A brief history of T cell help to B cells

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TIMELINE

A brief history of T cell help to B cellsspecificity’ paper by Nossal and Lederberg9. By 1972, the term ‘helper T cells’ was widely used to describe the thymus-educated cells that provide help to B cells5,8.

Discovery of interleukin?4. The nature of the ‘help’ was not immediately apparent5. Indeed, even today we are still trying to understand the process of T cell help to B cells. One early Shane Crottymodel was that helper T cells may secrete Abstract | In celebration of the 50th anniversary of the discovery of B cells, I take a one or more cytokines that are the molecular look back at the history of T cell help to B cells, which was discovered 47 years ago. embodiment of the ‘help’ to B cells. In 1982, In addition, I summarize and categorize the distinct molecules that are expressed interleukin-4 (IL-4) was discovered as the 10,11by CD4+ T cells that constitute ‘help’ to B cells, and particularly the molecules first B cell help factor (FIG. 1). The role of

expressed by T follicular helper (TFH) cells, which are the specialized providers of IL-4 was identified on the basis of its secretion from the mouse thymoma EL4 cell line and help to B cells.the in vitro ability of IL-4 in combination with B cell receptor (BCR) signalling to increase A timeline of B cell help discoveriesmice (consisting of predominantly mature the number of B cells. With the development Providing help to B cells was one of the earli-T cells obtained by cannulation) into adult of the TH1 cell–TH2 cell paradigm in 1986 est discovered functions of T cells, resulting thymectomized and irradiated CBA mice (REF. 12), it was generally inferred that as in the coining of the term ‘T helper (TH) cell’. that had been reconstituted for 2 weeks with there were two types of CD4+ T cells and The first indications came from Claman and CBA bone marrow and then immunized. only TH2 cells expressed IL-4, these must be colleagues in 1966 (REF. 1), but an unambigu-They made use of strain-specific antiserum the CD4+ T cells that help B cells. Although ous demonstration of a role for thymus-(H2-specific serum) to deplete CBA or the initial TH1 cell–TH2 cell paper had more derived helper cells in antibody responses C57BL/6 cells in vitro from spleen cell prepa-refined conclusions, the simple interpretation was made in a trio of back-to-back papers by rations from the immunized mice. Splenocyte that TH2 cells are the providers of B cell help Miller and Mitchell in 1968 (REFS 2–4) (FIG. 1). preparations depleted of C57BL/6-derived became the standard interpretation, ingrained Using cell transfer experiments, they showed cells (eliminating the thoracic duct-derived in textbooks and scientific papers alike. That that transfer of neither thymus (T) cells nor transferred cells but not the bone marrow-deduction based on in vitro data was errone-bone marrow (B) cells to irradiated mice was derived cells) did not lose antibody-secreting ous, but it was many years before the correct sufficient to result in the development of cells, whereas splenocyte preparations CD4+ T cell type would be identified. Along an antibody response after immunization depleted of CBA-derived cells (in which the the way, there were sporadic publications of mice with sheep erythrocytes. However, thoracic duct-derived cells and bone marrow-showing that deletion of TH2-associated genes co-transfer of both bone marrow-derived derived cells were eliminated) lost 97% of all did not result in a loss of germinal centres 3and thymus-derived cells led to robust antibody-secreting cells.in vivo13,14; these studies revealed a major gap antibody responses2,3. These experiments A rapid flurry of confirmatory studies in the understanding of T cell help to B cells. showed that the cells from the thymus were were published showing the requirement of Germinal centres are microanatomical struc-necessary for the antibody response to the T cell help for antibody responses against tures within the B cell regions of the lymph immunogen but that the thymus-derived many types of antigens in a plethora of nodes and the spleen. The germinal centres 5cells did not produce the antibodies them-experimental systems, including the impor-are the active sites of large-scale antigen-selves. Thus, two different cell types — B cells tant hapten-carrier systems that enabled specific B cell proliferation and mutation. It and T cells — were required to collaborate B cell and T cell antigens to be distinguished is primarily in germinal centres that B cells to induce an antibody response. The T cells at the molecular level5,6. One compelling evolve high-affinity BCRs via mutation and were recognized as a form of supporting cell experimental approach made use of T cell-selection by CD4+ T cells, and it is almost type and termed ‘antigen-reactive cells’ by depleting antiserum (θ-specific serum) to mainly via germinal centres that B cells 27the authors. The definitive nature of these eliminate T cells and thereby to prevent develop memory in the form of long-lived papers resulted from a series of careful and T cell help to B cells and antibody responses plasma cells and memory B cells15. Germinal clever controls — including using T cell-to immunogens8. However, of note, T cell centres depend on CD4+ T cells for their depleting antiserum, thymectomies and help was not required for antibody responses development and maintenance, and for the 2–4chromosomal markers. In one experiment, to Salmonella adelaide flagellin, which is production of plasma cells. Of particular note, Miller and Mitchell transferred thoracic duct the antigen that is used in the seminal and germline deletion of Il4 in mice resulted in no cells from CBA mice crossed with C57BL/6 brilliant 1958 ‘one cell — one antibody significant reduction of germinal centres or of IMMUNOLOGY

