br Materials and Methods br Results We characterized memory

Materials and Methods
Results We characterized memory B cell responses and HIV cross-neutralization potential in a cohort of ECs either positive or negative for the HLA-B*57 protective allele. We compared HLA-B*57+ and HLA-B*57− ECs with aviremic patients undergoing successful cART or HIV-negative individuals (Table 1).
Discussion In this study, we show that, in contrast to treated patients, ECs naturally preserve their memory B cell compartments and maintain HIV-specific memory B cell responses despite low to undetectable viral loads. HIV-specific B cells mainly express the IgG1+ Ab isotype and some ECs also express anti-HIV IgG2 and IgG3 Abs. The sera from a fraction of ECs exhibit a broad cross-neutralization capacity against difficult-to-neutralize tier-2 T/F viruses. Remarkably, the frequency of Env-specific B cells, in HLA-B*57+ ECs, correlates with a broader cross-neutralizing capacity. Our results suggest that, in HLA-B*57+ ECs, memory B cell responses might contribute to the maintenance of broad neutralization capacities and perhaps to the natural control of HIV infection. Ex vivo analysis of B cell compartments in ECs, cART and HIV-negative donors showed a global preservation of the B cell subsets. This is in contrast to other studies that have observed a slight decrease of RM B cells in cART patients (Pensieroso et al., 2013; Buckner et al., 2016) compared to HIV-negative donors and an increase of AM B cells in ECs (Pensieroso et al., 2013). However, we noticed a clear increase of TLM B cell proportion in HLA-B*57− compared to HLA-B*57+ ECs and to HIV-negative donors. This expansion of TLM B cells cannot be explained by a higher residual replication in HLA-B*57− ECs compared to HLA-B*57+ individuals, as neither the levels of plasma RNA nor cell associated viral DNA were significantly different between the two groups. Unfortunately, owing to the limited amount of cells available for this study, we could not directly analyze the phenotype of HIV-specific B cells using for instance gp140-fluorescent probes and flow cytometry. We analyzed the frequency of HIV Env-specific memory B cell responses using B-cell ELISPOT, that requires fewer cells but whose results correlate with the frequency obtained using fluorescent monocarboxylate transporters and flow cytometry (Buckner et al., 2016). Remarkably, despite very low to undetectable viral loads, the majority of ECs presented Env-specific memory B cell responses. We obtained similar results using HIV Gag p24 as antigens (not shown). In ECs, the magnitude of gp140-specific IgG+ memory B cell responses were 0.24%, a result slightly higher than reported by Bussman et al. (0.1% of memory B cell responses specific to gp120) using a cohort of 10 controllers with a median viral load above 400 RNA copy/ml (Bussmann et al., 2010). In contrast, we showed that memory B cells from cART patients, under successful virological control, rarely reacted to HIV antigens. This observation is consistent with other studies describing that upon initiation of cART the frequency of HIV-specific Ab secreting cells is strongly reduced to low or undetectable levels (Morris et al., 1998; Bussmann et al., 2010; Buckner et al., 2016; Fondere et al., 2004). Interestingly, using six ECs, Buckner et al. recently showed that the initiation of cART led to a decrease of HIV-specific memory B frequencies (Buckner et al., 2016). Therefore, although in our study, the frequency of HIV-specific memory B cells in ECs did not correlate with any virological parameters (Table 1), the maintenance of HIV-specific memory B cell responses might be due to a persistent low viral replication in the blood and/or tissues (Hatano et al., 2009; Pereyra et al., 2009). Several studies have highlighted that ECs present heterogeneous cross-neutralizing Ab responses, with some ECs exhibiting broad cross-neutralizing capacities while others show minimal or no neutralization (Deeks et al., 2006; Scheid et al., 2009; Pereyra et al., 2008; Bailey et al., 2006; Sajadi et al., 2011; Doria-Rose et al., 2009; Braibant et al., 2008). In this work, we also observed some heterogeneity between ECs and we identified 8% of ECs with broader cross-neutralizing activities. This value is slightly lower than the 12% of ECs with broad cross-neutralization responses reported in previous studies (Sajadi et al., 2011; Scheid et al., 2009). As previously observed in ECs (Palmer et al., 2016; Ranasinghe et al., 2015), we did not find a correlation between residual viral loads and cross-neutralization potentials. In two studies on ECs (defined as controllers with viral loads below 2000RNAcopy/ml), the breadth of cross-neutralization was linked to an expansion of terminally differentiated CD57+CD8+ T cells (Palmer et al., 2016) and an enhancement of HIV-specific CD4+ T cell responses (Ranasinghe et al., 2015). Recently, Martin-Gayo et al. observed in controllers a correlation between the enrichment of CXCR5+CXCR3+PD-1low CD4+ Tfh-like cells and the neutralization breadth (Martin-Gayo et al., 2017) while Dugast et al. identified a unique inflammatory profile that might be linked to the evolution of the neutralization breadth (Dugast et al., 2017).