Cells were stained with Live/Dead for 10 minutes at room temperature (RT) and then incubated with antibodies targeting surface proteins for 20 minutes at 4C. AHI also correlated with gp120-specific IgG antibody levels after 48 weeks of ART, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and increased antibody binding to infected cells after ART. Importantly, while beneficial for antibody development, CXCR3+Tfh cells were also infected by HIV-1 at higher frequencies than their CXCR3counterparts and may contribute to the initial dissemination of HIV-1 in follicles. Together, these data suggest that activation of CXCR3+Tfh cells is associated with induction of the germinal center response and subsequent antibody development, making these cells an important target for future therapeutic interventions. == IMPORTANCE == Early initiation of antiretroviral therapy (ART) is important to limit the seeding of the long-lasting HIV-1 reservoir; however, it also precludes the development of HIV-specific antibodies that can help control the virus if ART is stopped. Antibody development occurs within germinal centers in the lymph node and requires activation of both antigen-specific B cells and T follicular helper cells (Tfh), a specialized CD4+cell that provides B cell help. To understand how early ART initiation may prohibit antibody development, we analyzed the frequencies and activation status of Tfh and B cells in lymph node biopsies collected in the different stages of acute HIV-1 infection. Our data suggest that decreased antibody development after early ART initiation may be due to limited germinal center development at the time of treatment and that new interventions that target activation of CXCR3+Tfh may be beneficial to increase long-term HIV-specific antibody levels. KEYWORDS:human immunodeficiency virus, Tfh cells, B-cell responses, humoral immunity, lymph node == INTRODUCTION == The antibody response is delayed in acute HIV-1 infection (AHI): autologous neutralizing antibodies are not detectable until about 6 months after HIV-1 infection (1,2), and broadly neutralizing antibodies only develop in a fraction of individuals over the course of several years of uncontrolled viremia (26). By contrast, neutralizing antibodies can be detected within 12 weeks of symptom onset for other viral infections or vaccination (710). CHUK HIV-1 Env has developed a number of mechanisms to evade the antibody response, including extensive glycosylation, conformational masking, escape mutations, and low expression on virions and virus-infected cells; however, early impaired immune activation during acute HIV-1 infection could also contribute to delayed antibody development (1,1115). Changes in lymph node structure and B-cell populations have been reported in early and chronic HIV-1 infection, but little is known about the germinal center (GC) response during the earliest stages of HIV-1 infection. HIV-1 DNA can be detected PNPP in lymph nodes during the earliest stage of detectable HIV-1 in the blood, Stage 1 (S1), and reaches maximal levels as early as S2 (16). HIV-specific plasma antibodies are detected by immunoassay in S3 of AHI and by western blot thereafter. The first antibodies detected are gp41-specific antibodies followed by gp120-specific antibodies, but these initial antibodies do not contribute to viral control or the development of escape mutants during AHI (17). Analysis of antibody development in participants treated in different stages of AHI showed that participants who initiated antiretroviral therapy (ART) in S1 or S2 of AHI had limited development of gp120-specific antibodies, with lower levels of these antibodies than those who initiated treatment in S4/5 even after 2448 weeks of ART (18). This analysis also suggested that only participants in S4/5 of AHI had more mature antibody development, with most participants having developed antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) during AHI and all participants developing cross-clade ADCC PNPP activity after 1 year of ART (18). However, participants treated in these early acute time points do not develop autologous neutralizing antibodies in the absence of further antigen exposure through viral blips (19). The stage-specific development of HIV-specific antibodies in AHI called for an analysis of the development of the GC response in lymph nodes. After activation, nave B cells migrate to the periphery of the follicle where they interact with activated CD4+T cells that have upregulated expression of CXCR5, a chemokine receptor that mediates trafficking toward the CXCL13-rich B-cell follicle (2025). These interactions provide the signals PNPP necessary for the CD4+T cells to differentiate into T follicular helper (Tfh) cells and the B cells to either differentiate into short-lived plasmablasts, which produce PNPP the first wave of antibodies, or to migrate to the GC for affinity maturation (2628). Within the GC, Tfh cells provide B cells with survival signals that promote proliferation, somatic hypermutation, and selection to produce long-lived plasma cells and memory B cells with higher affinity receptors (24,29,30). While Tfh cells express CXCR5 for trafficking to the CXCL13-rich follicle (2325), they also express other PNPP chemokine receptors including CXCR3, which differentiates T helper 1 (Th1)-like Tfh cells from Th2/Th17-like Tfh cells (31). Frequencies of both CXCR3and CXCR3+circulating Tfh (cTfh) cells have been associated.