Alloreactive cytotoxic T cells. I. Alloreactive and allorestricted cytotoxic T cells

Nylon wool-nonadherent spleen cells from three inbred mouse strains of H-2k (CBA), H-2d (BALB/c) and H-2b (C57BL/6) haplotype were co-cultured with 2,4,6-trinitrophenyl (TNP)-modified or nonmodified allogeneic stimulator cells in a limiting dilution system. Using a recently described restimulation protocol, a surprisingly large number of splenic cytotoxic lymphocyte precursors (CLP) was clonally expanded in this primary in vitro response to allo-H-2 plus TNP determinants; measured CLP frequencies ranged from 1/30 to 1/300. The lytic specificity patterns of individual microcultures (selected for a high probability of clonality) were defined by split well analysis, and were furthermore followed up in time by sequentially reassaying microcultures at different time points of in vitro incubation. This analysis revealed the following: a large fraction of cytotoxic T lymphocyte clones lysed TNP-modified but not nonmodified allogeneic concanavalin A blast targets, i.e., were allorestricted; this was found in all 6 allogeneic strain combinations set up with b, k and d haplotype mice; allorestricted lytic patterns predominated in microcultures with low numbers of responder cells per well, and at late time points of in vitro culture; allorestricted lytic cultures were specific for the stimulating allogeneic H-2 plus TNP determinant(s); and allorestricted lytic patterns were also found in microcultures stimulated by nonmodified allogeneic cells. To our knowledge, these are the highest CLP frequencies yet reported in limiting dilution systems that used a specific (re)stimulation protocol and measured the lytic responses obtained in a specificity-controlled readout.