The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

SUMMARY HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5–1 gag-pol mRNA+/Gag protein+ infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia, and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally-suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

Highlights d Three weeks after the first BNT162b2 dose, weak neutralizing antibodies are elicited d These antibodies have robust Fc-mediated effector functions d Vaccination of individuals previously infected boosts humoral and cellular responses d Strong correlations between T helper cell and humoral responses are observed

Highlights d Spike-specific IgM and IgA wane more rapidly than IgG after recovery d Fc-effector functions, but not neutralization, are sustained over time d SARS-CoV-2-specific B cell immunity persists despite overall antibody decline