5+PD-1+Bcl6hi

IFN-γ+CXCR3+T-bet+CXCR5hiPD-1hiBcl6hi

IFN-γ–CXCR3–T-bet–CXCR5+PD-1+Bcl6lo

IFN-γ+CXCR3+T-bet+CXCR5+PD-1–/loBcl6lo

IFN-γ+CXCR3+T-betlo

CXCR5–Bcl6–

IFN-γ+CXCR3+T-bethi

CXCR5+Bcl6+

IFN-γ–CXCR3–T-bet–

CXCR5+Bcl6+

IFN-γ+CXCR3+T-bet+

2° TFH

2° TFH1

2° TH1

2° TFH

2° TFH1

2° TH1

CXCR5–Bcl6–

IFN-γ+CXCR3+T-bethi

CXCR5+Bcl6+


mTFH GC TFH

Transitioning TFH

TH1inflammatoryenvironment

MemoryTFH 2° TFH

+ Ag

TransitioningTFH1

MemoryTFH1

CXCR5+Bcl6+


CXCR5+PD-1+Bcl6lo

IFN-γ+CXCR3+T-bet+

Model 3. Early generated memory cells have greater plasticity

mTFH GC TFH

Transitioning TFH

MemoryTFH

?

?

?

?

Transitioning TFH

MemoryTFH 2° TFH

+ Ag

+ Ag

+ Ag

?

?

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





partial plasticity, or de novo partial plasticity.CXCR3 is expressed by TH1 cells and is a directtarget of T-bet, and the CXCR3þ CXCR5þ

memory CD4 T cells in human peripheralblood cells are almost all capable of producingIFN-g on stimulation (Morita et al. 2011;Bentebibel et al. 2013; Locci et al. 2013;Obeng-Adjei et al. 2015). Thus, CXCR3þ

CXCR5þ memory CD4 T cells could havepotentially derived from CXCR3þ GC TFH cellsor mTFH cells (Iyer et al. 2015), or CXCR32 GCTFH cells or mTFH cells that were exposed toa TH1 environment while transitioning tomemory TFH cells.

CD4 T-cell biology usually does not fit tidysingle pathway models; heterogeneity of pheno-types and differentiation patterns are common,as this is likely important to confound pathogenimmune evasion strategies (Crotty 2012). Thus,a new report is surprising, but perhaps shouldnot have been. Development of TH2 cells (IL-5þ

IL-13þ CXCR52) in the lungs in response to asecond exposure to house dust mite antigenswas observed to be dependent on effector TFH

cells, with multiple lines of evidence pointing todifferentiation of GC TFH cells (CXCR5þPD-1hi

IL-21þ) into TH2 cells (Ballesteros-Tato et al.2016). These appeared to be fully differentiatedactive GC TFH cells, to the best of the ability ofthe authors to sort a pure cell population, withthe previously stated caveats. In contrast, adifferent group, using a different IL-21 reportermouse, did not observe TFH cells to be precur-sors to TH2 cells (IL-33Rþ IL-5þ IL-13þ) in asimilar house dust mite model (Coquet et al.2015), but they did not gate on CXCR5þ cellsfor the cell sorts. The two groups also trans-ferred cells at different times after the primaryantigen exposure, which may result in trackingcells at different points of cell-fate commitment.When TFH!TH2 plasticity occurred, the TFH

cells were taken 6 d after the primary immuni-zations (Ballesteros-Tato et al. 2016). In neitherstudy were resting memory CD4 T cells used(cells without activation marker expressiontaken at .30 d after the last antigen exposure).Lineage-tracking models that do not depend oncell transfers are likely to be the only means ofresolving such disparate observations.

MEMORY CD4 T-CELL PHENOTYPEHETEROGENEITY AND STABILITY DURINGRESTING MEMORY

Resting memory CD4 T cells appear to be largelystable over time by major lineage-defining phe-notypic markers, as shown for antigen-specificTFH, TH1, and TH2 cells in mouse models(Harrington et al. 2008; Hale et al. 2013;Hondowicz et al. 2016; Tubo et al. 2016). Humandata support the same conclusion but the anti-gen-specific data are limited (Locci et al. 2013;Bancroft et al. 2016; Da Silva Antunes et al.2017). No longitudinal data on antigen-specificresting memory CD4 T-cell phenotypes from in-dividual human donors are available at single-cell flow-cytometric resolution, which is muchneeded to demonstrate memory CD4 T-cell sub-set stability.

