T follicular helper cells in cancer

Increased frequencies of Tfh cells in solid organ tumors of non-lymphocytic origin are often associated with a better T follicular helper (Tfh) cells provide essential help to B cells for effective antibodymediated immune responses. Although the crucial function of these CD4 T cells in infection and vaccination is well established, their involvement in cancer is only beginning to emerge. Increased numbers of Tfh cells in Tfh cell-derived or B cellassociated malignancies are often associated with an unfavorable outcome, whereas in various solid organ tumor types of non-lymphocytic origin, their presence frequently coincides with a better prognosis. We discuss recent advances in understanding how Tfh cell crosstalk with B cells and CD8 T cells in secondary and tertiary lymphoid structures (TLS) enhances antitumor immunity, but may also exacerbate immune-related adverse events (irAEs) such as autoimmunity during immune checkpoint blockade (ICB) and cancer immunotherapy. prognosis.


T follicular helper cells in cancer
Nicolás Gutiérrez-Melo 1 and Dirk Baumjohann 1, * T follicular helper (Tfh) cells provide essential help to B cells for effective antibodymediated immune responses. Although the crucial function of these CD4 + T cells in infection and vaccination is well established, their involvement in cancer is only beginning to emerge. Increased numbers of Tfh cells in Tfh cell-derived or B cellassociated malignancies are often associated with an unfavorable outcome, whereas in various solid organ tumor types of non-lymphocytic origin, their presence frequently coincides with a better prognosis. We discuss recent advances in understanding how Tfh cell crosstalk with B cells and CD8 + T cells in secondary and tertiary lymphoid structures (TLS) enhances antitumor immunity, but may also exacerbate immune-related adverse events (irAEs) such as autoimmunity during immune checkpoint blockade (ICB) and cancer immunotherapy.

Tfh cells in immunity and autoimmunity
Tfh cells (see Glossary and Box 1) are the specialized CD4 + T cell subset that provides essential help to B lymphocytes for effective antibody responses against various pathogens, including viruses, bacteria, fungi, and helminths [1,2]. These functions of Tfh cells are also being utilized during vaccination through their ability to drive immunoglobulin class-switching and affinity maturation in germinal centers (GCs), resulting in the generation of long-lived plasma cells and memory B cells [1,2] (Figure 1, Key Figure). In contrast, Tfh cell dysregulation may be associated with the development of autoimmunity [3]. Although most attention in cancer and immunooncology research has been given to effector CD4 + and in particular CD8 + T cells [4], Tfh cells are now emerging as another highly relevant immune cell population in various cancer types. However, the participation of Tfh cells in oncological settings differs significantly across different cancer entities such as Tfh cell-derived neoplasms, B cell lymphomas, and solid organ tumors of non-lymphocytic origin. In this review we discuss all three scenarios, with a focus on the recently fast-paced discoveries describing their involvement in solid tumors. In this regard, the role of Tfh cells in TLS, in ICB therapies, and as a potential link for the development of irAEs following ICB are of particular importance.
Tfh cells in cancer: the good, the bad, and the ugly One of the main challenges in assessing the involvement of Tfh cells in cancer has been the broad biological diversity of cancer entities because each of them presents unique genetic and phenotypic characteristics, as well as different disease progression and clinical outcomes. It is therefore not surprising that the role of Tfh cells varies depending on the type of cancer. To approach such diversity, it is useful to make a distinction between those instances in which Tfh cells (or Tfh-like cells) are the cancer entities themselves, and those in which Tfh cells participate in the disease pathobiology of other cancer entities derived from a different cell of origin (Table 1).
Tfh cell-derived lymphocytic neoplasms These malignancies contain T cell neoplasms in which the cells are derived from and/or have a Tfh cell phenotype and genetic signature, usually defined by the expression of Tfh cell-related markers such as CD279/PD-1, CD10, BCL6, CXCL13, or ICOS [5]. This category includes CD4 + T follicular helper (Tfh) cells provide efficient help to B cells and can be found in tertiary lymphoid structures of tumors.
Increased frequencies of Tfh cells in Tfh cell-derived or B cell malignancies are often associated with an unfavorable outcome.
Increased frequencies of Tfh cells in solid organ tumors of non-lymphocytic origin are often associated with a better prognosis.
The beneficial function of Tfh cells in solid tumors may extend beyond their primary role of providing help to B cells, for example, by fueling cytotoxic CD8 + T cell responses.
