- Email: [email protected]
PD-1hiCXCR5−CD4+ TFH Cells Play Defense in Cancer and Offense in Arthritis
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PD-1hiCXCR5CD4+ TFH Cells Play Defense in Cancer and Offense in Arthritis Chunyan Gu-Trantien1,2 and Karen Willard-Gallo2,* T follicular helper (TFH) cells are characteristically defined by their CXCR5 positivity and homing to B cell follicles in secondary lymphoid organs (SLO). An expanded subpopulation of functionally comparable and phenotypically similar PD-1hi[176_TD$IF]CXCR5CD4+ T cells were recently identified in breast cancer (BC) and rheumatoid arthritis (RA) to have beneficial or detrimental roles, respectively, but are they inflammatory tissue effector TFH cells? PD-1hiCXCR5CD4+ T cells, phenotypically resembling TFH cells, were previously identified in pathological tissues from patients with BC [1] and RA [2,3]. Two recent independent studies found that infiltrating PD-1hiCXCR5CD4+ T cells (Figure 1) induce B cell responses in BC [4] and RA [5] and, thus, are functionally similar to conventional PD1hiCXCR5+CD4+ TFH cells in SLOs. The Letter published in Nature by Rao et al. [5] defined all PD-1hi[17_TD$IF]CXCR5CD4+ T cells as peripheral helper T or TPH cells, finding this subpopulation to be predominantly expanded in synovial fluid and inflamed tissues from patients with seropositive RA (sRA). The article in JCI Insight [4] focused on a significant CXCL13-producing PD1hiCXCR5CD4+ T cell subpopulation infiltrating BC, named TFHX13 cells. The coincidence of these concurrent findings in distinct diseases merits a comparative analysis of PD-1hiCXCR5CD4+ TPH and TFHX13 cells (Table 1).
TFH cells, first identified in human tonsils, are the principal CD4+ T cell subpopulation providing essential help to B cells. TFH cells are necessary for B cell differentiation and maturation in reactive SLO germinal centers (GC) and for sustained antibody production in the periphery. Antigen-specific antibody responses are not only critical for controlling viral infections, but also contribute significantly to the severity of autoimmune diseases [6]. GC TFH cells are defined phenotypically as PD-1hiCXCR5hiICOShiBCL6+CD4+ T cells producing significant amounts of IL-21. These cells migrate to B cell follicles and GCs in response to CXCL13, a B cell chemoattractant that is the ligand for CXCR5. Human tonsillar TFH cells, but not their murine counterparts, have been shown to specifically produce CXCL13 [7]. The potential clinical importance of PD-1hiCXCL13+CXCR5CD4+ T cells is now clear because their expansion and B cell helper activities have been associated with two distinct pathologies [1–5]. The BC and sRA studies both found that, similar to tonsillar TFH cells, the highest levels of CXCL13 and IL-21 are produced by the patient’s PD-1hiCXCR5 TFHX13 or TPH cells [4,5]. While the expansion of PD-1hiCXCR5 TFHX13 cells in patients with BC was associated with long-term survival and an increase in tumor-infiltrating lymphocytes (TIL), their TPH cell counterparts in patients with sRA were linked with progressive disease [1,4,5]. TPH cells increased with disease severity in both synovial fluid and blood from patients with sRA and decreased following treatment [5]. It was found that blood PD-1hiCD4+ T cells are at low frequencies in patients with BC [4]; however, analysis with other PD-1 antibodies [5,8,9] should be performed to validate this observation. The current view is that blood CXCR5+ CD4+ T cells are GC-experienced circulating memory TFH cells distinguished by
reduced BCL6 expression (the master transcription factor directing their GC differentiation) and downregulation of PD-1 and ICOS. Subsets of CXCR5+ TFH cells, defined by differential chemokine receptor expression, are linked with distinct effector functions [6]. Despite these benchmarks, activated PD-1hi TFH cells are detectable in both healthy individuals and patient’s blood [8]. Rao et al. analyzed both PD-1hiCXCR5 and PD1hiCXCR5+ CD4+ T cells from healthy donors, finding that they both supported B cell responses [5]. Previous functional studies compared total CXCR5+ (or specific CXCR5+ subpopulations) to the heterogeneous collection of CXCR5CD4+ T cells [8], suggesting that helper activity by a PD-1hiCXCR5 subpopulation(s) could have gone undetected. Analyses of PD1hiCXCR5 cells are limited [9], signifying a need for detailed evaluation of blood PD-1/CXCR5-defined CD4+ T cell subpopulations from patients and healthy individuals. The BC study identified distinct, activated (high % [179_TD$IF]Ki-67+[178_TD$IF]) PD-1hiICOSint and PD1intICOShi subpopulations in [172_TD$IF]CD4+[180_TD$IF] TIL [4]. The PD-1hiICOSint TIL contained CXCL13-expressing TFHX13 cells together with other effector cells, while PD-1intICOShi TIL were predominantly FoxP3hi regulatory T cells (Treg). The PD-1loICOSlo TIL were primarily CXCR5+ cells with only a few proliferative and/or activated cells, suggesting their more recent recruitment to the tumor. Interestingly, exposure of tonsillar CXCR5hi TFH cells to primary BC tumor supernatant downregulated CXCR5 [1,4], suggesting that the tumor microenvironment inhibits CXCR5 upregulation on activated CD4+ T cells before they differentiate to CXCR5hi GC TFH cells. Alternatively, GC-experienced TFH cells may lose CXCR5 and/ or activated TFHX13 TIL may more readily expand in the immunosuppressive BC microenvironment. TFHX13 and TPH cells express limited amounts of BCL6 and
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TFHX13
(A)
TPH in seroposive rheumatoid arthris
(B)
in breast cancer PD-1hi
ICOSint
TIGIT hi
PD-1hi
ICOS+ hi
CD200
FAShi
TIGIThi CCR2+
+
MHCII
iCTLA-4+ MHCII? CCR2? BLIMP1?
