Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), is a leading cause of death worldwide, particularly in people with HIV. The current vaccine against TB, BCG, is a live-attenuated strain of M. bovis that is administered intradermally at birth in endemic countries. BCG protects children against disseminated TB but fails to protect adolescents and adults against pulmonary TB, the most transmissible form. Thus, more effective TB vaccines are urgently needed. We previously demonstrated that delivering BCG intravenously (IV) elicits a high number of Mtb-specific T cells in the airway, the site of infection, and confers robust protection against Mtb challenge 6 months later in rhesus macaques. While whole organism vaccines provide broad antigenic breadth, safety concerns may limit clinical development of a replication-competent IV BCG regimen, especially in immunocompromised individuals. To address this, we vaccinated macaques IV with either irradiation-inactivated (Ird) BCG or a live, replication-incompetent auxotroph (Aux) of BCG and compared their immunogenicity in the bronchoalveolar lavage (BAL) to wild type (WT) BCG. Early results indicate that Aux BCG elicits a similar magnitude of Mtb-specific T cell responses in the BAL compared to WT BCG; in contrast, Ird BCG is less potent in generating such responses. These studies provide data on whether whole organism vaccines need to be live, persist, or replicate to confer durable immunity against TB. Funded by the NIH Intramural Program

Tuberculosis (TB) causes 1.6 million deaths annually and the standard human vaccine, intradermal (ID) Bacille-Calmette Guérin (BCG), does not prevent pulmonary TB. Previously, we showed that intravenous (IV) BCG immunization elicited robust TB-specific lung-resident T cell responses compared to ID BCG in rhesus macaques (RMs). Following Mycobacterium tuberculosis (Mtb) challenge, 6 out of 10 RMs had no detectable disease as demonstrated by bacterial burden, granuloma formation, and primary Mtb-specific immune responses. Together, these data suggest that Mtb is cleared rapidly from the lungs of IV BCG-vaccinated RMs. To investigate the vaccine-induced lung environment and anamnestic response that might mediate protection against Mtb early after challenge, we developed a high-dose Mtb infection model in RMs that allowed us to locate and live-image areas of Mtb infection in the lungs using confocal microscopy. RMs immunized for 5 months with either IV or ID BCG were challenged with Mtb Erdman mCherry and euthanized 4 or 11 days later to characterize the lung microenvironment. We observed reductions in bacterial burdens in the lungs of RMs that received IV BCG compared to ID BCG-vaccinated and unvaccinated controls by CFU plating, flow cytometry, and confocal microscopy despite using a high challenge dose. Increased T cells were quantified in the lungs of both IV and ID BCG-vaccinated RMs compared to unvaccinated controls by flow cytometry and confocal microscopy. This research provides an opportunity to mine immune correlates of protection in a preclinical model, at the site of infection, and at a post-challenge time point that has been minimally characterized. NIH Intramural Program

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector-target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.

In contrast to conventional natural killer (NK) cells, adaptive NK cells lack expression of the Fc receptor γ chain and transcription factor PLZF. A higher proportion of adaptive NK cells is correlated with protection from malaria. Adaptive NK cells have shown enhanced degranulation in response to opsonized targets, as measured by CD107A and interferon-γ (IFN-γ) expression. We hypothesize that people with a higher frequency of adaptive NK cells are protected from malaria because of the enhanced degranulation of adaptive NK cells. However, it is unclear why adaptive NK cells degranulate better than conventional NK cells. To answer this question, we developed a CRISPR/cas9 protocol to ablate genes associated with the antibody-dependent cellular cytotoxicity (ADCC) signaling pathway and evaluated the effect of these ablations on NK cell degranulation. Individual gene targets included the Fc receptor γ chain, CD3ζ, Syk, ZAP70, and PLZF. Combinations of these targets were also ablated. After ablating gene(s) of interest, mutant NK cells were incubated with an opsonized target and evaluated five hours later for CD107A and IFN-γ expression. As a control, we found that the combined ablation of the Fc receptor γ and CD3ζ chain severely diminishes NK cell degranulation. Our results from other gene targets will be shown. Furthermore, details of our CRISPR/cas9 method will be shared. The method is the first to effectively ablate multiple primary NK cell targets using the Amaxa 4D nucleofector. On average, we obtained 82.8% protein ablation across all targets. Overall, this work will give insight into why adaptive NK cells function better at ADCC and help the field effectively ablate genes in primary NK cells.