Analysis of the frequencies of cytokine producing lymphocytes at different developmental stages

The study of the frequencies of cytokine producing T cells at different developmental stages under different stimulation was performed at the single cell level. It was demonstrated that the frequency of production of cytokines by naive (CD45RA+) T cells ciepends on the stimulation provided. Thus, there were liigh frequencies of lL-2, IFNγ and IL-4 producing CD45RA+ T cells derived form cord blood (CB) with T cell co-receptor independent signalling (using phorbol esters and calcium ionophore stimulation). MCSF, present in CB was found to be partially responsible for a reduced proliferative response to alloantigens. When co-receptor inciependent stimulation by PMA and ionomycin was used in established T cell lines and clones, there was unrestricted production of IFNγ and IL-4, resulting in high frequencies of all type 1 (defined by their exclusive production of IFNγ),type 2 (defined by their exclusive production of IL-4) and type 0 (defined by their simultaneous prociuction of IFNγ and IL-4) T cells. Thus, stimulation with phorbol ester and calcium ionophore bypasses gene regulation and induces the expression of IFNγ and IL-4, even in differentiated T cells. The frequency of cytokine producing alloantigen specific T cells was then determined using phorbol ester and CD3 stimulation. Using this stimulus, all samples had similar frequencies of type 1 cells regardless of the alloantigen (mismatch) used. Conversely, the enzyme linked imniunospot assay (ELISPOT) was able to cietermine frequencies of different IFNγ producing T cells with single mismatches. The expression of the Th2 marker CCR3 was considered to define T helper subsets. However it was found expressed in Thl lines and discarded as a Th2 marker. These results suggest that: A) CD45RA+ T cells from CB are not defective in their production of IL-2 or IFNγ, B) Soluble factors may contribute to the low response to alloantigens in CB cells, B) The use of non-T cell restricted mitogens to determine frequencies of cytokine prociucing cells may produce artificial profiles of cytokine producing cells and C) ELISPOT maybe able to ciistinguish between different mismatches, which asses the frequencies of IFNγ producing cells, suggesting that T cell restricted stimulation should be used to study frequencies of cytokine producing alloreactive T cells. D) CCR3 is not a Th2 cell marker