Tfh cells may contribute to the development of immune-related adverse events (irAEs) following checkpoint immune blockade (ICB)-based cancer immunotherapy. different entities previously known as angioimmunoblastic T cell lymphoma (AITL), follicular T cell lymphoma, and nodal peripheral T cell lymphomas, which have now been grouped as subtypes under the umbrella designation of nodal T follicular helper cell lymphoma (nTFHL) in the most recent World Health Organization (WHO) classification of lymphoid neoplasms [5]. Mutations in TET2 and DVMT3A are regularly found in nTFHL patients, although they can also be present in other types of lymphoma [5,6]. Efforts to discover early diagnostic markers specific for nTFHL led to the identification of the RHOA Gly17Val (c.50G>T) mutation that can be detected on average 7.87 months earlier than the lymphoma diagnosis; nonetheless, this detection method is not able to discriminate among the three nTFHL subtypes [6]. Differential diagnosis among the subtypes remains challenging because it has largely relied thus far on histopathological characteristics rather than on robust biological markers [5,7]. Some even argue that the significant clinical and molecular overlap between the three nTFHL subtypes suggests that these lymphomas are not separate entities and are instead a continuum or differential manifestations of the same disease [8]. Using genetic multivariate analyses, distinct molecular profiles have been identified that support the current WHO classification [9]. However, these profiles were only identified under supervised analyses, whereas unsupervised analyses did not show any clear subtype-defining clustering, suggesting that genetic differences might not be as strong as expected [9]. Tfh cell-associated genetic markers [10] or immunohistological findings [11] have also been reported in histiocyte-rich large B cell lymphoma or in nodal marginal zone B cell lymphoma, calling for extra caution when interpreting results using such markers. Despite this biomarker heterogeneity, Tfh cell-defining markers remain crucial for nTFHL progression, as shown in an AITL-like mouse model in which ablation of BCL6 or loss of SLAM-associated protein (SAP) led to a significant reduction of tumor growth [12]. Studies addressing nTFHL with specific mutations have also revealed a strong association between Tfh cell markers and this type of cancer. For instance, IDH2 R172 tumors present higher expression of CXCL13 and CD10 [13], whereas RHOA Gly17Val tumors were more likely to present two or more Tfh cell markers including PD-1, ICOS, CD10, or CXCL13 compared to wild-type RHOA (RHOA WT ) [14]. Although no difference in proliferation was detected between RHOA Gly17Val and RHOA WT , it is interesting that the former had significantly higher B symptoms and splenomegaly [14]. Altogether, the evidence suggests that Tfh cell markers in nTFHL not only serve for classification purposes but also play an important role in disease pathogenesis. Investigating how normal Tfh cell function is dysregulated in each of these entities will be essential for the design of clinical strategies and the identification of novel therapeutic targets.
Tfh cells that provide help to malignant B cells As participants in other types of cancer, the contribution of Tfh cells to disease progression is widely variable, and can either promote malignancies owing to their intrinsic B cell helper activity or participate in the immune response against solid tumors. Therefore, it is again useful to differentiate between those instances in which Tfh cells have been shown to be detrimental by promoting disease progression, and those in which they have been associated with positive clinical outcomes or improved immune control (Table 1). Box

What are T follicular helper (Tfh) cells?
For many years type 2 T helper (Th2) cells were regarded as the main provider of help to B cells, and it was not until the turn of the millennium that CXCR5 + CD4 + T cells residing in CXCL13 + follicles of SLOs were named Tfh cells [89,90]. Over the years it was then clarified that, after their priming by dendritic cells (DCs), Th2 cells emigrate into the periphery to coordinate type 2 immune responses in peripheral tissues, and were not in close proximity to B cells after all [91], and that Tfh cell differentiation required the transcriptional repressor Bcl6 [92][93][94]. Tfh cells also express high levels of costimulatory (e.g., ICOS, CD40L) and coinhibitory (e. g., PD-1) receptors on their cell surface and secrete important cytokines (e.g., IL-21, IL-4), all of which are tailored to their B cell helper activity [1,2,95]. Tfh cell differentiation is characterized by stepwise differentiation that requires continuous interactions with antigen-presenting cells in different microanatomical locations within SLOs, namely priming by DCs in the T zone, and subsequent interactions with B cells at the T-B zone border and within GCs [96].

Glossary
Germinal center (GC): a T cell-dependent anatomical structure in which B cells mutate their antigen receptors to generate high-affinity antibodies. Immune checkpoint blockade (ICB): inhibitors that interfere with immune checkpoint molecules (e.g., PD-1) to reinvigorate antitumor immune responses. Immune-related adverse events (irAEs): a complication of cancer immunotherapy (e.g., induced by ICB) in which serious side effects can arise (e.g., the development of autoimmunity). T peripheral helper (Tph) cells: CD4 + T cells first described in rheumatoid arthritis patients that share several characteristics with Tfh cells but do not reside in SLOs. Secondary lymphoid organ (SLO): complex organized immune cell-containing structures in which T and B cells are activated and differentiate into effector cells. T follicular helper (Tfh) cells: the primary CD4 + T cell subset that provides essential help to B cells for the generation of potent antibody responses. Although this review mostly focuses on the involvement of Tfh cells in solid organ tumors of nonlymphocytic origin, it is relevant to briefly discuss the role of Tfh cells in B cell lymphomas because this is perhaps the most prominent case in which these cells are associated with a negative  Although some cellular and molecular mechanisms have been elucidated (unbroken arrows), the ontogeny of these cell subpopulations in tumors and tertiary lymphoid structures (TLS) remains largely unknown (broken arrows with question marks). (Bottom left) After priming by dendritic cells (DCs) in tumor-draining lymph nodes (dLNs), activated CD4 + and CD8 + T cells differentiate into effector T cells such as type 1/2/17 T helper (Th1/Th2/Th17) cells and cytotoxic T lymphocytes (CTLs), respectively, which then leave the dLN and migrate to peripheral tissues such as the tumor. Activated CD4 + T cells differentiate into early Tfh cells that interact with B cells to initiate the early extrafollicular antibody response. Some of these Tfh cells leave the dLN to become circulating Tfh (cTfh) cells, and others join antigen-specific B cells and enter the follicle to form germinal centers (GCs). In these microanatomical structures, high-affinity antibodies, long-lived plasma cells, and memory B cells are formed. GCs also harbor follicular dendritic cells (FDCs) that present native antigen to B cells. (Top right) Interestingly, similar GC-like structures consisting of B cells, FDCs, and Tfh-like cells can be found in TLS that form adjacent to or within tumor tissues. TLS-resident Tfh cells may be differentially polarized depending on the environmental context of the tumor to Tfh1, Tfh2, or Tfh17 cell types. IL-21 produced by Tfh cells supports B cells but also CTL function. Other Tfh-like cells are TfhX13 cells and microbiota-specific Tfh cells that produce CXCL13 but lack CXCR5 expression and may contribute to TLS formation. Upon ICB with anti-PD-1, the function of Tfh cells is boosted but may also contribute to the development of immune-related adverse events (irAEs). The precise kinetics and dynamics of this phenomenon are still unknown, but current evidence indicates a positive correlation between antitumor activity and the development of irAEs (bottom right).

