Heterogeneity, subsets, and plasticity of T follicular helper cells in allergy

Antibody responses are critical for protection against pathogens. However, diseases such as allergic rhinitis or food allergy result from aberrant production of IgE antibodies against otherwise innocuous environmental antigens. The production of allergen-specific IgE requires interaction between B cells and CD4+ T cells, and a granular understanding of these interactions is required to develop novel therapies for allergic disease. CD4+ T cells are exceptionally heterogeneous in their transcriptional, epigenetic, and proteomic profiles, which poses significant challenges when attempting to define subsets relevant to the study of allergy among a continuum of cells. Defining subsets such as the T follicular helper (TFH) cell cluster provides a shorthand to understand the functions of CD4+ T cells in antibody production and supports mechanistic experimentation for hypothesis-driven discovery. With a focus on allergic disease, this Rostrum article broadly discusses heterogeneity among CD4+ T cells and provides a rationale for subdividing TFH cells into both functional and cytokine-skewed subsets. Further, it highlights the plasticity demonstrated by TFH cells during the primary response and after recall, and it explores the possibility of harnessing this plasticity to reprogram immunity for therapeutic benefit in allergic disease. Antibody responses are critical for protection against pathogens. However, diseases such as allergic rhinitis or food allergy result from aberrant production of IgE antibodies against otherwise innocuous environmental antigens. The production of allergen-specific IgE requires interaction between B cells and CD4+ T cells, and a granular understanding of these interactions is required to develop novel therapies for allergic disease. CD4+ T cells are exceptionally heterogeneous in their transcriptional, epigenetic, and proteomic profiles, which poses significant challenges when attempting to define subsets relevant to the study of allergy among a continuum of cells. Defining subsets such as the T follicular helper (TFH) cell cluster provides a shorthand to understand the functions of CD4+ T cells in antibody production and supports mechanistic experimentation for hypothesis-driven discovery. With a focus on allergic disease, this Rostrum article broadly discusses heterogeneity among CD4+ T cells and provides a rationale for subdividing TFH cells into both functional and cytokine-skewed subsets. Further, it highlights the plasticity demonstrated by TFH cells during the primary response and after recall, and it explores the possibility of harnessing this plasticity to reprogram immunity for therapeutic benefit in allergic disease. Antibody responses are a central feature of adaptive immunity. High-affinity antibodies of various isotypes (IgM, IgG, IgA, IgE) are generated against pathogens to restrict their ability to infect cells or invade tissues. However, antibodies that target otherwise harmless antigens in the environment result in allergies, the symptoms of which range in severity from excess mucous production and sneezing in the context of allergic rhinitis to life-threatening anaphylactic reactions against food allergens. The identification of follicular helper T (TFH) cells catalyzed an era of rapid discovery relating to the induction of antibody responses. TFH cells are a subset of CD4+ T cells classically defined by their expression of the lineage-defining transcription factor Bcl6, as well as PD1, ICOS, and CXCR5.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar CXCR5 drives TFH migration by binding CXCL13 produced in the B-cell follicle, the location where TFH cells interact with B cells and orchestrate a critical role in germinal center (GC) reactions.2Crotty S. T Follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar Recent research has revealed significant heterogeneity among the CD4+ T cells that support B-cell functions. These include pre-GC TFH cells at the T-cell zone/B-cell follicle (T/B) border and novel populations of TFH-like cells that provide B-cell help.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,3Walker L.S.K. The link between circulating follicular helper T cells and autoimmunity.Nat Rev Immunol. 2022; 22: 567-575Crossref PubMed Scopus (34) Google Scholar, 4Tonti E. Fedeli M. Napolitano A. Iannacone M. Andrian UH von Guidotti L.G. et al.Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4+ T cell help.J Immunol. 