TH17 cells may be an exception to memory.There remains little evidence showing cleardemonstration of in vivo–generated TH17memory cells in mice. TH17 memory was absentin intact mice in one longitudinal antigen-spe-cific CD4 T-cell study (Pepper et al. 2010). Alater paper reported TH17 memory, but it de-pended on transfer of in vitro–generated TH17cells (Muranski et al. 2011). Although an Il17alineage marking reporter mouse has beenavailable for many years (Hirota et al. 2011),there is a lack of publications on in vivo–gen-erated TH17 memory. In humans, presence ofantigen-experienced TH17 cells to Candida al-bicans has been clearly demonstrated (Zielinskiet al. 2012); however, recurrent or continualexposure was not excluded, and a resting mem-ory TH17 phenotype (e.g., Ki672) was notshown for C. albicans–specific cells. Thus,although the existence of resting stable memoryTH17 cells seems biologically reasonable andthere is indirectly supportive literature (Linden-strøm et al. 2012; McGeachy 2013), data show-ing antigen-specific resting memory TH17 cellswith single-cell analysis are currently quitelimited.

Although there has been evidence of hetero-geneity within CD4 T-cell subsets, going back toearly descriptions of TH1 and TH2 cells (e.g.,TH2 cells producing some or all of IL-4, IL-5,

S. Crotty




IL-13, and IL-10), the vastness of the dimen-sional space of memory T-cell phenotypic het-erogeneity was first made evident by the humanmemory CD8 T-cell mass spectrometry study ofNewell and Davis (Newell et al. 2012). There wassuch phenotypic variety of memory CD8 T cellsto influenza and cytomegalovirus (CMV) thatthe authors did not even attempt to put a num-ber on the total range of memory CD8 T-cellphenotypes observed; instead, the diversity wasbest calculated as large spaces of phenotypicvariation in three-dimensional principal com-ponent analysis (PCA) plots (Newell et al.2012). Subsequent mass spectrometry analysisof human CD4 T cells has shown even moreheterogeneity (Wong et al. 2015), consistentwith the diversity of TH1, TH2, TH17, TFH,TH9, TH1�, CD4 CTL, and iTreg biology. Impor-tantly, phenotypic diversity in human memoryCD4 T cells is seen even at the level of individualTCR clonotypes (Becattini et al. 2015). One ex-ample of heterogeneity is the presence or ab-sence of PD-1 expression by resting memoryTFH cells (Locci et al. 2013). Heterogeneity isclearly present among chemokine receptor ex-pression by memory CD4 T cells. A populationof CCR6þCXCR5þ resting memory CD4 T cellsis present in human peripheral blood, and it hasbeen suggested those cells represent “TFH17”memory cells (Morita et al. 2011). However,CCR6 expression is not specific to TH17 cellsand does not correlate well with TH17 program-ming in many cases. One report found no IL-17a expression by stimulated CCR6þ CXCR5þ

memory CD4 cells in single-cell analysis (Wonget al. 2015). Therefore, most CCR6 expressionby memory TFH cells may be unrelated to TH17biology and simply reflective of preferentialchemotaxis needs. A similar situation existsfor “TFH2” memory cells. “TFH2” memoryCD4 T cells in human peripheral blood arecommonly defined only on the basis of negativemarkers (CXCR32CCR62) (Morita et al. 2011;Jacquemin et al. 2015), based on the assump-tion that all TFH memory cells must have a TH1,TH2, or TH17 bias (which has certainly notbeen demonstrated at the single-cell level forGC TFH cells or resting memory TFH cells);thus, the concept of TFH2 cells remains poorly

defined. Given that TFH cells produce IL-4 in aTFH-specific manner (independent of the TH2program) (Crotty 2011), identification of aTFH2 cell requires demonstration of a restingmemory CXCR5þ CD4 T cell capable ofproducing IL-5 and/or IL-13 at the single-celllevel—a criterion not reached in the currentliterature. Such cells may exist, but they havenot been shown directly, and they may be aminor fraction of the CXCR32CCR62 memoryTFH cells. There is clearly vast phenotypic het-erogeneity among resting memory CD4 T cells,including resting memory TFH cells. However,at the level of cytokine production, it is stillunknown how rare memory TFH cells with cy-tokine production attributes of other CD4 T-cell subsets are, except for overlap between TH1and TFH programs in memory CD4 T cells,which has been clearly shown by multiplegroups.