Trends in Cancer
OPEN ACCESS impact (Table 1). These malignancies, such as follicular lymphoma, present an increased number of Tfh cells that overexpress genes such as TNF, LTA, IL4, or CD40L which alter the tumor microenvironment (TME) and promote malignant B cell survival [15]. Interactions between B cells and Tfh cells are mediated through a diverse set of membrane receptors, including ICOS, CD40L, SLAM, BTLA, PD-1, and FASL, whose impact on follicular lymphoma has been thoroughly reviewed elsewhere [16]. Pathological B cell-Tfh cell interactions are also mediated by epigenetic mechanisms that lead to repression of Tfh cell-associated genes such as Bcl6 [17]. Interestingly, single-cell analysis has recently shown that different follicular lymphoma sites from the same patient present site-restricted B cell receptor (BCR) clonotypes, and found a positive correlation between site heterogeneity and Tfh cell abundance [18]. In chronic lymphocytic leukemia (CLL) there is also a significant increase in the number of Tfh cells, particularly of the Tfh1 subset, whose frequency is associated with disease burden and which are phenotypically different from those in healthy controls in that they display higher levels of CD40L, IL-21, IFN-γ, and TIGIT [19]. Histological analysis of active CLL lymph nodes revealed a tight spatial association between CLL and Tfh cells that was not seen for any other CD4 + T cell subset, and whose expansion correlated with CLL proliferation [20]. Targeting CLL cells with the tyrosine kinase inhibitor ibrutinib led to a reduction of Tfh cell frequencies as well as a change in their phenotype, and partially restored the frequencies of Tfh subpopulations (Tfh1, Tfh2, Tfh17) to those of healthy controls [19]. Together, these findings support the idea that Tfh cells play a major role in disease development and the progression of B cell malignancies, suggesting that Tfh cells could be important targets for diagnosis and treatment.
Tfh cells as markers of positive clinical outcome in solid organ tumors In most solid organ tumors of non-lymphocytic origin, Tfh cells correlate with a better immune response against cancer, improved clinical outcomes, and increased therapy responsiveness. Although most evidence in humans is limited to observed associations, recent studies in animal models have started to elucidate the causal links for such observations (Table 1). Tfh cell gene signatures in tumor tissue are tightly correlated with immune activation and infiltration, tumor burden score, and overall survival, and have proved to be useful for patient stratification and clinical outcome prediction [21][22][23]. Research in the past decade has unraveled a broad number of mechanisms through which Tfh cells can have a positive impact on the immune response against cancer. For instance, Tfh cells have been identified as the main producers of IL-21 in the TME of different human cancers and mouse models [24,25] (Figure 1). This cytokine has a major role in mediating humoral responses by promoting B cell activation, class-switch recombination, and antitumor IgG1 and IgG3 secretion [24,26,27]. IL-21 blockade alone was able to drastically reduce B cell activation induced by coadministration of anti-PD-1 and anti-CTLA-4 therapy, highlighting the importance of Tfh cell-secreted effector molecules in cancer immunotherapy [26]. IL-21 also regulates CD8 + T cells derived from a MC38 cancer model by enhancing the expression of IFN-γ and granzyme B (GzmB), as well as the expression of surface markers such as Tigit and Lag3 [27]. Inhibition of IL-21 not only induced a reduction in CD8 + T cells with this phenotype but also abrogated the antitumor effect seen upon adoptive transfer of Tfh cells [27]. Likewise, deletion of Il21r caused a drastic drop in the frequency of mouse PD-1 hi GZMB hi CD8 + tumorinfiltrating lymphocytes (TILs) [25]. Surprisingly, a study with non-small cell lung carcinoma (NSCLC) samples found decreased levels of IL-21 production despite elevated numbers of Tfh cells, particularly in patients with advanced stages of the disease [28]. This anomaly was explained by an expansion of abnormal PD-1 + Tfh2 and PD-1 + Tfh17 subtypes which induced the differentiation of regulatory B cells [28]. By contrast, infiltrating type 1 T helper cell (Th1)polarized PD-1 hi ICOS int Tfh cells were associated with higher expression of IL-21 in the TME of breast cancer and were able to promote immunoglobulin and IFN-γ production by providing help to B and CD8 + TILs, respectively [29]. These findings highlight the need for robust phenotypic characterization of each Tfh cell subtype when assessing their impact in cancer because each might lead to a different clinical outcome (Box 2). Manipulating the balance between these subtypes could be an interesting therapeutic target, although the exact mechanisms that determine the frequencies of each population in the TME remain largely unknown.