2012; 188: 3217-3222Crossref PubMed Scopus (88) Google Scholar, 5King I.L. Fortier A. Tighe M. Dibble J. Watts G.F.M. Veerapen N. et al.Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner.Nat Immunol. 2011; 13: 44-50Crossref PubMed Scopus (183) Google Scholar, 6Chang P.P. Barral P. Fitch J. Pratama A. Ma C.S. Kallies A. et al.Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses.Nat Immunol. 2011; 13: 35-43Crossref PubMed Scopus (233) Google Scholar Heterogeneity is further observed in TFH cells expressing transcriptional programs characteristic of polarized effector TH (Teff) populations (eg, TH1, TH2, TH17), as well as the potential for regulatory functions such as those observed in T follicular regulatory (TFR) cells.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,7Gowthaman U. Chen J.S. Zhang B. Flynn W.F. Lu Y. Song W. et al.Identification of a T follicular helper cell subset that drives anaphylactic IgE.Science. 2019; 365eaaw6433Crossref PubMed Scopus (249) Google Scholar, 8Chan J.A. Loughland J.R. de Labastida Rivera F. SheelaNair A. Andrew D.W. Dooley N.L. et al.Th2-like T follicular helper cells promote functional antibody production during plasmodium falciparum infection.Cell Rep Med. 2020; 1100157Google Scholar, 9Kim C.J. Lee C.G. Jung J.Y. Ghosh A. Hasan S.N. Hwang S.M. et al.The transcription factor Ets1 suppresses T follicular helper type 2 cell differentiation to halt the onset of systemic lupus erythematosus.Immunity. 2018; 49: 1034-1048.e8Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Weinstein J.S. Laidlaw B.J. Lu Y. Wang J.K. Schulz V.P. Li N. et al.STAT4 and T-bet control follicular helper T cell development in viral infections.J Exp Med. 2018; 215: 337-355Crossref PubMed Scopus (74) Google Scholar, 11Fang D. Cui K. Mao K. Hu G. Li R. Zheng M. et al.Transient T-bet expression functionally specifies a distinct T follicular helper subset.J Exp Med. 2018; 215: 2705-2714Crossref PubMed Scopus (54) Google Scholar, 12Lu Y. Craft J. T follicular regulatory cells: choreographers of productive germinal center responses.Front Immunol. 2021; 12: 2267Crossref Scopus (15) Google Scholar Teff cells express transcriptional regulator Blimp-1 and lack chemokine receptors that home to lymph nodes (CD62L–).13Sallusto F. Lenig D. Förster R. Lipp M. Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4749) Google Scholar,14Johnston R.J. Poholek A.C. DiToro D. Yusuf I. Eto D. Barnett B. et al.Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (1213) Google Scholar As a result, they are most commonly localized in peripheral tissues. These cells express additional transcription factors and cytokines based on their polarization (discussed later in the context of TFH polarization). Evidence of plasticity between and within TFH and Teff clusters questions the degree of commitment that these cells have to their lineage fate.15Ballesteros-Tato A. Randall T.D. Lund F.E. Spolski R. Leonard W.J. León B. T follicular helper cell plasticity shapes pathogenic T helper 2 cell-mediated immunity to inhaled house dust mite.Immunity. 2016; 44: 259-273Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,16Hale J.S. Ahmed R. Memory T follicular helper CD4 T cells.Front Immunol. 2015; 6: 16Crossref PubMed Scopus (104) Google Scholar With such diversity and plasticity, it is not surprising that novel functions continue to be uncovered for TFH cells. The functions of TFH cells in general will be discussed in this Rostrum article only superficially, as they will be covered in depth by other Rostrum articles in this series. Here, we will highlight the critical roles that TFH cells play in allergic diseases. These functions differ from those reported for Teff cells, namely, for TH2 cells. The production of allergen-specific IgE is mediated by help from TFH cells in a manner that is critically dependent on IL-4.17Meli A.P. Fontés G. Leung Soo C. King I.L. T follicular helper cell-derived IL-4 Is required for IgE production during intestinal helminth infection.J Immunol. 2017; 199: 244-252Crossref PubMed Scopus (63) Google Scholar, 18Kobayashi T. Iijima K. Dent A.L. Kita H. Follicular helper T cells mediate IgE antibody response to airborne allergens.J Allergy Clin Immunol. 2017; 139: 300-313.e7Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Xie M.M. Chen Q. Liu H. Yang K. Koh B. Wu H. et al.T follicular regulatory cells and IL-10 promote food antigen-specific IgE.J Clin Invest. 2020; 130: 3820-3832Crossref PubMed Scopus (37) Google Scholar, 20Zhang B. Liu E. Gertie J.A. Joseph J. Xu L. Pinker E.Y. et al.Divergent T follicular helper cell requirement for IgA and IgE production to peanut during allergic sensitization.Sci Immunol. 