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Figure 1 | A timeline of discoveries about T cell help to B cells. T cell help to B cells was discovered only a few years after the discovery of B cells. Subsequent discoveries lead to coining of the term T follicular helper (TFH) cells ~30 years later. BCL-6, B cell lymphoma 6; CXCR5, CXC-chemokine receptor 5; ICOS, inducible T cell co-stimulator; SAP, SLAM-associated protein.

total IgG in response to immunizations; the effects of loss of Il4 were generally restricted to loss of IgE and IgG1, and a bias in the ratios of different IgG subtypes13,16. These results indicated that IL-4 uniquely contributes to IgE class-switch recombination but that most other aspects of T cell help to B cells primarily depend on other molecules.

Discovery of CD40. In the meantime, the importance of CD40 and CD40 ligand (CD40L) was discovered, creating interest in the help to B cells that occurs via direct interactions between CD4+ T cells and B cells, in addition to the role of the secretion of cytokines (which may act at a distance). The first step to recognizing the role of CD40 came in 1986 with the generation of an antibody specific for human CD40, which induced B cell proliferation when combined with BCR signals17. Furthermore, stimulation of human germinal centre B cells with a CD40-specific antibody prevented apoptosis18. Clearly, CD40 was an important molecule on the surface of B cells, but what was the ligand? CD40L was cloned in 1992 and was found to be highly expressed by activated CD4+ T cells19. Strikingly, treatment of naive mouse or human B cells with a CD40L–Fc fusion protein could induce B cell proliferation in the absence of any additional co-stimulation, which indicates that CD40L signalling to CD40 is a dominant mechanism of T cell help to B cells19. Shortly thereafter, it was determined by several research groups that the severe human genetic immuno-deficiency X-linked hyper-IgM syndrome is

frequently caused by mutations in CD40LG (the gene encoding CD40L)20–24, which reinforced the concept that CD40L signals from CD4+ T cells are a primary component of T cell help to B cells. Individuals with X-linked hyper-IgM syndrome who had mutations in CD40LG lacked germinal cen-tres. In 1994, it was shown that a CD40L- specific monoclonal antibody could prevent the formation of germinal centres in mice25. In addition, germinal centres were known to contain CD4+ T cells25,26, which implied that CD4+ T cells provide help to germinal centre B cells, and at least one component of that T cell help was contact-dependent CD40L.Discovery of the role of IL?21. In 2000, the cytokine IL-21 was cloned and shown to help B cell proliferation27,28. IL-21 has since been shown to be the most potent cytokine for stimulating plasma cell differentiation29,30. Mice that are deficient for both IL-4 and the IL-21 receptor (Il4?/?Il21r?/? mice) were found to have severe defects in antibody production, class-switch recombination and germinal cen-tres, which indicated that the combination of these two cytokines was important for help to B cells31. However, the source of the IL-4 and IL-21 remained unclear.Contact dependency. At this time, the evidence for additional contact-dependent help functions of T cells to B cells was also accumulating. Both inducible T cell co-stimulator (ICOS) and SLAM-associated protein (SAP; also known as SH2D1A) are expressed by CD4+ T cells and deletion of the corresponding genes (Icos or Sh2d1a, respec-tively) results in severe defects in germinal centres and B cell memory32–35. Mutations in SH2D1A36 (which result in the clinical dis-order X-linked lymphoproliferative disease)or ICOSL (which encodes ICOS ligand)37 result in immunodeficiency. A mutation in SH2D1A frequently leads to child mortality as a result of increased susceptibility to cer-tain infections36. As SAP binds cytoplasmic tails of signalling lymphocytic activation molecule (SLAM) family receptors, it was proposed that SAP was involved in adhesion and/or co-stimulation of B cells by T cells38. Nevertheless, discovering the importance of ICOS and SAP in T cell help to B cells raised more questions than answers about B cell–T cell interactions. The crucial importance of colocalization of CD4+ T cells and B cells for T cell help was highlighted when tech-nological advances in micro scopy enabled intravital microscopy imaging. Intravital microscopy studies revealed extensive cog-nate interactions between CD4+ T cells and B cells in the border region between the T cell zone and the B cell follicle early dur-ing antigen-specific immune responses39–41 and later in germinal centres42. Later, it would become clear that ICOS has roles in T follicular helper (TFH) cell differentiation43, migration44 and cytokine production45.Defining TFH cells. Although the TH1 cell–TH2 cell paradigm held sway for many years, cracks in that oversimplification emerged over time and, ultimately, discoveries showed that the catalogue of CD4+ T cell © 2015 Macmillan Publishers Limited. All rights reserved