PLASTICITY WHEN MEMORY CD4 T CELLSARE RECALLED?

A first study using an IL-21-GFP reportermouse observed extensive plasticity of IL-21-GFPþ CXCR5þ TFH or IL-21-GFP2 CXCR5þ

cells after transfer into new hosts, with ,50%of the memory cells maintaining CXCR5 ex-pression, and a majority of the cells observedafter recall by influenza infection wereCXCR52 (Luthje et al. 2012). A newer IL-21fluorescent protein reporter mouse model ob-served robust stability of IL-21+ CXCR5+ TFH

CD4 T cells after transfers into new hosts, butdid not test memory time points (Weinsteinet al. 2016). In the context of an acute LCMVinfection, memory CD4 T cells appear to largelymaintain their TH1 or TFH programming upon2˚ response. TCR transgenic memory CD4 Tcells with a resting TH1 phenotype all becameeffector TH1 cells (T-bethiBcl6loCXCR52

Granzyme Bþ) when transferred into a newhost that was then infected with LCMV. In thesame study, the major of memory CD4 T cellswith a resting TFH phenotype became effectorTFH and GC TFH cells on rechallenge (T-betloBcl6þCXCR5þGranzyme B2). A fractionof the memory TFH cells did lose CXCR5 in





the 2˚ response, but because those experimentsdepended on cell transfers, it is unknownwhether all of the memory TFH cells wouldhave maintained their TFH program on 2˚ chal-lenge under physiological conditions in an in-tact animal where they were allowed to maintaintheir normal localization. It is, of course, alsoformally possible that the memory cells wouldhave showed even more plasticity if studying inan intact host. Another longstanding challengewith sorted cells that have high proliferative ca-pacity such as lymphocytes is that even a 1%CXCR52 contamination of sorted cells couldsubsequently expand extensively during the ex-ponential proliferation following 2˚ challenge,thus confounding cell-fate interpretations ofcell-transfer experiments.

Resting central memory CD4 T cells showless polarized features than actively respondingeffector cells. Resting central memory TH1 orTFH cells have less active transcription andprotein expression of many signature featuresof activated TH1 cells or activated GC TFH cells.For example, central memory TH1 cells expresslow levels of T-bet compared with effector cells,and resting memory TFH cells express Bcl6 butat levels indistinguishable from other centralmemory cells. Intuitively, one expects suchcells to be prone to plasticity. Programmedchromatin modifications may prevent plasticity.Memory TFH cells rapidly up-regulate Bcl6 invivo on restimulation (Ise et al. 2014). It is un-known whether memory TFH cells with mixedTFH–TH1 phenotypes (CXCR3þCXCR5þ)differentiate into TFH (CXCR32CXCR5þBcl6þ

T-bet2), TFH1 (CXCR3þCXCR5þBcl6þT-betþ),and/or conventional TH1 cells (CXCR3þ

CXCR52Bcl62T-betþ) in vivo in an intactmouse or human (Fig. 1). Evidence of plasticitywas not observed in humans at the level of theoverall CD4 T-cell response to pertussis, where-in the whole-cell pertussis vaccine and the acel-lular pertussis vaccine elicit predominantly TH1and TH2 polarizing responses, respectively, andreimmunization with the acellular pertussisvaccine elicits a CD4 T-cell recall responsewith the same TH1 or TH2 characteristics forwhichever vaccine the individual was initiallyimmunized (Bancroft et al. 2016).

