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

The balance between follicular helper T cells (T_FH cells) and follicular regulatory T (Treg) cells (T_FR cells) is one of the key factors for the development of humoral immune responses. How the balance is regulated, however, is largely unknown. In a recent issue of Nature Immunology, Sage et al. revealed a critical role of the programmed death-1 (PD-1)/PD-L1 pathway in T_FR cell generation and function,¹ which provides a novel insight into the mechanisms how T_FH and T_FR cells regulate antibody production.

Germinal centers (GCs) are sites within lymph nodules, where B lymphocytes rapidly proliferate and differentiate into plasma cells and memory B cells during humoral immune responses. These events are precisely regulated by interactions between cognate B cells, T_FH cells, T_FR cells and follicular dendritic cells.^2,3,4,5 T_FH cells are a specialized CD4⁺ helper subset, which provides help to B cells. T_FR cells are a newly identified CD4⁺ Treg subset in GCs, which downregulates the GC response.^6,7 T_FH and T_FR cells are defined by expression of the CXC-chemokine receptor 5 (CXCR5) which directs them to B-cell follicles in GCs via gradients of the CXC-chemokine ligand 13 (CXCL13).^1,2,8 T_FH and T_FR cells are present not only in lymph nodes but also in peripheral blood. PD-1 is a well-known inhibitory receptor for humoral immune responses⁹ and can be expressed by T cells, B cells, macrophages and dendritic cells.¹⁰ T_FR cells express more PD-1 than T_FH cells.¹ B cells and dendritic cells express the PD-1 ligands, PD-L1 and PD-L2.¹⁰

To uncover how the balance between T_FH and T_FR cells controls humoral immunity, Sage et al. attempted to clarify the individual role of PD-1 expressed on T_FH and T_FR cells. Table 1 summarizes the major cells, molecules, antigens and knockout/transgenic mice and Figure 1 outlines the three main approaches and the findings of this work.

Table 1. Cell types, molecules, antigens and mice.

Name		Feature
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
	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
	CD4+ Treg cell	Treg cell, CD4+CXCR5−Foxp3+ and GITR+
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-DCPD-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
	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
	Pdcd1−/−	Deficient in PD-1
	Cd274−/−	Deficient in PD-L1
	Pdcd1lg2−/−	Deficient in PD-L2
	Cxcr5−/−	Deficient in CXCR5, lacking a follicular program
	Cd28−/−	Deficient in CD28, lacking TFH and TFR cells
	Icos−/−	Deficient in ICOS, lacking TFH and TFR cells
	Tcra−/−	Deficient in TCR α-chain, lacking TFH and TFR cells
	2D2	Transgenic mice, expressing a MOG-specific TCR and Foxp3-GFP
	Pdcd1−/−2D2	With the properties of both Pdcd1−/− 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.

Figure 1.

The major experimental approaches and findings which reveal the critical role of the PD-1/PD-L1 pathway in T_FR generation and function. *, T_FR-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; −, no significant difference.

In the first approach, the authors determined the frequency and number of T_FH cells (CD4⁺CXCR5⁺Foxp3⁻ICOS⁺CD19⁻) and T_FR cells (CD4⁺CXCR5⁺Foxp3⁺ICOS⁺CD19⁻) in 4 strains of C57BL/6 mice: wild-type (WT), PD-1-deficient (Pdcd1^−/−), PD-L1-deficient (Cd274^−/−) and PD-L2-deficient (Pdcd1lg2^−/−), these mice being immunized with MOG. Deficiency of PD-1 or PD-L1, but not PD-L2, resulted in more T_FR cells than T_FH cells in lymph nodes and blood. Compared to WT T_FR cells, PD-1-deficient T_FR cells displayed a potent capacity to inhibit both the proliferation of responder T and B cells and the T_FH cell-mediated IgG production in vitro. The expressions of activation markers (CD25, CD69 and Ki67) and transcription factors (Bcl-6, Blimp-1, RORγt and IRF4) by T_FH and T_FR cells were also compared between Pdcd1^−/− and WT mice, as shown in Figure 1. The data indicate that the PD-1/PD-L1 pathway controls not only the generation and function of T_FR cells but also the ratio of T_FH to T_FR cells. In addition, Cd28^−/− and Icos^−/− mice showed considerable deficiency of T_FH and T_FR cells, suggesting that CD28 and ICOS provide essential costimulatory signals for T_FH and T_FR cell development. Like WT T_FR cells, PD-1-deficient T_FR cells can home to GCs.

In the second approach, the authors investigated the role of PD-1/PD-L1 pathway in the differentiation of T_FR cells from CD4⁺Foxp3⁺CXCR5⁻ Treg cells. They used 2D2 and Pdcd1^−/−2D2 mice that obtained transgenic expressions of a MOG-specific TCR and Foxp3-green fluorescent protein (GFP). The MOG-specific GFP⁺ Treg cells purified from these mice were transferred into WT mice and the recipients were further immunized with MOG. In the mice receiving PD-1⁻ Treg cells, the GFP⁺ T_FR cells (CD4⁺Foxp3⁺CXCR5⁺) in draining lymph nodes were found at higher frequency and higher absolute number than in the mice received PD-1⁺ Treg cells. These data confirm that the PD-1/PD-L1 pathway controls the differentiation of CD4⁺Foxp3⁺CXCR5⁻ Treg cells into T_FR cells.

In the third approach, the authors evaluated whether T_FR cells in blood could suppress antibody production in vivo. WT, Pdcd1^−/− and Cxcr5^−/− mice were immunized with NP-OVA. WT T_FH cells (CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻), WT and PD-1-deficient T_FR cells (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻) and CXCR5-deficient Treg cells (CD25⁺CD62L⁺) were sorted from blood 8 days later. The isolated cells, in various combinations, were transferred into Cd28^−/− mice and into TCR α-chain knockout (Tcra^−/−) mice. These mice cannot produce their own T_FH and T_FR cells, and so any follicular T cells in their bodies must have come from the transferred T_FH and T_FR cells. The recipients were then immunized with NP-OVA and the titers of NP-specific IgG were measured. In both recipient mice, the transferred WT T_FH cells promoted antibody production, whereas the transferred WT T_FR cells strongly inhibited the antibody production induced by the T_FH cells. Particularly, the transferred PD-1-deficient T_FR cells displayed much stronger suppressive ability than the WT T_FR cells. The T_FR-mediated inhibition depends on the ‘follicular program' because CXCR5-deficient Treg cells failed to inhibit the antibody production induced by the WT T_FH cells. The T_FR cell-mediated inhibition was further confirmed by quantification of CD138⁺ plasma cells in Cd28^−/− and Tcra^−/− mice after the transfer of WT T_FH and T_FR cells. These in vivo results demonstrate that T_FR cells in blood can home to lymph nodes and potently inhibit antibody production.

Taken together, the PD-1/PD-L1 pathway plays a critical role in T_FR generation and function. Signaling via PD-1/PD-L1 inhibits T_FR cell differentiation from CD4⁺Foxp3⁺ Treg cells and controls their suppressive ability, thus changing the balance between T_FH and T_FR cells. These findings provide new insights into the regulation of humoral immunity, and suggest that T_FR cells and associated molecules could be used as targets for the treatment of autoimmune diseases.