CXCR5- CD4+ T cell FOXP3lo%+
FOXP3lo%+
hi%+
hi%+
T-bet
T-bet
IFNG
IL21hi
IFNGhi hi
IL10int IL10
BLIMP1hi
BCL6neg/int
BCL6int
IFNGhi
int
FAS? CD200? iCTLA-4?
CXCR5- CD4+ T cell
CXCL13hi CXCL13hi
IL21
hi
IL21hi
CXCL13hi
Correlated with GC+TLS and B TIL: increased disease-free and overall survival
IFNGhi IFNG
IL21hi
IFNGhi hi
IL10hi IL10
hi
IL10hi
CXCL13hi CXCL13hi
IL21hi IL21hi
CXCL13hi
Correlated with GC/memory B cells: increased autoimmune disease severity
Figure 1. Tissue-infiltrating PD-1hiCXCR5CD4+ T Cells Have Phenotypic Similarities Associated with Divergent Disease Activities in Breast Cancer (BC) (A) and Seropositive Rheumatoid Arthritis (sRA) (B). PD-1hiCXCR5CD4+ T cells functionally and phenotypically resemble conventional PD-1hiCXCR5+ T follicular helper (TFH) cells lacking CXCR5, the chemokine receptor for germinal center (GC) homing. Two recent studies focused on these PD-1hiCXCR5CD4+ T cells in: (A) BC, where Gu-Trantien et al. [4] identified distinct CD4+ T cell subpopulations based on PD-1 and ICOS expression and focused on the CXCL13-producing cells (named TFHX13 cells) within the PD-1hiICOSintCXCR5 effector subpopulation; and (B) sRA where Rao et al. [5] investigated PD-1hiICOS+CXCR5CD4+ T cells (called TPH cells) in patients and healthy donors. TFHX13 and TPH cells from pathological tissues were phenotypically similar to one another and conventional GC TFH cells from secondary lymphoid organs. PD-1hiCXCR5CD4+ T cells expressed CXCL13 (the unique ligand for CXCR5 and an important mediator of GC responses), IL21 (the key cytokine supporting antibody production) and other markers, including TIGIT and CD200. Other characteristics of TFHX13 and TPH cells, compared with conventional GC TFH cells, included reduced BCL6 expression and increased T-bet, BLIMP1, and IFNG. The data presented in these studies suggest that PD1hiCXCR5CD4+ T cells participate in mediating active immune responses in diseased tissues. The expression levels indicated are for protein (normal capital letters) or mRNA (italicized capital letters); if both were analyzed, only the protein data are shown.
(CCR5, CXCR3, CXCR6, and CX3CR1)related receptors in the PD-1hiCXCR5 TPH cells. Expression of a distinct chemokine receptor profile may dictate the migration of TPH cells to specific anatomic Rao et al. performed detailed gene locations and/or reflect influences in local expression profiling of PD-1hiCXCR5 tissue microenvironments. and PD-1hiCXCR5+ CD4+ T cell subpopulations purified from blood from In blood from patients with sRA, CXCL13 patients with sRA [5]. PD-1hiCXCR5 transcripts were elevated in both cells expressed a TFH-related gene profile PD-1hiCXCR5 and PD-1hiCXCR5+ cells, similar to PD-1hiCXCR5+ cells; however, although they were significantly higher in the authors found a major difference in the former [5]. This is in contrast to previtheir chemokine receptor patterns, char- ous studies showing that circulating acterized by expression of inflammatory PD-1+CXCR5+ TFH cells specifically (CCR2 and CCR3) and Th1/cytotoxicity secrete CXCL13 after stimulation neither is confined to the GCs of ectopic or tertiary lymphoid structures (TLS), suggesting that they are migratory within inflamed tissue sites [1–5].
