Table 1.
Characteristics of the study population.
Fig 1.
Significantly higher frequencies of IL-10-expressing B cells and granzyme A, granzyme B and perforin-expressing CD4+ T cells were observed in HCC patients than healthy controls.
(A) Representative gating strategy of IL-10-expressing B cells and granzyme A, granzyme B and perforin-expressing CD4+ T cells in one HCC patient. B cells were gated as CD19+CD3- lymphocytes while CD4+ T cells were gated as CD19-CD3+CD4+ lymphocytes. (B) Frequencies of IL-10-expressing (IL-10+) B cells in total B cells in the peripheral blood of healthy controls and HCC patients. (C) Frequencies of granzyme A, granzyme B and perforin-expressing CD4+ T cells in total CD4+ T cells in the peripheral blood of healthy controls and HCC patients. Welch’s t test. Mean ± SD. *** P < 0.001.
Fig 2.
The frequencies of granzyme A, granzyme B and perforin-expressing CD4+ T cells were negatively correlated with the frequencies of IL-10-expressing B cells in the peripheral blood of HCC patients.
(A) The correlations between the peripheral blood frequencies of IL-10-expressing B cells and granzyme A-, granzyme B- and perforin-expressing CD4+ T cells in HCC patients. (B) The correlations between the peripheral blood frequencies of IL-10-expressing B cells and granzyme A-, granzyme B- and perforin-expressing CD4+ T cells in healthy controls. Pearson correlation test. P < 0.05 was considered significant.
Fig 3.
IL-10 is preferentially expressed by Tim-1+ B cells in HCC patients.
(A) Representative IL-10 expression by Tim-1+ and Tim-1- B cells in one HCC patient, before or after stimulation with 1 μg/mL SEB for 72 h. (B) IL-10 expression by Tim-1+ vs. Tim-1- B cells in all HCC patients, before or after SEB stimulation. Two-way ANOVA followed by Sidak’s multiple comparisons test. Mean ± SD. *** P < 0.001.
Fig 4.
Tim-1+ B cells suppressed CD4+ T cell granzyme A, granzyme B, and perforin expression through IL-10-mediated mechanism.
105 purified Tim-1+ or Tim-1- B cells were incubated with 105 autologous purified CD4+ T cells with 1 μg/mL SEB for 72 h. 1 μg/mL sIL-10R was added in a subset of cultures to antagonize IL-10. The (A) granzyme A, (B) granzyme B, and (C) perforin expression by CD4+ T cells were then measured by flow cytometry. RM one-way ANOVA followed by Sidak’s multiple comparisons test. ** P < 0.01. * P < 0.05.
Fig 5.
Compared to peripheral blood, tumor-infiltrating IL-10-expressing B cells were further upregulated and CD4+ cytotoxic T cells were downregulated.
(A) The frequencies of IL-10-expressing B cells in the peripheral blood and resected tumor of the same subject. (B) The frequencies of granzyme A, granzyme B and perforin-expressing in the peripheral blood ans resected tumors of the same subject. Paired t test. *** P < 0.001. ** P < 0.01. (C) The correlations between the frequencies of tumor-infiltrating IL-10-expressing B cells and tumor-infiltrating granzyme A, granzyme B and perforin-expressing CD4+ T cells in HCC patients. Pearson correlation test. P < 0.05 is considered significant.