Another key Tfh cell-derived molecule that orchestrates the immune response to cancer is the chemokine CXCL13. Although several cell types such as CD8 + T cells [30,31], follicular dendritic cells (FDCs) [32], and even cancer-associated fibroblasts [33] secrete CXCL13 in the TME, Tfh cells have been shown to be a major source of this chemokine in different cancer types [22][23][24]29,32,[34][35][36] (Figure 1). CXCL13 is essential for the recruitment of different cell types to the TME, including B cells [24] and CD8 + T cells [32], as well as for the proper formation of tumor-associated TLS [37]. In breast cancer, CXCL13 is mainly produced by a very particular PD-1 hi ICOS int CD4 + T cell subset named TfhX13 [35]. These cells display an overall Tfh-like phenotype but are characterized by a lack of the canonical Tfh cell marker CXCR5 [35]. TfhX13 cells have the capacity to recruit B cells and promote their maturation as well as functional GC formation [35]. Similar findings were reported in muscle-invasive bladder cancer, where CXCL13 was produced by central memory Tfh cells and was significantly expressed after administration of anti-PD-1 therapy [34]. These observations are consistent with another study in an ovarian cancer mouse model in which blockade of CXCL13 disrupted TLS formation and led to higher tumor volume [38]. It is important to note that CXCL13 expression by Tfh or Tfh-like cells might not be the same in all types of cancer, and therefore caution must be taken in generalizing different findings.
For instance, although Tfh-like cells were identified as the main source of CXCL13 in breast cancer [35], an inverse correlation was described between this chemokine and Tfh cell enrichment in a clear cell renal carcinoma study [39]. Whether this divergence is due to differences in activation states or phenotypic subtypes remains to be determined. High levels of CXCL13 in the TME are not always associated with a better prognosis. In different types of cancer such as breast cancer, NSCLC, colorectal cancer (CRC), and prostate cancer, high levels of CXCL13 can be correlated with more advanced disease stages and poorer prognosis [40]. Whether CXCL13 is the cause or the consequence of tumor progression remains an open question. CXCL13 has also been associated with metastases by promoting the migration, proliferation, and invasion of CXCR5 + cancer cells [40]. Nevertheless, recent data in ovarian cancer showed that CXCL13 expression shifted Box 2. Tfh cell nomenclature in cancer One particularity regarding Tfh cells in cancer is that there is great variety in the definition of Tfh and Tfh-like cells used in various studies. Usually determined by a combination of markers including CXCR5, BCL6, PD-1, CD200, CD38, and/or ICOS, a consistent approach to define these cells in the TME is still lacking (see Table 1 in main text). A previous proposal for uniform nomenclature for canonical Tfh cells is compatible with most of the variation observed in tumor-associated Tfh cells, and is therefore a useful system to implement [1]. Although most studies agree on defining bona fide Tfh cells as CD4 + CXCR5 + PD-1 + ICOS + BCL6 + IL-21 + , classification of Tfh cells into groups (or subtypes) requires assessment of at least one of the following: surface expression of CXCR3 and CCR6, cytokine expression of IFN-γ, IL-4, and IL-17, or expression of the master transcription factors T-BET, GATA3, and RORγt [1,95]. In addition to systematically capturing and classifying Tfh cell plasticity, committing to a more robust characterization will facilitate the assessment of each group in different cancer entities. For instance, Tfh1 cells, but not all Tfh or CD4 + PD-1 + cells, have been reported to be predictive of better disease-free survival in NSCLC [28]. By contrast, Tfh2 and Tfh17 cells, but not all Tfh cells, were associated with increased overall survival in clear cell renal cell carcinoma [39]. These observations also highlight how a lack of robust characterization may lead to the loss of relevant biological information. Another interesting case is the classification of CXCR5 − Tfh-like cells in cancer, such as TfhX13 cells in breast cancer [22]. CXCR5 − Tfh-like cells have been shown to be crucial for tumor control and TLS formation; however, their origin remains uncertain [81]. Lastly, moving towards a more systematic characterization and classification of Tfh and Tfh-like cells will also allow better comparison with results in other fields, particularly in autoimmunity where Tfh-like cells such as Tph cells have also been described to participate in TLS formation and B cell response stimulation [97]. To conclude, shifting towards a more uniform classification system for Tfh cells in cancer will not only simplify the interpretation of results from different types of cancer or immune conditions but will also unveil relevant biological mechanisms that would otherwise remain undiscovered. from CD4 + T cells (presumably Tfh or Tfh-like cells) in early stages of the disease to CD21 + FDCs in mature TLS [32]. This opens new questions regarding how Tfh cell function in the TME might vary over time, and how this change might impact on disease progression. It should be pointed out that, although CXCL13 is strongly produced by tumor-associated Tfh cells in humans, mouse Tfh cells do not express large amounts of CXCL13 [2].
