506 FcγRIIB expressed on CD8 T cells limits responsiveness to PD-1 checkpoint inhibition in cancer (2022)

<h3>Background</h3> Immune checkpoint inhibition (ICI) using Fc-containing monoclonal antibodies has emerged as a powerful therapeutic approach to augment anti-tumor immunity. While ICI has drastically improved patient outcomes in melanoma, there is still variability in patient response.<sup>1, 2</sup> We recently showed that FcγRIIB, the only inhibitory IgG-Fc receptor, is expressed on differentiated effector CD8 T cells in mice and humans <sup>3, 4</sup>, raising the possibility that CD8 T cell responses may be directly modulated by checkpoint inhibitor binding to T cell-expressed FcγRIIB. <h3>Methods</h3> Flow cytometric phenotyping was performed on PBMCs isolated from melanoma patients and healthy donors. For <i>in vitro</i> functional experiments, healthy human PBMCs were stimulated with CD3/28 Dynabeads and/or PMA/ionomycin. Effector function was assessed through intracellular cytokine staining. Anti-PD1 (clone J116) or anti-CTLA4 (clone BN13) were used where mentioned and the anti-PD1 F(ab) was generated from Nivolumab. <i>In vivo</i> experiments were performed in mice with B16-hgp100, B16-OVA, or LLC-OVA tumors. Where mentioned, WT OT-I, <i>Fcgr2b</i><sup>-/-</sup> OT-I, or WT pmel-17 CD8 T cells were adoptively transferred into these mice. For treatment, 250 μg of anti-PD1 (clone RMP1-14) and anti-FcγRIIB (clone 2.4G2) or isotype antibodies were administered for blockade experiments. <h3>Results</h3> Here, we show that despite exhibiting strong proliferative and cytokine responses at baseline, human FcγRIIB<sup>+</sup> CD8 T cells exhibited reduced responsiveness to both PD-1 and CTLA-4 checkpoint inhibition compared to FcγRIIB<sup>-</sup> CD8 T cells <i>in vitro</i> (p&lt;0.05). Moreover, frequencies of FcγRIIB<sup>+</sup> CD8 T cells were reduced following treatment of human melanoma patients with nivolumab <i>in vivo</i> (p&lt;0.05). This reduced responsiveness was FcγRIIB-dependent, because conditional genetic deletion of FcγRIIB on tumor-specific CD8 T cells improved response to checkpoint blockade in a B16 mouse melanoma model (p&lt;0.01). The limited responsiveness of FcγRIIB<sup>+</sup> CD8 T cells was dependent on an intact Fc region of the checkpoint inhibitor, in that treatment with Fc-devoid anti-PD-1 F(ab) fragments resulted in a significant increase in proliferation of FcγRIIB<sup>+</sup> CD8 T cells, without altering the response of FcγRIIB<sup>-</sup> CD8 T cells(p&lt;0.05). Finally, blocking FcγRIIB in the context of PD-1 blockade significantly improved anti-tumor CD8 T cell responses in B16 melanoma and in Lewis lung cancer mouse models (p&lt;0.05, p&lt;0.001). <h3>Conclusions</h3> These results illuminate an FcγRIIB-mediated, cell-autonomous mechanism of CD8 T-cell suppression which limits the efficacy of checkpoint inhibitors <i>in vivo</i>. The data presented here support the novel conclusion that CD8-expressed FcγRIIB is both a factor to consider in the development of therapeutic antibodies, and a new potential target for immunotherapeutic intervention. <h3>References</h3> Lugowska I, Teterycz P, Rutkowski P. Immunotherapy of melanoma. <i>Contemp Oncol (Pozn)</i>. 2018;<b>22</b>(1A):61–7. Epub 2018/03/05. doi: 10.5114/wo.2018.73889. PubMed PMID: 29628796. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob J-J, Rutkowski P, Lao CD, Cowey CL, Schadendorf D, Wagstaff J, Dummer R, Ferrucci PF, Smylie M, Hogg D, Hill A, Márquez-Rodas I, Haanen J, Guidoboni M, Maio M, Schöffski P, Carlino MS, Lebbé C, McArthur G, Ascierto PA, Daniels GA, Long GV, Bastholt L, Rizzo JI, Balogh A, Moshyk A, Hodi FS, Wolchok JD. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. <i>N Engl J Med</i>. 2019;<b>381</b>(16):1535–46. doi: 10.1056/NEJMoa1910836. Morris AB, Farley CR, Pinelli DF, Adams LE, Cragg MS, Boss JM, Scharer CD, Fribourg M, Cravedi P, Heeger PS, Ford ML. Signaling through the Inhibitory Fc Receptor FcgammaRIIB Induces CD8(+) T Cell Apoptosis to Limit T Cell Immunity. <i>Immunity</i>. 2020;<b>52</b>(1):136–50 e6. Epub 2020/01/16. doi: 10.1016/j.immuni.2019.12.006. PubMed PMID: 31940267; PMCID: PMC7326381. Farley CR, Morris AB, Tariq M, Bennion KB, Potdar S, Kudchadkar R, Lowe MC, Ford ML. Fc?RIIB is a T cell checkpoint in antitumor immunity. <i>JCI Insight</i>. 2021;<b>6</b>(4). doi: 10.1172/jci.insight.135623. <h3>Ethics Approval</h3> Patients undergoing treatment at Emory University Hospital for advanced stage II-IV melanoma between 2009 and 2019 were enrolled in an immune monitoring protocol approved by Emory University9s Institutional Review Board (IRB #00046593). Healthy controls were enrolled after informed consent. This study was also carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol (PROTO201700558) was approved by the Institutional Animal Care and Use Committee of Emory University. All surgery was performed under general anesthesia with maximum efforts made to minimize suffering.