Extracellular calcium ion concentrations ([Ca2+]o) nonlinearly affected the capacity of CD4+ T cells in human peripheral blood (HPB) to polyclonal activation in vitro. The dependencies of the tested parameters of CD4+ T cell activation on [Ca2+]o values corresponded to a curve with two maxima and one local minimum in between. One of the maxima was in the area of the HPB lower [Ca2+]o normal level, whereas the other was even lower, which is characteristic for pathological states of various origin. Such a pattern could be explained by some differences in T cell surface receptors. For the confirmation of this hypothesis we compared receptor-dependent (a mixture of monoclonal antibodies (mAbs) to CD3 and CD28 molecules) and receptor-independent (a mixture of phorbol myristate acetate and ionomycin) T cell activation pathways in HPB at varied [Ca2+]o values. The results evidenced a key role of the T cell intracellular signaling system for the differentiation of their sensitivity to calcium ions. Another reason can be related to the different in vitro rates of CD4+ T cell activation and differentiation stages. To check this assumption, we measured the time dependencies of activated CD4+CD69, CD4+(Tyr-P)high, CD4+CD29+ and CD4+(HLA-DR)+ T cell portions as well as their [Ca2+]o dependencies. The [Ca2+]o concentration was shown to influence the activation and differentiation stages of CD4+ T cell activation in vitro to the same degree. Hence, the earliest induction stages play a key role for the unequal sensitivity to calcium ions of CD4+ T cells in vitro. It was additionally supported by the analysis of the relative accumulation rates of activated CD4+CD69+ T cells in the presence of 0.4, 0.8, and 1.2 mM EGTA. A comparative role of CD4+ T memory cells and their naive precursors was studied in a separate series of experiments. First, we performed the phenotypical analysis of CD4+CD69+ T cells within the curve maxima. The first maximum of the CD4+ T cell portion dependence on calcium ion concentrations was formed on average by 85% CD4+CD45R0highCD69+ memory T cells, whereas the remaining 15% was constituted by naive CD4+CD45RAlowCD69+ T cells. The second maximum was nearly completely formed by CD4+CD45R0+CD69+ memory T cells. A portion of naive CD4+CD45RAlowCD69+ T cells rarely exceeded 2%. The content of CD4+CD69 T cells in these maxima was proportional to the donor age and was expressed as a straight line with R2 = 0.9077. Ionomycin-resistant (IR) memory T lymphocytes are the most probable candidates for the role of CD4+ T cells capable of being activated in the conditions of hypocalcemia. For the confirmation of this assumption we isolated mononuclear cells and their IR fraction from donor peripheral blood. Both cell populations were activated with a mAbs mixture at varied EGTA concentrations. In the first maximum only a small portion of the IR fraction of CD4+CD45RA-CD45R0+ memory T cells was activated. It was the second maximum that demonstrated the activation of the majority of CD4+CD45RA-CD45R0+ memory T cells. Hence, the presence of two activation maxima of CD4+ T cell in the conditions of hypocalcemia could be accounted for by the presence in HPB of two CD4+ memory Tcell populations differing in the calcium-dependent systems of intracellular signaling.
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