Impaired development of memory B cells and antibody responses in humans and mice deficient in PD-1 signaling

Abstract

T follicular helper (Tfh) cells abundantly express the immunoreceptor programmed cell death protein 1 (PD-1), and the impact of PD-1 deficiency on antibody (Ab)-mediated immunity in mice is associated with compromised Tfh cell functions. Here, we revisited the role of the PD-1-PD-L1 axis on Ab-mediated immunity. Individuals with inherited PD-1 or PD-L1 deficiency had fewer memory B cells and impaired Ab responses, similar to Pdcd1^-/- and Cd274^-/-Pdcd1lg2^-/- mice. PD-1, PD-L1, or both could be detected on the surface of human naive B cells following in vitro activation. PD-1- or PD-L1-deficient B cells had reduced expression of the transcriptional regulator c-Myc and c-Myc-target genes in vivo, and PD-1 deficiency or neutralization of PD-1 or PD-L1 impeded c-Myc expression and Ab production in human B cells isolated in vitro. Furthermore, B cell-specific deletion of Pdcd1 prevented the physiological accumulation of memory B cells in mice. Thus, PD-1 shapes optimal B cell memory and Ab-mediated immunity through B cell-intrinsic and B cell-extrinsic mechanisms, suggesting that B cell dysregulation contributes to infectious and autoimmune complications following anti-PD-1-PD-L1 immunotherapy.

Keywords: IL-21; PD-1; PD-L1; T follicular helper cells; antibody; c-Myc; humoral immunity; memory B cells.

Figure 1.. Impaired B-cell memory and antibody responses in children with inherited PD-1 or PD-L1 deficiency.

(A-B) VirScan analysis for serum IgG samples from the PD-1-deficient patient (aged 10 and 11 years; two sampling points), his healthy brother (aged 6 and 7 years) and parents, five healthy age-matched controls and 13 patients with biallelic CARMIL2 mutations. (A) Principal component analysis (PCA). Left: sample distribution. Right: variable loadings. (B) Scores for representative microbes. (C-D) Mass cytometry showing (C) B cells among total leukocytes and (D) memory B cells. (E-L) PBMCs from the PD-1-deficient patient (aged 10 and 11 years; two sampling points), two PD-L1-deficient patients (aged 11 and 10 years), their healthy relatives, IEI controls, and healthy adults and age-matched controls were analyzed for (E) memory B cells and (F) plasmablasts. Graphs depict proportions of (G) B cells among total leukocytes, (H) B-cell subsets, (I) IgA⁺ and IgG⁺ memory B cells. (J) UMAP representation of B-cell flow cytometry data. (K) CD21 and T-bet expression in B cells. (L) CD21^loCD23^loT-bet⁺ B cells. Graphs represent mean ± SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Figure 2.. Human PD-1:PD-L1 axis enhances c-Myc-driven transcriptional signatures in B cells.

(A-E) scRNASeq analysis for naïve and memory B cells in PBMCs from the PD-1-deficient patient, a healthy brother (aged 10 and 6 years), PD-L1-deficient patients (aged 11 and 10 years), and healthy adult and age-matched controls. (A) UMAP representation of B cells from all donors. Color codes indicate cluster assignments. (B) Top 10 marker genes for each cluster. (C) Genotypes. (D) Memory B cells defined by clustering (mean ± SEM). (E) Geneset overrepresentation analysis. (F-J) Transcriptomic profiling of naïve and memory B cells sorted from PD-1-deficient patient (aged 11 years), his healthy brother (aged 6 years), age-matched controls, through bulk RNASeq. (F) Representative FACS plots. (G) PCA of global transcriptomic profiles. (H) MA plot. Significantly downregulated c-Myc-targeted genes (defined by Hallmark c-Myc target v1 geneset) are shown in blue. (I, J) Transcription factor activity inference. (I) PCA. (J) Normalized activity of 21 TFs known to regulate MYC mRNA levels among the top 30 TFs according to the PC1 loading in (I). (K-M) Geneset enrichment analysis (GSEA) of memory B cells and Ab-secreting cells in cancer patients on PD-1 blockade. (K) Memory B cells at baseline. (L) Leading-edge genes for the c-Myc target geneset in (K). (M) Responses to influenza vaccine in Ab-secreting cells.

Figure 3.. Impaired B-cell memory and antibody responses in mice deficient for PD-1 signaling.

(A-F) Analysis in young (3–4 mo) and aged (11–12 mo) unimmunized Pdcd1^−/− C57BL/6 (B6) mice. (A) Absolute B-cell counts. (B) Proportions of memory B cells among total B cells (n=5 mice per group). (C) Ki-67 Expression by BM memory B cells. N=5 and 7 for young and aged mice for each genotype. (D-F) Bulk RNASeq analysis in aged Pdcd1^−/− B6 mice. N=4 mice for the spleen. For LNs, cells from three mice were pooled due to the small number of cells available. (D) PCA. (E) GSEA. NES, normalized enrichment score. (F) Heatmap of normalized mRNA levels for leading-edge genes for Hallmark c-Myc target v1 geneset in (E). (G, H) Impaired development of B-cell memory after oral exposure to bacterial antigens. N=7 and 5 for WT and Pdcd1^−/− mice, respectively. % of (G) memory B cells and (H) IgM⁻IgA⁺ B cells. (I-L) Impaired Ab responses in mice with impaired PD-1 signaling (I) 12–15 weeks after primary immunization, or at the indicated times following the second immunization in (J) Pdcd1^−/− B6 mice, (K) Pdcd1lg2^−/− B6 mice, (L) Cd274^−/−Pdcd1lg2^−/− BALB/c mice. Results from at least two independent experiments are compiled. Graphs (A-C, G-H, and J-K) depict mean ± SEM. In (I-L), numbers alongside graphs indicate the number of mice tested. In (I and L), dots represent outliers, as defined by 1.5 times the interquartile range (IQR) above the third quartile. **** P < 0.0001; ** P < 0.01; * P < 0.05; ns, not significant.

