Fig. 2.

SERV-K1 Env-specific T cell responses are rare, but can be high frequency. a Fifty-nine Indian rhesus macaques were screened for SERV-K1 Env T cell responses using overlapping 15-mer peptides spanning SERV-K1 Env in IFN-γ ELISPOT. Table shows summarized results. b Raw IFN-γ ELISPOT images of SERV-K1 Env LL15-specific T cell response identified in r02120 performed in duplicate alongside positive control (Concanavalin A, Con A) and negative control (no stimulation, no stim) wells. c Intracellular cytokine staining (ICS) flow plots showing TNF-α and IFN-γ cytokine induction in SERV-K1 Env LL15-responding T cells. Gating scheme shown on left displays the CD3+ cell population, demonstrating that the SERV-K1 Env LL15-specific T cell response is CD4+ T cell-mediated. d ELISPOT was performed on CD8-depleted PBMC from r02120 stimulating with either SERV-K1 Env LL15 peptide, or a pool of overlapping 15-mer peptides spanning the entire SIVmac239 proteome. Graphs display summed responses performed in duplicate. SFCs = IFN-γ spot forming cells. e Flow plots display memory subset staining of either bulk CD4+ T cells (left) or SERV-K1 Env LL15-responding CD4+ T cells (right, gated by TNF-α/IFN-γ induction shown in part c). Memory subsets are labeled as follows: N naïve, CM central memory, EM effector memory. Note SERV-K1 Env LL15-specific T cells are uniformly effector memory in phenotype. f ICS flow plots showing functionality of SERV-K1 Env LL15-specific CD4+ T cells based on induction of TNF-α in conjuction with IFN-γ, MIP-1β, and CD107a staining. Plots are gated on CD3+CD4+ as in part c. Note frequency of response of double positive cells is similar among plots, suggesting LL15-specific CD4+ T cells are polyfunctional. Of interest, screening of two half-siblings of r02120’s dam and four siblings of r02120’s sire revealed no SERV-K1 Env-specific T cell responses