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receptor 5 (CXCR5), which was shown a decade earlier to be required by B cells for entry into follicles61 and therefore it was logical that TFH cells would need to express the same chemokine receptor. It was also determined that TFH cells express IL-21

(REF. 50) and that TFH cells are the primary producers of IL-4 in lymphoid tissue62,63. The regulation of Il4 differs between TFH cells and TH2 cells — in TFH cells Il4 is regulated by SAP and protein kinase Cθ (PKCθ), and in TH2 cells it is regulated by GATA-binding protein 3 (GATA3) — and it is the IL-4 secreted from TFH cells that is necessary for class-switch recombination64–67.Following the identification of BCL-6, the study of TFH cells and T cell help to B cells has markedly increased. Stages of TFH cell differentiation, inductive signals, migration patterns, memory, associations with human autoimmune diseases, and BCL-6+ TReg cells (also known as T follicular regulatory (TFR) cells) have since been discovered and have recently been reviewed68. Briefly, TFH cell differentiation is independent of the differen-tiation of TH1 cells, TH2 cells or TH17 cells54, and induction of BCL-6 expression and TFH cell differentiation can occur within the first 48 hours of CD4+ T cell priming, by the second cell division43,69–71, by dendritic cells or by other myeloid antigen-presenting cells43,72. Further-more, our understanding of the nature of T cell help to B cells has become more refined.

What is T cell help?‘Help’ to B cells is not a single product of TFH cells and not even a single process. T cell help to B cells can be divided into seven distinct functions, as illustrated in FIG. 2: proliferation, survival, plasma cell differen-tiation, somatic hypermutation, class-switch recombination, adhesion and attraction. These seven different forms of help are all contributors to TFH cell–B cell interactions, and each process consists of multiple path-ways, with only a minority shown in FIG. 2 for simplicity. Furthermore, some molecules have a role in several different forms of help.The simplest B cell help function that is provided by TFH cells is the induction of B cell proliferation. CD40L is the most prominent protein expressed by TFH cells that contributes to pro-mitotic signalling in B cells64. Survival signals from TFH cells are also crucial, as germinal centre B cells are exquisitely pro-apoptotic73. IL-4 produced by TFH cells triggers pro-survival signals to germinal centre B cells via the IL-4 receptor complex64. Somatic hypermutation is central to germinal centre biology and the primary purpose of germinal centres is to facilitate

| Immunologytypes included many more than just TH1 cells and TH2 cells. This started with the firm establishment of regulatory T (TReg) cells in 2000–2003 — catalysed by the discovery of forkhead box P3 (FOXP3)46. The catalogue of CD4+ TH cell types then expanded to include TH17 cells in 2005–2006 (REF. 47). These revelations opened the door for seri-ous consideration that there may be a subset of CD4+ T cells that are specialized in B cell help. TFH cells were first proposed in 2000 and 2001 (REFS 48–50) (FIG. 1). However, that proposal was mainly ignored as shown by the lack of mention of TFH cells in almost all CD4+ T cell reviews and textbook chapters in the years thereafter. Nevertheless, some savvy scientists recognized the importance of the TFH cell concept and forded key areas51–57. TFH cells were not widely accepted until 2009 when the transcrip-tional repressor B cell lymphoma 6 (BCL-6) was identified as a lineage-defining tran-scription factor of TFH cells58–60. A range of experiments — including the use of Bcl6?/? CD4+ T cells, constitutive expression of BCL-6 in antigen-specific CD4+ T cells and manipulation of the expression of B lymphocyte-induced maturation protein 1 (BLIMP1; a potent antagonist of BCL-6) in CD4+ T cells — showed that the expression of BCL-6 by CD4+ T cells is necessary for TFH cell differentiation and that TFH cells are the unique providers of T cell help to B cells for the development of germinal centres and for the generation of most class-switched antibodies58–60. A central marker of TFH cells is CXC-chemokine