Cell-to-cell interactions of Tfh cells with other immune cell subsets play an important role in mounting a coordinated response against cancer. Deficiency in ICOS or CD40L, hallmark molecules of Tfh cells and key mediators of interactions with B cells, have been shown to severely compromise immune control, leading to greater tumor volume over time [25]. Coculture of Tfh cells with nurse-like cells, a subset of blood mononuclear cells that are characteristic of CLL patients, induced downregulation of CD40L on the former, contributing to disease progression [20]. Studies in non-human primates reported a significant drop in the number of circulating Tfh (cTfh) cells upon administration of the anti-ICOS monoclonal antibody KY1044 [41]. Although such therapy resulted in tumor regression, it remains unclear whether local Tfh cells in the TME were also affected by it and how [41]. By contrast, silencing of the SAT1B regulator has recently been shown to increase the expression of ICOS and to induce greater Tfh cell differentiation, thereby driving enhanced infiltration and activation of B cells to the tumor site, as well as promoting TLS formation and tumor control [38]. Moreover, shorter distances between CD20 + B cells and Tfh cells in TLS have been demonstrated to correlate with longer patient survival in oral squamous cell carcinoma [42].
Altogether, participation of Tfh cells in the immune response to cancer is highly diverse and context-dependent. It relies on the secretion of different effectors as well as on surface interactions with other cells. Despite significant advances in our knowledge of the mechanisms involved, inconsistent definitions of Tfh cells across studies (Table 1) remain a significant difficulty when generalizing or comparing results from different studies (Box 2). This complication may also derive from the limited number of markers that could be simultaneously assessed until recently with conventional flow cytometry and immunohistochemistry (IHC) techniques. New high-dimensional technologies such as codetection by indexing (CODEX) have now been adapted to study the TME, thus improving the definition of different tissue cell types and introducing the concept of cell neighborhoods to TLS [43]. Robust in situ characterization of Tfh cells with such technologies will allow important gaps to be filled, particularly those regarding how different subpopulations localize and develop, and how they are regulated at the molecular level.

Tfh cells and the tumor-associated TLS architecture
One of the most important features of Tfh cells in cancer is their role in the formation and function of tumor-associated TLS, which are ectopic lymphoid aggregates that form in the context of chronic inflammation, and which resemble secondary lymphoid organs (SLOs) in function and structure to varying degrees [44] (Box 3). Current research efforts aim to determine what drives TLS formation, how it happens, and which cells and molecules are implicated. Enhanced Tfh cell differentiation in an ovarian cancer mouse model has been shown to induce larger TLS and B cell recruitment accompanied by increased FAS and GL7 expression, suggesting an increase in GC B cells [38]. In fact, transfer of Tfh cells from tumor-bearing mice alone was sufficient to induce enhanced TLS formation [38]. This is consistent with findings in a CRC mouse model in which ablation of Tfh cells led to a loss of TLS, decreased immune infiltration, and loss of tumor control [45]. These features were restored upon adoptive transfer of pathogen-specific CD4 + T cells, indicating that cells other than tumor-specific T cells can participate in the immune response against cancer [45]. In human breast cancer, activated Tfh cells were found to be indicative of overall TLS activity, characterized by B cell proliferation, immunoglobulin production, and Th1-skewed cytotoxic activity by CD8 + T cells [29]. Moreover, weaker interactions between Tfh cells and B cells through the CXCL13-CXCR5 axis were recently shown to lead to smaller TLS in NSCLC [46]. Since they are critical for TLS formation and function, Tfh cells and B cells are often correlated with positive outcomes in multiple cancer settings [44]. In this context, Tfh cells have been found to be associated with GC B cells [29,42,47,48] and memory B cells [35,49], and across different B cell maturation stages [26,50,51]. Nevertheless, conclusions in this regard remain limited given that the depth of characterization of both Tfh and B cells across studies remains highly heterogeneous (Box 2).
Numerous studies have assessed the value of B cells and CD8 + T cells within TLS as predictive biomarkers of clinical prognosis or therapy responsiveness [37,52,53]; however, it is intriguing that less is known about which CD4 + T subsets within TLS may be associated with the positive correlation between TLS and antitumor immune responses. For instance, high BCL6 expression in tumor-associated tissue is associated with TLS development in a CRC mouse model; nonetheless, it is unclear whether this master regulator is being expressed by B cells, Tfh cells, or both [54]. The prognostic value of TLS in lung squamous cell carcinoma has been shown to be determined by GCs within those TLS; however, which cells mediate GC formation and function within these structures remains to be further studied [55]. Furthermore, Tfh cell-associated gene signatures have long been recognized as indicators of TLS activity and predictors of clinical outcomes [22]. Better characterization of the CD4 + subsets in TLS and their interactions within these structures remains a pressing issue to be further addressed. Of particular importance is the interplay between TLS-Tfh cells and T follicular regulatory (Tfr) cells. The latter, which can be broadly defined as CXCR5 + FOXP3 + CD4 + T lymphocytes, are well-known regulators of Tfh cell activity (reviewed in more detail in [56]). In the context of cancer, Tfr cells can inhibit TLS Tfh cell function [29] and can curtail cancer immunotherapy efficacy [57]. In fact, the ratio between Tfr and Tfh cells, rather than the presence or absence of these subsets in TLS, has been shown to negatively correlate with CD8 + T cells in TLS [58]. The tight relationship between Tfh cell function and location has been one of the biggest challenges in characterizing this subset within TLS, given that conventional approaches largely rely on disruption of tissue structures for cell isolation. New technologies are now emerging that allow such complications to be circumvented. Spatial transcriptomics has been recently used to determine B cell gene expression and BCR repertoires within TLS in human clear cell renal cell carcinoma [51]. Applying such technologies to CD4 + T cells will also reveal a clearer picture of T helper cell networks in TLS.

