Technical advance: decreased helper T cells and increased natural killer cells in chronic periodontitis analyzed by a novel method for isolating resident lymphocytes

Abstract

During CP, the gingival environment is primed to recruit and activate homing lymphocytes. However, detailed phenotypic and functional characterization of gingival tissue resident lymphocytes has been challenging as a result of limitations associated with available isolation methods and limited availability of human samples. This study aimed to develop a novel explant culture technique for effectively isolating human gingival lymphocytes. This technique takes advantage of the natural tendency of MNCs to migrate toward a chemokine gradient generated by the gingival fibroblasts. The explant system allowed isolation of MNCs with ∼95× higher yield relative to conventional approaches. The MNC yield correlates directly with wet weights of the tissues, and maximal MNCs are isolated during the 2nd day of the culture. The explant culture method and conventional approach produce similar MNC subpopulations such as Th, Tc, and B cells. Chemokines associated with MNC migration but not cytokines associated with MNC proliferation and differentiation were produced in the explant culture. Moreover, MNC migration in response to the secreted chemokines was inhibited by PTX. T cells did not undergo proliferation during the culture. However, the isolated T cells responded to mitogenic stimulation ex vivo. A statistically, significantly decreased Th cell with reduced CD25 expression along with increased NK and NKT cells in CP are shown. However, the number of naïve NK cells was decreased significantly in MNCs, suggesting activation of NK cells in CP.