PD-1 Controls Follicular T Helper Cell Positioning and Function - PubMed (original) (raw)
PD-1 Controls Follicular T Helper Cell Positioning and Function
Jingwen Shi et al. Immunity. 2018.
Abstract
Follicular T helper (Tfh) cells highly express the programmed cell death-1 (PD-1) molecule. Whereas inhibition of T cell receptor (TCR) signaling and CD28 co-stimulation is thought to be the primary mode of PD-1 functions, whether and how PD-1 regulates Tfh cell development and function is unclear. Here we showed that, when engaged by the ensemble of bystander B cells constitutively expressing PD-1 ligand 1 (PD-L1), PD-1 inhibited T cell recruitment into the follicle. This inhibition involved suppression of PI3K activities downstream of the follicle-guidance receptor CXCR5, was independent of co-signaling with the TCR, and necessitated ICOS signaling to overcome. PD-1 further restricted CXCR3 upregulation on Tfh cells, serving to concentrate these cells toward the germinal center territory, where PD-L1-PD-1 interactions between individual Tfh and B cells optimized B cell competition and affinity maturation. Therefore, operating in both costimulation-independent and -dependent manners, PD-1 controls tissue positioning and function of Tfh cells.
Keywords: CXCR3; ICOS; ICOSL; PD-1; PD-L1; Tfh cells; affinity maturation; follicular helper T cells; germinal center; motility.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
Graphical abstract
Figure 1
Costimulation-Independent Suppression of PI3K Activities and Follicular Recruitment of T Cells by PD-1 (A) Surface staining of PD-L1 or PD-L2 expression on follicular B cells. Grey histograms: isotype control staining. (B) CD4+ T cells retrovirally co-transduced with CXCR5 and PD-1 were starved in serum-free media for 3 hr. AKT phosphorylation was probed after 30 min CXCL13 stimulation at indicated concentrations in the presence or absence of PD-L1-Fc crosslinked by anti-human IgG. Data represent two independent experiments. (C) AKT phosphorylation was probed after CD4+ T cells were starved in serum-free media for 3 hr and then stimulated with anti-ICOS in the presence or absence of PD-L1-Fc at indicated concentration for 30 min. Data represent two independent experiments. (D) Splenic distribution patterns of CD4+ T cells retrovirally co-transduced with a combination of CXCR5 or control GFP and PD-1 or control RFP 24 hr after being injected into B6 mice (2–3 × 106 sorted transduced cells per mouse). (E) Scatterplots of the homing coefficients of the four groups in (D), with each symbol indicating one section. Data are pooled from four independent experiments, with each experiment contributing 10–20 sections. Scale bar, 50 μm. ∗∗p < 0.01; ns, not significant.
Figure 2
PD-1 Abrogation on Activated T Cells Rescues Follicular Homing Defect due to ICOSL-ICOS Deficiency (A and B) CXCR5- or GFP-transduced wild-type (top) or _Pdcd1_KI/KI (bottom) CD4+ T cells were transferred into Icosl +/+ (left) or Icosl −/− (right) mice (2–3 × 106 cells per mouse). Representative splenic distribution patterns (A) and homing coefficients (B) of T cells 24 hr after adoptive transfer. (C and D) Representative splenic distribution patterns (C) and homing coefficients (D) of CXCR5-transduced Icos+/+, _Icos_−/−, or _Pdcd1_KI/KI_Icos_−/− CD4+ T cells 24 hr after being adoptive transfer into B6 mice. Each symbol denotes one section. Data are pooled from five (B) or three (D) independent experiments. Scale bar, 100 μm. ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant.
Figure 3
PD-L1 on Bystander B Cells Suppresses T Cell Recruitment Splenic distribution patterns (A) and homing coefficients (B) of CXCR5-transduced Icos+/+ (top) or _Icos_−/− (bottom) T cells 24 hr after adoptive transfer into mixed bone-marrow chimeric hosts of indicated types (2–3 × 106 cells per mouse). Each symbol denotes one tissue section. Data are pooled from three experiments. Scale bar, 50 μm. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.
Figure 4
PD-1-Mediated Suppression of Follicular T Cell Recruitment Implicates ITSM and SHP-2 (A) Splenic distribution patterns of CD4+ T cells that were co-transduced with a vector expressing CXCR5 and another vector expressing control RFP (top left) or wild-type PD-1 (top right) or ITIM-mutated PD-1Y225F (bottom left) or ITSM-mutated PD-1Y248F (bottom right) 24 hr after being transferred into B6 recipients (2–3 × 106 per mouse). (B) Scatterplots of homing coefficients of the four groups in (A). Each symbol denotes one tissue section. Data are pooled from three experiments. Scale bar, 100 μm. ∗∗∗∗p < 0.0001; ns, not significant. (C) SHP-2 phosphorylation in CD4+ T cells after 30 min anti-ICOS stimulation in the presence or absence of PD-L1-Fc. Cells were pre-starved for 3 hr. Data represent two independent experiments.
