Optimizing the Depletion of innate immune Effector Cells in Nonhuman Primates

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PROJECT SUMMARY Innate immunity plays an important role in the host response against HIV infection. Crucial to this response are innate immune effector cells including NK cells, monocytes, macrophages and granulocytes, which possess numerous anti-HIV activities. Notably, monocytes and macrophages may also contribute to HIV persistence during suppressive therapy by harboring integrated copies of replication-competent HIV proviral DNA. Cytotoxic NK cells and other Fcγ-RIIIA (CD16) expressing cells can bind to specific antibodies to enable antibody- dependent cellular cytotoxicity (ADCC), which can mediate killing of HIV-infected cells. However, the nonredundant role of innate immune effector activity is often difficult to define in vivo. A key barrier is that modalities to deplete these innate immune cells for loss-of-function studies (e.g., Janus kinase 3 inhibitors, anti- IL-15 monoclonal antibodies [mAb], anti-MHC-II mAb) can also impact cells of the adaptive immune system, namely T cells. For example, we previously used anti-IL-15 mAbs to neutralize IL-15 signaling in vivo in rhesus macaques (RM) to assess the role of NK cells during acute and chronic infection, using the CCR5-tropic SIVmac239. We observed a massive depletion of NK cells in blood and tissues (DeGotarrdi et al., J. Immunol. 2016), surprisingly minimal impact on SIV replication, and a significant reactivation of oncogenic gammaherpes viruses (Okoye et al., J. Immunol. 2019). In particular, we noted a reactivation of rhesus RM rhadinovirus (RRV), a simian γ-herpesvirus closely related to human herpesvirus type 8/Kaposi's sarcoma–associated herpesvirus, which may have been directly related to the depletion of NK cells, or alternatively due to the impact of IL-15 signaling blockade on other lymphocyte populations such as CD4+ and CD8+ effector memory T cells. Likewise, use of an anti-CD16 depleting mAb to assess the role of CD16/Fcγ-RIIIA in SIV-infected RM resulted in transient and often incomplete loss-of-function (Choi et al., J. Virol. 2008). The experiments proposed here will overcome these limitations by using chimeric antigen receptor (CAR) T cells designed to deplete NKG2A+ and CD16+ cells in RM. Our preliminary data clearly shows our expertise in this approach using CD20-directed CAR T cells to deplete B cells in blood and lymphoid tissues of RM. We will use similar techniques to optimize two CAR molecules, which we have already designed and preliminarily validated. Each is designed to target markers that are expressed on the vast majority of NK cells in RM (NKG2A) or cells that can mediate ADCC activity (CD16), which will in turn induce their specific functional deficiency in vivo. CD16 CAR T cells may provide added value as an approach to deplete myeloid reservoirs. If successful, this project has the potential to substantially advance the utility of nonhuman primate models to study the specific role of innate immune effector activity in HIV pathogenesis, vaccine development and cure strategies.