PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells

Abstract

Memory B and plasma cells (PCs) are generated in the germinal center (GC). Because follicular helper T cells (T(FH) cells) have high expression of the immunoinhibitory receptor PD-1, we investigated the role of PD-1 signaling in the humoral response. We found that the PD-1 ligands PD-L1 and PD-L2 were upregulated on GC B cells. Mice deficient in PD-L2 (Pdcd1lg2(-/-)), PD-L1 and PD-L2 (Cd274(-/-)Pdcd1lg2(-/-)) or PD-1 (Pdcd1(-/-)) had fewer long-lived PCs. The mechanism involved more GC cell death and less T(FH) cell cytokine production in the absence of PD-1; the effect was selective, as remaining PCs had greater affinity for antigen. PD-1 expression on T cells and PD-L2 expression on B cells controlled T(FH) cell and PC numbers. Thus, PD-1 regulates selection and survival in the GC, affecting the quantity and quality of long-lived PCs.

Figure 1. Differential expression of PD-1 and its ligands by B cell subsets

Representative flow cytometry analysis of B6 mice immunized with NP-CGG in alum (n = 3–5) 12 days post-immunization. Splenic CD19⁺EMA⁻ cells were analyzed for NIP-binding and expression of Fas (a), and were gated as follows: NIP⁻Fas⁻ (naïve, thin solid histogram), NIP^intFas^hi (GC, dashed histogram) and NIP⁺Fas⁺ (emerging memory, thick solid histogram). B cell populations were then examined for expression of PD-L2 (b), PD-L1 (c) and PD-1 (d). Frequency (e–i) and kinetics (j–n) of B7 family members and PD-1 on B cell subsets 12 days, and 4 and 14 wks post-immunization. The CD19⁺NIP⁺IgG1⁺CD38⁺kappa^lo phenotype was used to identify memory B cells 14 wks post-immunization due to the very low frequency of detectable cells. (e–n): points are means and error bars are SEM. Data are representative of at least three independent experiments.

Figure 2. Long-lived PCs are decreased in the absence of PD-1 signaling

Analysis of long-lived PC and memory responses at least 12 weeks post-immunization with alum (n ≥ 5) or NP-CGG. (a–c) ELISpot analysis of spleen and BM from Pdcd1lg2^−/− (a; n ≥ 16), CD274^−/−Pdcd1lg2^−/− (b, n ≥ 10), Pdcd1^−/− (c; n ≥ 14) (open bars) or wild-type (WT) controls (black bars). (d) Live splenocytes were gated on CD19⁺NIP⁺IgG1⁺CD38⁺kappa^lo to assess memory B cell frequency. The CD38 marker was included to gate out any residual GC B cells (which are CD38⁻). Because naïve B cells are also CD38⁺, IgG1 was included to separate memory B cells from naïve B cells, although this precludes analysis of any present IgM⁺ memory B cells. (e–g) Frequency of memory B cells in Pdcd1lg2^−/− (e), CD274^−/−Pdcd1lg2^−/− (f) and Pdcd1^−/− (g) mice (open symbols) compared to WT controls (closed symbols). *P < 0.05, **P < 0.01, ***P < 0.001. Data are combined from two (CD274^−/−Pdcd1lg2^−/−) or seven (Pdcd1lg2^−/− and Pdcd1^−/−) independent experiments.

Figure 3. The reduction in PC numbers occurs during the late GC response, and affects both IgG1 and IgM Ab

(a–c) ELISpot analysis of NP⁺IgG1⁺ AFCs in spleen and BM from WT controls (black bars) or Pdcd1lg2^−/− (a, n ≥ 6 for WT and mutant strain), CD274^−/−Pdcd1lg2^−/− (b, n ≥ 10) or Pdcd1^−/− (c, n ≥ 12; open bars) mice immunized with alum (n = 3–9 for all genotypes) or NP-CGG ~4 wks post-immunization. Data is combined from at least two independent experiments. Pdcd1lg2^−/− (d), CD274^−/−Pdcd1lg2^−/− (e), Pdcd1^−/− (f) or WT controls were immunized with NP-CGG or alum alone and NP⁺IgG1⁺ AFCs in spleen and BM assessed at multiple time-points post-immunization (d28 includes d27-d31, as shown also in a–c; d12 also shown in Supplementary Fig. 1, >d84 in Fig. 2). For all time-points except for CD274^−/−Pdcd1lg2^−/− d18 and d21, data are combined from multiple independent experiments. (g,h) Circulating IgG1 and IgM in Pdcd1lg2^−/− (g; n = 6–8), Pdcd1^−/− (h; n = 6) or WT at wk4 and wk3 post-immunization, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of at least two independent experiments from wks 3–4.

