Response of human T lymphocytes to phytohemagglutinin (PHA) after sequential depletion of monocytes, HLA-DR+, Leu11a+, and Leu7+ cells

The experiments presented in this paper deal with the question of whether there is an absolute requirement for alpha-naphtylacetate esterase (ANAE)-positive monocytes, HLA-DR+, Leu11a+, and/or Leu7+ cells to stimulate human peripheral blood T lymphocytes by phytohemagglutinin (PHA). Purified (p) T lymphocytes containing less than 0.1% ANAE-positive monocytes were isolated from human peripheral blood mononuclear cells (MNC) by sequential removal of carbonyl-iron phagocytic cells and of low-density cells by density gradient centrifugation and isolation of E-rosette-forming cells (E-RFC). These pT-cells were further depleted of HLA-DR+, Leu11a+, and/or Leu7+ cells using monoclonal antibodies and cell sorting. The T lymphocytes were stimulated by PHA in an ultra-micro culture in glass capillaries at a volume of 1 microliter or 2 microliters, containing 1000 cells per culture. With this method, the accessory cell requirement could be studied under limiting cell number conditions. The results show that pT-cells can be stimulated by PHA in the absence of ANAE-positive monocytes. No ANAE-positive monocytes were found in the culture after stimulation, indicating the lack of differentiation into ANAE-positive monocytes from ANAE-negative precursors. A rabbit antiserum against leukocytic pyrogen (LP, also containing anti-IL 1 activity) only reduced but did not abrogate the stimulation of pT-lymphocytes by PHA. Addition of adherent cells resulted in an enhancement or in an inhibition of the response of pT-lymphocytes to PHA, depending on cell concentration and culture time: The lower the number of cultured T lymphocytes and the shorter the culture time, the higher was the enhancing activity by additional adherent cells, and vice versa. Further purification of the pT-cells using monoclonal antibodies and cell sorting led to the finding that depletion of either HLA-DR+, Leu11a+, or Leu7+ from pT-cells only reduced but did not abrogate the stimulation of the pT-cells by PHA. However, in absence of HLA-DR+ and Leu7+ cells, the pT-lymphocytes totally failed to respond to PHA. This abrogation of the response was not observed when pT-cells were depleted of HLA-DR+ and Leu11a+ cells. In addition, T11+/HLA-DR- T lymphocytes isolated from E-RFC by positive selection in a cell sorter also responded to PHA.(ABSTRACT TRUNCATED AT 400 WORDS)