Flt3L therapy increases the abundance of Treg-promoting CCR7+ cDCs in preclinical cancer models

Abstract

Conventional dendritic cells (cDCs) are at the forefront of activating the immune system to mount an anti-tumor immune response. Flt3L is a cytokine required for DC development that can increase DC abundance in the tumor when administered therapeutically. However, the impact of Flt3L on the phenotype of distinct cDC subsets in the tumor microenvironment is still largely undetermined. Here, using multi-omic single-cell analysis, we show that Flt3L therapy increases all cDC subsets in orthotopic E0771 and TS/A breast cancer and LLC lung cancer models, but this did not result in a reduction of tumor growth in any of the models. Interestingly, a CD81⁺migcDC1 population, likely developing from cDC1, was induced upon Flt3L treatment in E0771 tumors as well as in TS/A breast and LLC lung tumors. This CD81⁺migcDC1 subset is characterized by the expression of both canonical cDC1 markers as well as migratory cDC activation and regulatory markers and displayed a Treg-inducing potential. To shift the cDC phenotype towards a T-cell stimulatory phenotype, CD40 agonist therapy was administered to E0771 tumor-bearing mice in combination with Flt3L. However, while αCD40 reduced tumor growth, Flt3L failed to improve the therapeutic response to αCD40 therapy. Interestingly, Flt3L+αCD40 combination therapy increased the abundance of Treg-promoting CD81⁺migcDC1. Nonetheless, while Treg-depletion and αCD40 therapy were synergistic, the addition of Flt3L to this combination did not result in any added benefit. Overall, these results indicate that merely increasing cDCs in the tumor by Flt3L treatment cannot improve anti-tumor responses and therefore might not be beneficial for the treatment of cancer, though could still be of use to increase cDC numbers for autologous DC-therapy.

Keywords: Flt3L; breast cancer; combination therapies; dendritic cell; immunotherapy; lung cancer.

Figure 1

Flt3L treatment significantly increases DCs in the E0771 tumor microenvironment. (A) Frequency of immune populations within E0771 tumors treated for 9 days with vehicle or Flt3L. (B) Tregs expressed as percentage of T cells of E0771 tumors collected after 9 days of vehicle or Flt3L treatment. (C) Graphical representation of the experimental setup and UMAP plot of 9838 cells (vehicle) or 10341 cells (Flt3L) isolated from pools of 3 E0771 tumors at a volume of 789 ± 221 mm³ (vehicle) or 773 ± 116 mm³ (Flt3L). (D) Pie charts representing the frequency of distinct immune populations identified in (C). (E) Pie charts of subclustered T-cell subsets from the CITE-seq dataset obtained in (C). (F) Violin plots showing the expression of Tcf7, Tox, Pdcd1, Gzmb, Cd69, Cd44 and Ifng in CD8⁺ T cells identified in (C). (G) E0771 tumor growth upon 9 days of vehicle or FLt3L treatment. Representative data from 2 experiments (n=7). *, P < 0.05; ****, P < 0.0001.

Figure 2

Flt3L therapy induces a CD81⁺migcDC1 activation status. (A, B) Merged (A) and split (B) UMAP plots of subclustered DCs from Figure 1C . (C) Volcano plot showing genes upregulated in Cd81 ⁺migcDC1 and migDC with red dots representing significantly upregulated genes. (D) Velocity estimates projected onto the UMAP plot of (B) The arrows visualize the observed and extrapolated future states of the cells. (E) Gating strategy to identify different cDC subsets in E0771 tumors. (F) Percentages of CD81⁺migcDC1, CD81^-migcDC1 and migcDC2 within migDCs. Representative data from 2 independent experiments (n=7). **, P < 0.01.

Figure 3

Flt3L-induced tumor-associated CD81⁺migcDC1 are potent Treg inducers. (A, B) Gene ontology analysis of differentially upregulated (A) or downregulated (B) genes in Cd81 ⁺migcDC1 versus all other DCs from Figure 2A . (C, D) Violin plots of Il12b (C) and Ccl22 (D) in DC subsets from Figure 2A . (E) Percentage of Tregs within CD45⁺ cells after coculture of sorted CD81⁺migcDC1 and migDCs (CD81^-migcDC1 and migcDC2) derived from vehicle or Flt3L-treated animals with naive CD44^-CD62L⁺CD4⁺T cells. Representative data from 2 independent experiments.

Figure 4

Flt3L therapy does not improve the αCD40-mediated tumor-growth reduction. (A) Graphical representation of the experimental design. (B) Delta mean fluorescence intensity (ΔMFI) of CD80 in CD81⁺migcDC1 two days after αCD40 as depicted in (A). (C) Percentage of CD81⁺migcDC1 within DCs two days after αCD40 as depicted in (A). (D) E0771 tumor growth upon Flt3L+αCD40 therapy. (E, F) Tumor growth (E) and survival (F) of E0771 tumor-bearing mice treated with Flt3L +αCD40+αCD25. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.