2020; 5eaay2754Crossref Scopus (34) Google Scholar, 21Dolence J.J. Kobayashi T. Iijima K. Krempski J. Drake L.Y. Dent A.L. et al.Airway exposure initiates peanut allergy by involving the IL-1 pathway and T follicular helper cells in mice.J Allergy Clin Immunol. 2018; 142: 1144-1158.e8Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar In contrast, tissue effector functions such as airway hyperresponsiveness in asthma are mediated by resident effector TH2 cells and persist in the absence of TFH cells.18Kobayashi T. Iijima K. Dent A.L. Kita H. Follicular helper T cells mediate IgE antibody response to airborne allergens.J Allergy Clin Immunol. 2017; 139: 300-313.e7Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar,22Hondowicz B.D. An D. Schenkel J.M. Kim K.S. Steach H.R. Krishnamurty A.T. et al.Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma.Immunity. 2016; 44: 155-166Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar However, TFH cells may be the precursors of lung-homing effector TH2 cells,15Ballesteros-Tato A. Randall T.D. Lund F.E. Spolski R. Leonard W.J. León B. T follicular helper cell plasticity shapes pathogenic T helper 2 cell-mediated immunity to inhaled house dust mite.Immunity. 2016; 44: 259-273Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar and the presence of intermediates between TFH and TH2 cells suggests that these populations are more closely related than was originally thought.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar We will discuss the heterogeneity of TFH cells, the subsets that are useful to define this repertoire, the flexibility of TFH cells during initial antigen exposures, their multipotency during recall responses, and the potential for polarized TFH cells to be reprogrammed. Immunologists have often applied simplistic classifications to navigate the complexities of the immune system. This is certainly the case regarding CD4+ T cells, the classification of which began with Mosmann and Coffman’s proposed TH1/TH2 dichotomy.23Mosmann T.R. Coffman R.L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.Annu Rev Immunol. 1989; 7: 145-173Crossref PubMed Scopus (7140) Google Scholar Teff cells such as TH1 and TH2 cells express oppositional transcriptional programs, resulting in unique functions driven by the context of their activation. For example, infection with viruses (eg, influenza A) or some bacteria (eg, Salmonella enterica) will strongly induce antigen-specific TH1 cells, which classically express Tbet and CXCR3 and secrete IFN-γ.24Krueger P.D. Goldberg M.F. Hong S.W. Osum K.C. Langlois R.A. Kotov D.I. et al.Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.Immunity. 2021; 54: 687-701.e4Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Conversely, disruptions to tissue homeostasis such as helminth infections or venoms from insect stings reliably induce TH2 cells, which express GATA3 at a high level and secrete IL-4, IL-5, and IL-13.25Bruton K. Koenig J.F.E. Phelps A. Jordana M. Perturbations to homeostasis in experimental models revealed innate pathways driving food allergy.Front Immunol. 2020; 11: 3229Crossref Scopus (8) Google Scholar As our knowledge of CD4+ Teff cells has expanded, so too has the repertoire of named helper phenotypes. Certain phenotypes are traditionally characterized by “master” transcription factor expression, such as TH17 cells (RORγt),26Ivanov II, McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. et al.The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (4136) Google Scholar regulatory T (Treg) cells (FoxP3),27Sakaguchi S. Mikami N. Wing J.B. Tanaka A. Ichiyama K. Ohkura N. Regulatory T cells and human disease.Annu Rev Immunol. 2020; 38: 541-566Crossref PubMed Scopus (446) Google Scholar and TFH cells (Bcl6),28Choi J. Crotty S. Bcl6-mediated transcriptional regulation of follicular helper T cells (TFH).Trends Immunol. 2021; 42: 336-349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar yet the identification of cells that coexpress master transcription factors indicates that their expression is not mutually exclusive and therefore questions the idea of hermetic phenotypes. Conventional subsets fall short of explaining the extensive heterogeneity observed among CD4+ T cells. For example, Wambre et al identified a subset of mature effector TH2 cells that distinguish individuals with allergy from individuals without allergy. This subset, termed TH2A, expresses the natural killer cell marker CD161, a canonic TH2 marker CRTH2, and produces TH2 cytokines.29Wambre E. Bajzik V. DeLong J.H. O’Brien K. Nguyen Q.-A. Speake C. et al.A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders.Sci Transl Med. 2017; 9eaam9171Crossref PubMed Scopus (255) Google Scholar More detailed analyses of TH2A cells revealed diverse cytokine profiles, with some cells secreting a single TH2 cytokine and others expressing an array of TH2 and non-TH2 cytokines.29Wambre E. Bajzik V. DeLong J.H. O’Brien K. Nguyen Q.-A. Speake C. et al.A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders.Sci Transl Med. 2017; 9eaam9171Crossref PubMed Scopus (255) Google Scholar What mediates such heterogeneity, even among a population carefully curated by multiple surface markers is not yet known. Further, Teff populations can possess characteristics of various CD4+ T-cell subsets. Bcl6+ TFH cells can coexpress the lineage-defining transcription factors of other subsets, namely Tbet, GATA3, RORγt, and FoxP3.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,7Gowthaman U. Chen J.S. Zhang B. Flynn W.F. Lu Y. Song W. et al.Identification of a T follicular helper cell subset that drives anaphylactic IgE.Science. 2019; 365eaaw6433Crossref PubMed Scopus (249) Google Scholar, 8Chan J.A. Loughland J.R. de Labastida Rivera F. SheelaNair A. Andrew D.W. Dooley N.L. et al.Th2-like T follicular helper cells promote functional antibody production during plasmodium falciparum infection.Cell Rep Med. 2020; 1100157Google Scholar, 9Kim C.J. Lee C.G. Jung J.Y. Ghosh A. Hasan S.N. Hwang S.M. et al.The transcription factor Ets1 suppresses T follicular helper type 2 cell differentiation to halt the onset of systemic lupus erythematosus.Immunity. 2018; 49: 1034-1048.e8Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Weinstein J.S. Laidlaw B.J. Lu Y. Wang J.K. Schulz V.P. Li N. et al.STAT4 and T-bet control follicular helper T cell development in viral infections.J Exp Med. 2018; 215: 337-355Crossref PubMed Scopus (74) Google Scholar, 11Fang D. Cui K. Mao K. Hu G. Li R. Zheng M. et al.Transient T-bet expression functionally specifies a distinct T follicular helper subset.J Exp Med. 2018; 215: 2705-2714Crossref PubMed Scopus (54) Google Scholar, 12Lu Y. Craft J. T follicular regulatory cells: choreographers of productive germinal center responses.Front Immunol. 2021; 12: 2267Crossref Scopus (15) Google Scholar Further, FoxP3+ T cells can express GATA3 at a high level, and, in allergic disease, they can produce functional amounts of TH2 cytokines.30Noval Rivas M. Chatila T.A. Regulatory T cells in allergic diseases.J Allergy Clin Immunol. 2016; 138: 639-652Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar Indeed, allergic sensitization often involves the production of cytokines from the full array of TH cell subsets, not only from TH2 cells.31Chu D.K. Llop-Guevara A. Walker T.D. Flader K. Goncharova S. Boudreau J.E. et al.IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization.J Allergy Clin Immunol. 2013; 131: 187-200.e8Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar Classical Teff subsets poorly segregate when their transcriptomes are analyzed by single-cell RNA sequencing or when their epigenetic profile is characterized by using an assay for transposase-accessible chromatin sequencing. In fact, human circulating CD4+ T cells form a transcriptional continuum from naive to central/effector memory phenotypes.32Cano-Gamez E. Soskic B. Roumeliotis T.I. So E. Smyth D.J. Baldrighi M. et al.Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines.Nat Commun. 2020; 11: 1-15Crossref PubMed Scopus (102) Google Scholar Rather than identifying unique clusters that match TH1, TH2, TH17, and TFH cells, CD4+ T cells tend to cluster as a “cloud,” some corners of which loosely map to a profile consistent with the aforementioned subsets.33Monian B. Tu A.A. Ruiter B. Morgan D.M. Petrossian P.M. Smith N.P. et al.Peanut oral immunotherapy differentially suppresses clonally distinct subsets of T helper cells.J Clin Invest. 2022; 132e150634Crossref PubMed Scopus (33) Google Scholar, 34Kiner E. Willie E. Vijaykumar B. Chowdhary K. Schmutz H. Chandler J. et al.Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes.Nat Immunol. 2021; 22: 216-228Crossref PubMed Scopus (77) Google Scholar, 35Eizenberg-Magar I. Rimer J. Zaretsky I. Lara-Astiaso D. Reich-Zeliger S. Friedman N. Diverse continuum of CD4+ T-cell states is determined by hierarchical additive integration of cytokine signals.Proc Natl Acad Sci U S A. 2017; 114: E6447-E6456Crossref PubMed Scopus (42) Google Scholar, 36Zemmour D. Kiner E. Benoist C. CD4+ teff cell heterogeneity: the perspective from single-cell transcriptomics.Curr Opin Immunol. 