Research Grants 1997-1998

in vitro Assay for Hapten-Specific Priming of Human T Lymphocytes

Wayne J. Streilein, MD
Schepens Eye Research Institute, Boston, Massachusetts

Our initial goal has focused on generation and characterization of hapten-specific sensitized T cells with human PBMC. We have recruited 5 DNCB-sensitized donors and 10 naive volunteers for the present study. Sensitized donors had been previously immunized in vivo with DNCB; naive donors were never exposed to this hapten. 50 ml peripheral blood was collected from these donors. Each blood sample was diluted and overlayered on lymph-opaque, then centrifuged (at 1600 rpm for 30 min). Low density mononuclear cells at interface were harvested and divided in two samples for preparation of purified T cells and adherent PBMC respectively. One sample was subjected to human T cell-enrichment column. The other sample was either used directly as fresh PBMC or cultured overnight in RPMI 1640 with human recombinant GM-CSF (10 ng/ml) to generate cultured nonadherent PBMC. The fresh and cultured PBMC were then derivatized with 5mM DNCB in vitro. For hapten-specificity control, some fresh PBMC were derivatized with 0.1% oxazolone; negative control cells were treated with acetone alone.

Autologous mixed lymphocyte reactions (AMLR) were then set up using purified T cells as responder cells (10,000 cells/well) and fresh or cultured PBMC that had been derivatized or not, and irradiated (2000 rads) were used as stimulator cells. The AMLRs were maintained for 5 days. 48-hr cultured supernatants were collected and assayed for production of IFN-y and IL-4. During the terminal 18-hr of the 5 day culture period, 3H-thymidine was added to measure incorporation as indication of T cells proliferation. To prove that in vitro primed T cells are hapten-specific, we set up secondary cultures using in vitro activated T cells and freshly prepared hapten-derivatized PBMC. T cell proliferation and cytokine production were also detected in same way.

The results from representative experiments are presented at the end of this section of application. From Figure 1, we have defined that blood T cells from sensitized donors specifically respond to stimulation of fresh PBMC derivatized with the same hapten and secrete large amounts of IFN-y and little IL-4; From Figure 2, we have determined that after cultured with GM-CSF, DNCB-derivatized PBMC acquire capacity of activating hapten-specific autologous naive T cells; From Figure 1 and Figure 3, we have proved that in vitro primed T cells display functional properties similar to T cells from peripheral blood of hapten-sensitized donors. Based on these results, we can come to the following conclusions:

T cells prepared from peripheral blood of DNCB-sensitized donors display proliferative responses in hapten-specific fashion upon restimulation with heptenated fresh PBMC. In addition, the cells' specific cytokine profile can be characterized: large amounts of of IFN-y are produced, by contrast, little if any IL-4 is found. However, fresh PBMC, whether DNCB-derivatized or not, fail to activate T cells harvested from naive donors.
After in vitro culture with human recombinant GM-CSF, PBMC acquire potent accessory function. Whether derivatized or not, they can significantly activate autologous naive T cells. However, DNCB-conjugated PBMC induce significantly greater T cell proliferation than their non-derivatized counterparts. Similarly, significant amounts of IFN-y are detected in cultures stimulated by cultured PBMC which have been derivatized or not, although T cells activated by haptenated PBMC produced significantly more IFN-y.