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Title:   Author  item
                                            Livingston, Kimberlyitem
                                            Jiang, Xiaowenitem
                                            Stephensen, Charles
  Submitted to: Journal of Immunological Methods
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/2/2013
                                    Publication  Date: 1/11/2013
                                    Citation: Livingston, K.A., Jiang, X., Stephensen, C.B. 2013. CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization. Journal of Immunological Methods. 390:18-29. DOI: 10.1016/j.jim.2013.01.001.Interpretive Summary: Poor nutritional status can affect the immune system, including the response to standard vaccinations.  In healthy subjects, T lymphocytes (a type of white blood cell) respond to vaccination by producing different types of cytokines.  For example, T lymphocytes that produce interferon-gamma are called T-helper type 1 (Th1) cells and respond to viral infections or vaccines.  Th2 cells produce interleukin-4 and respond to simple protein antigens, Th17 cells produce interleukin-17A and respond to extracellular bacteria, and regulatory T-cells produce interleukin-10, which helps with the termination of an immune response.  However, many of these cell types may occur at very low frequency and are thus difficult to detect in peripheral blood.  In the present study we validated a method for characterizing the development of Th1, Th2, Th17 and regulatory T cells in response to immunization with the tetanus toxoid (TT) vaccine.  The method is more sensitive than previous methods in that it can identify these cell types even if they occur at very low frequency in the peripheral blood.  As a result of the testing of this new method it is now possible to evaluate the impact of nutritional interventions (e.g., with vitamin D) that are thought to affect the development of T lymphocytes.
                                Technical Abstract: 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 d after vaccination and peripheral blood mononuclear cells (PBMC) were cultured for 7 d 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 d, and intracellular cytokine staining (ICS) of proliferated cells (identified by dye dilution) after 7d (n = 6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3+CD4+ lymphocytes, and TT-specific proliferation at 7, 14 and 28 d post vaccination. Vaccination induced TT-specific T-helper type 1 (Th1) (interferon-gamma, tumor necrosis factor-alpha, and interleukin [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.


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