PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+ CD25(Hi) regulatory T cells

Abstract

Regulatory CD4(+)CD25(Hi) T cells (Treg) and programmed death-1 (PD-1) molecule have emerged as pivotal players in immune regulation. However, the underlying mechanisms by which they impact antigen-specific CD8(+) immune responses in cancer patients and how they interact with each other under physiologic conditions remain unclear. Herein, we examined the relationship of PD-1 and its abrogation to the function of Treg in patients with melanoma using short-term in vitro assays to generate melanoma-specific T cells. We identified Treg in the circulation of vaccinated melanoma patients and detected PD-1 expression on vaccine-induced melanoma antigen-specific CTLs, as well as on and within Treg from patients' peripheral blood. Programmed death ligand (PD-L) 1 expression was also detected on patients' Treg. PD-1 blockade promoted the generation of melanoma antigen-specific CTLs and masked their inhibition by Treg. The mechanisms by which PD-1 blockade mediated immune enhancement included direct augmentation of melanoma antigen-specific CTL proliferation, heightening their resistance to inhibition by Treg and direct limitation of the inhibitory ability of Treg. PD-1 blockade reversed the increased expression of PD-1 and PD-L1 on melanoma antigen-specific CTL by Treg, rescued INF-gamma and IL-2 or INF-gamma and tumor necrosis factor-alpha co-expression and expression of IL-7 receptor by melanoma antigen-specific CTL which were diminished by Treg. PD-1 blockade also resulted in down-regulation of intracellular FoxP3 expression by Treg. These data suggest that PD-1 is importantly implicated in the regulation of Treg function in melanoma patients.

Fig. 1.

Detection and characterization of Treg. (A) The phenotype of Treg from melanoma patients (A, left), Memory/naïve phenotype of the Treg (A, middle and right). Dot plots showed one representative profile from 14 patients. (B) CD27-, CTLA-4-, GITR-, PD-1- and PD-L1-specific staining on gated CD4⁺CD25^Hi Treg (solid black lines), CD4⁺CD25^Low T cells (gray dashed lines) and CD4⁺CD25^Negative T cells (gray dotted lines) overlaid with isotype-matched control antibodies (shaded gray histograms). (C) Intracellular FoxP3, PD-1 and CTLA-4 staining on gated CD4⁺CD25^Hi Treg (solid black lines), CD4⁺CD25^Low T cells (gray dashed line) and CD4⁺CD25^Negative T cells (gray dotted line) overlaid with isotype-matched control antibodies (shaded gray histograms). (D) PD-1 and PD-L1 expression on melanoma antigen-specific CD8⁺ cells, shown in the upper right quadrant was the percentage of PD-1/PD-L1-positive tetramer + CD8⁺ cells.

Fig. 2.

PD-1 blockade augmented generation of melanoma antigen-specific CTL and masked inhibition by Treg. (A) IgG4 isotype control antibody was added to all culture conditions in upper row, and anti-PD-1 antibody was added at all conditions continuously in lower row. Fig. 2A was a representative experiment of 14. (B) Summary of the frequency of melanoma antigen-specific CTL from 14 individual experiments; + represented the median and − represented the mean frequency of melanoma antigen-specific CTL generated from each culture condition. (C) The fold difference of the yield of tetramer gp 1002M+CD8⁺ cells from each culture was shown. Fig. 2C was a representative experiment from 11. (D) The summary of the fold difference of the yield of melanoma antigen-specific CTL from 11 melanoma patients. The dotted line was one; + represented the median and − represented the mean of each ratio. (E) Impact of anti PD-1 on the frequency of melanoma antigen-specific CTL generated from each culture condition from 14 melanoma patients. The doted line was one; + represented the median and − represented the mean of each ratio. (F) Summary of the impact of PD-1 blockade on the absolute yield of melanoma antigen-specific CTL generated from each culture condition from 11 individual experiments. The dotted line was one; + represented the median and − indicated the mean of each ratio. (G) CSFE-labeled CD8⁺ cells were cultured with autologous mature gp 100-2M-loaded DCs. Human IgG4 isotype control antibodies (upper rows) or anti PD-1 antibody (lower rows) were added into the culture. The numbers represented the percentage of dividing CFSE-labeled CD8⁺ T cells relative to total CFSE-labeled CD8⁺ T cells.

Fig. 3.

PD-1 blockade increased the resistance of CD8 T cells to inhibition by Treg and directly limited the suppressive ability of Treg. (A) The impact of 4-h pretreatment of PD-1 blockade on the frequency of gp100-2M-specific CD8⁺ T cells. (B) Total yield of MART 27L tetramer + CTLs generated from different culture conditions; (B) Showed one representative experiment of four. (C) Dot plot summarized three experiments. The yield of melanoma antigen-specific CTL generated from Teff co-cultured with Treg and control antibody was arbitrarily set as 1 (the dotted line). Each circle indicates the index number; + was the median and * was the mean of the index number from each culture condition.

Fig. 4.

PD-1 blockade augmented the function of melanoma antigen-specific CTLs and reversed the impairment of effector function by Treg. (A) PD-1 blockade reduced PD-1 expression on melanoma antigen-specific CTL induced by Treg; + represented the median and − represented the mean PD-1 expression on melanoma antigen-specific CTL. Data were summarized from five separate experiments. (B) PD-1 blockade decreased PD-L1 expression on melanoma antigen-specific CTL up-regulated by Treg; + was the median and * was the mean of PD-L1 expression on melanoma antigen-specific CTL. The data shown as scatter dot plots from four individual experiments. (C) Anti PD-1 antibody reversed the impairment of INF-γ secretion by CTL induced by Treg. INF-γ secretion was presented as mean ± SD. This figure showed one representative experiment from six. (D) The impact of PD-1 blocking antibody on intracellular INF-γ and TNF-α secretion by melanoma antigen-specific CTL. The upper row represents cultures treated with control antibody and the lower row was the cultures treated with PD-1-blocking antibody for 10 days. (E) PD-1 blockade increased intracellular INF-γ and IL-2 co-expression in melanoma antigen-specific CTLs under different culture conditions. Dot plot is gated on tetramer gp100-2M+CD8⁺ T cells. (F) PD-1-blocking antibody treatment up-regulated the IL-7R expression on melanoma antigen-specific CTL; + was the median and * was the mean of the IL-7R expression on melanoma antigen-specific CTL. Data were shown as scatter plots from three individuals experiments.

Fig. 5.

The impact of anti PD-1 antibody on FoxP3 expression by Treg; + represented the median of FoxP3 expression and − indicated the mean of FoxP3 expression in Treg.