CONCLUDING REMARKS

There remains no direct demonstration of invivo–generated resting memory CD4 T-cellconversion to a different subtype on secondaryantigen challenge in vivo in an intact animal atthe single-cell level. Lineage tracing experimentsare needed to directly test whether plasticityoccurs and, more importantly, how commonor rare the process is under physiological con-ditions. Unfortunately, proper lineage-trackinggenetic models are difficult to generate for TFH,TH17, and TH2 cells. The lineage-definingtranscription factors for TFH, TH17, and TH2cells are Bcl6, RORgT, and GATA3. GATA3 isconstitutively expressed by all CD4 T cells; TH2cells are defined by high GATA3 expression.Thus, a conventional GATA3 lineage reporterwould mark all T cells. Therefore, developmentof a high-fidelity TH2 lineage genetic marker isa difficult challenge. A successful TH2 lineagegenetic marker would probably need to de-pend on transcription from a gene or locusother than GATA3. Bcl6 is highly expressedby thymocytes, and thus a Bcl6 lineage report-er mouse constructed based on standard de-signs would be expected to mark most T cells.RORgt is also expressed by thymocytes and,thus, an RORgt lineage reporter mouse basedon standard designs would be expected tomark most T cells. Therefore, a successfulTFH or TH17 lineage genetic marker wouldprobably need to depend on transcriptionfrom a gene or locus other than GATA3; IL-17a is one candidate for TH17 cells (Hirotaet al. 2011). CXCR5 may be a good candidatefor TFH cells. As for TH1 cells, a T-bet lineagereporter should be useful, but a key caveat isthat transient expression of T-bet early on by anaıve CD4 T cell after activation is commonand may have no influence on the future his-tory of the cell. The same caveat applies for allconstitutively active lineage marker systems,and contributed to controversy over the ontog-eny of “exTregs.” Transient expression of Foxp3by some cells may result in erroneous conclu-sions on the basis of very transient Cre expres-sion (Rubtsov et al. 2008; Zhou et al. 2009;Miyao et al. 2012). This concern may be

S. Crotty




avoided by using estrogen-responsive Cre pro-tein (Rubtsov et al. 2010).

As an alternative to lineage-tracking geneticmarkers, single-cell transfers into infection-matched hosts may be the best test of stabilityversus plasticity in a recall response. Such ex-periments would need to show that transferredcells (not using single-cell transfers) would lo-calize in the new host to the same regions oflymph node (LN) and spleen as untransferredantigen-specific resting memory CD4 T cells ofthat subtype (e.g., proper microanatomical lo-calization of memory TFH cells posttransfer).

In the end, it is unknown whether the ap-pearance of plasticity by memory CD4 T cells atthe population level is predominantly because ofheterogeneity and outgrowth of subpopulationsor predominantly attributable to plasticity.

It remains possible that true memory CD4T-cell plasticity may be primarily of interest forpurely academic reasons, as a memory recallresponse with apparent plasticity at the whole-cell population level could presumably be ac-complished via rapid outgrown of a very minorpopulation of memory cells. Because essentiallyall in vivo CD4 T-cell responses involve a mix-ture of subtypes (TH1, TH2, TH CTL, and TFH,for example), that scenario is plausible.

ACKNOWLEDGMENTS

This work is funded by the National Institutes ofHealth (NIH) National Institute of Allergy andInfectious Diseases (NIAID) R01 (S.C.). Thanksto Daniel DiToro for helpful discussions.

REFERENCES

Ballesteros-Tato A, Randall TD, Lund FE, Spolski R, Leo-nard WJ, Leon B. 2016. T follicular helper cell plasticityshapes pathogenic T helper 2 cell-mediated immunity toinhaled house dust mite. Immunity 44: 259–273.

Bancroft T, Dillon MBC, Da Silva Antunes R, Paul S, PetersB, Crotty S, Lindestam Arlehamn CS, Sette A. 2016. Th1versus Th2 T cell polarization by whole-cell and acellularchildhood pertussis vaccines persists upon re-immuni-zation in adolescence and adulthood. Cell Immunol 304 –305: 35–43.

Becattini S, Latorre D, Mele F, Foglierini M, De Gregorio C,Cassotta A, Fernandez B, Kelderman S, Schumacher TN,Corti D, et al. 2015. Functional heterogeneity of human

memory CD4þ T cell clones primed by pathogens orvaccines. Science 347: 400–406.

Bentebibel SE, Lopez S, Obermoser G, Schmitt N, MuellerC, Harrod C, Flano E, Mejias A, Albrecht RA, Blanken-ship D, et al. 2013. Induction of ICOSþCXCR3þCXCR5þ

TH cells correlates with antibody responses to influenzavaccination. Sci Transl Med 5: 176ra32.

Choi YS, Yang JA, Yusuf I, Johnston RJ, Greenbaum J, PetersB, Crotty S. 2013. Bcl6 expressing follicular helper CD4T cells are fate committed early and have the capacity toform memory. J Immunol 190: 4014–4026.

