Response to Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

A technique to efficiently identify Ag-specific GC Tfh cells had not been available, due to intrinsically stingy cytokine production by human and nonhuman primate GC T follicular helper (Tfh) cells, as described in our two papers (1, 2). Since this has been a major impediment for the field, we performed an RNA-seq experiment to identify non-cytokine proteins that are upregulated by GC Tfh cells in response to Ag. That RNA-seq identified CD25 and PD-L1 as candidate molecules for TCR activation induced marker (AIM) identification of Ag-specific GC Tfh cells (1). While upregulation of CD25 on GC Tfh cells upon in vitro stimulation was surprising, given that IL-2 is inhibitory for Tfh cell differentiation (3, 4), it was highly reproducible (1, 2). Separately, we observed OX40 was upregulated by GC Tfh cells in proliferation assays (2). We then used the combination of CD25, PD-L1, and OX40 to identify and quantify Ag-specific human and NHP GC Tfh cells, while also performing a series of controls. The results are the most robust to date for identification of Ag-specific GC Tfh cells in humans (2) and non-human primates (1). We then noted, and cited, that Zaunders and colleagues (5, 6) previously determined that upregulation of OX40 and CD25 could be used as activation markers to identify Ag-specific CD4 T cells in whole anti-coagulated blood, and that approach had been used in additional studies (7, 8). We therefore examined whether using CD25, OX40, and/or PD-L1 could be successfully applied for identification of Ag-specific CD4 T cells in blood for our Ags of interest, which would allow for quantitation of Ag-specific GC Tfh cells and non-Tfh cells using the same experimental technique. We confirmed the Zaunders et al. (5) results detecting Ag-specific CD4 T cells in human peripheral blood, but we modified the approach to use isolated PBMCs (allowing the assay to be used with frozen cells, which is a common need), modified to use a shorter incubation period (discussed below), and incorporating the information from the RNA-seq study to add the flexibility of the assay to use PD-L1 or other activation markers instead of OX40 (hence the moniker AIM) (2). We then extended the results to dengue virus infection (2), pertussis immunization (2), and HIV Env immunization (1). The new quality control data presented by Cook, Zaunders, and Kelleher in Figs. 1 and 2 of their letter to the editor are excellent. We use an 18-h stimulation as the optimal time for GC Tfh cell AIM assays, and 18–24 h for PBMCs (1, 2). We did not see improved Ag-specific signal to noise at 44 h compared with 18–24 h, for either lymph node or peripheral blood samples (J.M. Dan, C. Havenar-Daughton, and S. Crotty, unpublished data), which for the blood samples may relate to the use of PBMCs instead of whole anti-coagulated blood. The data presented by Cook, Zaunders, and Kelleher in Figs. 1 and 2 of their letter are a valuable addition to the literature and will hopefully encourage more laboratories to adopt this general AIM approach for identification of Ag-specific CD4 T cells, which was first pioneered by Kelleher and Zaunders, and which we have found to be extremely useful for identification of Ag-specific GC Tfh cells in lymphoid tissues of humans and non-human primates.