Expanding ICB targets
One of the most significant advances in the treatment of cancer has been the development of ICB therapies that aim at blocking particular inhibitory receptors on T lymphocytes to overcome Box 3. B cells in TLS: partners in crime TLS consist of different immune cell types, including GC and memory B cells, plasma cells, CD8 + T cells, and CD4 + T cells such as Tfh and Tfr cells [37,44,98]. Mesenchymal stromal cells, particularly FDC, are also present in TLS [99], as well as a complex network of endothelial cells and cancer-associated fibroblasts that are required for TLS formation [33]. Among the cells that make up TLS, B lymphocytes have been particularly widely studied and, similar to Tfh cells, have also been strongly correlated with a better prognosis and response to ICB in various solid tumor entities [44,53,98,100]. The mechanisms underlying the B cell effector function in cancer include cytokine production, antigen presentation, antibody-dependent cell-mediated phagocytosis and cytotoxicity, as well as antibody-mediated signaling interference [53,101]. Expression of Ki67 and activation-induced cytidine deaminase (AID) by B lymphocytes in TLS supports the idea that BCR affinity maturation and immunoglobulin class-switching also occur within these structures [102]. However, the spatial organization of these cell populations varies widely, and is believed to be related to the maturity and function of a given TLS [103]. In fact, TLS do not always exhibit GC-like follicle structures, nor clearly defined dark and light zones, suggesting that Tfh-B cell interactions in these structures might not necessarily be the same as those in GCs in SLOs [101]. A closer look at these interactions will reveal how the development and maturation of antitumor responses in TLS differs from that in SLOs.
tumor-induced cell exhaustion and to promote effective antitumor immune responses [59,60]. Numerous blocking antibody therapies have been developed that target receptors such as TIM-3, CTLA-4, LAG-3, and, in particular, PD-1 or its ligands PD-L1/PD-L2 [60]. Owing to their crucial role in the antitumor response, infiltrating CD8 + T cells have been the focus of most of the research assessing the effects of ICB on the immune system [34,61]. Paradoxically, despite their characteristic high expression of PD-1 and other inhibitory surface ligands, the effects of ICB on Tfh cells have remained only poorly covered. Over recent years, however, compelling evidence has started to point to Tfh cells as a key determinant and predictor of ICB success. For instance, cTfh cells from a syngeneic NSCLC mouse model treated with anti-PD-1 displayed an enhanced helper capacity defined by a higher expression of CD38, as well as by increased secretion of IL-21 and IL-4 [62]. Likewise, upon coadministration of anti-PD-1/anti-CTLA4 in a breast cancer mouse model, Tfh cell-associated transcriptional signatures were elevated in ICBsensitive tumors compared to resistant tumors, and increased expression of IL-21 was identified in cells exhibiting such expression profiles [26]. Depletion of IL-21 in this experimental setting caused a dramatic decrease in the number of tumor-associated IgG + cells [26].
In humans, the frequencies of infiltrating Tfh, B, and CD8 + T cells were significantly increased in different types of cancer after ICB administration [26,[62][63][64][65][66], and the overall cTfh cell concentration was shown to be higher in those patients that respond to therapy compared to those who do not [39,62]. Similar results were observed in melanoma samples in which patients with high Tfh cell infiltration were found to be more likely to positively respond to anti-PD-1 treatment [21]. Using this and other validating cohorts, it was demonstrated that the Tfh cell score, rather than an overall immune score, was able to predict favorable clinical outcomes of anti-PD-1 therapy and a higher proportion of complete response or partial response (CR/PR) in patients [21]. By contrast, Zappasodi et al. described a CD4 + Foxp3 − PD-1 hi population that presents a Tfh-like phenotype and which actively limits the antitumor immune response in melanoma and NSCLC models [67]. The identity of these cells and their relationship to bona fide Tfh cells remains to be determined; nonetheless, these results highlight how distinct Tfh and/or Tfh-like cell subsets might behave differently. Moreover, the authors found opposing effects of anti-CTLA-4 and anti-PD-1 therapies for this specific cell population. While anti-PD-1 therapy had a positive effect on the antitumor response, anti-CTLA-4 therapy had an overall negative effect that was able to abrogate that of anti-PD-1 when administered in combination [67]. Binding of the anti-PD-1 antibody pembrolizumab was shown to be particularly intense in CD38 + Tfh-like cells among all PD-1 + CD4 + T cells, especially for those residing in TLS, indicating that Tfh cells are arguably the main target of ICB within the CD4 + compartment [34]. Strikingly, Escherichia coli-specific memory Tfh and B cell responses, but not those for other resident bacteria, were shown to be predictive of clinical responses to neoadjuvanted anti-PD-1 therapy [34]. In the past few years it has been demonstrated that the gut microbiota can have a significant effect on ICB outcomes [68]. The exact mechanisms that define this association remain unclear, although it is believed that the gut microbiome can either produce metabolites that stimulate T cells or promote pre-existing immunity that would then be amplified by ICB [68] (Figure 1). The presence of E. coli in bladder cancer is unsurprising given that colonization of this tissue by gut-derived bacteria is frequently observed in urinary tract infections [34]. However, how bacteria-specific Tfh cells are able to promote antitumor responses remains unknown. IgA transcytosis has been previously demonstrated to be a key process that can explain antigen-independent responses against ovarian cancer [69]. Nonetheless, it remains puzzling that, in the study by Goubet et al., a clinical response was seen for IgG but not for IgA antibodies [34], suggesting that other unknown mechanisms might trigger antitumor responses in a pathogen-specific fashion (Figure 1).
