CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases.

The frequencies of antigen-specific memory cells are often low in chronic disease states related to infection and autoimmunity, making detection of such populations difficult, even with high sensitivity assays such as the cytokine enzyme-linked immunospot (ELISPOT). The spectrum and function of antigen presenting cells (APC) in the peripheral compartment can differ considerably from the inflamed target organ. In order to approximate the costimulatory environment of the target organ, we measured T cell responses with and without the addition of agonistic anti-CD28 antibody in the ELISPOT assay. CD4 and CD8 IFN-gamma responses to viral (hepatitis C) and autoimmune antigens (islet cell) were tested in 10 hepatitis C and 8 type 1 diabetic as well as healthy control subjects. IFN-gamma responses to tetanus toxoid, mumps and cytomegalovirus (CMV) protein antigen, as well as Epstein-Barr virus and CMV peptides were also measured in healthy control subjects. We found higher frequencies of T cells reactive to protein and peptide antigens when anti-CD28 antibody was present, often detecting responses only in the presence of anti-CD28 antibody. These results demonstrate that anti-CD28 antibody signal enhanced ELISPOT assays can facilitate the identification of low precursor frequency T cells in chronic infectious and autoimmune disease states where suboptimal costimulatory environment may exist in the periphery. The use of such costimulation may also enable a more quantitative assessment of circulating memory effector T cell frequency.