2020; 63: 61-67Crossref PubMed Scopus (13) Google Scholar, 37Sharif H. Acharya S. Dhondalay G.K.R. Varricchi G. Krasner-Macleod S. Laisuan W. et al.Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy.J Allergy Clin Immunol. 2021; 147: 663-676Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 38Ma J. Tibbitt C.A. Georen S.K. Christian M. Murrell B. Cardell L.O. et al.Single-cell analysis pinpoints distinct populations of cytotoxic CD4+T cells and an IL-10+CD109+TH2 cell population in nasal polyps.Sci Immunol. 2021; 6eabg6356Crossref Scopus (25) Google Scholar, 39Tibbitt C.A. Stark J.M. Martens L. Ma J. Mold J.E. Deswarte K. et al.Single-cell RNA sequencing of the T helper cell response to house dust mites defines a distinct gene expression signature in airway Th2 cells.Immunity. 2019; 51: 169-184.e5Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar This understanding of CD4+ T-cell heterogeneity has been termed the cloud model (Fig 1).36Zemmour D. Kiner E. Benoist C. CD4+ teff cell heterogeneity: the perspective from single-cell transcriptomics.Curr Opin Immunol. 2020; 63: 61-67Crossref PubMed Scopus (13) Google Scholar TFH cells cluster within the island of Teff cells despite their unique function and localization, which has led some to postulate that TFH cells are a state of differentiation rather than a distinct subset.40Ruterbusch M. Pruner K.B. Shehata L. Pepper M. In vivo CD4+ T cell differentiation and function: revisiting the Th1/Th2 paradigm.Annu Rev Immunol. 2020; 38: 705-725Crossref PubMed Scopus (199) Google Scholar The difficulties in segregating TFH cells might also be attributed to their plasticity. The degrees of commitment model (Fig 1) proposed by Hale and Ahmed suggests that following their initial activation, individual CD4+ T-cell clones will display varying levels of commitment or polarization to the TFH cell lineage and to the Th cell lineages (eg, TH2) depending on the collection of activation signals that they experienced (eg, T-cell receptor [TCR] stimulation strength and longevity, cytokine milieu).16Hale J.S. Ahmed R. Memory T follicular helper CD4 T cells.Front Immunol. 2015; 6: 16Crossref PubMed Scopus (104) Google Scholar Transcriptional and epigenetic programming mediates this polarization and thus generates a heterogeneous antigen-specific memory pool that includes highly committed TFH and non-TFH memory cells as well as more plastic (or stem-like) TFH and non-TFH memory cells with multipotent potential subsequent to recall.16Hale J.S. Ahmed R. Memory T follicular helper CD4 T cells.Front Immunol. 2015; 6: 16Crossref PubMed Scopus (104) Google Scholar This model is attractive, as it accounts for the transcriptional and epigenetic similarities among the classical CD4+ T-cell subsets, as well as for the plasticity observed between them subsequent to recall. The continuum of CD4+ T cells may render a definitive delineation of Teff and TFH populations futile. It may be desirable to drop these delineations altogether. What experimental avenues or approaches, which are currently limited by our collective tendency to divide cells into groups, might emerge from viewing CD4+ T cells as a continuum? As a counterpoint, there are practical reasons for defining subsets. Computational approaches can consider myriad parameters to define the CD4+ T-cell continuum, but that same multidimensionality eludes the human mind. Subsetting allows for a shorthand that can, for the most part, be applied accurately to a heterogeneous population of cells. This allows for the generation of hypotheses that can be experimentally tested to understand the influence of individual genes, proteins, pathogens, etc, on the overall CD4+ T-cell compartment. Shifts in subset composition can be used as a readout for conventional binary immunologic tools (ie, knockout vs wild type) to uncover the mechanisms underlying CD4+ T-cell biology. We therefore posit that while subsetting the TFH compartment is an oversimplification of reality, it still is of significant utility. Here we propose 2 non–mutually exclusive ways to subset TFH cells. Firstly, the localization and surface phenotype of a TFH cell provides a framework to understand its role in providing B-cell help (Fig 2). Second, the cytokine polarization of that TFH cell tends to dictate the antibody isotypes that they elicit, thereby tailoring the downstream effector functions of the humoral response. Following activation, TFH cells upregulate CXCR5 to migrate to the T/B border. At this stage, TFH cells express Bcl6, CXCR5, and PD1 at low levels and are sometimes referred to as pre-GC TFH cells or pre-TFH cells.1Eisenbarth S.C. Baumjohann D. Craft J. Fazilleau N. Ma C.S. Tangye S.G. et al.CD4+ T cells that help B cells – a proposal for uniform nomenclature.Trends Immunol. 