ICB also has a strong impact on tumor-associated TLS. Anti-PD-1 monotherapy or in combination with anti-CTLA-4 has shown a significant increase in the size and number of TLS in mouse models, and also promoted a more classical microanatomical organization characterized by distinct T cell and B cell/FDC regions [33]. Although both treatments led to an increase in the number of intratumoral T cells, a reduction in tumor size was only seen in intraperitoneal tumors, whereas subcutaneous tumors remained unchanged [33], suggesting that both anatomical location and TME composition are crucial for ICB effectiveness. The specific cell types that increased in TLS upon ICB administration remain to be defined. TLS also act as a robust predictor of therapy outcome through the creation of a TLS gene signature that is associated with increased survival in melanoma patients treated with anti-CTLA4 [52]. Noteworthy, this TLS signature was independent of tumor mutational burden, and validation with previously published datasets demonstrated that it outperformed other immune-related signatures [52]. Switched B cells are also enriched in responders versus non-responders, and they locate within TLS [70]. These cells present higher clonal expansion, greater BCR diversity, and a more activated phenotype than those found in non-responders [70]. It is tempting to speculate that Tfh cells within TLS provide the necessary help to B cells for their activation during ICB; however, further investigation is needed. Regarding anti-PD-L1 treatment (atezolizumab), single-cell analysis from a triple-negative breast cancer cohort showed that CXCL13 + CD4 + T cells were expanded in ICB responders, and had positive predictive value for therapy responsiveness [65]. These cells presented features of both Th1 and Tfh exhausted cells, and were reduced upon administration of chemotherapy regime (paclitaxel), highlighting the fact that coadministration of chemotherapy regimens might blunt the positive effects of ICB [65]. Everything considered, the current evidence supports the idea that Tfh cells are a major target of ICB therapy, and shows that these cells can be predictive for clinical responses. Further investigation will be necessary to assess how different therapies affect specific Tfh cell subsets in different cancer entities.
irAEs in cancer immunotherapythe dark side of Tfh cells?
Despite the uncontestable success of ICB in the treatment of cancer, recurrent identification of irAEs associated with this type of therapy has become a major concern in the medical community [71][72][73]. The precise causes, drivers, and mechanisms of ICB-induced irAEs remain somewhat elusive [74], and the development of clinical management guidelines has been challenging [71,[75][76][77]. Although further work will be necessary to clearly define the specific role of the different Tfh cell subtypes and phenotypes in cancer (Box 2), it is very intriguing that cells with similar characteristics and functions have been extensively studied in the context of autoimmune disorders [78,79]. These cells, which have been named T peripheral helper (Tph) cells, are strongly associated with a proinflammatory milieu and the secretion of autoantibodies, and can be broadly defined as PD-1 hi CXCR5 − CD4 + T cells with a robust capacity to secrete CXCL13 [79,80]. It is remarkable how these cells resemble Tfh-like cell populations that can be found in multiple types of cancer (Table 1 and Figure 2), not only in their phenotype but also in their function [81], as Tph cells have been shown to mediate B cell help through production of IL-21, and are believed to participate in the formation of TLS in rheumatoid arthritis [80].
Considering their high expression of inhibitory receptors such as PD-1 and their resemblance to Tph cells, it is possible that administration of ICB therapies could promote a dysregulated Tfh cell response, leading to the development of irAEs, as previously hypothesized [82] (Figure 1). Importantly, more evidence supporting this proposition is starting to emerge. Vaccination studies in melanoma patients showed that subjects treated with anti-PD-1 had a significant increase in cTfh cell and plasmablast responses, as well as increased CXCL13 secretion, compared to the control group [83]. Transcriptomic analysis also revealed increased expression of cell proliferation and activation-related pathways in cTfh cells from anti-PD-1-treated patients. Strikingly, cell-cycle and proliferation pathways were also identified in cTfh cells from patients who developed irAEs compared to those from healthy subjects [83], suggesting that anti-PD-1-induced dysregulation of cTfh cells might contribute to the development of irAEs. Moreover, despite similar antibody titers at later time-points after vaccination, reduced antibody galactosylation, sialylation, and overall affinity at baseline were found in the ICB-treated group only, indicating that the quality of the immune response might be compromised upon anti-PD-1 treatment, despite higher activation of Tfh cells [83]. A multi-omic approach on >18 000 patient samples also seems to suggest a correlation between Tfh cells and irAEs [84]. This correlation did not reach the threshold of statistical significance; however, this could have been due to the high heterogeneity of the samples given that they were derived from 26 different types of cancer. When considered independently, the relationship between Tfh cell and ICB-induced irAEs might differ across different cancer types. For instance, a strong correlation between Tfh cells and irAEs was found in renal cell and urothelial carcinomas [83], whereas in two independent cohorts of melanoma patients such an association was absent [85]. Considering that tumor patients often undergo preconditioning chemotherapy or radiation treatment, the observation that T cell lymphopenia may Circulating Tfh (cTfh) cells derive from early Tfh cell precursors that are generated before GC entry and possess several features of memory T cells. They express CXCR5, but mostly lack BCL6 expression and only express higher amounts of PD-1 and ICOS upon (re)activation. TfhX13 cells were first described in human breast cancer and similar cells have now also been described in other human solid tumors. They produce large amounts of CXCL13 and share several other features of Tfh cells (IL-21 production, PD-1 expression, etc.), but do not express CXCR5. TfhX13 cells share striking similarities with T peripheral helper (Tph) cells that were first described in the inflamed joints of rheumatoid arthritis patients. In addition, Tph cells are characterized by elevated levels of BLMP-1, a transcriptional repressor that counteracts BCL6, and they express chemokine receptors that mediate migration to inflamed sites (e.g., CCR2).