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(PD-1+CXCR5 T cells were not analyzed) [8]. CXCR5+ TFH cells in SLOs could downregulate CXCL13 expression as active GC responses attenuate and TFH cells move into the periphery. Alternatively, CXCL13+ GC TFH cells may modulate their chemokine receptor expression to enable migration to other tissues. Functionally, this would direct CXCR5+ memory TFH cells to designated sites, where they could contribute to TLS formation in cancer, arthritis, and other diseases [10]. Data from [4] clearly demonstrated that follicular dendritic cells are not potent
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Table 1. A Detailed Comparison of TFHX13 Cells in Breast Cancer and TPH Cells in Seropositive Rheumatoid Arthritis.a[173_TD$IF] Characteristic
Subpopulation studied
Human [174_TD$IF]disease Cancer
Autoimmune [174_TD$IF]disease
BCb
sRAc TPH: PD-1hiCXCR5 CD4+ T cellse
TFHX13: PD-1hiICOSintCXCL13+ CD4+ T cellsd f
Additional phenotypic markers (flow cytometry)
Unstimulated fresh TFHX13 cells from BC tissue : CXCR5 (majority), CD45RA (majority), CD200hi, TIGIThi, FAShi, intracellular CTLA-4+, BCL6int, CD25lo, CD62L, CCR7, high %T-bet+, high %Ki67+, low % FOXP3+[175_TD$IF]. CXCL13+TFHX13 TIL uniquely in PD-1hiICOSint effector CD4+ subpopulation
Cryopreserved Tph cells from synovial tissue/fluidg: unstimulated: high % MHCII+, high % ICOS+, high % CCR2+, MAFhi, BLIMP1hi, subpopulation was BCL6int, most analyses gated on CD45RA. Stimulated: IL-21hi, CXCL13hi, IL-2lo. Blood: TIGIThi, low % FOXP3+, increased % CX3CR1+ and % CCR5+
Gene expression (RT-qPCR)
High expression: CXCL13, IL21, IFNG, TBX21, BCL6; intermediate expression: IL10; low expression: FOXP3h
High expression: CXCL13 IL21, IFNG, IL10, MAF, SAP, BATF, BLIMP1; no significant increase: BCL6; low expression: IL2h
Helper roles for B cells: CXCL13+TFHX13 in BC versus IL-21+TPH in RA
Flow cytometry: total CXCL13+ TIL correlated with CXCR5+ B TIL; PD-1hiCXCR5hiCD4+ GC TFH TIL correlated with GC B TIL; CXCL13+CD4+ TFHX13 TIL correlated with GC B plus PC TIL; high levels of TFHX13 TIL associated with Ki67+ proliferating B TIL with different maturation phenotypes. Confocal microscopy: CXCL13+ (majority CD4+) TIL co-localized with rare, tumor bedmigrating B TIL and are likely positioned to recruit B TIL and promote formation of functional TLS in BC tissues
In vitro T-B cell co-cultures: PD-1hiCXCR5CD4+ T cells from sRA synovial tissue/fluid supported antibody production and plasma cell differentiation in healthy donor blood memory B cells; PD-1hiCXCR5 and PD-1hiCXCR5+ CD4+ T cells from healthy donor blood provided comparable help (as above) to autologous memory B cells (IL-21R and SLAMF5 dependent) Confocal microscopy: PD-1+CXCR5 cells co-localized with B cells in lymphoid aggregates and diffuse infiltrates in RA synovial tissues
Correlation with disease progression
CXCL13 gene expression: positive correlation with clinical outcomes in BC; intratumoral B TIL presence: positive prognosis; % TFHX13 TIL: increase in triple-negative and all Grade 3 BC; CXCL13 gene prognostic value is independent from other clinicopathological parameters
% PD-1hi in synovial fluid CD4+ T cells: increase in sRA; % PD-1hiCXCR5 in blood CD4+ T cells: increase in sRA and positive correlation with serum anti-CCP antibody titer in sRA; % PD-1hiCXCR5 in blood CD4+ T cells and % plasmablast in blood B cells: decrease after treatment in patients with sRA showing reduced disease activity
Other important findings
Follicular dendritic cells are not potent CXCL13 producers in BC. In addition to PD-1hiICOSint effector subpopulation, a distinct PD1intICOShi subpopulation (principally FoxP3hi activated Treg) identified in BC CD4+ TILi. Both subpopulations positively correlated with equilibrium attained in TFHX13 TIL-rich BC. IL-2 deprivation induced CXCL13 expression in vitro from blood CD4+ T cells without affecting IFNg expression. TGFb1 provided synergistic effect for CXCL13 induction and selectively reduced IFNg expression in FoxP3l CD4+ T cells. Large public data set analysis revealed homogenous absence of IL2 together with high TGFB1, low IFNG, and a broad range of CXCL13 gene expression in total BC tumor tissues
Protein (mass cytometry) and mRNA (RNA sequencing) expression levels in PD-1hiCXCR5 and PD-1hiCXCR5+ memory CD4+ T cell subpopulations from sRA blood. Results: PD-1hiCXCR5 cells expressed high levels of ‘TIGIT, T-bet, CD38, CXCR3, MHCII, and Perforin’, and low levels of ‘CCR7, CD25 and CD1270 protein, with some degree of similarity to PD-1hiCXCR5+ cells. TFH cell-related genes (SAP, ICOS, PDCD1, MAF, CD200, BATF, TOX2, TIGIT, SLAMF6, NFATC1, SOSTDC1 and CTSB) highly expressed by both PD-1hiCXCR5 and PD-1hiCXCR5+ subpopulations. Expression patterns of chemokine receptors distinct with CCR2, CCR3, CX3CR1, CCRL2, CCR5 and CXCR3, together with CXCL13 more specifically associated with PD-1hiCXCR5 subpopulation
a
