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Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
Elevation of intracellular cAMP in T lymphocytes, induced by agents such as IL-1α, prostaglandins or forskolin, inhibits Th1-type cytokine production but stimulates Th2-type cytokine production. The signaling pathway engaged in cAMP-mediated induction of Th2 lymphokines remains obscure and therefore my doctoral work was focused on the elucidation of cAMP pathway in primary Th lymphocytes. While forskolin treatment of EL-4 cells led both to an activation of Th2 lymphokines and inhibition of Th1 lymphokines, ectopic expression of catalytically active PKA stimulated Th2 lymphokines but failed to inhibit Th1 lymphokine expression. Thus, the PKA activity is selectively involved in the stimulation of Th2 lymphokine expression whereas other cAMP-dependent pathway(s) appears to downregulate Th1 lymphokines. By investigating different types of primary murine Th cells, it was found that active PKA enhanced IL-5 expression only in Th0 and Th2 but not in Th1 cells. This is likely due to the different levels of GATA-3 whose expression is high in Th2, moderate in Th0 and very low in Th1 cells. Ectopic expression of GATA-3 in Th1 cells induced Th2 lymphokine expression which could be further enhanced by increased cAMP levels or PKA activity. Investigations on the role of increased cAMP levels on Th2 lymphokines in D10 cells, a Th2-type cell line, led to the conclusion that elevated cAMP concentrations do not stimulate PKA but p38 activity which, through phosphorylation of GATA-3, appeared to induce IL-5 and IL-13 expression (Chen et al., 2000). While focusing on primary Th lymphocytes, it was observed that expression of the catalytic subunit α of PKA is sufficient for optimal IL-5 expression in primary Th0 cells. In addition, downregulation of IL-5 production in primary Th2 cells by the treatment with low concentrations of H-89, a PKA specific inhibitor, as well as by the ectopic expression of a negatively acting version of regulatory PKA subunit I demonstrates that active PKA plays an important role in IL-5 gene regulation. These findings using different types of primary CD4+ T lymphocytes, including Th2 cells, the one likely to represent the native IL-5 producers in vivo, demonstrates that the adenylyl cyclase/cAMP/PKA signaling pathway plays an important role in IL-5 gene expression in primary Th2 cells. Thus the importance of cAMP/PKA signaling pathway in Th2 effector function was established during this doctoral research work.