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promote impaired antigen-specific antibody responses, hypergammaglobulinemia and autoantibody production [86], could provide another link for a potential predisposition to developing irAEs following ICB treatment. Another factor that seems to be a determinant of the development of ICB-induced irAEs is age. Aged mice in an anti-PD-1-treated melanoma model showed a significant increase in multi-organ pathology, mostly due to excessive IgG accumulation [87]. Paradoxically, aged mice presented features characteristic of good prognosis such as larger T and B cell infiltration, an increased number of TLS, and higher production of IL-21 and CXCL13 [87]. Depletion of CD4 + T cells or blockade of IL-21 was sufficient to prevent IgG deposition and organ damage, which is intriguing considering that most of the CD4 + TILs were BCL6 + CXCR5 − IL-21 + cells [87]. IgG transfer from aged mice was sufficient to induce multi-organ pathology in aged recipients but not in young mice, suggesting that ageassociated changes on the immunological milieu (also known as immunosenescence) have a pivotal role in the occurrence of irAEs [87]. Finally, no difference in CXCL13 levels before anti-PD-1 therapy was found between patients and healthy donors, although a strong correlation with irAEs was observed after treatment administration [87].
Regarding anti-CTLA-4 therapy, the number of T cells with a Tfh cell phenotype appears to increase upon its administration [3]. Direct proof that this type of therapy induces Tfh-mediated irAEs is still lacking; however, deficiencies in the CTLA4 gene have been widely associated with several autoimmune manifestations, and antibody-mediated blockade of this receptor promotes spontaneous Tfh cell differentiation as well as GC formation [3]. Together, these observations hint that dysregulation of Tfh cells during anti-CTLA-4 immunotherapy could in turn promote autoimmune manifestations. Further studies will be necessary to determine the causal links that may exist between Tfh cells and immunotherapy-induced irAEs [82], as well as the impact of other factors such as age or tumor type.

Concluding remarks and future perspectives
Tfh cells represent a promising target in human diseases and vaccination [88]. Reducing Tfh cell numbers or their function may be beneficial in settings in which Tfh cells are either the origin of T cell malignancies or in cases where Tfh cells provide help to malignant B cells. By contrast, in many solid organ tumor entities that exhibit TLS, promoting Tfh cell numbers or their function may help to boost the antitumor immune response. While new possibilities in personalized medicine hold promise to enable the development and fine-tuning of individual therapy strategies, for example, through concerted combination therapies consisting of various biologics and/or small-molecule drugs, it will be important to balance the positive and negative effects of these treatments to avoid the development of irAEs. To facilitate the applicability of targeting tumor-associated Tfh cells or boosting their function in cancer immunotherapy, it will be particularly important to further elucidate the identity and ontogeny of these cells as well as their functions beyond the classical help to B cells (see Outstanding questions). In summary, a better understanding of the cellular and molecular mechanisms driving Tfh cell responses in cancer and cancer immunotherapy will be necessary to improve the efficacy and safety of existing therapies and to determine the full potential of this subset as a novel therapeutic target.

Outstanding questions
What is the origin and function of different Tfh-like cell populations in tumor tissues, in particular in TLS? An answer to this question should provide a foundation for reaching a consensus on what is and what is not a tumorassociated Tfh cell subset.
What are the precise functions of Tfhlike cells in tumors? Most studies on Tfh cells in cancer have so far been addressed directly in cancer patients, thus remaining largely descriptive. Novel sophisticated genetic in vivo models will provide complementary mechanistic insights into the role of Tfh cells in cancer.
How can the beneficial effects of ICB therapy on Tfh cell function be isolated from the detrimental effects that may contribute to the development of irAEs, particularly autoimmunity?
Can personalized omics or other approaches be used to predict therapy outcome, thus facilitating the development of tailored therapy regimens that could enable increased response rates?
Recent evidence has shown that tissue-resident, pathogen-specific Tfh cells can take part in the antitumor immune response upon ICB. Could the tissue-resident microbiota be modulated to boost ICB-induced responses? Further research in this field could open up new avenues for clinical interventions that improve immunotherapy outcomes.