Critical Role of PD-1/PD-L1 Pathway in Generation and Function of Follicular Regulatory T Cells

RESEARCH HIGHLIGHT

Critical role of PD-1/PD-L1 pathway in generation and function of follicular regulatory T cells

Zhi-Qing Hu and Wei-Hua Zhao

Cellular & Molecular Immunology (2013) 10, 286–288; doi:10.1038/cmi.2013.15; published online 29 April 2013

he balance between follicular PD-1 is a well-known inhibitory receptor only the generation and function of TFR 9 T helper T cells (TFH cells) and folli- for humoral immune responses and can cells but also the ratio of TFH to TFR cells. 2/2 2/2 cular regulatory T (Treg) cells (TFR cells) be expressed by T cells, B cells, macro- In addition, Cd28 and Icos mice 10 is one of the key factors for the develop- phages and dendritic cells. TFR cells showed considerable deficiency of TFH 1 ment of humoral immune responses. express more PD-1 than TFH cells. B cells and TFR cells, suggesting that CD28 and How the balance is regulated, however, and dendritic cells express the PD-1 ligands, ICOS provide essential costimulatory sig- 10 is largely unknown. In a recent issue of PD-L1 and PD-L2. nals for TFH and TFR cell development. Nature Immunology, Sage et al. revealed a To uncover how the balance between Like WT TFR cells, PD-1-deficient TFR cells critical role of the programmed death-1 TFH and TFR cells controls humoral can home to GCs. (PD-1)/PD-L1 pathway in TFR cell gen- immunity, Sage et al. attempted to clarify In the second approach, the authors eration and function,1 which provides the individual role of PD-1 expressed on investigated the role of PD-1/PD-L1 path- a novel insight into the mechanisms TFH and TFR cells. Table 1 summarizes way in the differentiation of TFR cells from 1 1 2 how TFH and TFR cells regulate antibody the major cells, molecules, antigens and CD4 Foxp3 CXCR5 Treg cells. They production. knockout/transgenic mice and Figure 1 used 2D2 and Pdcd12/22D2 mice that Germinal centers (GCs) are sites within outlines the three main approaches and obtained transgenic expressions of a lymph nodules, where B lymphocytes the findings of this work. MOG-specific TCR and Foxp3-green rapidly proliferate and differentiate into In the first approach, the authors fluorescent protein (GFP). The MOG- plasma cells and memory B cells during determined the frequency and number specific GFP1 Treg cells purified from 1 1 2 humoral immune responses. These events of TFH cells (CD4 CXCR5 Foxp3 thesemiceweretransferredintoWTmice 1 2 1 are precisely regulated by interactions ICOS CD19 ) and TFR cells (CD4 and the recipients were further immu- 1 1 1 2 between cognate B cells, TFH cells, TFR cells CXCR5 Foxp3 ICOS CD19 )in4 nized with MOG. In the mice receiving 2–5 2 1 and follicular dendritic cells. TFH cells strains of C57BL/6 mice: wild-type PD-1 Treg cells, the GFP TFR cells are a specialized CD41 helper subset, (WT), PD-1-deficient (Pdcd12/2), PD- (CD41Foxp31CXCR51) in draining 2/2 which provides help to B cells. TFR cells L1-deficient (Cd274 )andPD-L2-defi- lymphnodeswerefoundathigherfre- are a newly identified CD41 Treg subset cient (Pdcd1lg22/2),thesemicebeing quency and higher absolute number than in GCs, which downregulates the GC res- immunized with MOG. Deficiency of in the mice received PD-11 Treg cells. 6,7 ponse. TFH and TFR cells are defined by PD-1 or PD-L1, but not PD-L2, resulted These data confirm that the PD-1/PD-L1 expression of the CXC-chemokine recep- in more TFR cells than TFH cells in lymph pathway controls the differentiation of 1 1 2 tor 5 (CXCR5) which directs them to B- nodes and blood. Compared to WT TFR CD4 Foxp3 CXCR5 Treg cells into cell follicles in GCs via gradients of the cells, PD-1-deficient TFR cells displayed a TFR cells. CXC-chemokine ligand 13 (CXCL13).1,2,8 potent capacity to inhibit both the proli- In the third approach, the authors TFH and TFR cells are present not only in feration of responder T and B cells and the evaluated whether TFR cells in blood lymph nodes but also in peripheral blood. TFH cell-mediated IgG production in vitro. could suppress antibody production in The expressions of activation markers vivo. WT, Pdcd12/2 and Cxcr52/2 mice Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo, (CD25, CD69 and Ki67) and transcription were immunized with NP-OVA. WT 1 1 1 2 Japan factors (Bcl-6, Blimp-1, RORctandIRF4) TFH cells (CD4 ICOS CXCR5 GITR Correspondence: Dr ZQ Hu, Department of 2 by TFH and TFR cells were also compared CD19 ), WT and PD-1-deficient TFR Microbiology and Immunology, Showa University between Pdcd12/2 and WT mice, as cells (CD41ICOS1CXCR51GITR1CD192) School of Medicine, 1-5-8 Hatanodai, 1 Shinagawa-ku, Tokyo 142-8555, Japan. showninFigure1.Thedataindicatethat and CXCR5-deficient Treg cells (CD25 E-mail: [email protected] the PD-1/PD-L1 pathway controls not CD62L1) were sorted from blood 8 days Research Highlight 287

