HDAC6 inhibition by ITF3756 modulates PD-L1 expression and monocyte phenotype: insights for a promising immune checkpoint blockade co-treatment therapy

Abstract

Introduction: Tumor immunotherapy has revolutionized cancer treatment, particularly through the use of immune checkpoint inhibitors targeting the PD-L1/PD-1 axis. While PD-L1 expression on tumor cells is an established predictive biomarker for therapeutic response, emerging evidence highlights the importance of PD-L1 expression on myeloid cells, both in the periphery and within the tumor microenvironment (TME). This study explores the immunomodulatory effects of the selective HDAC6 inhibitor ITF3756 on monocytes and dendritic cells (DCs).

Methods: Monocytes were stimulated with the pro-inflammatory cytokine TNF-α and treated with ITF3756. PD-L1 and CD40 expression levels were assessed by flow cytometry. Transcriptomic and proteomic analyses were performed to characterize changes in gene and protein expression profiles. T cell proliferation was evaluated in co-culture assays. Additionally, the impact of ITF3756 was assessed in an in vivo murine model of colon cancer.

Results: ITF3756 effectively downregulated PD-L1 expression in TNF-α-activated monocytes and enhanced their costimulatory capacity by increasing CD40 expression. Transcriptomic and proteomic analyses revealed that ITF3756 counteracted TNF-α pathway activation and downregulated multiple inhibitory immune checkpoint molecules, promoting a less immunosuppressive phenotype. In co-culture assays, ITF3756-treated monocytes and DCs significantly enhanced T cell proliferation. In vivo, ITF3756 treatment led to reduced tumor growth in a colon cancer model.

Discussion: These findings demonstrate that selective HDAC6 inhibition by ITF3756 modulates myeloid cell functionality by diminishing inhibitory signals and promoting T cell activation. Thus, ITF3756 represents a promising immunomodulatory agent that could enhance the efficacy of immune checkpoint blockade in cancer immunotherapy.

Keywords: HDAC6; TNF-α; dendritic cells; immuno-checkpoints; monocytes.

Figure 1

ITF3756 treatment significantly upregulates CD40 and downregulates PD-L1. Human purified monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml) ON. (A) Percentage of CD40 positive cells and expression of CD40 measured as the fluorescence intensity geometric mean (GMFI). For both the percentage of CD40 positive cells and for CD40 GFMI, the graphs on the left show the analysis of CD40 in all experimental conditions where statistical analysis was performed using RM one-way ANOVA followed by Dunnetts multiple comparison test, while the graphs on the right show the paired analysis of TNF-α-stimulated versus TNF-α stimulated and ITF3756-treated monocytes, where paired student’s t-test was used for statistical analysis. (B) Percentage of PD-L1 positive cells and PD-L1 GMFI. For both the percentage of PD-L1 positive cells and for PD-L1 GFMI, the graphs on the left show the analysis of PD-L1 in all experimental conditions where statistical analysis was performed using RM one-way ANOVA followed by Dunnetts multiple comparison test, while the graphs on the right show the paired analysis of TNF-α-stimulated versus TNF-α stimulated and ITF3756-treated monocytes, where paired student’s t-test was used for statistical analysis. Values on the graphs are expressed as mean ± SD. *p<0,05, **p<0,001, ***p<0,0005,****p<0,0001.

Figure 2

ITF3756 dose-dependently downregulates PD-L1 positive cells and expression in TNF-α stimulated monocytes and the effective PD-L1 suppression needs long exposure to the inhibitor. (A) Dose dependent PD-L1 inhibition by ITF3756 (1,5μM-0,0625μM). The percentage of PD-L1 positive cells and PD-L1 expression measured as the fluorescence intensity geometric mean (GMFI), together with a representative histogram of the flow cytometry results obtained, are reported. Values on the graphs represent the mean of 2 experiments carried out on 3 different donors (n=3). (B) Human monocytes were stimulated with TNF-α (100ng/ml) and ITF3756 1μM for 2h, 4h, 6 h, 10h and 18h. After 2h, 4h, 6 h and 10h medium was removed and replaced with fresh medium without the cytokine and ITF3756. Expression of PD-L1 was analyzed for all conditions at 18h. The left panel shows the GMFI of PD-L1 at the different time points; the right panel displays the percentage of PD-L1 inhibition at the different time points. Values on the graphs are expressed as mean ± SD. n=6 for 18h; n=4 for 2, 4 and 6h; n=2 for 10h. P-values were calculated by one-way ANOVA test followed by Dunnetts multiple comparison test. *p<0,05, **p<0,001, ***p<0,0005.

