CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

Whiteside SK ，Grant FM ，Gyori DS ，Conti AG ，Imianowski CJ ，Kuo P ，Nasrallah R ，Sadiyah F ，Lira SA ，Tacke F ，Eil RL ，Burton OT ，Dooley J ，Liston A ，Okkenhaug K ，Yang J ，Roychoudhuri R ... -  《-》

Therapeutic depletion of CCR8(+) tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.

Modulation and depletion strategies 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, indicating the need for a ti-Treg specific biomarker. We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. 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 antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. We were able 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-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. 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 in a natural killer (NK) cell-dependent fashion 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.

Van Damme H ，Dombrecht B ，Kiss M ，Roose H ，Allen E ，Van Overmeire E ，Kancheva D ，Martens L ，Murgaski A ，Bardet PMR ，Blancke G ，Jans M ，Bolli E ，Martins MS ，Elkrim Y ，Dooley J ，Boon L ，Schwarze JK ，Tacke F ，Movahedi K ，Vandamme N ，Neyns B ，Ocak S ，Scheyltjens I ，Vereecke L ，Nana FA ，Merchiers P ，Laoui D ，Van Ginderachter JA ... -  《Journal for ImmunoTherapy of Cancer》

Targeting tumor-infiltrating CCR8(+) regulatory T cells induces antitumor immunity through functional restoration of CD4(+) T(convs) and CD8(+) T cells in colorectal cancer.

Chemokine (C-C motif) receptor 8 (CCR8) is a chemokine receptor selectively expressed on tumor-infiltrating regulatory T cells (Tregs). Strong immunosuppression mediated by CCR8+ Tregs observed in breast and lung malignancies suggest for their functional significance in cancer therapy. To date, detailed characterization of tumor-infiltrating CCR8+ Tregs cells in colorectal cancer (CRC) is limited. To study the presence and functional involvement of CCR8+ Tregs in CRC, we analyzed the proportions of CCR8-expressing T cells in different T cell subsets in tumor and adjacent normal tissues and peripheral blood mononuclear cells (PBMCs) from CRC patients by Flow cytometry. Also, we compared the distribution of CCR8+ T cells in malignant tissues and peripheral lymphoid organs from a subcutaneous CRC murine model. Bioinformatic analysis was performed to address the significance of CCR8 expression levels in CRC prognosis, immune regulatory gene expression profiles and potential molecular mechanisms associated with CCR8+ Tregs in CRC tumors. Further, we administrated an anti-CCR8 monoclonal antibody to CT26 tumor-bearing mice and examined the antitumor activity of CCR8-targeted therapy both in vivo and in an ex vivo confirmative model. Here, we showed that Tregs was predominantly presented in the tumors of CRC patients (13.4 ± 5.8, p < 0.0001) and the CRC subcutaneous murine model (35.0 ± 2.6, p < 0.0001). CCR8 was found to be preferentially expressed on these tumor-infiltrating Tregs (CRC patients: 63.6 ± 16.0, p < 0.0001; CRC murine model: 65.3 ± 9.5, p < 0.0001), which correlated with poor survival. We found that majority of the CCR8+ Tregs expressed activation markers and exhibited strong suppressive functions. Treatment with anti-CCR8 antibody hampered the growth of subcutaneous CRC tumor through effectively restoring the anti-tumor immunity of CD4+ conventional T cells (CD4+ Tconvs) and CD8+ T cells, which was confirmed in the ex vivo examinations. Collectively, these findings illustrate the importance of CCR8+ Tregs for an immunosuppressive microenvironment in CRC tumors by functional inhibition of CD4+ Tconvs and CD8+ T cells, and suggest for the applicable value of CCR8-targeted therapy for CRC.

Chen Q ，Shen M ，Yan M ，Han X ，Mu S ，Li Y ，Li L ，Wang Y ，Li S ，Li T ，Wang Y ，Wang W ，Wei Z ，Hu C ，Jin A ... -  《Journal of Translational Medicine》

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Kidani Y ，Nogami W ，Yasumizu Y ，Kawashima A ，Tanaka A ，Sonoda Y ，Tona Y ，Nashiki K ，Matsumoto R ，Hagiwara M ，Osaki M ，Dohi K ，Kanazawa T ，Ueyama A ，Yoshikawa M ，Yoshida T ，Matsumoto M ，Hojo K ，Shinonome S ，Yoshida H ，Hirata M ，Haruna M ，Nakamura Y ，Motooka D ，Okuzaki D ，Sugiyama Y ，Kinoshita M ，Okuno T ，Kato T ，Hatano K ，Uemura M ，Imamura R ，Yokoi K ，Tanemura A ，Shintani Y ，Kimura T ，Nonomura N ，Wada H ，Mori M ，Doki Y ，Ohkura N ，Sakaguchi S ... -  《-》

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon T(reg) depletion.

Whiteside SK ，Grant FM ，Alvisi G ，Clarke J ，Tang L ，Imianowski CJ ，Zhang B ，Evans AC ，Wesolowski AJ ，Conti AG ，Yang J ，Lauder SN ，Clement M ，Humphreys IR ，Dooley J ，Burton O ，Liston A ，Alloisio M ，Voulaz E ，Langhorne J ，Okkenhaug K ，Lugli E ，Roychoudhuri R ... -  《Science Immunology》