2021; 42: 658-669Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,2Crotty S. T Follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar,41Kerfoot S.M. Yaari G. Patel J.R. Johnson K.L. Gonzalez D.G. Kleinstein S.H. et al.Germinal center B cell and T follicular helper cell development initiates in the interfollicular zone.Immunity. 2011; 34: 947-960Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar The pre-GC phase is often considered a checkpoint at which TFH cells briefly interact with noncognate B cells, using ICOS/ICOSL to migrate across the T/B border in search of antigen-presenting cognate B cells.2Crotty S. T Follicular helper cell biology: a decade of discovery and diseases.Immunity. 2019; 50: 1132-1148Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar,42Xu H. Li X. Liu D. Li J. Zhang X. Chen X. et al.Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility.Nature. 2013; 496: 523-527Crossref PubMed Scopus (306) Google Scholar When TFH cells meet cognate B cells, the exchange of activation signals (eg, peptide-MHC [pMHC]/TCR, CD40/CD40L, ICOS/ICOSL) drives B cells into a mature GC B-cell phenotype or toward extrafollicular memory B-cell and plasma cell differentiation.43Koenig J.F. Bruton K. Phelps A. Grydziuszko E. Jiménez-Saiz R. Jordana M. Memory generation and re-activation in food allergy.Immunotargets Ther. 2021; 10: 171-184Crossref PubMed Scopus (7) Google Scholar In this model, the ultimate fate of a TFH cell is to acquire a GC-TFH phenotype, which is instructed reciprocally by signaling from cognate B cells. This is supported by intravital microscopy experiments demonstrating TFH cells migrating with B cells into the B-cell follicle to seed or join GCs.44Okada T. Miller M.J. Parker I. Krummel M.F. Neighbors M. Hartley S.B. et al.Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells.PLoS Biol. 2005; 3: e150Crossref PubMed Scopus (458) Google Scholar Further, TFH cells decline rapidly and do not acquire a GC-TFH phenotype in B-cell–deficient mice.45Weinstein J.S. Bertino S.A. Hernandez S.G. Poholek A.C. Teplitzky T.B. Nowyhed H.N. et al.B cells in T follicular helper cell development and function: separable roles in delivery of ICOS ligand and antigen.J Immunol. 2014; 192: 3166-3179Crossref PubMed Scopus (49) Google Scholar However, some evidence suggests that the pre-GC phase of TFH activation may represent a subset of TFH cells that are not necessarily fated to enter GCs. Their localization at the T/B border is unique compared with GC-TFH cells and they have a distinct phenotype (eg, lower CXCR5, Bcl6).24Krueger P.D. Goldberg M.F. Hong S.W. Osum K.C. Langlois R.A. Kotov D.I. et al.Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.Immunity. 2021; 54: 687-701.e4Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar They can also be detected throughout a primary immune response and they persist following GC collapse.24Krueger P.D. Goldberg M.F. Hong S.W. Osum K.C. Langlois R.A. Kotov D.I. et al.Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.Immunity. 2021; 54: 687-701.e4Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar When a single naive T cell is transferred before immunization, some TCR clones preferentially generate a TFH phenotype whereas others generate a higher proportion of GC-TFH cells, suggesting that TCR specificity or affinity may regulate entry into either fate.46Tubo N.J. Pagán A.J. Taylor J.J. Nelson R.W. Linehan J.L. Ertelt J.M. et al.Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.Cell. 2013; 153: 785-796Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar Additionally, the functions performed at the T/B border are unique from those performed in the GC. Recent work demonstrated that GC B cells arrive in the GC following class switch recombination (CSR) and minimal switching happens within the GC, indicating that TFH cells at the T/B border may be the “subset” that mediates CSR.47Roco J.A. Mesin L. Binder S.C. Nefzger C. Gonzalez-Figueroa P. Canete P.F. et al.Class-switch recombination occurs infrequently in germinal centers.Immunity. 2019; 51: 337-350.e7Abstract Full Text Full Text PDF PubMe