Coquet JM, Schuijs MJ, Smyth MJ, Deswarte K, Beyaert R,Braun H, Boon L, Karlsson Hedestam GB, Nutt SL, Ham-mad H, et al. 2015. Interleukin-21-producing CD4þ

T cells promote type 2 immunity to house dust mites.Immunity 43: 318–330.

Crotty S. 2011. Follicular helper CD4 T cells (TFH). AnnuRev Immunol 29: 621–663.

Crotty S. 2012. The 1-1-1 fallacy. Immunol Rev 247: 133–142.

Crotty S. 2014. T follicular helper cell differentiation, func-tion, and roles in disease. Immunity 41: 529–542.

Dan JM, Lindestam Arlehamn CS, Weiskopf D, dda SilvaAntunes R, Havenar-Daughton C, Reiss SM, Brigger M,Bothwell M, Sette A, Crotty, S. et al.2016. A cytokine-independent approach to identify antigen-specific hu-man germinal center T follicular helper cells and rareantigen-specific CD4þ T cells in blood. J Immunol 197:983–993.

Da Silva Antunes R, Paul S, Sidney J, Weiskopf D, Dan JM,Phillips E, Mallal S, Crotty S, Sette A, Arlehamn CSL.2017. Definition of human epitopes recognized in teta-nus toxoid and development of an assay strategy to detectex vivo tetanus CD4þ T cell responses. PLoS ONE 12:e0169086.

DuPage M, Bluestone JA. 2016. Harnessing the plasticity ofCD4þ T cells to treat immune-mediated disease. Nat RevImmunol 16: 149–163.

Hale JS, Youngblood B, Latner DR, Mohammed AUR, Ye L,Akondy RS, Wu T, Iyer SS, Ahmed R. 2013. Distinctmemory CD4þ T cells with commitment to T follicularhelper- and T helper 1-cell lineages are generated afteracute viral infection. Immunity 38: 805–817.

Harrington LE, Janowski KM, Oliver JR, Zajac AJ, WeaverCT. 2008. Memory CD4 T cells emerge from effectorT-cell progenitors. Nature 452: 356–360.

Hatzi K, Nance JP, Kroenke MA, Bothwell M, Haddad EK,Melnick A, Crotty S. 2015. BCL6 orchestrates Tfh celldifferentiation via multiple distinct mechanisms. J ExpMed 212: 539–553.

Havenar-Daughton C, Reiss SM, Carnathan DG, Wu JE,Kendric K, Torrents de la Pena A, Kasturi SP, Dan JM,Bothwell M, Sanders RW, et al. 2016. Cytokine-indepen-dent detection of antigen-specific germinal center T fol-licular helper cells in immunized nonhuman primatesusing a live cell activation-induced marker technique.J Immunol 197: 994–1002.

He J, Tsai LM, Leong YA, Hu X, Ma CS, Chevalier N, Sun X,Vandenberg K, Rockman S, Ding Y, et al. 2013. Circulat-ing precursor CCR7loPD-1hi CXCR5þ CD4þ T cells in-dicate Tfh cell activity and promote antibody responsesupon antigen reexposure. Immunity 39: 770–781.





Hirota K, Duarte JH, Veldhoen M, Hornsby E, Li Y, Cua DJ,Ahlfors H, Wilhelm C, Tolaini M, Menzel U, et al. 2011.Fate mapping of IL-17-producing T cells in inflammato-ry responses. Nat Immunol 12: 255–263.

Hondowicz BD, An D, Schenkel JM, Kim KS, Steach HR,Krishnamurty AT, Keitany GJ, Garza EN, Fraser KA,Moon JJ, et al. 2016. Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma. Im-munity 44: 155–166.

Ise W, Inoue T, McLachlan JB, Kometani K, Kubo M, OkadaT, Kurosaki T. 2014. Memory B cells contribute to rapidBcl6 expression by memory follicular helper T cells. ProcNatl Acad Sci 111: 11792–11797.

Iyer SS, Gangadhara S, Victor B, Gomez R, Basu R, Hong JJ,Labranche C, Montefiori DC, Villinger F, Moss B, et al.2015. Codelivery of envelope protein in alum with MVAvaccine induces CXCR3-biased CXCR5þ and CXCR52

CD4 T cell responses in rhesus macaques. J Immunol 195:994–1005.