Abbreviations: TIL, tumor infiltrating lymphocyte; TLS, tertiary lymphoid structure; GC, germinal center; PC, plasmablast/plasma cell (antibody-secreting B cell). [4]. [5]. d TFHX13 cells defined as CXCL13+CD4+ T cells. TFHX13 cells are uniquely found in the PD-1hiICOSint effector CD4+ subpopulation of BC TIL but undetectable in blood or nontumor breast tissue. e TPH cells defined as PD-1hiCXCR5CD4+ T cells. TPH cells increase in synovial tissues and synovial fluid from patients with sRA and are detected in their blood. The TPH cell subpopulation in sRA is comparable to the PD-1hiICOSint effector subpopulation in BC. f Fresh (<2-h surgical specimens) tissue samples processed using nonenzymatic mechanical dissociation followed by immediate staining for all markers. g Tissue samples digested with LiberaseTL and DNaseI before cryopreservation. h Gene expression levels are relative among the analyzed CD4+ T cell subpopulations in each study; differences observed for IL10 and BCL6 may be because the PD1intICOShi Treg subpopulation was separately analyzed only in BC. i A distinct PD-1intICOShi subpopulation was not observed in RA synovial fluid, and unclear for synovial tissues (mass cytometry). b c
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CXCL13 producers in BC, with TFHX13 TIL functioning to attract and/or guide B cells and initiate TLS formation in malignant tissues. It is currently unknown whether they have the same role in SLOs. Data from a murine model detected lunginfiltrating PD-1+CXCR5 TFH-like cells together with abundant GC B cells despite a limited number of follicular dendritic cells [11], which supports the BC data [4]. TLS are important local sites of B cell maturation, suggesting that PD-1hiCXCR5CD4+ TFH cells (including CXCL13+TFHX13 and IL-21+TFH cells) contribute to initiating, maintaining, and modulating GC and post-GC responses in tissues. It is unknown whether PD-1hi and/or CXCL13+ cells differentiate in tissues following an antigen-specific response. The data reported in [4] suggest that Treg cell accumulation at the tumor site reduces IL-2 availability, which in turn induces TFHX13 cell differentiation [4] in a manner reflecting murine TFH cell development. TGFb1, abundant in tumors, could synergize with CXCL13 in the absence of IL-2. A growing body of data suggests that limiting IL-2 is important for promoting the TFH program [12]. Based on current data, PD-1intICOShi Treg are not likely involved in sRA; however, it is curious that PD-1hi cells express the
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lowest amounts of IL-2 in these tissues [5]. Finally, both studies detected increased IFNG T helper (Th)1 responses in tissue-infiltrating PD-1hiCXCR5CD4+ T cells, supporting the functional importance of type 1 effector cells in antitumor immunity and autoimmune disease [4,5].
Concluding Remarks PD-1hiCXCR5CD4+ TFH (TFHX13 and TPH) cells, which Rao et al. [5] and GuTrantien et al. [4] have shown expand under chronic inflammatory conditions, are functionally and phenotypically similar to conventional TFH cells lacking CXCR5. Future studies are necessary to determine whether conventional TFH cells are recruited from SLOs or differentiate into effector TFH cells upon arrival in diseased tissues. The plasticity of helper T cells suggests this could be another case where important CD4+ mediators of immune responses adapt to signals in their surrounding microenvironment. We propose that the role of PD1hiCXCR5CD4+ effector TFH cells (TFHX13 and TPH) in B cell differentiation and their potential activities in other human diseases merits their recognition as a subpopulation of genuine TFH cells. 1
Institute for Medical Immunology, Université Libre de Bruxelles, 6041 Charleroi, Belgium Molecular Immunology Unit, Institut Jules Bordet, Université Libre de Bruxelles, 1000 Brussels, Belgium
2
*Correspondence: [email protected] (K. Willard-Gallo). https://doi.org/10.1016/j.it.2017.10.003 References 1. Gu-Trantien, C. et al. (2013) CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J. Clin. Invest. 123, 2873–2892 2. Manzo, A. et al. (2008) Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint. Arthritis Rheum. 58, 3377–3387 3. Kobayashi, S. et al. (2013) A distinct human CD4+ T cell subset that secretes CXCL13 in rheumatoid synovium. Arthritis Rheum. 65, 3063–3072 4. Gu-Trantien, C. et al. (2017) CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer. JCI Insight 2, 91487 5. Rao, D.A. et al. (2017) Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature 542, 110–114 6. Ueno, H. (2016) T follicular helper cells in human autoimmunity. Curr. Opin. Immunol. 43, 24–31 7. Kim, C.H. et al. (2004) Unique gene expression program of human germinal center T helper cells. Blood 104, 1952– 1960 8. Locci, M. et al. (2013) Human circulating PD-1+CXCR3CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity 39, 758–769 9. He, J. et al. (2013) Circulating precursor CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity 39, 770–781 10. Dieu-Nosjean, M.C. et al. (2016) Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers. Immunol. Rev. 271, 260–275 11. Vu Van, D. et al. (2016) Local T/B cooperation in inflamed tissues is supported by T follicular helper-like cells. Nat. Commun. 7, 10875 12. Locci, M. et al. (2016) Activin A programs the differentiation of human TFH cells. Nat. Immunol. 17, 976–984
Forum
