A novel subset of T-helper cells: follicular T-helper cells and their markers

It has been known for a while that T-helper (T_H) cells are involved in the regulation of B-cell responses. The characterization of a precise subset of these specialized cells, called follicular T-helper cells (T_FH), was first reported in 2000.1 T_FH cells are characterized by their homing capacity in CXCL13-rich areas, such as B-cell follicles, through CXCR5 expression and their ability to support immunoglobulin production. The differentiation and homing of T_FH, as well as their interactions with B cells, have been the subject of intense research and several markers have been identified as components of the T_FH signature. Thanks to gene expression profiling carried out on peripheral T-cell lymphomas, a T_FH signature has been reported in angioimmunoblastic T-cell lymphoma (AITL) and in follicular T-cell lymphoma.2–4 Several markers that identify T_FH cells are now used in hematopathology to reinforce the diagnostic criteria of AITL. In this issue of the Journal, Marafioti et al.5 describe that inducible T-cell co-stimulatory molecule (ICOS), a CD28 homolog implicated in the regulation of T-cell differentiation and function, appears to be a relevant marker for the diagnosis of T_FH-derived lymphoma such as AITL and follicular T-cell lymphoma. This review first summarizes the recent evidence on T_FH cell differentiation and the present state of knowledge on molecules expressed by T_FH cells. Then, from this perspective, the potential implications of T_FH cells in lymphomagenesis and as part of the tumor microenvironment are covered.

Lineage and differentiation of follicular T-helper cells

T_H cell responses are heterogeneous and effector function choice is imposed during initial activation of the naïve T_H cells through the expression of transcription factors. It is now clear that T_FH cells belong to a subset of T_H cells that differ from the other T_H cell subsets. Indeed, T_H1 cells express STAT4, STAT1 and T-box transcription factor/T-bet, T_H2 cells express GATA3, T_H17 cells express retinoid related orphan receptor (RORγt) and regulatory T cells (Treg) express FOXP3. It has recently been shown that the T_FH cell differentiation program is controlled by BCL6.6,7 T_FH cells upregulate BCL6 which in turn blocks T_H1, T_H2 and T_H17 cell differentiation by repressing their selective transcription factors.6 Moreover, BCL6 antagonizes the expression of Blimp-1 transcription factor, which is preferentially expressed by T_H cells in the T zone area.6,8 BCL6 also represses the expression of miR-17-92, a micro RNA that down-regulates CXCR5 expression, which is essential for T_FH cell function. Finally over-expression of BCL6 in activated T_H cells induces expression of interleukin (IL)-6 receptor and IL-21 receptor, which are both required for T_FH cell generation as shown by the role of IL-6 and IL-21 in T_FH cell differentiation.9 ICOS is also implicated in the regulation of T_FH cell differentiation. Once engaged with its ligand (ICOS-L), expressed on antigen-presenting cells including B cells, ICOS induces the production of helper cytokines such as IL-2, IL-4 and especially IL-10 and IL-21.10 ICOS deficiency is associated with a reduction of germinal center formation and fewer T_FH cells, suggesting that ICOS signaling is essential in T_FH cell generation.10 Recent reports described that T_FH cells can produce significant amounts of IL-4, interferon-γ and IL-17 which are normally associated with T_H2, T_H1 and T_H17 cells, respectively.1,11 Thus, T_FH cells share phenotypic features with other T_H cell lineages raising the question of whether T_FH cells really do constitute a distinct lineage or whether they result from a conversion of effector T_H cells that acquire T_FH cell functions.

The development of follicular homing ability by activated T_H cells is the first event in the process of T_FH generation. Indeed, naïve T cells that express CD62L and CCR7 enter secondary lymphoid organs in the T-cell zones.8,11 Up-regulation of CXCR5, the receptor for CXCL13, a chemokine produced by follicular dendritic cells which promotes B-cell entry into the follicle, occurs after the interaction between antigen-presenting cells and naïve T_H cells.8 Thanks to the expression of CXCR5 and down-regulation of CCR7, T_FH cells move into the B follicle where they regulate antigen-specific B-cell responses via co-stimulatory signals delivered through CD28, OX40 and ICOS.11

Cell functions and markers of follicular T-helper cells

In addition to CXCR5, T_FH cells express markers such as CD25, CD69, CD95, CD57 (in humans only), OX40 (CD134) and CD40L (CD154) and induce over-expression of activation-induced cytidine deaminase in B cells. T_FH also produce cytokines such as IL-10 and IL-21 that promote B-cell survival and antibody production.10 An antigen-dependent T-B cell interaction is a critical initiating event in the development of effective B-cell immunity. This cognate interaction induces B cells to become short-lived plasma cells or to enter the germinal center where B cells will undergo maturation processes (immunoglobulin class-switching and somatic hypermutation) which will lead in the end to the development of high-affinity long-lived plasma cells and memory B cells.1,2,8 Mutations of CD40L or ICOS genes in mice or humans are associated with T_FH cell deficiency and affect B-cell differentiation and humoral responses, indicating the important role of the co-stimulatory molecules in regulating B-cell responses. Of note, T_FH cells express some molecules which play a key role in the stability of the antigen-dependent T cell-B cell interaction such as programmed cell death 1 (PD1), an immunoreceptor which favors strong cognate interactions with B cells in the germinal center, and the signaling lymphocytic activation molecule-associated protein (SAP) which is required to form stable contacts between B and T cells and supports T_FH cell generation and germinal center formation.10,12 A recent study demonstrated that T_FH can express high levels of B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor of the CD28 superfamily which negatively regulates the immune response in synergy with the PD1/PDL1 inhibitory pathway.13

