P12. NK cells recognize multiple regions of HIV envelope through antibody dependent mechanisms.

Kerri G. Lal1,2, Margaret C. Costanzo1,2, Matthew Creegan1,2, Johan K. Sandberg3, Kristopher Paolino4, Merlin L. Robb1,2, Nelson L. Michael1, Julie Ake1, and Michael A. Eller1,2

Affiliates: 1U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD; 2Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD; 3Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 4Clinical Trials Center, Walter Reed Army Institute of Research, Silver Spring, MD, USA

Background
Natural killer (NK) cells are an important component of the innate immune system and play a critical role in controlling viral infections. NK cells may indirectly recognize infected cells through antibody cross-linking of CD16 (Fcγ receptor IIIa), initiating antibody dependent cellular cytotoxicity (ADCC) or via direct cell recognition through an array of activating and inhibitory receptors. The exact mechanism that NK cells use to respond to HIV-1 infection remains insufficiently characterized.

Methods
Cryopreserved PBMC and autologous plasma from HIV positive volunteers residing in the Washington, D.C. area were used for this study. HIV-specific responses were measured by intracellular cytokine staining after stimulation with pools of peptides against HIV-1 Env and Gag proteins across clades A, B, and C, and regulatory proteins from clade B in the presence or absence of autologous plasma. The polychromatic flow cytometry panel consisted of monoclonal antibodies for lymphocyte lineage markers CD3, CD4, CD8, CD56, CD16, CD14, and CD19 and function measured by degranulation (CD107a) and IFNγ production. Further studies were performed using individual peptides to de-convolute NK cell HIV specificity; and antibodies were depleted from autologous plasma using antibody-binding columns to determine the role of antibodies in NK cell responses.

Results
Eight adult volunteers with chronic HIV-1 infection, most of who were not on ART (6/8 individuals), were studied. HIV-1 viral loads varied from below detection to 12,568 copies/ml while CD4 T cell counts ranged from 423–1070 cells/μl. HIV-1 specific lymphocyte responses were observed in all 8 individuals. CD8 T cell responses were most diverse with specificities seen against Env, Gag, and regulatory proteins. The most dominant and consistent CD8 T cell response was directed toward Gag. CD4 T cells show lower frequency of response but were mostly Gag-specific. NK cells demonstrated robust HIV-specific responses in 7/8 (88%) participants, predominantly against HIV-1 Env. The range of the NK cell response was 2.6-31.1% of CD56+ NK cells. The nature of these responses included all combinations of IFNγ and CD107a, individually or together. Dominant and subdominant NK cells response were mapped throughout the Env protein in 3 donors. NK cell responses required autologous plasma while the CD8 and CD4 T cell responses did not. Through antibody column depletion of autologous plasma, IgG was found to be the subclass of antibody needed to direct the HIV-specific NK cell response.

Conclusion
Together, our data are consistent with a significant role for NK cell-mediated ADCC in HIV-1 infection. Better understanding of ADCC and HIV-specificities associated with more favorable outcomes will help us identify immune correlates of protection involving NK cells and may further inform HIV-1 preventative and therapeutic strategies.

Disclaimer: The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense.