Toxicity-specific peripheral blood T and B cell dynamics in anti-PD-1 and combined immune checkpoint inhibition

Abstract

Immune checkpoint inhibitors (ICI) have revolutionized the treatment landscape of advanced malignancies, but come with a diverse spectrum of immune-related adverse events (irAEs). Mechanistic studies can aid the transition from expert-opinion to evidence-based irAE treatment strategies. We aimed to longitudinally characterize peripheral blood T and B cell dynamics in ICI-treated patients by multicolor flow cytometry and serum multiplex immunoassay at baseline, ± 3 weeks and ± 6 weeks or upon clinically relevant irAEs. We analyzed samples from 44 ICI-treated patients (24 anti-PD-1 monotherapy, 20 combined anti-PD-1/anti-CTLA-4; cICI), of whom 21 developed irAEs, and 10 healthy donors. IrAEs after cICI were characterized by significantly enhanced proliferation of Th1-associated, mainly (CD4⁺) CD27^- effector memory T cells, as well as Th17-associated immune responses and germinal center activation (reflected by CXCL13 and IL-21 increases). We observed no changes in CD21^lo, memory, class-switched or newly activated B cell subsets. Particularly double-positive PD-1⁺LAG-3⁺ CD8⁺ T cells showed enhanced cytotoxic capacity in patients with irAEs after cICI. Within anti-PD-1 monotherapy, irAEs were associated with modestly enhanced Th1-associated responses reflected by increased serum CXCL9 and CXCL10. In conclusion, ICI-induced toxicity is dominated by enhanced Th1-associated responses, but in cICI we also found evidence for Th17-associated responses and germinal center activation. Together, our data add to the growing body of evidence that irAEs may be driven by newly activated CD4⁺ helper T cells, specifically after cICI. This study also supports tailored irAE treatment, based on ICI regimen, and to deploy specific strategies such as Th17 inhibition especially in cICI-associated irAEs.

Keywords: Immune checkpoint inhibitors; Immune-related adverse event; Peripheral blood, immunophenotyping; T and B lymphocytes.

Fig. 1

Design of multicolor longitudinal flow cytometry study. Peripheral blood mononuclear cell (PBMC) and serum samples from immune checkpoint inhibitor (ICI)-treated patients developing (TOX) or remaining free (NOTx) of clinically relevant immune-related adverse events (irAEs) were included at baseline, ± 3 weeks into treatment and at ± 6 weeks or upon irAE onset (for which median time-to-onset and inter-quartile range are shown). Healthy donor PBMCs and serum were included for comparison. Created with BioRender.com

Fig. 2

CD27⁻ effector memory CD4⁺ T cell proliferation increases over time in combined-ICI treated patients with toxicity, relative to other groups, and is at baseline associated with early irAE onset. a Plot showing clustering of different groups in principal component analysis (PCA) with pooled flow cytometric data including all 167 readout parameters across panels. b Individual patient trajectories of the percentage Ki67⁺ of CD27⁻ CD4⁺ effector memory (CD27⁻ CD4_EM) T cells. Comparison by unpaired Wilcoxon test because of missing paired data for some patients. c,d Percentage proliferating of total CD27⁻ CD4_EM (c) and CD27⁻ CD8_EM (d) T cells over time. Only significant coefficients from mixed-effects models for the interaction term with time (‘B’), indicating statistically significant change compared to other groups, are shown. Gray solid and dashed lines indicate mean healthy donor level with 95% confidence interval. e Correlation between baseline CD27⁻ CD_EM proliferation rate and time-to-toxicity, stratified by ICI-regimen (by Spearman correlation). irAE: immune-related adverse event, NS: not significant, TOX: with irAEs, NOTx: without irAEs, *P < 0.05

Fig. 3

Both Th1- and Th17-associated immune responses are observed in combined-ICI treated patients with toxicity, whereas only enhanced Th1-associated immunity is associated with anti-PD-1 associated toxicity. a,b Percentage IFN-γ⁺ of total CD4⁺ memory T cells (a) and percentage proliferating of CXCR3⁺CCR4⁻ Th1-associated CD4⁺ T cells (b) over time. Only significant coefficients from mixed-effects models for the interaction term with time (‘B’), indicating statistically significant change compared to other groups, are shown. Gray solid and dashed lines indicate mean healthy donor level with 95% confidence interval. c Heatmap showing serum levels of cytokines and chemokines after ± 6 weeks (T3) for NOTx, upon irAE onset for TOX, or in healthy donors. Data are scaled by individual proteins across timepoints (see Supplementary Fig. 2b,c), enabling direct comparison of individual proteins between three timepoints. Unsupervised clustering of patients by Manhattan distance. d Volcano plot showing relative increase in serum protein levels at Timepoint 3/Toxicity in all patients with irAEs (TOX; anti-PD-1 and cICI combined) versus all patients without irAEs (NOTx). Full protein names are in Supplementary Table 2. e Fold-change in percentage IL-17⁺ of CD4⁺ T cells relative to baseline in cICI-treated patients without irAEs (left; NOTx) and with (right; TOX) irAEs. irAE: immune-related adverse event, NS: not significant, TOX: with irAEs, NOTx: without irAEs

Fig. 4

PD-1⁺LAG-3⁺ double positive CD8⁺ memory T cells retain their inflammatory potential regardless the development of toxicity. a Representative flow cytometry plot of CD95 and CD45RO expression in CD8⁺ T cells. b t-SNE visualizations for a random 10% subset of all CD3⁺ T cells from all patients at Timepoint 3/Toxicity and healthy donors, with relative abundance shown in density plots (top) and expression of lineage markers, IFN-γ and granzyme B in overlay plots (bottom). c Baseline percentages of CD45RO⁺ CD4⁺ (left) and CD95⁺ CD8⁺ (right) T cells positive for PD-1 alone or PD-1 and LAG-3. Comparisons by Kruskal–Wallis tests. (d, e) Percentages of CD95⁺CD8⁺ T cells producing IFN-γ after PMA/ionomycin stimulation, depending on inhibitory receptor expression pattern, in patients without (NOTx; d) or with irAEs (TOX; e) stratified by timepoint; anti-PD-1- and cICI-treated patients are combined. Comparisons by pairwise paired Wilcoxon tests with Benjamini–Hochberg correction for multiple testing. f Percentage of granzyme B producing PD-1⁺LAG-3⁺ CD95⁺CD8⁺ T cells over time. Significant coefficient (interaction term with time; ‘B’) from mixed-effects model is shown. Gray solid and dashed lines indicate mean healthy donor level with 95% confidence interval. irAE: immune-related adverse event, NS: not significant, TOX: with irAEs, NOTx: without irAEs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Increasing serum CXCL13 levels and higher baseline plasmablast abundance in patients with early toxicity suggest a role for adaptive humoral immunity in toxicity after combined ICI. a-c Percentages of CD21^lo (a) and CD27⁺ memory (b) B cells of total B cells and serum level (in pg/ml) of CXCL13 (c) over time. Only significant coefficients (interaction terms with time; ‘B’) from mixed-effects models are shown. Gray solid and dashed lines indicate mean healthy donor level with 95% confidence interval. d Correlation between baseline percentage CD27⁺CD38^hi plasmablasts of total B cells and time-to-toxicity, stratified by ICI-regimen (by Spearman correlation). irAE: immune-related adverse event, TOX: with irAEs, NOTx: without irAEs

Fig. 6

Peripheral regulatory T cell abundance and function are generally preserved upon toxicity. a Percentage of CD25⁺FOXP3⁺ CD4⁺ Treg cells of total CD4⁺ T cells over time. Significant coefficient (interaction term with time; ‘B’) from mixed-effects model is shown. Gray solid and dashed lines indicate mean healthy donor level with 95% confidence interval. b Comparison of median fluorescence intensity (MFI) of T-bet in Tregs at baseline between healthy donors and all ICI-treated patients, by Wilcoxon test. c Expression levels (by MFI) of inhibitory and costimulatory receptors in Tregs (for patients Timepoint 3/Toxicity), hierarchically clustered by Euclidean distance. cICI TOX patients with grade 4 irAEs (n = 2) are indicated by red arrows. irAE: immune-related adverse event, TOX: with irAEs, NOTx: without irAEs