This year, the lab has continued to study the mechanisms and consequences of self tolerance, the mechanisms maintaining T cell memory, and the nature of the thymic stem cell. TOLERANCE: 1) Neonatal tolerance: Our discovery that virgin T cells are tolerized by B cells hints that neonatal mice may not be uniquely tolerizable. If the number of T cells is low; if they are all virgin; and if the injection contains many B cells, then most T cells will encounter antigen on a B cell and be tolerized, yet they should respond to professional APCs. We therefore immunized neonatal female mice with male dendritic cells. Some responded as early as day 2 after birth. Thus, neonatal T cells are simply virgin T cells tolerized by B cells and immunized by professional APCs. 2) Maternal tolerance: We analyzed cell traffic across the placenta. Using quantitative PCR for the Y chromosome, we found male cells in the thymus, spleen, and lymph nodes of pregnant mice, but by limiting dilution analysis, we found that female mice are not immunized by their male progeny. We are now testing whether they are tolerized. 3) Results of tolerance in T~ mice: We are testing thymic """"""""positive selection"""""""" by asking which haplotypes support the development of a Tgamma TCR derived from an F1 mouse. Positive selection models predict that only one of the parental haplotypes permits development whereas we predict that these T cells will develop in both. THE THYMIC STEM CELL: 1) Is it precommitted? Eleven-day-old fetuses (one day after stem cell colonization begins) contain about 2000 cells divided into two distinct populations: one of them rapidly proliferating. We measure the developmental potential of these cells using thymus and spleen organ cultures. By measuring the mature cell types produced by a stem cell, we hope eventually to determine the nature and timing of commitment signals. 2) How long does it last? Some fetal thymuses transplanted into SCID mice are producing T cells months later. We cannot predict which particular thymuses will be producing at any time, and our current hypothesis is that the stem cell may cycle and that we sometimes catch the thymuses at the low ebb of the cycle and sometimes at its peak. T CELL MEMORY: We have previously shown that T cell memory in both rats and mice requires the persistence of antigen. If Ag is deposited as Ag-Ab complexes on follicular dendritic cells in germinal centers, than T cell memory should require the presence of B cells. To test this, we created B cell-deficient mice by grafting SCID mice with fetal thymuses. Such B-less mice responded to soluble protein antigens but did not maintain long-term memory. Using limiting dilution assays, we find that the frequency of responding cells drops steadily from two weeks after immunization, whereas SCIDs containing B cells maintain their ability to mount a memory response for months. Thus, T memory requires the presence of B cells.
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