Fig. 5.

Global transcriptomic changes and T cell polarization upon combined inhibition of HIF-1α and CPT1a

A. Scatter plots (percent positive cells) and bar graphs (MFI) showing the cytokine levels of IL-10 and IL-27 upon combined treatment with etomoxir (50μM) + KC7F2 (10μM) in 6h CpG activated control and NCoR1 depleted tolerogenic DCs. (n = 5)

B. Heatmap showing the differentially expressed genes of glycolytic and FAO pathway and their regulators in the RNAseq data before and after treatment with combined inhibitors KC7F2 and etomoxir in 6h CpG stimulated control and NCoR1 KD DCs. (n = 3)

C. Heatmap with bar plots showing the enriched GO terms associated with genes differentially regulated in 6h CpG activated NCoR1 KD before and after combined inhibition with HIF-1α and CPT1a. The asterisk in the heatmap denotes the significantly up or down regulated genes (p-adjusted <0.05) from DESeq2 results in CpG activated NCoR1 KD + Inhibitor treated as compared to only NCoR1 KD conditions. (n = 3)

D. Box plots depicting the distribution of gene expression (z-score) belonging to tolerogenic and inflammatory pathways with and without combined inhibition of HIF-1α and CPT1a in 6 h CpG activated control and NCoR1 KD DCs. (n = 3)

E. Bar graphs showing the normalized counts (DESeq2) of Il10, Il27, Ido1, Ctla4, Cd274 and Socs3 in 6h CpG stimulated control and NCoR1 KD DCs before and after combined inhibition of HIF-1α and CPT1a. (n = 3)

F. Schematic outline showing the control and NCoR1 KD DCsT cell co-culture experiment with the naive CD4⁺T cells isolated from OT-II transgenic mice ex vivo in the presence and absence of combined inhibitors KC7F2 and etomoxir.

G. Bar graph and histogram plot showing MFI levels and shifts respectively of eF670 labelled CD4⁺ T cells upon co-culturing with 6h CpG activated control and NCoR1 KD DCs before and after treatment with combined inhibitors of HIF-1α and CPT1a. Changes in the cell proliferation levels were measured for CD3⁺CD4⁺CD44⁺ gated effector T cells by flow cytometry after 72 h of co-culture. (n = 6)

H. Scatter plots (percent positive cells) and bar graphs (MFI) showing the IFN-γ⁺ and T-bet⁺ cells gated on CD3⁺CD4⁺CD44⁺ effector T cells upon co-culturing with control and NCoR1 KD DCs, with and without combined inhibition of HIF-1α and CPT1a. Percent positive cells and MFI levels were analyzed by flow cytometry after 96 h of co-culture. (n = 3)

I. Scatter plot showing the percent CD25⁺ and FOXP3⁺ double positive cells (Tregs) gated on CD3⁺CD4⁺CD44⁺ effector T cells upon co-culturing with control and NCoR1 KD DCs, with and without combined inhibition of HIF-1α and CPT1a. (n = 3)

J. Schematic showing the outline of an in vivo experiment for polarization of naive T cells upon adoptive transfer of NCoR1 KD DCs intraperitoneally in OT-II transgenic mice with and without combined inhibition of HIF-1α and CPT1a. Nearest draining lymph nodes (DLNs) were harvested after 96 h to profile the Th1 and Tregs.

K. Scatter plots (percent positive cells) and bar graphs (MFI) showing the IFN-γ⁺ and T-bet⁺ cells, gated on CD3⁺CD4⁺CD44⁺ effector T cells in DLNs after 96 h, representing the Th1 subtype in OT-II mice upon adoptive transfer of NCoR1 KD DCs with and without combined inhibition of HIF-1α and CPT1a. (n = 7)

L. Scatter plot showing the percent CD25⁺ and FOXP3⁺ double positive cells (Tregs), gated on CD3⁺CD4⁺CD44⁺ effector T cells in DLNs after 96 h, representing the Treg subtype in the OT-II mice upon adoptive transfer of NCoR1 KD DCs, with and without combined inhibition of HIF-1α and CPT1a. (n = 7).

*p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001 and ****p ≤0.0001. p-value has been calculated using two tailed paired [A, G, H and I] and unpaired [K and L] student’s t-test. Error bars represent SEM.