Frank E. M. Stiekema

IL-12-induced reversal of human Th2 cells is accompanied by full restoration of IL-12 responsiveness and loss of GATA-3 expression.

IL‐12 is a potent inducer of IFN‐γ production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling β2‐subunit of the IL‐12R and, therefore, do not signal in response to IL‐12. The question was raised as to what extent the loss of the IL‐12Rβ2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL‐12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA‐3 expression and induction of T‐bet expression. These reversed cells are further characterized by a marked IL‐12Rβ2 chain expression and fully restored IL‐12‐inducible STAT4 activation. The IL‐12‐induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL‐4 and in the absence of IL‐12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen‐specific Th2 cell clones. These findings suggest the possibility of restoring IL‐12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL‐12, and that IL‐12‐promoting immunotherapy can be beneficial for Th2‐mediated immune disorders, targeting both naive and memory effector T cells.

A simple method for immunoselective cell separation with the Avidin-Biotin system

A new method for immunoselective cell separation is described which uses biotinylated antibodies and avidin-coupled sheep erythrocytes for rosette formation, and a density gradient to separate the rosetting from the non-rosetting cells. The utility of the technique is illustrated by separating subpopulations of rabbit, mouse and rat lymphocytes. The method offers a rapid, sensitive and easily reproducible means for obtaining purified cell populations.

Accessory cell requirements in mitogen-induced rabbitt lymphocyte proliferation in an improved tissue culture system

SummaryThe impact of an improved culture medium (IMDM+), consisting of Iscove’s modified Dulbecco’s medium supplemented with albumin, transferrin, insulin, zinc, 2-mercapthoethanel, and 0.1% fetal bovine serum was investigated in phytohemagglutinin (PHA)-induced rabbit T cell proliferation. At the number of 2×105 cells/well purified T lymphocytes cultured in IMDM+ responded 3 to 10 times better than lymphocytes cultured in serum-supplemented RPMI 1640 medium. In these conditions, PHA-induced proliferation seemed not to require the presence of accessory cells. However, at lower cell numbers, T cell proliferation was more efficient when calculated on a per cell basis. At these low cell numbers, optimal proliferation required accessory cells like macrophages or dendritic cells. The appraent absence of this requirement for accessory cells at high T cell concentrations may be explained by the contribution of contaminating macrophages and dendritic cells in the purified T cell fractions.

Dendritic cells as accessory cells in antigen-specific murine T lymphocyte proliferation in vitro

The role of dendritic cells in antigen-induced murine T lymphocyte activation was studied by addition of purified dendritic cells to purified lymph node T lymphocytes from ovalbumin-primed mice. In the presence of the priming antigen T cells generated an antigen-specific response. The response was at least 3-fold higher with the use of a modified IMDM culture medium. The complete requirement for accessory cells was demonstrated only when nylon wool-purified T lymphocytes were thoroughly depleted of Ia antigen-expressing cells. Dendritic cells as well as peritoneal exudate macrophages were equally effective as antigen-presenting cells.

Macrophage support and suppression in rabbit T cell mitogenesis

The role of macrophages in mitogen-induced rabbit T cell proliferation has been investigated. The blastogenic response to the 3 mitogens, PHA, ConA and oxidative treatment by neuraminidase and galactose oxidase (NaGo) was tested. T cell proliferation was reduced by removal of low density or plastic adherent cells, including macrophages, and could be enhanced by the addition of peritoneal resident macrophages, indicating a macrophage requirement for rabbit T cell proliferation. However, PHA-induced proliferation could not be raised to the level expected. It was found that catalase and especially 2-ME could considerably enhance macrophage dependent proliferation, even at low macrophage concentrations. It is concluded therefore, that macrophages not only support but also suppress lymphocyte proliferation, namely by non-specific damage to lymphocytes through release of radicals and hydrogen peroxide. In addition, peritoneal, but not lymph node macrophages were found to suppress lymphocyte proliferation by prostaglandin production, although to a lesser extent. Experiments, done in the presence of blockers of macrophage-mediated suppression, showed that macrophages were able to magnify the PHA-induced T cell proliferation to the expected values. The experiments thus show that unactivated macrophages support and suppress lymphocyte proliferation at the same time.

Dendritic cells and macrophages as antigen presenting cells in ovalbumin-induced T lymphocyte proliferation in vitro

A variety of cells has been reported as having the capacity to act as antigen presenting cells in antigen specific activation of helper T cells. They include dendritic cells, macrophages and B cells. Dendritic cells (DC) in particular were shown to be potent antigen presenting cells in the proliferative T cell response to soluble antigens (1). In this volume we (2) have shown that DC most likely represent an in vitro subpopulation of interdigitating cells. Interdigitating cells are localized in T cell areas of peripheral lymphoid organs. Previous experiments have suggested that these cells play a role as inducers of T cells in responses to subcutaneously administered paratyphus vaccin (3). Since both in vitro and in vivo observations stress that DC are obvious antigen presenting cells, we have studied the role of DC as antigen presenting cells in the secondary response to ovalbumin in more detail.