Table 1 Cell types, molecules, antigens and mice Name Feature

1 1 2 1 1 2 1 2 Cell TFH cell Follicular helper T cell, CD4 CXCR5 Foxp3 ICOS Bcl-6 CD19 and PD-1 GITR .TFH cells are essential for GC formation and for B cell activation/differentiation into plasma cells 1 1 1 1 1 2 1 1 TFR cell Follicular regulatory T cell, CD4 CXCR5 Foxp3 ICOS Bcl-6 CD19 and PD-1 GITR .TFR cells can inhibit TFH cell function and naive T- and B-cell activation CD41 Treg cell Treg cell, CD41CXCR52Foxp31 and GITR1 Molecule PD-1 PD-1 (CD279), also called PDCD-1. Well-known inhibitory receptor and expressed by T cells, B cells, macrophages and dendritic cells PD-L1 Ligand 1 of PD-1 (CD274), also called B7 homolog-1 (B7-H1) PD-L2 Ligand 2 of PD-1 (CD273), also called B7-DC PD-L1 and PD-L2 are type I transmembrane proteins related to the CD28 ligands, but do not bind CD28 or CTLA-4 (CD152). B cells and dendritic cells in GCs express both PD-L1 and PD-L2

CXCR5 CXC-chemokine receptor 5 (CD185), a marker of TFH and TFR cells in a follicular program 1 CXCL13 CXC-chemokine ligand 13, also called BLC. CXCL13 can direct the migration and relocation of CXCR5 B cells, TFH

cells and TFR cells into follicles GITR Glucocorticoid-induced tumor-necrosis factor receptor (CD357, TNFRSF-18), a type 1 transmembrane protein of

the TNF receptor superfamily. GITR is expressed on TFR cells but not on TFH cells, and is used as a marker to isolate

TFR cells for functional study CD28 Well-known T-cell costimulatory molecule. Its natural ligands are CD80 (B7-1) and CD86 (B7-2). CD28 provides

essential costimulatory signals for TFH and TFR cells ICOS Inducible T-cell costimulator (CD278). Its ligand is CD275 (B7H). ICOS provides essential costimulatory signals for

TFH and TFR cells Antigen MOG Myelin oligodendrocyte glycoprotein peptide (amino acids 35–55) emulsified in complete Freund’s adjuvant. NP-OVA The hapten NP (4-hydroxy-3-nitrophenylacetyl) conjugated to ovalbumin. Mouse WT WT C57BL/6 Pdcd12/2 Deficient in PD-1 Cd2742/2 Deficient in PD-L1 Pdcd1lg22/2 Deficient in PD-L2 Cxcr52/2 Deficient in CXCR5, lacking a follicular program 2/2 Cd28 Deficient in CD28, lacking TFH and TFR cells 2/2 Icos Deficient in ICOS, lacking TFH and TFR cells 2/2 Tcra Deficient in TCR a-chain, lacking TFH and TFR cells 2D2 Transgenic mice, expressing a MOG-specific TCR and Foxp3-GFP Pdcd12/22D2 With the properties of both Pdcd12/2 and 2D2 mice

Abbreviations: BLC, B lymphocyte chemoattractant; GC, germinal center; GFP, green fluorescent protein; PD, programmed death; PDCD-1, programmed cell death-1; TCR, T-cell receptor; Treg, regulatory T; WT, wild-type. later. The isolated cells, in various com- on the ‘follicular program’ because cells. These findings provide new insights binations, were transferred into Cd282/2 CXCR5-deficient Treg cells failed to into the regulation of humoral immun- mice and into TCR a-chain knockout inhibit the antibody production induced ity, and suggest that TFR cells and assoc- 2/2 (Tcra ) mice. These mice cannot pro- by the WT TFH cells. The TFR cell- iated molecules could be used as targets duce their own TFH and TFR cells, and so mediated inhibition was further con- for the treatment of autoimmune diseases. any follicular T cells in their bodies must firmed by quantification of CD1381 2/2 2/2 have come from the transferred TFH and plasma cells in Cd28 and Tcra TFR cells. The recipients were then mice after the transfer of WT TFH and 1 Sage PT, Francisco LM, Carman CV, Sharpe AH. The receptor PD-1 controls immunized with NP-OVA and the titers TFR cells. These in vivo results dem- follicular regulatory T cells in the lymph of NP-specific IgG were measured. In onstrate that TFR cells in blood can home nodes and blood. Nat Immunol 2013; both recipient mice, the transferred WT to lymph nodes and potently inhibit 14: 152–161. TFH cells promoted antibody produc- antibody production. 2 Crotty S. Follicular helper CD4 T cells tion, whereas the transferred WT T Taken together, the PD-1/PD-L1 (TFH). Annu Rev Immunol 2011; 29: FR 621–663. cells strongly inhibited the antibody pro- pathway plays a critical role in TFR gen- 3 Nutt SL, Tarlinton DM. Germinal center B duction induced by the TFH cells. eration and function. Signaling via PD- and follicular helper T cells: siblings, cousins or just good friends? Nat Immunol Particularly, the transferred PD-1-defi- 1/PD-L1 inhibits TFR cell differentiation cient T cells displayed much stronger from CD41Foxp31 Treg cells and con- 2011; 12: 472–477. FR 4 Ramiscal RR, Vinuesa CG. T-cell subsets in suppressive ability than the WT TFR cells. trols their suppressive ability, thus chan- the germinal center. Immunol Rev 2013; The TFR-mediated inhibition depends ging the balance between TFH and TFR 252: 146–155.

Cellular & Molecular Immunology Research Highlight 288

Approach 1 Approach 2 Approach 3 MOG MOG MOG MOG NP-OVA NP-OVA NP-OVA WT

Pdcd1−/− Cd274−/− Pdcd1lg2−/− 2D2 Pdcd1−/−2D2 WT Pdcd1−/− Cxcr5−/− Compared to WT

TFH number in LN/B –/↑ –/↑ –/– Tregreg Treg TT TTFR TTFR TTreg TFR number in LN/B ↑↑/↑↑↑/↑ –/– FHFH FR FR

Ratio of T /T FH FR ↓ ↓ – Transfer Transfer NP-OVA NP-OVA T -mediated suppression* ↑↑ ND ND FR MOG MOG

MOG Cd28−/− Tcra−/− WT WT Pdcd1−/− Cells transferred NP-specific IgG Compared to WT None + ± Activation marker Transcription factor

WT TFH alone ++++ ++++ CD25 CD69 Ki67 Bcl-6 Blimp-1 RORg t IRF4 TTFRFR TTFRFR WT TFH+ WT TFR ++ ++ T ––↓↓ ––– FH −/− WT TFH+ Pdcd1 TFR + + T ++++ FR ↓↓– –– ↑ ↑ WT T + Cxcr5−/− Treg Numbers in LN FH ND ++++

Figure 1 The major experimental approaches and findings which reveal the critical role of the PD-1/PD-L1 pathway in TFR generation and function. *,TFR-mediated suppression includes the inhibition of responder T- and B-cell proliferation, and the inhibition of IgG production. See Table 1 for the mice and antigens used. B, blood; LN, lymph nodes; MOG & NP-OVA, antigens; ND, not detected; PD-1, programmed death-1; 2, no significant difference.

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