Figure 4.. Analysis of Tfh cell-dependent and B-cell-intrinsic mechanisms governed by PD-1 and PD-L1.

(A) Immunophenotyping of circulating Tfh (cTfh) cells among peripheral blood leukocytes from the PD-1-deficient patient (aged 10 or 11 years; three sampling time points), his healthy brother (aged 6 years), two PD-L1-deficient patients (aged 10 and 11 years), and healthy age-matched and adult controls. (B) cTfh cell subsets. (C) IL21 mRNA levels in PBMCs. TPM, transcript per million. (D) IL-21 secretion by sorted naïve and memory CD4⁺ αβ T cells. (E) IL-21 secretion by sorted memory CD4⁺ αβ T cells expanded for 2 weeks in vitro. (F) IL-21 production by memory CD4⁺ T cells sorted from LNs or spleen of aged WT or Pdcd1^−/− mice (N=10 and 9, respectively) and expanded in vitro. (G) Expression of PD-1 on blood B cells at baseline. The same data were previously published . (H) Upregulation of PD-1 in activated B cells sorted from the PBMCs of healthy donors. Results compiled from three independent experiments. (I and J) Expression of PD-L1 on blood B cells in PBMCs stimulated in vitro. (I) Representative plots. (J) Proportions of PD-L1⁺ cells in total B cells. (K) Effect of IFN-γ neutralization on the induction of PD-L1 on B cells in PBMCs from healthy donors (N=6). (L) Induction of PD-1, PD-L1, and PD-L2 on sorted naïve B cells from healthy donors (N=3~5) stimulated in vitro. Graphs (A-F and H) depict the mean ± SEM. **** P < 0.0001; * P < 0.05; ns, not significant.

Figure 5.. Cell-intrinsic impairment of antibody production in PD-1-deficient naïve and memory B cells.

(A) Schematic of the in vitro naïve B-cell stimulation assay. (B) Ig secretion by naïve B cells from PD-1-deficient patient (aged 10 years; four technical replicates), healthy adults (N=13) and age-matched (N=2) donors. (C) Two additional experiments similar to (B) for the PD-1-deficient patient (aged 11 years), his healthy brother (aged 6 years), and healthy adult (N=4) and age-matched (N=5) donors. Fold-changes in secreted Ig levels were calculated against the mean of healthy donors. (D) flow cytometric analysis of surface Ig expression at the end of culture in (C). (E-F) In vitro expansion of sorted memory B cells from the PD-1-deficient patient (aged 11 years), healthy brother (aged 6 years), and healthy age-matched (N=2) and adult (N=2) controls. Technical replicates were prepared for all controls and the PD-1-deficient patient (R=6). (E) Number of viable cells at day 14. (F) Secreted Ig levels. (G-I) In vitro PD-1 blockade assay in naïve and memory B cells from healthy donors. (G) Functional validation of nivolumab and pembrolizumab biosimilars. Six technical replicates were prepared. Representative results from two independent experiments. (H) IgM and IgA secretion. (I) IgA and IgG secretion in the presence of mouse anti-human PD-1 neutralizing mAb. Graphs depict mean ± SEM. ** P < 0.01; * P < 0.05; ns, not significant.

Figure 6.. Cell-intrinsic impairment of c-Myc expression and proliferation in PD-1-deficient naïve B cells.

(A-C) Bulk RNASeq of naïve B cells from the PD-1-deficient patient, his brother, IEI controls, and healthy adults and age-matched controls. (A) Three representative co-expressing gene modules differentially regulated in PD-1-deficient cells. (B) Top 3 Hallmark genesets overrepresented for each module. (C) Heatmap showing normalized mRNA levels for a subset of M2 genes upregulated in cells of age-matched controls following CD40L + IL-21 stimulation. (D, E) CFSE assay in naïve B cells from the PD-1-deficient patient (aged 11 years), his brother (aged 6 years), multiple age-matched and adult controls. (D) CFSE dilution. (E) Proportions of CFSE^lo cells among total B cells or in Ig⁺ B-cell subsets. (F, G) Proportions of total, naïve, and memory B cells co-expressing c-Myc and IRF4. (H, I) Expression of c-Myc and IRF4 in naïve B cells cultured for 7 days. Graphs depict mean ± SEM.

Figure 7.. Altered immunophenotypes in mice with B-cell-specific deletion of PD-1.

(A) Schematic of cre-lox-mediated targeted disruption of Pdcd1 gene in B6 mice (Pdcd1^fl/fl mb1-Cre mice). (B-D) Unimmunized young and aged Pdcd1^fl/fl mb1-Cre or flox control (Pdcd1^fl/fl) mice (N=6 per group). (B) PD-1 expression on LPS-stimulated blood B cells. (C) Spleen weight. (D) Absolute cell counts. (E-J) B-cell phenotypes. N=6 for all groups; 2 experiments for BM were compiled. Absolute counts of (E) total and (F) memory B cells. (G) Proportions of age-associated B cells (ABCs). (H-J) BM B cells. In (J), samples with too few cells were excluded from the analysis (PD-1, N=3 for Subset F from aged Pdcd1^fl/fl mb1-Cre mice; Ki-67, N=5 and N=4 for Subsets E and F from aged Pdcd1^fl/fl mb1-Cre mice, respectively). In (D-G and I), * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.