Figure 4. Increased cell death but normal proliferation in the GCs of Pdcd1^−/− mice

WT (closed symbols) and Pdcd1^−/− B6 (open symbols) mice immunized with NP-CGG, were pulsed with 3 mg/mouse of BrdU 2hr before analysis. Splenic CD19⁺NIP^intFas^hi cells undergoing cell death were revealed by detection of activated caspases using CaspGLOW immediately after cell harvest at the timepoints: d10, 12 (also shown in Supplementary Fig. 1), 14, 15, 18 and 21 post-immunization. (a) Sample flow cytometry data of CaspGLOW and BrdU staining from an immunized B6 mouse at d12. (b–d) Summary of frequencies derived from flow cytometry analysis of splenic GC B cells (b) that are CaspGLOW⁺ (c) and BrdU⁺ (d) over time. (e) Flow cytometry analysis of emerging memory cells and plasmablasts/PCs that are CaspGLOW⁺; n ≥ 4 per time point. (f) NP₂-BSA or NP₁₇-BSA was used as capture Ag for ELISpot and the ratio of NP₂ versus NP₁₇-specific IgG1⁺ BM AFCs for knockout (open histogram; n ≥ 6) and WT mice (closed histogram) were plotted. *P < 0.05, **P < 0.01, ***P < 0.001. Data are combined from, or are representative of, at least two independent experiments.

Figure 5. An increase of cells of a T_FH phenotype correlates with a decrease in cytokine production in the absence of PD-1 signaling

(a–c) CD274^−/−Pdcd1lg2^−/−, Pdcd1lg2^−/−, Pdcd1^−/− and WT mice were immunized with NP-CGG and CD4⁺CD90.2⁺EMA⁻ cells were gated for the T_FH markers of PD-1 expression and CCR7 downregulation, or ICOS expression and CCR7 downregulation for Pdcd1^−/− and corresponding WT mice. (a,b) Flow cytometry analysis of T_FH frequency among CD4 cells (a) and number (b) in CD274^−/−Pdcd1lg2^−/− and WT mice d12, d15 and d18 post-immunization. (c) T_FH frequency in Pdcd1lg2^−/−, Pdcd1^−/− and WT mice d15 post-immunization (n = 3–5). Data are representative of at least two independent experiments during the late GC response. (d) Pdcd1^−/− and WT mice were immunized with NP-CGG in alum and sorted for ICOS expression and CCR7 downregulation (d12, n = 3), in conjunction with CXCR5 expression (d15, n = 2; d18, n = 3). Il4, Il21 and Ifng expression was assessed by qPCR. Data are shown as fold change as calculated by 2^{(actin Ct − cytokine Ct)}. *P < 0.05, **P < 0.01, ***P < 0.001. n.s. = no significance. Each time point is a separate independent experiment.

Figure 6. Decrease in AFCs is due to impaired interactions between PD-ligands on B cells and PD-1 on T cells

(a,b) Schematic representation of the B cell transfer system (a) and T cell transfer system (b). (c,d) ELISpot analysis of NP⁺IgG1⁺ AFCs in spleen and BM from AM14 Vκ8R recipient mice of transferred control B cells (black bar) or B cells from Pdcd1lg2^−/−B1-8^+/−(c; >day 63; n = 16) or Pdcd1lg2^−/− (d; day 26–28; n = 14–15) mice (open bar) immunized with alum or NP-CGG. Data are combined from three (c) or four (d) independent experiments. (e–g) Analysis of Pdcd1^−/− B and T cell transfers 4 wks post-immunization: (e) ELISpot analysis of splenic NP⁺IgG1⁺ AFCs in either AM14 Vκ8R recipients of transferred B cells (n = 8), or OT-II recipients of transferred T cells (n = 14), from B6 WT mice (black bar) or Pdcd1^−/− mice (open bar). (f,g) Frequency and number of NIP⁺IgG1⁺CD38⁺kappa^lo B cells after transfer of WT (closed symbols) or Pdcd1^−/− (open symbols) T cells (f) or B cells (g). *P < 0.05, **P < 0.01, ***P < 0.001. Data are combined from two (B cell transfer) or three (T cell transfer) independent experiments.

Figure 7. AFC production, memory B cell formation and T_FH are altered in mixed BM chimeras

Mixed BM chimeras were made by mixing Igh-J^−/− BM with Pdcd1lg2^−/−, CD274^−/− Pdcd1lg2^−/−, Pdcd1^−/− or BALB/c BM at a 80:20% ratio, and injecting into lethally irradiated BALB/c recipients (white bars). Black bars represent mice that received BM only from WT or knockout mice (without Igh-J^−/− BM). Mice were assessed for degree of chimerization, which approximated the above ratio (not shown). Mice were rested for 6 wks before immunization with NP-CGG. Mice were assessed at d25–26 post-immunization (Igh-J^−/−:BALB/c: n = 19, Igh-J^−/−:Pdcd1lg2^−/−: n = 12, Igh-J^−/−:CD274^−/−Pdcd1lg2^−/−: n = 8, Igh-J^−/−:Pdcd1^−/−: n = 5). (a,b,d) ELISpot analysis of mixed BM chimeras in the spleen (a,c) and BM (b). (c, e–g) flow cytometry analyses of memory B cell frequency (of CD19⁺ cells; c) and T_FH cell frequency (of CD4⁺CD90.2⁺ cells; Igh-J^−/−: BALB/c: n = 15, Igh-J^−/−:Pdcd1lg2^−/−: n = 9, Igh-J^−/−:CD274^−/−Pdcd1lg2^−/−: n = 4; Igh-J^−/−: Pdcd1^−/−: n = 5; e,f) as described in the text. *P < 0.05, **P < 0.01, ***P < 0.001. Data are combined from or are representative of two independent experiments.