Jacquemin C, Schmitt N, Contin-Bordes C, Liu Y, Nara-yanan P, Seneschal J, Maurouard T, Dougall D, DavizonES, Dumortier H, et al. 2015. OX40 ligand contributes tohuman lupus pathogenesis by promoting T follicularhelper response. Immunity 42: 1159–1170.

Johnston RJ, Poholek AC, DiToro D, Yusuf I, Eto D, BarnettB, Dent AL, Craft J, Crotty S. 2009. Bcl6 and Blimp-1 arereciprocal and antagonistic regulators of T follicularhelper cell differentiation. Science 325: 1006–1010.

Kitano M, Moriyama S, Ando Y, Hikida M, Mori Y, KurosakiT, Okada T. 2011. Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helperT cell heterogeneity. Immunity 34: 961–972.

Liang HE, Reinhardt RL, Bando JK, Sullivan BM, Ho IC,Locksley RM. 2012. Divergent expression patterns of IL-4and IL-13 define unique functions in allergic immunity.Nat Immunol 13: 58–66.

Lindenstrøm T, Woodworth J, Dietrich J, Aagaard C, Ander-sen P, Agger EM. 2012. Vaccine-induced th17 cells aremaintained long-term postvaccination as a distinct andphenotypically stable memory subset. Infect Immun 80:3533–3544.

Liu X, Yan X, Zhong B, Nurieva RI, Wang A, Wang X, Mar-tin-Orozco N, Wang Y, Chang SH, Esplugues E, et al.2012. Bcl6 expression specifies the T follicular helpercell program in vivo. J Exp Med 209: 1841–1852.

Locci M, Havenar-Daughton C, Landais E, Wu J, KroenkeMA, Arlehamn CL, Su LF, Cubas R, Davis MM, Sette A,et al. 2013. Human circulating PD-1þCXCR3-CXCR5þ

memory Tfh cells are highly functional and correlatewith broadly neutralizing HIV antibody responses. Im-munity 39: 758–769.

Lu KT, Kanno Y, Cannons JL, Handon R, Bible P, ElkahlounAG, Anderson SM, Wei L, Sun H, O’Shea JJ, et al. 2011.Functional and epigenetic studies reveal multistep differ-entiation and plasticity of in vitro–generated and invivo–derived follicular T helper cells. Immunity 35:622–632.

Luthje K, Kallies A, Shimohakamada Y, Belz GT, Light A,Tarlinton DM, Nutt SL. 2012. The development and fateof follicular helper T cells defined by an IL-21 reportermouse. Nat Immunol 13: 491–498.

Ma CS, Suryani S, Avery DT, Chan A, Nanan R, Santner-Nanan B, Deenick EK, Tangye SG. 2009. Early commit-ment of naıve human CD4þ T cells to the T follicularhelper (TFH) cell lineage is induced by IL-12. ImmunolCell Biol 87: 590–600.

McGeachy MJ. 2013. Th17 memory cells: Live long andproliferate. J Leukoc Biol 94: 921–926.

Miyao T, Floess S, Setoguchi R, Luche H, Fehling HJ, Wald-mann H, Huehn J, Hori S. 2012. Plasticity of Foxp3þ

T cells reflects promiscuous Foxp3 expression in conven-tional T cells but not reprogramming of regulatory Tcells. Immunity 36: 262–275.

Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bour-dery L, Zurawski G, Foucat E, Dullaers M, Oh S, Sabz-ghabaei N, et al. 2011. Human blood CXCR5þCD4þ Tcells are counterparts of T follicular cells and containspecific subsets that differentially support antibody secre-tion. Immunity 34: 108–121.

Muranski P, Borman ZA, Kerkar SP, Klebanoff CA, Ji Y,Sanchez-Perez L, Sukumar M, Reger RN, Yu Z, Kern SJ,et al. 2011. Th17 cells are long lived and retain a stem cell-like molecular signature. Immunity 35: 972–985.

Nakayamada S, Kanno Y, Takahashi H, Jankovic D, Lu KT,Johnson TA, Sun HW, Vahedi G, Hakim O, Handon R, etal. 2011. Early Th1 cell differentiation is marked by a Tfhcell-like transition. Immunity 35: 919–931.

Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM. 2012.Cytometry by time-of-flight shows combinatorial cyto-kine expression and virus-specific cell niches within acontinuum of CD8þ T cell phenotypes. Immunity 36:142–152.

