Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality

Abstract

The extent to which T-cell-mediated immune surveillance is impaired in human cancer remains a question of major importance, given its potential impact on the development of generalized treatments of advanced disease where the highest degree of heterogeneity exists. Here, we report the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). Using multi-parameter fluorescence-activated cell sorting analysis, we quantified the cumulative frequency of regulatory T cells (Treg), exhausted CD4(+) helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concurrent views on the overall level of immune dysfunction in these inoperable patients. We documented augmented numbers of Tregs, MDSC, PD-1(+)-exhausted T cells, and increased levels of immunosuppressive cytokines in patients with HCC, compared with normal controls, revealing a network of potential mechanisms of immune dysregulation in patients with HCC. In dampening T-cell-mediated antitumor immunity, we hypothesized that these processes may facilitate HCC progression and thwart the efficacy of immunotherapeutic interventions. In testing this hypothesis, we showed that combined regimens to deplete Tregs, MDSC, and PD-1(+) T cells in patients with advanced HCC restored production of granzyme B by CD8(+) T cells, reaching levels observed in normal controls and also modestly increased the number of IFN-γ producing CD4(+) T cells. These clinical findings encourage efforts to restore T-cell function in patients with advanced stage disease by highlighting combined approaches to deplete endogenous suppressor cell populations that can also expand effector T-cell populations.

Figure 1. Increased numbers of GARP and CTLA-4 expressing Tregs in HCC patients

Flow cytometric analysis was performed on PBMCs from HCC patients (n=23) and healthy controls (n=20). (A, B) Representative staining from an individual HCC patient and normal healthy donor for the frequency of CD3⁺CD4⁺Foxp3⁺ T cells. (C) Frequency and (D) absolute number of cells/ml of CD4⁺Foxp3⁺ Tregs in peripheral blood of HCC patients and normal healthy subjects. (E, F) Representative staining of GARP on CD3⁺CD4⁺Foxp3⁺ T cells. (G) Frequency of GARP⁺ Tregs and (H) GARP expression levels measured by mean fluorescent intensity (MFI) on Tregs. (I, J) Representative staining of CTLA-4 on CD3⁺CD4⁺Foxp3⁺ T cells. (K) Frequency of CTLA-4⁺ Tregs and (L) CTLA-4 expression levels. Each symbol represents an individual HCC patient () or normal healthy subjects (); lines represent median values for the group. * P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney U test; † P < 0.05 Hochberg adjustment for multiple comparison.

Figure 2. Accumulation of MDSCs in HCC patients

(A, B) Representative staining of HLA-DR⁻CD14⁻CD11b⁺CD33⁺ MDSC from one HCC patient and one normal healthy donor. (C) Frequency and (D) absolute number of cells/ml of MDSC in the peripheral blood of HCC patients and healthy donors. (E) Correlation of CD11b⁺CD33⁺ MDSC frequency and CD4⁺Foxp3⁺ Treg frequency. Each symbol represents an individual HCC patient () or normal healthy subjects (); lines represent median values for the group. * P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney U test; † P < 0.05 Hochberg adjustment for multiple comparison.

Figure 3. Elevated levels of immunosuppressive cytokines in HCC patients

(A–C) Cytokine-specific sandwich ELISA of plasma from HCC patients and healthy normal subjects were assayed in order to measure levels of circulating IFN-γ, IL-10, and TGF-β1. (D) Correlation of TGF-β1 plasma levels and frequency of CD4⁺Foxp3⁺ Tregs. Each symbol represents an individual HCC patient () or normal healthy subjects (); lines represent median values for the group. ** P < 0.01, *** P < 0.001, Mann-Whitney U test; † P < 0.05 Hochberg adjustment for multiple comparison.

Figure 4. Exhausted T cells from HCC patients exhibit defective proliferation

(A) Frequency of PD-1⁺CD4⁺ T cells and (B) PD-1 expression levels on CD4⁺ T cells. (C) Representative CD4⁺ T cell and (D) CD8⁺ T cell proliferation measured by CFSE dilution in an HCC patient pre-depletion () and post-depletion () of suppressor cells and a normal healthy donor (). (E) CD4⁺ T cell and (F) CD8⁺ T cell proliferation index for PHA and (G) CD4⁺ T cell and (H) CD8⁺ T cell proliferation index for anti-CD3/anti-CD28 stimulation. Each symbol represents an individual HCC patient pre-depletion (), post-depletion (), or normal healthy subjects (); lines represent median values for the group. * P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney U test; † P < 0.05 Hochberg adjustment for multiple comparison.

Figure 5. Diminished IFN-γ and granzyme B production by HCC patient T cells

Frequency of (A, B) CD4⁺ T cells and (C, D) CD8⁺ T cells producing IFN-γ upon (A, C) PHA or (B, D) anti-CD3/anti-CD28 stimulation. Frequency of CD8⁺ T cells granzyme B upon (E) PHA or (F) anti-CD3/anti-CD28 stimulation. Each symbol represents an individual HCC patient pre-depletion (), post-depletion (), or normal healthy subjects (); lines represent median values for the group. n.s. not significant, * P < 0.05, ** P < 0.01, Mann-Whitney U test; † P < 0.05 Hochberg adjustment for multiple comparison.