Figure 3

ITF3756 upregulates CD40 and decreases PD-L1 mRNA expression in TNF-α stimulated monocytes after 4h. Human purified monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml) for 1, 2 and 4h. (A) Time course analysis of CD40 gene expression. The analysis was performed by qPCR on 3 different donors (n=3). Values on the graph are expressed as mean± SEM. (B) Time course analysis of PD-L1 gene expression. The analysis was performed by qPCR on 3 different donors (n=3). Values on the graph are expressed as mean± SEM. Gene expression analysis on further donors was conducted only at 4h for (C) CD40 (n=4) and for (D) PD-L1 (n=7). For (C, D) panel, the graph on the left shows the analysis of CD40 and PD-L1 expression in all experimental conditions in which RM one-way ANOVA test was used for the statistical analysis, while the graph on the right shows the paired analysis of TNF-α-stimulated versus TNF-α-stimulated and ITF3756-treated monocytes, in which paired student’s t-test was used for the statistical analysis. Values on the graphs are expressed as mean ± SD. *p<0,05, **p<0,001.

Figure 4

Transcriptomic analysis of TNF−α stimulated monocytes treated or not with ITF3756 for 4h. Purified human monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml) for 4h. (A) Volcano plot displaying significantly up- and down-regulated genes (orange and blue, respectively) in monocytes stimulated with TNF-α. Fold changes (FC) are calculated versus the unstimulated control cells. (B) Volcano plot displaying significantly up- and down-regulated genes (orange and blue, respectively) in monocytes stimulated with TNF-α and treated with ITF3756. Fold changes (FC) are calculated versus the TNF-α; stimulated cells. (C, E) Pathways analyses of significantly up- and down-regulated genes (orange and blue, respectively) in TNF-α; stimulated monocytes were performed with the EnrichR software. (D, F) Pathways analyses on significantly up- and down-regulated genes (orange and blue, respectively) in monocytes stimulated with TNF-α; and treated with ITF3756 were performed with the EnrichR software.

Figure 5

Proteomics analysis of TNF-α stimulated monocytes treated or not with ITF3756 for 18h. Purified human monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml) for 18h. (A) Volcano plot displaying significantly up- and down-regulated proteins (orange and blue, respectively) in monocytes stimulated with TNF-α. Fold changes (FC) are calculated versus the unstimulated control cells. (B) Volcano plots displaying significantly up- and down-regulated proteins (orange and blue, respectively) in monocytes stimulated with TNF-α and treated with ITF3756. Fold changes (FC) are calculated versus the TNF-α stimulated cell. (C, E) Pathways analyses on significantly up- and down-regulated proteins (orange and blue, respectively) in TNF-α stimulated monocytes were performed with the EnrichR software. (D, F) Pathways analyses on significantly up- and down-regulated proteins (orange and blue, respectively) in monocytes stimulated with TNF-α and treated with ITF3756 were performed with the EnrichR software.

Figure 6

ITF3756 downregulates monocytes activation and differentiation markers activated by TNF-α and promotes a less immunosuppressive phenotype in TNF-α stimulated monocytes. Purified human monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml) for 4h. RNAseq data obtained as described before were used for this analysis. (A) Analysis of the modulation of specific markers of monocytes-derived cell population by TNF-α (left panel) and by the combination of TNF-α and ITF3756 (right panel). Fold changes (FC) are calculated versus the unstimulated control cells or versus the TNF-α stimulated cells, respectively. (B–D) Analysis of the modulation by TNF-α and by the combination of TNF-α and ITF3756 of a list of inhibitory immune checkpoints (31). Fold changes (FC) are calculated versus the unstimulated control cells in (B), versus the TNF-α stimulated cells in (C) and between ITF3756 and unstimulated control cells in (D). Significant differentially expressed genes are represented as circles, while non-significant genes are shown as triangles.