Figure 5
PD-1 and PD-L1 Promote Tfh Cell Concentration toward the GC Area (A and B) GFP-transduced wild-type or _Pdcd1_KI/KI OT-II T cells were transferred into B6 mice (5 × 105 sorted GFP+ cells per mouse) together with 5 × 105 dsRed-expressing MD4 B cells. OT-II distribution patterns (A) and density ratios between the GCs and the follicle (B) in draining lymph nodes 5 days after subcutaneous HEL-OVA immunization. (C and D) Wild-type or _Cd274_−/− mice received 5 × 105 GFP-transduced wild-type or _Pdcd1_KI/KI OT-II T cells together with 5 × 105 dsRed-expressing MD4 B cells. OT-II distribution patterns (C) and density ratios between the GCs and the follicle (D) in draining lymph nodes 5 days after subcutaneous HEL-OVA immunization. Scale bar, 100 μm. All data are pooled from three independent experiments involving 3–4 mice per condition per experiment. ∗∗∗∗p < 0.0001; ns, not significant.
Figure 6
PD-1 Restricts CXCR3 Expression and Promotes Confinement of Tfh Cells in GCs (A and B) GFP-transduced wild-type or _Pdcd1_KI/KI OT-II T cells were transferred into B6 mice (5 × 105 sorted GFP+ cells per mouse) together with 5 × 105 dsRed-expressing MD4 B cells, and the recipients were subcutaneously immunized with HEL-OVA. (A) CXCR5hi OT-II T cells were sorted from the two groups on day 5 and subjected to transcriptomic analysis by RNA-seq. Shown is unsupervised clustering of genes differentially expressed as defined by an adjusted p value < 0.01. (B) CXCR3 expression on CXCR5hi wild-type and _Pdcd1_KI/KI OT-II T cells. Left, CXCR5hi gate; middle, histograms; right, representative CXCR3 histogram overlay and MFI values of CXCR5hi OT-II cells in three mice; data representative of three independent experiments; ∗p < 0.05. (C) CXCR3 expression on CXCR5hi_Pdcd1_KI/KI OT-II T cells that were transduced with scramble control shRNA or two separate CXCR3-targeting shRNAs. Grey histogram, isotype control. (D and E) Wild-type (left) or _Pdcd1_KI/KI (right) OT-II T cells were transduced with scramble control shRNA or CXCR3-targeting shRNA and then were transferred into B6 mice (5 × 105 Ametrine+ transduced cells per mouse) together with 5 × 105 dsRed-expressing MD4 B cells. OT-II distribution patterns (D) and density ratios between the GCs and the follicle (E) in draining lymph nodes 5 days after subcutaneous HEL-OVA immunization. Scale bar, 100 μm. Each symbol denotes one tissue section. Data are pooled from three experiments involving three and four mice per condition per experiment. ns, not significant; ∗p < 0.05; ∗∗∗∗p < 0.0001.
Figure 7
Compromised Affinity Maturation in the Absence of PD-1-PD-L1 Interactions between GC Tfh and B Cells Mixed BM chimeras were constructed using 50% CD45.2 Cd274+/+ or _Cd274_−/− BM cells and 50% CD45.1 BM cells. Chimeric mice were immunized with NP-KLH. (A) Gating strategies for total B cells, plasma cells, GC B cells, NP-specific GC B cells, and memory B cells (top row) and for CD45.1 and CD45.2 cells within each indicated population (bottom row). The example is from _Cd274_−/−:CD45.1 chimera. (B) Scatterplots of competitive competencies of CD45.2 cells in the two sets of mixed chimeras contributing to GC B cells, NP-specific GC B cells, NP-specific memory B cells and plasma cells, and BM plasma cells at day 8, day 13, or day 21 after immunization. The competitive competency is defined as the CD45.2/CD45.1 ratio in the indicated compartment normalized against the same ratio in the total B cell compartment or the GC compartment in the case of NP-specific GC cells. Data are pooled from three independent experiments involving 3–4 mice per group per experiment. Each symbol denotes one mouse. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. (C) Total mutation rates and frequencies of affinity-enhancing W33L mutation in NP-specific, VH186.2-carrying GC B cells isolated on day 13 after NP-KLH immunization. Numbers in the center of the pie chart indicate total numbers of clones analyzed, and numbers of W33L+ or W33L− cells are indicated. Numbers of total mutation are presented as mean ± SEM. Frequencies of W33L mutations were compared by the Fisher’s exact test. Data are pooled from three independent experiments, each involving 3–4 mice for each group.
Comment in
- Location, Location, Location.
Ueno H. Ueno H. Immunity. 2018 Aug 21;49(2):197-199. doi: 10.1016/j.immuni.2018.08.006. Immunity. 2018. PMID: 30134195
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