Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Abstract

Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-γ. Interferon-γ release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4 + T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-γ ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination

“…For ex vivo culture, fresh PBMC isolated from buffy coat fractions of blood collected in 2× acid citrate dextrose solution were plated into 96-well plates (2 × 10 5 ) and stimulated with PPD (5 μg/ml), Ag85A/TB10 4 (1 μg/ml of each protein), ESAT-6:CFP10 (1 μg/ml), PWM (1 μg/ml), or medium alone for 16 h at 39°C/5% CO 2 with Brefeldin A (Sigma, 10 μg/ml) added at 4 h of culture ( 56 ).…”

Recent Advances in the Analysis of Single Cells

“…As the wells are coated with specific antibodies, the secreted cytokines are directly bound to the well plate surface at the location of their release. Subsequent enzymatic labeling results in conversion of a precipitating substrate to a colored product, and this corresponds to the cytokine expression of an individual cell. − Saletti et al recently published a protocol for a modified ELISPOT that is capable of detecting virtually any vaccine antigen after magnetic enrichment of circulating plasmablasts from blood samples …”