Steven Neben

A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules

T cell activation requires two signals: a signal from the TCR and a co‐stimulatory signal provided by antigen‐presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co‐stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co‐stimulatory capacity between CD28 and other molecules. Anti‐CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co‐immobilized with a submitogenic dose of anti‐CD3 mAb. [ 3 H]Thymidine incorporation determined 2 days after co‐stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co‐stimulation, co‐stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL‐2 production; CD28 co‐stimulation induced progressively increasing IL‐2 production, whereas co‐stimulation by other molecules produced limited amounts of IL‐2. Addition of recombinant IL‐2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co‐stimulation. These results indicate that a fundamental difference exists in the nature of co‐stimulation between CD28 and other molecules, which can be evaluated by the levels of IL‐2 production, but not simply by [ 3 H]thymidine incorporation.

SYNERGY BETWEEN CD28 AND CD9 COSTIMULATION FOR NAIVE T-CELL ACTIVATION

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb. The levels of costimulation induced by ligation of CD9 and CD28 were comparable. However, the costimulatory effect differed between soluble anti-CD9 and CD28 mAb. A soluble form of anti-CD28 mAb could costimulate anti-CD3-triggered T-cells, whereas soluble anti-CD9 mAb failed to costimulate. Although anti-CD28 costimulated naive T-cells treated with phorbol myristate acetate (PMA) instead of anti-CD3 mAb, a combination of PMA plus anti-CD9 mAb could not induce T-cell activation. The combined costimulation of anti-CD3-triggered T-cells with anti-CD9 and anti-CD28 mAbs resulted in strikingly enhanced [3H]TdR uptake and lymphokine (IL-2 and IFN-gamma) production when compared to those induced by each costimulation. These results suggest that CD9 and CD28 induce T-cell costimulation using different signaling pathways, thereby inducing synergy in T-cell activation.

Differential Involvement of a Fas-CPP32-Like Protease Pathway in Apoptosis of TCR/CD9-Costimulated, Naive T Cells and TCR-Restimulated, Activated T Cells

Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells. AICD was observed at 12 h after the restimulation of activated T cells with anti-CD3 and reached a peak level at 24 h after this restimulation. CPP32-like protease activity was detected during AICD. Although TCR/CD9 stimulation-associated apoptosis was observed at 24 h after the stimulation of naive T cells and reached a peak level at 36 h after this stimulation, CPP32-like protease activity in these T cells was only marginal at all time points. Nevertheless, both forms of apoptosis were prevented similarly by two different peptide-based caspase inhibitors. These results indicate that the apoptosis that follows the T cell activation which is induced as a result of CD9 costimulation does not involve a Fas-CPP32-like protease pathway, but suggest that different caspase members are likely to be critical in this form of apoptosis.

Effects of Interleukin 12 on Hematopoietic Stem and Progenitor Cells

Interleukin-12 (IL-12) has been shown to possess potent immunomodulatory activity. It has a unique structure among cytokines, consisting of two covalently linked subunits, one with homology to other members of the cytokine superfamily, the other being highly homologous to gp130, the signalling subunit of a number of cytokine receptors. Here we summarize studies showing that IL-12 is a hematopoietic growth factor with potent activity on hematopoietic stem and progenitor cells. In clonal and liquid culture assays, IL-12 synergizes with IL-3 and Steel Factor to increase the number of colonies as well as to expand both stem and progenitor cell content in the cultures. In stroma-dependent long-term bone marrow cultures, IL-12 addition causes a decrease in cell production in the first week after inoculation of whole bone marrow cells, followed by an increase in both mature cells and progenitor cells over the next 3 weeks. The initial decrease appears to be mediated by IL-12-induced production of IFN-γ, possibly by natural killer cells and/or T cells which do not persist in these cultures. Studies in naive mice demonstrate a similar acute decrease in peripheral leukocyte count, mediated by IFN-γ, upon administration of IL-12. In contrast, despite a significant decrease in peripheral platelet count, reticulated platelets become elevated and mean megakaryocyte ploidy in the bone marrow shifts from 16N to 32N during IL-12 treatment. These IL-12-mediated effects on megakaryopoiesis are abrogated by simultaneous treatment of mice with antibodies against IFN-γ. These studies provide further information on the potential physiological role and applications of IL-12 outside the immune system.