GSE162621
Single cell RNA-seq analysis of the LLC-OVA tumor-infiltrating T-cell compartment.
Modulation and depletion of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population. We here employed single-cell RNA-sequencing to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-kB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an ADCC-deficient or an ADCC-prone Fc region. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs without affecting peripheral Tregs. Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy. Overall design: Single cell RNA Seq = Live CD45+CD11b-TCRß+ cells sorted from LLC-OVA tumors subcutaneously injected in WT C57BL/6 mice.