T_FH cells seem to play an important role in systemic autoimmunity. Mice with systemic autoimmune diseases such as systemic lupus erythematosus show a constant increase of T_FH cells.2 Furthermore, drugs that block ICOS or IL-21 signaling pathways improve autoimmune disorders such as diabetes, rheumatoid arthritis and murine models of systemic lupus erythematosus.11 T_FH cells could, therefore, represent an interesting therapeutic target in autoimmune diseases.

Implications of follicular T-helper cells in lymphoma

AILT is an aggressive lymphoma characterized by a polymorphic infiltrate, marked proliferation of endothelial venules and a dense meshwork of follicular dendritic cells.14 Gene expression profiling studies have identified a specific signature corresponding to T_FH cells.2–4 Furthermore, these studies have shown that T_FH cells represent the putative cell of origin of AILT. T_FH cells seem to be responsible for the clinico-biological aspects of AILT, including B-cell activation, hypergammaglobulinemia and proliferation of follicular dendritic cells. Typically the tumor cells of AILT have a T_FH phenotype with expression of CD3, CD4, CXCR5, CD57 and BCL6. CD10, which is not expressed in normal T_FH cells, but rather in a distinct subset of GC T_H cells, is frequently present at the cell membrane of lymphoma cells.3,4 In addition, the neoplastic T cells frequently lose CD5 and CD7.14 Most interestingly, CXCL13, PD1 and SAP are also markers of malignant cells in AILT.15,16 This signature is not completely specific to AILT since it has also been reported in peripheral T-cell lymphoma not otherwise specified.15,17 Rare cases of peripheral T-cell lymphoma with a follicular growth pattern display a T_FH phenotype and show some overlapping features with AILT, raising the question of a possible relationship between the two lymphoma types.5,18 In addition, in the study by Marafioti et al.,5 ICOS appeared to be expressed by tumor cells of both AILT and peripheral T-cell lymphoma not otherwise specified.

Figure 1.Human TFH in a lymph node from a patient with angio-immunoblastic T-cell lymphoma. Note the interactions between PD-1⁺ (in green) and CD4⁺ (in blue) TFH cells and CD20 (in red) B cells (confocal microscopy x500).

Nodular lymphocyte-predominant Hodgkin’s lymphoma is a monoclonal B-cell neoplasm characterized by nodular or nodular and diffuse proliferation of scattered large neoplastic B cells referred to as popcorn or lymphocyte-predominant cells.14 These cells interact with aggregates of follicular dendritic cells and immune cells such as small naïve B cells and T cells. Furthermore, nodules of this form of lymphoma are characterized by an increase of germinal center-derived CD57 T cells which are closely associated with neoplastic lymphocyte-predominant cells.19 These CD57 T cells also express markers reminiscent of germinal center T_H cells such as PD1, BCL6, IRF4/MUM1 and c-MAF.19 Altogether these data suggest that some of these reactive cells could be T_FH cells, but a more precise micro-dissection of the microenvironment is required to confirm this.

In follicular lymphoma, derived from germinal center B cells, tumor cells reside and proliferate in follicular structures in close association with T_H cells and follicular dendritic cells.19 In order to proliferate, follicular lymphoma cells require cellular interactions such as those in normal germinal centers, including contacts with germinal center T_H cells which express CD3, CD4, CD57, PD1 and CXCL13.19 Proliferation is stimulated by CD40-CD40L interaction, markers expressed on malignant B cells and T_FH cells, respectively.19 This is further supported by the presence of significant amounts of PD1 in the germinal centers of patients with follicular lymphoma, as shown in a recent study.20 As for FOXP3 cells, high numbers of PD1 cells seem to have a favorable impact on progression in follicular lymphoma.20 Nevertheless, the exact nature of those PD1 cells remains unclear and further studies are needed to determine whether or not they correspond to T_FH cells.

The increasing attention paid to T_FH cells has revealed notable plasticity in the differentiation pathways of these cells. Beside canonical T_H markers, the minimal T_FH signature requires expression of CXCR5, PD1, BCL6, SAP and ICOS. These markers can also be individually expressed by other T_H cell subsets. The use of immunohistochemistry to detect T_FH cells on tissue sections is, therefore, questionable since only one or two markers can be labeled simultaneously. Hence, identification of T_FH cells relies on both phenotype and function, which are closely related to the specific microenvironment in which T_FH cells localize, requiring further studies based on multicolor cell imaging.

Footnotes

• Dr Laurent is an Assistant Professor of Pathology and works with Dr Brousset, Professor of Pathology in the University of Medicine in Toulouse.
• Dr Laurent (MD, PhD), Pr Brousset (MD, PhD) and Dr Fazilleau (PhD, Researcher) work in the Centre de Physiopathologie de Purpan, Inserm U563, in Toulouse.
• Related Original Article on page 356
• No potential conflicts of interests relevant to this article were reported.