Human Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies Do Not Recruit Non-Human Primate CD20+ NK Cells Stephanie Asdell, R. Whitney Edwards, Shalini Jha, Dr. Guido Ferrari Department of Surgery and Molecular Genetics and Microbiology

Introduction Results Results

• The antibody-dependent cell-mediated cytotoxicity 1) How does the % NK in the effector population 4) Do CD20+ NK populations play a role in the ADCC (ADCC) response represents one of mechanisms affect ADCC activity? response? Figure 3: We through which the immune system destroys HIV-1 NK Purity measured levels of infected cells. CD107a in 4 NHP • ADCC responses in non primate (NHP) models, PBMC and 5 which are used to study protection from HIV-1 in splenocyte samples. preclinical trials to translate for human use, have not The threshold of been well characterized to date. positivity was 0.396%. Results: Using • In ADCC, natural killer (NK) cells are recruited by C11_WT human Ab natural infection- or vaccine-induced antibodies (Abs) and JR4 NHP Ab, the bound to the HIV-1 envelope glycoproteins expressed difference in the on infected CD4+ T-cells via Fc-γ Receptor IIIA . Figure 1: Population of NK was determined from live lymph cells proportion of CD8- that are CD3-, CD20-, CD16+. The x-axes represent one or more • NK cells’ recognition of the infected cells leads to the NKG2a- CD107A+ human (left figure) or NHP (right two figures) donors, and the y- cells was not release granzymes and perforin by degranulation, axes represent the % of live lymph cells that are NK. statistically significant triggering apoptotic signal pathways in the infected Results: In previous experiments, we showed that the NHP between CD20- and cells and ultimately causing their elimination. splenocyte population has %NK within the known range of NHP CD20+ populations • We have developed an Ab panel to perform an assay PBMC. While human PBMC have higher %NK than NHP according to a non- that detects the level of degranulation, and thus splenocytes, we found that this is simply not correlated to the parametric Wilcoxon range of ADCC activity. recruitment of effectors, via cellular marker CD107a test. This suggests that the two NK present on NK lysosomes, in order to better populations play a characterize the types of cells recruited in the NHP 2) How can the degranulation of effector cell populations be measured? similar role in the ADCC response. ADCC response. A B C Objectives Conclusions

• Compare the recruitment of NHP peripheral blood • NHP splenocytes have approximately the same mononuclear cells (PBMC) and NHP splenocyte percentage of NK as NHP PBMC, which is lower effector cells using human and NHP antibodies than %NK of human PBMC, but was shown to not affect ADCC responses. • Examine the activity of CD20+ subsets of NHP NK D E compared to more traditional CD20- subsets of NHP • With human Ab C11 and NHP Ab JR4, CD20- and splenocytes CD20+ NK CD20+ NK populations appear to play a similar role Range 0.25-6.34% in the NHP ADCC response. • Human Ab C11 did not recruit a higher percentage of Methods CD20- NK CD20+ NK than JR4 Ab. Range 0.09-4.42% • Further transcriptomic analysis could elucidate specific CD20+ NK functions at the RNA level. • NHP splenocyte/PBMC pairs from donors R711, 6755, 6761, 6762 and splenocytes from NHP R729 Figure 2: Gating strategy for NHP splenocytes. We first References were used as sources of effector cells. identified single cell events (A), then lymphocytes (B) using side • For Abs, C11_4A human mAb and JR4 NHP mAb scatter vs. forward scatter. Lymphocytes were gated for live 1Alter G., Malenfant, J.M., Altfield M. (2004). CD107a as a functional marker for the identification of cells by presence of BV510-A (C). Then, we gated for CD8- natural killer cell activity. J Immunol Methods: 294(1-2), 15-22. were isolated from chronically-infected humans and 2 Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, Komoriya A, Oschenbauer C, Kappes NKG2A- cells (D). To measure degranulation, the CD3-CD8- JC, Roederer M, Huang Y, Weinhold K, Tomaras GD, Haynes BF, Montefiori DC, Ferrari G. (2011). rhesus macaques, with anti-influenza Ab CH65 used NKG2A- subset was gated for presence of CD107a and then High-Throughput Quantitative Analysis of HIV-1 and SIV-Specific ADCC-Mediating Antibody Responses. J Cytometry: 79A; 603-612. as negative control. could be separated into CD20- and CD20+ subsets (E). • After staining cells with fluorochrome-conjugated Abs, Results: In the 5 NHP donors tested, the percentage of CD3- Acknowledgements presence of CD107a was detected by flow cytometry CD8-NKG2A- live cells that were CD20+CD107a+ ranged from according to previously developed methodologies.1-2 0.25% to 6.34%. The percentage of CD20-CD107a- cells I would like to thank the Ferrari Lab, CFAR-ID teams, Whitney ranged from 0.09% to 4.42%. Edwards for technical assistance, and Shalini Jha for statistical assistance.