PD-1hiCXCR5CD4+ TFH Cells Play Defense in Cancer and Offense in Arthritis Chunyan Gu-Trantien1,2 and Karen Willard-Gallo2,* T follicular helper (TFH) cells are characteristically defined by their CXCR5 positivity and homing to B cell follicles in secondary lymphoid organs (SLO). An expanded subpopulation of functionally comparable and phenotypically similar PD-1hi[176_TD$IF]CXCR5CD4+ T cells were recently identified in breast cancer (BC) and rheumatoid arthritis (RA) to have beneficial or detrimental roles, respectively, but are they inflammatory tissue effector TFH cells? PD-1hiCXCR5CD4+ T cells, phenotypically resembling TFH cells, were previously identified in pathological tissues from patients with BC [1] and RA [2,3]. Two recent independent studies found that infiltrating PD-1hiCXCR5CD4+ T cells (Figure 1) induce B cell responses in BC [4] and RA [5] and, thus, are functionally similar to conventional PD1hiCXCR5+CD4+ TFH cells in SLOs. The Letter published in Nature by Rao et al. [5] defined all PD-1hi[17_TD$IF]CXCR5CD4+ T cells as peripheral helper T or TPH cells, finding this subpopulation to be predominantly expanded in synovial fluid and inflamed tissues from patients with seropositive RA (sRA). The article in JCI Insight [4] focused on a significant CXCL13-producing PD1hiCXCR5CD4+ T cell subpopulation infiltrating BC, named TFHX13 cells. The coincidence of these concurrent findings in distinct diseases merits a comparative analysis of PD-1hiCXCR5CD4+ TPH and TFHX13 cells (Table 1).
TFH cells, first identified in human tonsils, are the principal CD4+ T cell subpopulation providing essential help to B cells. TFH cells are necessary for B cell differentiation and maturation in reactive SLO germinal centers (GC) and for sustained antibody production in the periphery. Antigen-specific antibody responses are not only critical for controlling viral infections, but also contribute significantly to the severity of autoimmune diseases [6]. GC TFH cells are defined phenotypically as PD-1hiCXCR5hiICOShiBCL6+CD4+ T cells producing significant amounts of IL-21. These cells migrate to B cell follicles and GCs in response to CXCL13, a B cell chemoattractant that is the ligand for CXCR5. Human tonsillar TFH cells, but not their murine counterparts, have been shown to specifically produce CXCL13 [7]. The potential clinical importance of PD-1hiCXCL13+CXCR5CD4+ T cells is now clear because their expansion and B cell helper activities have been associated with two distinct pathologies [1–5]. The BC and sRA studies both found that, similar to tonsillar TFH cells, the highest levels of CXCL13 and IL-21 are produced by the patient’s PD-1hiCXCR5 TFHX13 or TPH cells [4,5]. While the expansion of PD-1hiCXCR5 TFHX13 cells in patients with BC was associated with long-term survival and an increase in tumor-infiltrating lymphocytes (TIL), their TPH cell counterparts in patients with sRA were linked with progressive disease [1,4,5]. TPH cells increased with disease severity in both synovial fluid and blood from patients with sRA and decreased following treatment [5]. It was found that blood PD-1hiCD4+ T cells are at low frequencies in patients with BC [4]; however, analysis with other PD-1 antibodies [5,8,9] should be performed to validate this observation. The current view is that blood CXCR5+ CD4+ T cells are GC-experienced circulating memory TFH cells distinguished by
reduced BCL6 expression (the master transcription factor directing their GC differentiation) and downregulation of PD-1 and ICOS. Subsets of CXCR5+ TFH cells, defined by differential chemokine receptor expression, are linked with distinct effector functions [6]. Despite these benchmarks, activated PD-1hi TFH cells are detectable in both healthy individuals and patient’s blood [8]. Rao et al. analyzed both PD-1hiCXCR5 and PD1hiCXCR5+ CD4+ T cells from healthy donors, finding that they both supported B cell responses [5]. Previous functional studies compared total CXCR5+ (or specific CXCR5+ subpopulations) to the heterogeneous collection of CXCR5CD4+ T cells [8], suggesting that helper activity by a PD-1hiCXCR5 subpopulation(s) could have gone undetected. Analyses of PD1hiCXCR5 cells are limited [9], signifying a need for detailed evaluation of blood PD-1/CXCR5-defined CD4+ T cell subpopulations from patients and healthy individuals. The BC study identified distinct, activated (high % [179_TD$IF]Ki-67+[178_TD$IF]) PD-1hiICOSint and PD1intICOShi subpopulations in [172_TD$IF]CD4+[180_TD$IF] TIL [4]. The PD-1hiICOSint TIL contained CXCL13-expressing TFHX13 cells together with other effector cells, while PD-1intICOShi TIL were predominantly FoxP3hi regulatory T cells (Treg). The PD-1loICOSlo TIL were primarily CXCR5+ cells with only a few proliferative and/or activated cells, suggesting their more recent recruitment to the tumor. Interestingly, exposure of tonsillar CXCR5hi TFH cells to primary BC tumor supernatant downregulated CXCR5 [1,4], suggesting that the tumor microenvironment inhibits CXCR5 upregulation on activated CD4+ T cells before they differentiate to CXCR5hi GC TFH cells. Alternatively, GC-experienced TFH cells may lose CXCR5 and/ or activated TFHX13 TIL may more readily expand in the immunosuppressive BC microenvironment. TFHX13 and TPH cells express limited amounts of BCL6 and
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TFHX13
(A)
TPH in seroposive rheumatoid arthris
(B)
in breast cancer PD-1hi
ICOSint
TIGIT hi
PD-1hi
ICOS+ hi
CD200
FAShi
TIGIThi CCR2+
+
MHCII
iCTLA-4+ MHCII? CCR2? BLIMP1?
CXCR5- CD4+ T cell FOXP3lo%+
FOXP3lo%+
hi%+
hi%+
T-bet
T-bet
IFNG
IL21hi
IFNGhi hi
IL10int IL10
BLIMP1hi
BCL6neg/int
BCL6int
IFNGhi
int
FAS? CD200? iCTLA-4?
CXCR5- CD4+ T cell
CXCL13hi CXCL13hi
IL21
hi
IL21hi
CXCL13hi
Correlated with GC+TLS and B TIL: increased disease-free and overall survival
IFNGhi IFNG
IL21hi
IFNGhi hi
IL10hi IL10
hi
IL10hi
CXCL13hi CXCL13hi
IL21hi IL21hi
CXCL13hi
Correlated with GC/memory B cells: increased autoimmune disease severity
Figure 1. Tissue-infiltrating PD-1hiCXCR5CD4+ T Cells Have Phenotypic Similarities Associated with Divergent Disease Activities in Breast Cancer (BC) (A) and Seropositive Rheumatoid Arthritis (sRA) (B). PD-1hiCXCR5CD4+ T cells functionally and phenotypically resemble conventional PD-1hiCXCR5+ T follicular helper (TFH) cells lacking CXCR5, the chemokine receptor for germinal center (GC) homing. Two recent studies focused on these PD-1hiCXCR5CD4+ T cells in: (A) BC, where Gu-Trantien et al. [4] identified distinct CD4+ T cell subpopulations based on PD-1 and ICOS expression and focused on the CXCL13-producing cells (named TFHX13 cells) within the PD-1hiICOSintCXCR5 effector subpopulation; and (B) sRA where Rao et al. [5] investigated PD-1hiICOS+CXCR5CD4+ T cells (called TPH cells) in patients and healthy donors. TFHX13 and TPH cells from pathological tissues were phenotypically similar to one another and conventional GC TFH cells from secondary lymphoid organs. PD-1hiCXCR5CD4+ T cells expressed CXCL13 (the unique ligand for CXCR5 and an important mediator of GC responses), IL21 (the key cytokine supporting antibody production) and other markers, including TIGIT and CD200. Other characteristics of TFHX13 and TPH cells, compared with conventional GC TFH cells, included reduced BCL6 expression and increased T-bet, BLIMP1, and IFNG. The data presented in these studies suggest that PD1hiCXCR5CD4+ T cells participate in mediating active immune responses in diseased tissues. The expression levels indicated are for protein (normal capital letters) or mRNA (italicized capital letters); if both were analyzed, only the protein data are shown.
(CCR5, CXCR3, CXCR6, and CX3CR1)related receptors in the PD-1hiCXCR5 TPH cells. Expression of a distinct chemokine receptor profile may dictate the migration of TPH cells to specific anatomic Rao et al. performed detailed gene locations and/or reflect influences in local expression profiling of PD-1hiCXCR5 tissue microenvironments. and PD-1hiCXCR5+ CD4+ T cell subpopulations purified from blood from In blood from patients with sRA, CXCL13 patients with sRA [5]. PD-1hiCXCR5 transcripts were elevated in both cells expressed a TFH-related gene profile PD-1hiCXCR5 and PD-1hiCXCR5+ cells, similar to PD-1hiCXCR5+ cells; however, although they were significantly higher in the authors found a major difference in the former [5]. This is in contrast to previtheir chemokine receptor patterns, char- ous studies showing that circulating acterized by expression of inflammatory PD-1+CXCR5+ TFH cells specifically (CCR2 and CCR3) and Th1/cytotoxicity secrete CXCL13 after stimulation neither is confined to the GCs of ectopic or tertiary lymphoid structures (TLS), suggesting that they are migratory within inflamed tissue sites [1–5].
