Follicular helper T cell exhaustion induced by PD-L1 expression in hepatocellular carcinoma results in impaired cytokine expression and B cell help, and is associated with advanced tumor stages

Abstract

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is one of the most common cancers in HBV-endemic regions, with irreversible progression and poor prognosis. HBV-related HCC patients lack effective antiviral/antitumor B cell antibody responses. We hypothesize that dysregulation of PD-1-expressing follicular helper T (Tfh) cell, induced by intrahepatic/intratumoral PD-L1 expression in HCC, could contribute to the defects in B cell immunity. The Tfh responses in healthy control (HC) subjects, chronic hepatitis B (HepB) patients, and HBV-related HCC patients were examined. Compared to HC and HepB individuals, HCC patients showed reduced ICOS expression, IL-10 and IL-21 secretion, and proliferation in Tfh cells. Tfh cells from stage III patients demonstrated increased impairment than those from stage I and stage II patients. Compared to Tfh cells from HC and HepB subjects, those from stage III HCC patients were significantly less effective at inducing the differentiation of naive B cells toward plasmablasts. HCC is known to upregulate hepatic PD-L1 expression, which could suppress Tfh responses. Blocking PD-1 partially rescued the Tfh functions in stage I and stage II HCC subjects but not in stage III HCC patients, while treatment with recombinant PD-L1 strongly suppressed Tfh functions in all HCC stages. Moreover, the level of IL-10 and IL-21 expression by Tfh cells was inversely correlated with the intensity of PD-L1 expression in resected tumors. Together, our results demonstrated an HCC-specific Tfh exhaustion, which might have resulted from elevated PD-1 and PD-L1 signaling.

Keywords: Follicular helper T cells; PD-L1; hepatocellular carcinoma.

Figure 1

Circulating Tfh cells in HC, HepB, and HCC subjects. A. Circulating Tfh cells are identified as CD4⁺CXCR5⁺ T cells in the representative gate. PBMCs were stained with CD3, CD4 and CXCR5 mAbs and Aqua dead cell stain for 30 min at 4°C. B. Frequencies of circulating Tfh cells in healthy control (HC), chronic HBV-infected (HepB), and HBV-related hepatocellular carcinoma (HCC) patients, stained directly ex vivo. Mean ± SD.

Figure 2

Expression of Tfh activation and function molecules in HC, HepB, and HCC subjects. PBMCs were labeled with CFSE and then cultured in plain media (blank), 2 µg/mL HBV-peptide pool (HBV-peptide), or 2 µg/mL staphylococcal enterotoxin B (SEB) for 72 h. At the end, flow cytometry of ICOS and CFSE was performed on half of the PBMCs. CD4⁺CXCR5⁺ T cells were positively selected from the other half of the PBMCs and were cultured alone with IL-10 and IL-21 luminex beads for an additional 12 h. A. Expression of ICOS on CD4⁺CXCR5⁺ circulating Tfh cells in one representative individual. B. The mean fluorescence intensity (MFI) of ICOS on circulating Tfh cells from 12 HC, 12 HepB and 20 HCC subjects, after 72 h stimulation with the stimulants. C. Secreted IL-10 and IL-21 concentration by purified CD4⁺CXCR5⁺ circulating Tfh cells, measured by luminex. D. The identification of proliferating CD4⁺CXCR5⁺ Tfh cells as CFSE-lo cells. E. The frequencies of proliferating cells in CD4⁺CXCR5⁺ Tfh cells after 72 h incubation with stimulants. Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparison test where applicable. *P < 0.05. ***P < 0.001. ****P < 0.0001.

Figure 3

Expression of Tfh activation and function molecules in HCC patients of different stages. The (A) ICOS MFI, (B) IL-10 and IL-21 secretion, and (C) proliferation of CD4⁺CXCR5⁺ Tfh cells in HCC patients are grouped according to the tumor stage. Mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05. **P < 0.01.