Figure 7

Signaling pathways involved in PD-L1 regulation by ITF3756 in TNF-α stimulated monocytes. Purified human monocytes were treated for 2h with ITF3756 (1μM) and then stimulated with TNF-α (100ng/ml). (A) Analysis of the modulation by TNF-α (left panel) and by the combination of TNF-α and ITF3756 (right panel) of specific pathways involved in PD-L1 regulation. RNAseq data obtained as described before were used for this analysis. Significant differentially expressed genes are represented as circles, while non-significant genes are shown as triangles. (B) Transcription factor activity inference upon treatment with TNF-α (left panel) and with the combination of TNF-α and ITF3756 (right panel). (C) Cytofluorimetric analysis of p65 phosphorylation on Ser536 (p-p65). Monocytes were pre-treated for 2h with ITF3756 and then stimulated or not with TNF-α for 15 minutes. RM one-way ANOVA followed by Dunnetts multiple comparison test was used for the statistical analysis. The graphs show the results obtained from 6 different donors. *p<0,05, **p<0,001, ***p<0,0005.

Figure 8

ITF3756 promotes T cells proliferation induced by TNF-α in monocytes. Human monocytes were pre-treated with ITF3756 (1μM) and stimulated with TNF-α (100ng/ml) ON. After incubation, monocytes were washed and co-cultured with allogeneic CFSE-labelled T cells at the indicated ratios. Proliferation was measured on day 6 as CFSE dilution. (A) Proliferation of T cells in co-culture with TNF-α-stimulated monocytes treated or not treated with ITF3756; values are expressed as mean ± SEM. Values on the graphs represent the mean of 6 experiments carried out on 12 different donors (n=12) (B) Paired analyses of T cell proliferation in the co-culture at 1:2, 1:4 and 1:8 monocytes/T cells ratios in TNF-α-stimulated monocytes treated or not with ITF3756. (C) Proliferation of T cells in co-culture with TNF-α stimulated monocytes treated or not treated with ITF3756 (dotted lines), and together with an anti-PD-L1 antibody (solid lines). Values are expressed as mean ± SEM. Values on the graphs are the mean of 2 experiments carried out on 4 different donors (n=4). (D) Paired analysis of T cell proliferation in the co-culture at 1:2 monocytes/T cells. P values were calculated by Student’s t-test, using a Paired t test. *p<0,05, **p<0,01.

Figure 9

ITF3756 enhances the APCs phenotype of immature and mature dendritic cells supporting allogenic T cells proliferation. Human monocytes were treated with ITF3756 (1μM and 0,5μM) and stimulated with GMCSF/IL-4 for 5 days. After incubation, iDC were collected and assessed by flow cytometer and by qPCR. (A) Percentage of CD86 positive cells and CD86 GMFI. (B) Percentage of PD-L1 positive cells and PD-L1 GMFI. (C, D) Differentiated immature dendritic cells were treated with ITF3756 (1μM and 0,5μM) and stimulated with LPS (1μg/ml) for 18h. After incubation mDC cells were collected and evaluated by flow cytometry. (C) Expression of CD86 in mDC reported as the GMFI. (D) Expression of PD-L1 in mDC reported as the GMFI. (E) Immature dendritic cells, differentiated in the presence of ITF3756 (1μM), were collected and mRNA extracted for CD86 and PD-L1 gene expression analysis by qPCR. Values on the graphs (A–E) represent the mean of 2 separate experiments carried out on at least 3 different donors. (F) Dendritic cells differentiated in the presence of ITF3756 1μM for 5 days, were collected and co-cultured with CFSE-labelled T cells (DC/T cells ratio 1:10) for 5 days. Proliferation was measured by flow cytometry on day 5 as CFSE dilution. Values on the graph represent the mean of 2 different donors (n=2).

Figure 10

ITF3756 is effective in a colon carcinoma model in vivo. (A) Tumor weight in animals treated with ITF3756 25mg/kg BID and TID. (B) Tumor weight in animals treated with ITF3756 50mg/kg QD, BID and TID. Data are reported as mean ± SEM. Statistical analysis was carried out by two-way ANOVA followed by Tukey’s multiple comparisons test. *p<0,05, **p<0,001, ***p<0,0005, ****p<0,0001.