Obeng-Adjei N, Portugal S, Tran TM, Yazew TB, Skinner J,Li S, Jain A, Felgner PL, Doumbo OK, Kayentao K, et al.2015. Circulating Th1-cell-type Tfh cells that exhibit im-paired B cell help are preferentially activated during acutemalaria in children. Cell Rep 13: 425–439.

Oestreich KJ, Mohn SE, Weinmann AS. 2012. Molecularmechanisms that control the expression and activity ofBcl-6 in TH1 cells to regulate flexibility with a TFH-likegene profile. Nat Immunol 13: 405–411.

Pepper M, Linehan JL, Pagan AJ, Zell T, Dileepan T, ClearyPP, Jenkins MK. 2010. Different routes of bacterial infec-tion induce long-lived TH1 memory cells and short-livedTH17 cells. Nat Immunol 11: 83–89.

Ray JP, Staron MM, Shyer JA, Ho P-C, Marshall HD, GraySM, Laidlaw BJ, Araki K, Ahmed R, Kaech SM, et al. 2015.The interleukin-2-mTORc1 kinase axis defines the sig-naling, differentiation, and metabolism of T helper 1 andfollicular B helper T cells. Immunity 43: 690–702.

Rubtsov YP, Rasmussen JP, Chi EY, Fontenot J, Castelli L, YeX, Treuting P, Siewe L, Roers A, Henderson WR, et al.2008. Regulatory T cell-derived interleukin-10 limits in-flammation at environmental interfaces. Immunity 28:546–558.

Rubtsov YP, Niec RE, Josefowicz S, Li L, Darce J, Mathis D,Benoist C, Rudensky AY. 2010. Stability of the regulatoryT cell lineage in vivo. Science 329: 1667–1671.

Tubo NJ, Pagan AJ, Taylor JJ, Nelson RW, Linehan JL, ErteltJM, Huseby ES, Way SS, Jenkins MK. 2013. Single naiveCD4þ T cells from a diverse repertoire produce differenteffector cell types during infection. Cell 153: 785–796.

S. Crotty




Tubo NJ, Fife BT, Pagan AJ, Kotov DI, Goldberg MF, JenkinsMK. 2016. Most microbe-specific naıve CD4þ T cells pro-duce memory cells during infection. Science 351: 511–514.

Vinuesa CG, Linterman MA, Yu D, Maclennan ICM. 2016.Follicular helper T cells. Annu Rev Immunol 34: 335–368.

Weinstein JS, Herman EI, Lainez B, Licona-Limon P, Es-plugues E, Flavell R, Craft J. 2016. TFH cells progressivelydifferentiate to regulate the germinal center response. NatImmunol 17: 1197–1205.

Wong MT, Chen J, Narayanan S, Lin W, Anicete R, KiaangHTK, De Lafaille MAC, Poidinger M, Newell EW. 2015.Mapping the diversity of follicular helper T cells in hu-man blood and tonsils using high-dimensional mass cy-tometry analysis. Cell Rep 11: 1822–1833.

Yu D, Rao S, Tsai LM, Lee SK, He Y, Sutcliffe EL, SrivastavaM, Linterman M, Zheng L, Simpson N, et al. 2009. The

transcriptional repressor Bcl-6 directs T follicular helpercell lineage commitment. Immunity 31: 457–468.

Yusuf I, Kageyama R, Monticelli L, Johnston RJ, DiToro D,Hansen K, Barnett B, Crotty S. 2010. Germinal centerT follicular helper cell IL-4 production is dependenton signaling lymphocytic activation molecule receptor(CD150). J Immunol 185: 190–202.

Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Mar-tınez-Llordella M, Ashby M, Nakayama M, Rosenthal W,Bluestone JA. 2009. Instability of the transcription factorFoxp3 leads to the generation of pathogenic memoryT cells in vivo. Nat Immunol 10: 1000–1007.

