CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization

Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vaccination. Blood was drawn before, 3, 7, 14, and 28 days after vaccination and peripheral blood mononuclear cells (PBMC) were cultured for 7 days with TT, negative control (diluent), and a positive control (Staphylococcus enterotoxin B [SEB]). Activation markers (CD25 and CD69) were measured after 44 h (n = 8), cytokines in supernatant after 3 and 7 days, and intracellular cytokine staining (ICS) of proliferated cells (identified by dye dilution) after 7 days (n = 6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3+CD4+ lymphocytes, and TT-specific proliferation at 7, 14 and 28 days post vaccination. Vaccination induced TT-specific Th1 (IFN-γ, TNF-α, and IL-2) Th2 (IL-13, IL-5, and IL-4), Th17 (IL-17A) and IL-10+ cells as measured by ICS. TT-specific Th1 cells were the most abundant (12–15% of all TT-specific CD4+ T-cells) while IL10+ (1.8%) Th17 (1.1%) and Th2 cells (0.2–0.6%) were less abundant. TT-specific cytokine concentrations in PBMC supernatants followed the same pattern where a TT-specific IL-9 response was also seen. In conclusion, TT booster vaccination induced a broad T-helper cell response. This method of evaluating cytokine phenotypes may be useful in examining the impact of nutrition and environmental conditions on the plasticity of T-helper cell memory responses.

► Expression of CD69 on CD4+ T-cells increases following tetanus immunization.
► CD4+ T-cell proliferation increases following tetanus immunization.
► TT-specific CD4+ T-cells express cytokine phenotypes with a predominance of Th1.
► PBMC cultures yield TT-specific cytokines, only IFN-γ, IL-2, and IL-13 relate to ICS.
► Antibody in lymphocyte supernatant (ALS) assay correlates closely with plasma TT-specific antibody concentration