HIV Quality and quantity of T FH cells are critical for broad antibody development in SHIV AD8 infection Takuya Yamamoto, 1 * Rebecca M. Lynch, 2 * Rajeev Gautam, 3 Rodrigo Matus-Nicodemos, 1 Stephen D. Schmidt, 2 Kristin L. Boswell, 1 Sam Darko, 4 Patrick Wong, 2 Zizhang Sheng, 5 Constantinos Petrovas, 1 Adrian B. McDermott, 1 Robert A. Seder, 6 Brandon F. Keele, 7 Lawrence Shapiro, 5 Daniel C. Douek, 4 Yoshiaki Nishimura, 3 John R. Mascola, 2 Malcolm A. Martin, 3 Richard A. Koup 1† Broadly neutralizing antibodies (bNAbs) protect against HIV-1 infection, yet how they are generated during chronic infection remains unclear. It is known that T follicular helper (T FH ) cells are needed to promote affinity maturation of B cells during an immune response; however, the role of T FH during HIV-1 infection is undefined within lymph node germinal centers (GCs). We use nonhuman primates to investigate the relationship in the early stage of chronic SHIV AD8 (simian-human immunodeficiency virus AD8) infection between envelope (Env)–specific T FH cells, Env-specific B cells, virus, and the generation of bNAbs during later infection. We found that both the frequency and quality of Env- specific T FH cells were associated with an expansion of Env-specific immunoglobulin G–positive GC B cells and broader neutralization across HIV clades. We also found a correlation between breadth of neutralization and the degree of somatic hypermutation in Env-specific memory B cells. Finally, we observed high viral loads and greater diversity of Env sequences in rhesus macaques that developed cross-reactive neutralization as compared to those that did not. These studies highlight the importance of boosting high-quality T FH populations as part of a robust vaccine regimen aimed at eliciting bNabs. INTRODUCTION Most anti–HIV-1 broadly neutralizing antibodies (bNAbs) show a high degree of somatic hypermutation (SHM) (1), suggesting that they have undergone extensive affinity maturation within germinal centers (GCs). Specialized follicular helper CD4 T (T FH ) cells support the formation of GCs and contribute to B cell differentiation, isotype switching, SHM, and the survival of high-affinity memory B cells and plasma cells through secretion of cytokines and chemokines [IL-4 (interleukin-4), IL-21, and CXCL13] and interactions with GC B cells via costimulatory receptors (2–5). T FH cells are characterized by high expression of Bcl6, CXCR5, and costimulatory receptors (PD-1 and ICOS) and low expres- sion of CCR7. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) alters the CD4 T cell dynamic in lymph nodes (LNs), leading to T FH cell accumulation and increased egress of non-T FH cells (3). This accumulation of T FH cells is associated with increased frequen- cy of activated GC B cells in LN and SIV-specific antibodies in plasma. However, a direct role of T FH cells in eliciting broadly neutralizing re- sponses against HIV remains unclear, and a deeper understanding may lead to new ways of analyzing vaccine potential in nonhuman primates. Simian-human immunodeficiency virus AD8 (SHIV AD8 ) consist- ently establishes sustained viremia, causes unremitting depletion of CD4 T cells, and induces clinical immunodeficiency and death in inocu- lated RMs (6). Many of the pathologic and immunologic consequences of HIV-1 infection can be observed in RMs infected with SHIV AD8 (6). In particular, potent cross-clade NAbs against tier 1 and tier 2 HIV- 1 isolates can be generated in some SHIV AD8 -infected RMs (7–9). Here, we investigated the relationship between the generation of broader cross-clade neutralizing activity and aspects of T FH cells, envel- ope (Env)–specific B cells, and viral replication during SHIV AD8 in- fection. We found that the frequency of Env-specific T FH cells and Env-specific immunoglobulin G–positive (IgG + ) GC B cells within the LN correlated with the subsequent development of NAbs in the chronic phase of infection. We also found that Env-specific T FH cells in monkeys with greater neutralizing activity expressed a gene profile skewed toward T FH and away from T helper 1 (T H 1) cells. Broader anti- body neutralization was also associated with greater affinity maturation in memory B cells. RESULTS Longitudinal analysis of T FH cells and neutralization breadth in SHIV AD8 -infected macaques Eight RMs were inoculated intrarectally with SHIV AD8 . All developed persistent infection (set point viremia: 10 2 to >10 5 RNA copies/ml) (Fig. 1A), and all but one had a gradual loss of total circulating CD4 T cells (Fig. 1B). We first determined the frequency of T FH cells in the LN of SHIV-infected RMs (3, 10, 11). The CD28 dim CD95 low (hereafter referred to as naïve) CD4 T cells were used to set gates for the identification of particular populations within the CD28 high CD95 high [hereafter referred to as central memory (CM)] compartment (fig. S1). CM CD4 T cells expressing a CXCR5 + PD-1 ++ phenotype were defined as T FH cells for further analyses. We observed variable accumulation of T FH cells in LN tissues beginning at 20 weeks and extending through 44 to 47 weeks post-infection (PI) 1 Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA. 2 Humoral Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA. 3 Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, MD 20892, USA. 4 Human Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA. 5 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. 6 Cellular Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA. 7 AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. *These authors contributed equally to this work. †Corresponding author. E-mail: rkoup@mail.nih.gov RESEARCH ARTICLE www.ScienceTranslationalMedicine.org 29 July 2015 Vol 7 Issue 298 298ra120 1 on July 30, 2015 Downloaded from