IL-2 produced by HBV-specific T cells as a biomarker of viral control and predictor of response to PD-1 therapy across clinical phases of chronic hepatitis B

Abstract

Background: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure.

Methods: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors.

Results: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration.

Conclusions: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.

FIGURE 1

Ex vivo detection of HBV-specific T cells in patient cohorts with chronic hepatitis B by means of multianalyte FluoroSpot assays. (A) IFN-γ+ SFU for HBV OLP (filled circles) and DMSO (empty circles) conditions were linked per patient in each cohort. (B) IFN-γ+ HBV-specific SFU for each patient were calculated by subtracting DMSO SFU from HBV OLP SFU. (C) IFN-γ+ signal:noise (S:N) ratios were calculated for each patient by dividing HBV OLP SFU by DMSO SFU. Ratios ≥2 were considered positive responses. Fractions beneath each cohort denote the number of patients with detectable responses. Black lines indicate sample means. (D) IL-2 + SFU for HBV OLP and DMSO conditions linked per patient in each cohort. (E) IL-2+ HBV-specific SFU for each patient from subtracting DMSO SFU from HBV OLP SFU. (F) IL-2+ S:N ratios for each patient by dividing HBV OLP SFU by DMSO SFU in each patient. (G) Multifunctional IFN-γ+IL-2+ SFU for HBV OLP and DMSO were linked per patient. (H) HBV-specific IFN-γ+IL-2+ SFU for each patient from subtracting DMSO SFU from HBV OLP SFU. Wilcoxon tests were conducted to compare SFU between HBV OLP and DMSO conditions (A, D, G). Mann-Whitney tests were used to compare HBV-specific SFU and S:N ratios between patient cohorts (B, C, E, F, H) (*p<0.05, **p<0.01, ***p<0.001). Abbreviations: FC, functionally cured; IA, immune-active; IC, inactive carrier; IFN-γ, interferon; IT, immunotolerant; OLP, overlapping peptide pool; PBMC, peripheral blood mononuclear cells; S:N ratio, signal-to-noise ratio; SFU, spot-forming unit.

FIGURE 2

Ex vivo α-PD-1 blocking leads to functional restoration of HBV-specific T cells among patients with chronic hepatitis B. (A) HBV-specific IFN-γ+ SFU for each patient were calculated by subtracting DMSO SFU from HBV OLP SFU for isotype- (empty circles) and α-PD-1 blocked (filled circles) conditions. (B) IFN-γ+ S:N ratios were calculated for each patient by dividing HBV OLP SFU by DMSO SFU for each blocking condition. (C) HBV-specific IL-2+ SFU for each patient from subtracting DMSO SFU from HBV OLP SFU per blocking condition. (D) IL-2+ S:N ratios for each patient by dividing HBV OLP SFU by DMSO SFU per blocking condition. (E) Multifunctional IFN-γ+IL-2+ HBV-specific SFU for each patient following background subtraction for each blocking condition. Wilcoxon tests were conducted to compare HBV-specific SFU and S:N ratios between isotype- and α-PD-1 blocked conditions in each cohort (A–E) (*p<0.05, **p<0.01, ***p<0.001). Abbreviations: FC, functionally cured; IA, immune-active; IC, inactive carrier; IFN-γ, interferon; IT, immunotolerant; PBMC, peripheral blood mononuclear cells; S:N ratio, signal-to-noise ratio; SFU, spot-forming unit; PD-1, programmed cell death ligand protein 1.

FIGURE 3

Functional restoration from ex vivo PD-1 blockade across patient cohorts with chronic hepatitis B. Functional restoration was calculated and tabulated per patient for (A) IFN-γ and (B) IL-2 responses. The dotted line represents 0%, while full black lines indicate means with SDs. Fractions beneath each cohort indicate the number of patients having a functional restoration percentage of ≥20% and considered treatment-responsive to ex vivo PD-1 blockade. Mann-Whitney tests were used to compare functional restoration between patient cohorts (A, B) (* p<0.05, ** p<0.01, *** p<0.001). Abbreviations: FC, functionally cured; FR, functional restoration; IA, immune-active; IC, inactive carrier; IFN-γ, interferon; IT, immunotolerant PD-1, programmed cell death protein 1.

FIGURE 4

Liver biopsy stains for PD-1/PD-L1 across patient cohorts with chronic hepatitis B. (A) Area of PD-1+ expression among intrahepatic immune cells across chronic hepatitis B cohorts. (B) Area of PD-L1+ expression among intrahepatic immune cells across chronic hepatitis B cohorts. (C) Correlation between PD-L1+ area among intrahepatic immune cells and PD-L1 scoring on hepatocytes among IC patients. (D) Representative PD-L1 staining on hepatocytes to demonstrate weak (left) and diffuse (right) stains, black scale bars represent specified lengths. Unpaired parametric t tests were conducted to compare marker expression between cohorts (A, B). Pearson correlation coefficient and p-values are listed (C). (*p<0.05). Abbreviations: IA, immune-active; IC, inactive carrier; IT, immunotolerant; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; Tx, treatment.

FIGURE 5

Ex vivo HBV antigen hierarchies are distinct between chronic hepatitis B phase. (A) IFN-γ+ SFU for IT (circles) and IC patients (triangles) were calculated by subtracting DMSO SFU from each HBV overlapping peptide pool antigen SFU. Black lines indicate sample means. (B) Total IFN-γ SFU are listed above each patient and the proportions of IFN-γ responses were calculated for each HBV antigen. (C) IL-2+ SFU for IT and IC patients were calculated by subtracting DMSO SFU from each HBV overlapping peptide pool antigen SFU. (D) Total IL-2 SFU are listed for each patient and the proportions of IL-2 responses were calculated for each HBV antigen. Mann-Whitney tests were used to compare antigen-specific SFU between IT and IC patients (A, C) (**p<0.01). Abbreviations: IC, inactive carrier; IFN-γ, interferon; IT, immunotolerant; n.d., not detected; SFU, spot-forming unit.

FIGURE 6

Differential functional restoration of HBV antigen-specific T cell responses by clinical phase and cytokine response. Ag-specific IFN-γ+ SFU for (A) IT and (B) IC patients were each calculated for isotype- (empty circles) and α-PD-1 blocked (filled circles) conditions. Ag-specific IL-2+ SFU for (C) immunotolerant and (D) inactive carrier patients between isotype- and α-PD-1 blocked conditions. Wilcoxon tests were conducted to compare Ag-specific SFU between isotype- and α-PD-1 blocked conditions (*p<0.05, **p<0.01). Abbreviations: IFN-γ, interferon; PBMCs, peripheral blood mononuclear cells; PD-1, programmed cell death protein 1; SFU, spot-forming unit.

FIGURE 7

IFN response capacity correlates with IL-2 restoration after PD-1 blockade. (A) UMAP projection of immune cell populations identified in the cytof staining panel. (B) Single-cell heatmap showing the expression pattern of 12 interferon-stimulated proteins across the immune populations defined in (A). The transformed expression of each protein is scaled as a percentage of maximum expression. (C) Fast-Phenograph was used to define distinct modules of interferon-stimulated proteins. The relative abundance of each module within a given cell subset (size of dots) and the enrichment of the cell subset within a module (color of dots) are shown for the 16 modules. (D) Frequency of CD4 Teff cells positive for interferon-stimulated protein module 6 between anti-PD1, IL-2+IFN-γ+ responder (n=6) and nonresponder (n=4) patients. Boxes show the median, upper, and lower quartile and whiskers extend to 1.5X the interquartile range. Patients were stratified to responder/nonresponders to anti-PD-1 based on their (E) IL-2 (yellow) (R n=13; NR n=12 ) or (F) IFN-γ (green) (R n=11 ; NR n=14 ) HBV-specific T cell FluoroSpot results. Fold change in ISG15 expression in CD4 T cells, CD8 T cells, and myeloid cells was calculated by dividing the mean fluorescent intensity of IFN-β-stimulated cells by that of the unstimulated sample. Nonparametric Mann-Whitney t test was used for the statistical analysis. (G) Correlation between the percent increase in IL-2 production after PD-1 blockade and fold change in ISG15 expression. (H) Correlation between the percent increase in IFN-γ production after PD-1 blockade and fold change in ISG15 expression. Abbreviations: BST2, bone marrow stromal cell antigen 2; CD274, cluster of differentiation 274; CXCL-10, C-X-C motif chemokine 10; DC, dendritic cell; DN, double negative; DP, double positive; IDO, Indoleamine 2,3-dioxygenase; IFI16, interferon gamma inducible protein 16; IFIT3, interferon induced protein with tetratricopeptide repeats 3; IFN-γ, interferon; IRF7, interferon regulatory factor 7; ISG15, interferon stimulated gene 15; MX1, Myxovirus resistance protein 1; NK, natural killer cell; NR, nonresponder; PD-1, programmed cell death protein 1; pDC, plasmacytoid dendritic cell; PKR, protein kinase R; R, responder SOCS1, suppressor of cytokine signaling 1; Treg, regulatory CD4 T cell; UMAP, uniform manifold approximation and projection.