Figure 4

B cell help by circulating Tfh cells in HC, HepB and HCC patients. A. CD4⁺CXCR5⁺ Tfh cells sorted from PBMCs were cocultured with autologous CD19⁺CD27^-IgD⁺ naive B cells for 12 d in varying concentrations of SEB (0-2 µg/mL). Ig concentrations in the supernatant were measured by ELISA. Experiment was performed on 7 HC and HepB subjects, as well as 7 stage III HCC patients. B. CD19⁺CD27⁺ memory B cells were cultured alone in varying concentrations of SEB for 12 d. Ig concentrations were measured in the supernatant by ELISA. Mean ± SD. Differences between HC, HepB and HCC samples at each SEB concentration were tested computed by one-way ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 5

PD-1 blockade partially increased circulating Tfh function in HCC patients. A. Expression of PD-1 on CD4⁺CXCR5⁺ cells compared to CD4⁺CXCR5^- cells, in one representative individual. B. PD-1 MFI on CD4⁺CXCR5⁺ circulating Tfh cells in HC, HepB, and HCC patients. The PD-1 expression on CD4⁺CXCR5^- cells from HC is also shown for comparison. C. Expression of PD-1 by circulating Tfh cells in different stages of HCC patients. Mean± SD. One-way ANOVA and Tukey’s multiple comparison test. *P < 0.05. D and E. PBMCs from HC or HCC patients were labeled with CFSE and then cultured with 2 µg/mL staphylococcal enterotoxin B (SEB) for 72 h, with anti-PD-1 blocking antibody or isotype control. At the end, CFSE was measured on half of the PBMCs, while the CD4⁺CXCR5⁺ T cells were positively selected from the other half and were cultured alone with IL-10 and IL-21 luminex beads for an additional 12 h. D. The frequencies of proliferating Tfh cells, with isotype control or with PD-1 blockade. E. The IL-10 and IL-21 production by purified Tfh cells, with isotype control or with PD-1 blockade. RM two-way ANOVA and Sidak’s test. Asterisk (*) indicates the P value between the isotype and the anti-PD-1 treatments of the same sample, connected by a solid line. Pound sign (#) indicates the P value between that set of data with the corresponding set from the HC group. One symbol: P < 0.05. Two symbols: P < 0.01. Three symbols: P < 0.001. Four symbols: P < 0.0001. F. CD4⁺CXCR5⁺ circulating Tfh cells from different staged of HCC patients were cocultured with autologous CD19⁺CD27^-IgD⁺ naive B cells for 12 d with 2 µg/mL SEB, with isotype control or with PD-1 blockade. Ig secretion in the supernatant was measured by ELISA. RM two-way ANOVA and Sidak’s test. *P < 0.05.

Figure 6

Increased suppression of Tfh function in HCC patients by PD-L1. PBMCs from HCC patients were labeled with CFSE and cultured with plain media (blank) or recombinant PD-L1, together with 2 µg/mL staphylococcal enterotoxin B (SEB) for 72 h. A. The proliferation of CD4⁺CXCR5⁺ T cells by the end of the 72 h incubation. B. The CD4⁺CXCR5⁺ T cells were sorted and counted with Trypan Blue. 10⁵ live cells per mL were cultured without PD-L1 for an additional 12 h, in the presence of IL-10 and IL-21 capture beads. The IL-10 and IL-21 secretion during this time was measured by luminex assay. RM two-way ANOVA and Sidak’s test. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 7

The correlation between circulating Tfh exhaustion and intrahepatic/intratumoral PD-L1 expression. Single cell preparations were obtained from the liver biopsies of HepB patients, or from the peritumor and tumor samples of HCC patients who had undergone resection. PD-L1 expression was examined by flow cytometry. (A) The MFI of PD-L1 in HepB patients’ liver cells and in HCC patients’ peritumor cells. (B) The MFI of PD-L1 in HCC patients’ tumor cells. Mean ± SD. One-way ANOVA and Tukey’s test. (C) The correlation between the IL-10 and IL-21 secretion by circulating Tfh cells after SEB stimulation, and the PD-L1 expression level in HCC tumor. P represent the Pearson correlation coefficient. (D) The frequency of CXCR5⁺ cells in CD4⁺ T cells and (E) the total number of CD4⁺CXCR5⁺ T cells in HepB liver biopsies or HCC tumor tissues.