Zielinski CE, Mele F, Aschenbrenner D, Jarrossay D, RonchiF, Gattorno M, Monticelli S, Lanzavecchia A, Sallusto F.2012. Pathogen-induced human TH17 cells produceIFN-g or IL-10 and are regulated by IL-1b. Nature 484:514–518.





published online April 21, 2017Cold Spring Harb Perspect Biol Shane Crotty CellsHeterogeneity of Memory CD4 T Cells, Including T Follicular Helperversus Fate Commitment?: Complexities of Interpretation due to the Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity

Subject Collection Immune Memory and Vaccines: Great Debates

and CD8 T Cells Switch Jobs?Harnessed by Vaccination?: Can Natural Killer Is There Natural Killer Cell Memory and Can It Be

Christine A. Biron and Marcus Altfeld Diseases and CancerImmunization Strategies against InfectiousHarnessed by Vaccination?: NK Cell Memory and Is There Natural Killer Cell Memory and Can It Be

Joseph C. Sun and Lewis L. Lanier

Strategies Based on NK Cell and ILC MemoryHarnessed by Vaccination?: Vaccination Is There Natural Killer Cell Memory and Can It Be

ColonnaMegan A. Cooper, Todd A. Fehniger and Marco

VaccinationinHarnessed by Vaccination?: Natural Killer Cells

Is There Natural Killer Cell Memory and Can It Be

al.Harold R. Neely, Irina B. Mazo, Carmen Gerlach, et

Vaccine Targets for CancerHow Can These Be Overcome?: Neoantigens asNeoantigens, What Are the Major Challenges, and Is It Possible to Develop Cancer Vaccines to

Haydn T. KissickRight Antigens in the Right PatientsHow Can These Be Overcome?: Targeting theNeoantigens, What Are the Major Challenges, and Is It Possible to Develop Cancer Vaccines to

Stephen P. Schoenberger

Nothing New in Spite of the NameHow Can These Be Overcome?: Neoantigens:Neoantigens, What Are the Major Challenges, and Is It Possible to Develop Cancer Vaccines to

Olivera J. Finn and Hans-Georg RammenseeOnly One True WinnerEnhanced Disease after Vaccination?: There IsPromising, and Should We Be Concerned about Which Dengue Vaccine Approach Is the Most

Scott B. Halstead

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved


http://cshperspectives.cshlp.org/cgi/collection/


http://cshperspectives.cshlp.org/cgi/adclick/?ad=53044&adclick=true&url=http%3A%2F%2Fwww.genelink.com%2Fnewsite%2Fproducts%2FmodoligosINFO.asp


Challenges of a Dengue VaccineEnhanced Disease after Vaccination?: ThePromising, and Should We Be Concerned about Which Dengue Vaccine Approach Is the Most

Gavin Screaton and Juthathip MongkolsapayaPasteur Tetravalent Dengue Vaccineof Dengue Vaccines: Example of the SanofiRaised by the Development and Implementation Enhanced Disease after Vaccination?: QuestionsPromising, and Should We Be Concerned about Which Dengue Vaccine Approach Is the Most

Bruno Guy

Dengue Infection Studies and Vaccine Trialsa Dengue Vaccine: Learning from Human Natural Enhanced Disease after Vaccination?: The Path toPromising, and Should We Be Concerned about Which Dengue Vaccine Approach Is the Most

Aravinda M. de Silva and Eva Harrisby Vaccination

Revealedof Incomplete Immunity to Dengue Virus Enhanced Disease after Vaccination?: The RisksPromising, and Should We Be Concerned about Which Dengue Vaccine Approach Is the Most

Stephen S. Whitehead and Kanta Subbarao

VaccineInfluenzaVirus Vaccine?: Potential for a Universal

Is It Possible to Develop a ''Universal'' Influenza

James E. Crowe, Jr.Hemagglutinin StemPan-Subtype Influenza A Vaccine Targeting the Underlying B-Cell Biology in the Development of aVirus Vaccine?: Immunogenetic Considerations Is It Possible to Develop a ''Universal'' Influenza

Mascola, et al.Sarah F. Andrews, Barney S. Graham, John R.

Influenza VaccinationImmunodominance on the Road to UniversalVirus Vaccine?: Outflanking Antibody Is It Possible to Develop a ''Universal'' Influenza

Davide Angeletti and Jonathan W. Yewdell

Critical Aspects for a Universal Influenza VaccineVirus Vaccine?: Potential Target Antigens and Is It Possible to Develop a ''Universal'' Influenza

PaleseFlorian Krammer, Adolfo García-Sastre and Peter

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved



http://cshperspectives.cshlp.org/cgi/adclick/?ad=53044&adclick=true&url=http%3A%2F%2Fwww.genelink.com%2Fnewsite%2Fproducts%2FmodoligosINFO.asp


do memory cd4 t cells keep their cell-type programming...

Documents