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(PD-1+CXCR5 T cells were not analyzed) [8]. CXCR5+ TFH cells in SLOs could downregulate CXCL13 expression as active GC responses attenuate and TFH cells move into the periphery. Alternatively, CXCL13+ GC TFH cells may modulate their chemokine receptor expression to enable migration to other tissues. Functionally, this would direct CXCR5+ memory TFH cells to designated sites, where they could contribute to TLS formation in cancer, arthritis, and other diseases [10]. Data from [4] clearly demonstrated that follicular dendritic cells are not potent
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Table 1. A Detailed Comparison of TFHX13 Cells in Breast Cancer and TPH Cells in Seropositive Rheumatoid Arthritis.a[173_TD$IF] Characteristic
Subpopulation studied
Human [174_TD$IF]disease Cancer
Autoimmune [174_TD$IF]disease
BCb
sRAc TPH: PD-1hiCXCR5 CD4+ T cellse
TFHX13: PD-1hiICOSintCXCL13+ CD4+ T cellsd f
Additional phenotypic markers (flow cytometry)
Unstimulated fresh TFHX13 cells from BC tissue : CXCR5 (majority), CD45RA (majority), CD200hi, TIGIThi, FAShi, intracellular CTLA-4+, BCL6int, CD25lo, CD62L, CCR7, high %T-bet+, high %Ki67+, low % FOXP3+[175_TD$IF]. CXCL13+TFHX13 TIL uniquely in PD-1hiICOSint effector CD4+ subpopulation
Cryopreserved Tph cells from synovial tissue/fluidg: unstimulated: high % MHCII+, high % ICOS+, high % CCR2+, MAFhi, BLIMP1hi, subpopulation was BCL6int, most analyses gated on CD45RA. Stimulated: IL-21hi, CXCL13hi, IL-2lo. Blood: TIGIThi, low % FOXP3+, increased % CX3CR1+ and % CCR5+
Gene expression (RT-qPCR)
High expression: CXCL13, IL21, IFNG, TBX21, BCL6; intermediate expression: IL10; low expression: FOXP3h
High expression: CXCL13 IL21, IFNG, IL10, MAF, SAP, BATF, BLIMP1; no significant increase: BCL6; low expression: IL2h
Helper roles for B cells: CXCL13+TFHX13 in BC versus IL-21+TPH in RA
Flow cytometry: total CXCL13+ TIL correlated with CXCR5+ B TIL; PD-1hiCXCR5hiCD4+ GC TFH TIL correlated with GC B TIL; CXCL13+CD4+ TFHX13 TIL correlated with GC B plus PC TIL; high levels of TFHX13 TIL associated with Ki67+ proliferating B TIL with different maturation phenotypes. Confocal microscopy: CXCL13+ (majority CD4+) TIL co-localized with rare, tumor bedmigrating B TIL and are likely positioned to recruit B TIL and promote formation of functional TLS in BC tissues
In vitro T-B cell co-cultures: PD-1hiCXCR5CD4+ T cells from sRA synovial tissue/fluid supported antibody production and plasma cell differentiation in healthy donor blood memory B cells; PD-1hiCXCR5 and PD-1hiCXCR5+ CD4+ T cells from healthy donor blood provided comparable help (as above) to autologous memory B cells (IL-21R and SLAMF5 dependent) Confocal microscopy: PD-1+CXCR5 cells co-localized with B cells in lymphoid aggregates and diffuse infiltrates in RA synovial tissues
Correlation with disease progression
CXCL13 gene expression: positive correlation with clinical outcomes in BC; intratumoral B TIL presence: positive prognosis; % TFHX13 TIL: increase in triple-negative and all Grade 3 BC; CXCL13 gene prognostic value is independent from other clinicopathological parameters
% PD-1hi in synovial fluid CD4+ T cells: increase in sRA; % PD-1hiCXCR5 in blood CD4+ T cells: increase in sRA and positive correlation with serum anti-CCP antibody titer in sRA; % PD-1hiCXCR5 in blood CD4+ T cells and % plasmablast in blood B cells: decrease after treatment in patients with sRA showing reduced disease activity
Other important findings
Follicular dendritic cells are not potent CXCL13 producers in BC. In addition to PD-1hiICOSint effector subpopulation, a distinct PD1intICOShi subpopulation (principally FoxP3hi activated Treg) identified in BC CD4+ TILi. Both subpopulations positively correlated with equilibrium attained in TFHX13 TIL-rich BC. IL-2 deprivation induced CXCL13 expression in vitro from blood CD4+ T cells without affecting IFNg expression. TGFb1 provided synergistic effect for CXCL13 induction and selectively reduced IFNg expression in FoxP3l CD4+ T cells. Large public data set analysis revealed homogenous absence of IL2 together with high TGFB1, low IFNG, and a broad range of CXCL13 gene expression in total BC tumor tissues
Protein (mass cytometry) and mRNA (RNA sequencing) expression levels in PD-1hiCXCR5 and PD-1hiCXCR5+ memory CD4+ T cell subpopulations from sRA blood. Results: PD-1hiCXCR5 cells expressed high levels of ‘TIGIT, T-bet, CD38, CXCR3, MHCII, and Perforin’, and low levels of ‘CCR7, CD25 and CD1270 protein, with some degree of similarity to PD-1hiCXCR5+ cells. TFH cell-related genes (SAP, ICOS, PDCD1, MAF, CD200, BATF, TOX2, TIGIT, SLAMF6, NFATC1, SOSTDC1 and CTSB) highly expressed by both PD-1hiCXCR5 and PD-1hiCXCR5+ subpopulations. Expression patterns of chemokine receptors distinct with CCR2, CCR3, CX3CR1, CCRL2, CCR5 and CXCR3, together with CXCL13 more specifically associated with PD-1hiCXCR5 subpopulation
a