IMMUNOLOGY affinity maturation of B cells via sequential rounds of immunoglobulin gene mutation and selection68,74,75. The enzyme activation-induced cytidine deaminase (AID; which is encoded by Aicda) induces the DNA damage in the immunoglobulin genes that is then converted into mutations by DNA repair enzymes73. BCL-6 must be co-expressed with AID by the germinal centre B cell to repress the DNA damage response programme that would otherwise trigger self-destruction of the cell76. The signals that induce AID and BCL-6 expression by B cells are not entirely defined, but CD40L, IL-4 and IL-21 contrib-ute77. Indeed, the combination of CD40L, IL-4 and IL-21 in different ratios seems to be the primary mix of T cell help signals that control B cell proliferation, somatic hypermutation and differentiation. Class-switch recombination can also be induced by instructive signals from TFH cells to B cells. AID is necessary for class-switch recombina-tion, but the specific target of the heavy chain constant region gene recombination depends on additional factors that are selectively activated by different cytokines, which pre-dominantly, but not exclusively, come from CD4+ T cells. Human IgM to IgG class-switch recombination is most efficiently induced by IL-21, whereas IgE recombination is induced by a high IL-4 to IL-21 ratio78,79.B cell help crucially depends on cell con-tact, probably because of a mixture of cell-surface co-stimulatory ligand interactions and directional cytokine production during cognate interactions. Therefore, adhesion molecules expressed by TFH cells and B cells (FIG. 2) are necessary components of T cell help to B cells, as they regulate the overall duration of the ‘pas de deux’. The most dra-matic example of this requirement is SAP, which is described above. SAP binds to the intracellular domains of SLAM family surface receptors, which are involved in cell–cell adhesion. In the absence of SAP, the duration of B cell–T cell adhesion is short and inade-quate for the TFH cell to provide sufficient help signals to the B cell. This leads to a general defect in SAP-dependent T cell help to B cells and thus a loss of antigen-specific B cell pro-liferation and survival, as well as a complete loss of germinal centres and of most memory B cells and long-lived plasma cells32,57,80.Finally, chemoattraction is another component of T cell help to B cells (FIG. 2). CXC-chemokine ligand 13 (CXCL13) is the ligand for CXCR5 and human germinal centre TFH cells constitutively secrete copious quantities of CXCL13 (REFS 81,82), which probably recruits B cells to colocalize with the TFH cells and to facilitate confinement of

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TGFβ andpossiblyothers

IL-4andIL-21

Figure 2 | Categories of T cell help to B cells. Help can come in many different forms and can have different consequences for different processes. T follicular helper (TFH) cells provide seven main forms of T cell help to B cells: signals that promote survival, proliferation, plasma cell differentiation, hyper-mutation, class-switch recombination, adhesion and chemoattraction (cell migration). For simplicity, only a few examples of factors that are important for each process are shown, although many more molecules are involved in the regulation of the processes. Several of these pathways are reviewed in detail elsewhere64,73,85. Some molecules have pleiotropic effects, resulting in

combinatorial possibilities and functional redundancies between molecules. | Immunology

AID, activation-induced cytidine deaminase; BAFF, B cell-activating factor; BCL, B cell lymphoma; BLIMP1, B lymphocyte-induced maturation protein 1; CD40L, CD40 ligand; CXCL13, CXC-chemokine ligand 13; CXCR5, CXC-chemokine receptor 5; IL, interleukin; MCL1, myeloid cell leukaemia 1; SAP, SLAM-associated protein; SLAMF, signalling lymphocytic activation mole cule F; TGFβ, transforming growth factor-β.

the B cells to the germinal centre. Notably,

CXCL13 signalling via CXCR5 also modifies B cell adhesion and lymphotoxin synthesis, which shows that CXCL13 also has cytokine-type functions83,84. Thus, chemoattraction is another form of T cell help to B cells.Conclusions and perspectives

T cell help to B cells is a complex interplay of many factors and processes. Particularly in the germinal centre, many signals (both stimulatory and inhibitory) are exchanged between TFH cells and germinal centre B cells (and other cells in the microenvironment) in an iterative manner, over many rounds of rapid B cell division, mutation and selection. These integrated interactions remain poorly understood at the molecular and temporal levels, as the features of the cells change and the availability of antigen becomes more and

more limiting. Furthermore, fundamental gaps remain in defining the signals that induce or that inhibit TFH cell differentiation68. Finally, it is important to better understand TFH cells in humans who have been immunized with vaccines to learn how to better boost vaccine responses, and an increased understanding of TFH cells in individuals with autoantibody-associated auto immune diseases or allergies is important for learning how to ameliorate or how to block these TFH cell responses. There is much to be learned in the next 47 years about T cell help to B cells!

Shane Crotty is at the Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, San Diego, California 92037, USA.

e-mail: shane@http://wendang.chazidian.com

doi:10.1038/nri3803

Published online 13 February 2015

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Competing interests statement

The author declares no competing interests.

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