Abbreviations: TIL, tumor infiltrating lymphocyte; TLS, tertiary lymphoid structure; GC, germinal center; PC, plasmablast/plasma cell (antibody-secreting B cell). [4]. [5]. d TFHX13 cells defined as CXCL13+CD4+ T cells. TFHX13 cells are uniquely found in the PD-1hiICOSint effector CD4+ subpopulation of BC TIL but undetectable in blood or nontumor breast tissue. e TPH cells defined as PD-1hiCXCR5CD4+ T cells. TPH cells increase in synovial tissues and synovial fluid from patients with sRA and are detected in their blood. The TPH cell subpopulation in sRA is comparable to the PD-1hiICOSint effector subpopulation in BC. f Fresh (<2-h surgical specimens) tissue samples processed using nonenzymatic mechanical dissociation followed by immediate staining for all markers. g Tissue samples digested with LiberaseTL and DNaseI before cryopreservation. h Gene expression levels are relative among the analyzed CD4+ T cell subpopulations in each study; differences observed for IL10 and BCL6 may be because the PD1intICOShi Treg subpopulation was separately analyzed only in BC. i A distinct PD-1intICOShi subpopulation was not observed in RA synovial fluid, and unclear for synovial tissues (mass cytometry). b c
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CXCL13 producers in BC, with TFHX13 TIL functioning to attract and/or guide B cells and initiate TLS formation in malignant tissues. It is currently unknown whether they have the same role in SLOs. Data from a murine model detected lunginfiltrating PD-1+CXCR5 TFH-like cells together with abundant GC B cells despite a limited number of follicular dendritic cells [11], which supports the BC data [4]. TLS are important local sites of B cell maturation, suggesting that PD-1hiCXCR5CD4+ TFH cells (including CXCL13+TFHX13 and IL-21+TFH cells) contribute to initiating, maintaining, and modulating GC and post-GC responses in tissues. It is unknown whether PD-1hi and/or CXCL13+ cells differentiate in tissues following an antigen-specific response. The data reported in [4] suggest that Treg cell accumulation at the tumor site reduces IL-2 availability, which in turn induces TFHX13 cell differentiation [4] in a manner reflecting murine TFH cell development. TGFb1, abundant in tumors, could synergize with CXCL13 in the absence of IL-2. A growing body of data suggests that limiting IL-2 is important for promoting the TFH program [12]. Based on current data, PD-1intICOShi Treg are not likely involved in sRA; however, it is curious that PD-1hi cells express the
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lowest amounts of IL-2 in these tissues [5]. Finally, both studies detected increased IFNG T helper (Th)1 responses in tissue-infiltrating PD-1hiCXCR5CD4+ T cells, supporting the functional importance of type 1 effector cells in antitumor immunity and autoimmune disease [4,5].
Concluding Remarks PD-1hiCXCR5CD4+ TFH (TFHX13 and TPH) cells, which Rao et al. [5] and GuTrantien et al. [4] have shown expand under chronic inflammatory conditions, are functionally and phenotypically similar to conventional TFH cells lacking CXCR5. Future studies are necessary to determine whether conventional TFH cells are recruited from SLOs or differentiate into effector TFH cells upon arrival in diseased tissues. The plasticity of helper T cells suggests this could be another case where important CD4+ mediators of immune responses adapt to signals in their surrounding microenvironment. We propose that the role of PD1hiCXCR5CD4+ effector TFH cells (TFHX13 and TPH) in B cell differentiation and their potential activities in other human diseases merits their recognition as a subpopulation of genuine TFH cells. 1
Institute for Medical Immunology, Université Libre de Bruxelles, 6041 Charleroi, Belgium Molecular Immunology Unit, Institut Jules Bordet, Université Libre de Bruxelles, 1000 Brussels, Belgium
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*Correspondence: [email protected] (K. Willard-Gallo). https://doi.org/10.1016/j.it.2017.10.003 References 1. Gu-Trantien, C. et al. (2013) CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J. Clin. Invest. 123, 2873–2892 2. Manzo, A. et al. (2008) Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint. Arthritis Rheum. 58, 3377–3387 3. Kobayashi, S. et al. (2013) A distinct human CD4+ T cell subset that secretes CXCL13 in rheumatoid synovium. Arthritis Rheum. 65, 3063–3072 4. Gu-Trantien, C. et al. (2017) CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer. JCI Insight 2, 91487 5. Rao, D.A. et al. (2017) Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature 542, 110–114 6. Ueno, H. (2016) T follicular helper cells in human autoimmunity. Curr. Opin. Immunol. 43, 24–31 7. Kim, C.H. et al. (2004) Unique gene expression program of human germinal center T helper cells. Blood 104, 1952– 1960 8. Locci, M. et al. (2013) Human circulating PD-1+CXCR3CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity 39, 758–769 9. He, J. et al. (2013) Circulating precursor CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity 39, 770–781 10. Dieu-Nosjean, M.C. et al. (2016) Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers. Immunol. Rev. 271, 260–275 11. Vu Van, D. et al. (2016) Local T/B cooperation in inflamed tissues is supported by T follicular helper-like cells. Nat. Commun. 7, 10875 12. Locci, M. et al. (2016) Activin A programs the differentiation of human TFH cells. Nat. Immunol. 17, 976–984