Graft-and 4C. cells CTL generated by immunizing B6 mice having a suspension of normal B/b spleen cells were shown to be strongly cytotoxic to B/b Con A blasts (80% at 50 : 1 E/T) but not to other allogeneic (H-2 incompatible) targets nor to syngeneic B6 (H-2 identical) blasts, thus supporting the hypothesis that T cells are specific to mHAgs expressed on B/b spleen cells (data not shown). Acid eluates extracted from the same weight of normal B/b spleens, livers, gut, skin, hearts and kidneys were separated by HPLC. The individual HPLC profiles revealed a variation between the tissues in the main peak position and in the relative amount of peptides: similar amounts of peptides were extracted from skin, liver, kidney and heart whereas the extraction from gut and especially spleen yielded more peptides (Fig. 1). Therefore, we tested the lytic capability of the CTL raised against B/b spleen cells for 51Cr-labelled RMA-S cells loaded with 20 with irradiated B/b spleen cells. The CTL obtained were incubated 4 h at an E : T = 50 : 1 with 51Cr-labelled RMA-S cells loaded MK-0822 irreversible inhibition with 20 with irradiated B/b spleen cells. The CTL obtained were incubated 4 h at an E : T = 50 : 1 with 51Cr-labelled RMA-S cells loaded with 20 secondary stimulation with spleen cells collected from GVHD mice instead of normal mice resulted in very low cytotoxic activity (less than 12%) (data not shown). Open in a separate window Fig. 5 Lytic activity of CTL generated against normal and GVHD tissues. B6 responders were primed subcutaneously with several normal or GVHD B/b homogenized tissues emulsified into IFA. Responding spleen cells had been restimulated with irradiated B/b spleen cells. CTL produced had been tested because of their lytic convenience of 51Cr-labelled B/b ConA blast goals at different E : T ratios. ?, Regular; MK-0822 irreversible inhibition ?, GVHD. These organ-specific CTL elevated against regular and GVHD tissue had been likened for the repertoire of prominent peptides known in the eluates ready from GVHD tissue (Fig. 6) and examined previously with CTL elevated against regular spleen cells (Fig. 3). The same fractions ready from kidneys had been recognized whether or not CTL had Rabbit Polyclonal to HNRPLL been generated against the standard or GVHD kidney. For all the tissues (spleen, epidermis and liver organ), a proclaimed loss of recognition of positive fractions happened when CTL had been elevated against GVHD tissue instead of regular tissues. For example, positivity of small fraction 57 in the spleen, small fraction 72 in the small fraction and liver organ 28 in epidermis ingredients disappeared. In contrast, CTL raised against GVHD epidermis MK-0822 irreversible inhibition recognized small fraction 73 strongly. These observations indicated a MK-0822 irreversible inhibition selective adjustment of mHAg immunogenicity that affected preferentially GVHD tissue. Open in another home window Fig. 6 Differential reputation of peptide fractions extracted from GVHD tissue by CTL produced against regular and GVHD B/b tissue. B6 responders had been primed subcutaneously with many GVHD (higher component) or regular (lower component) B/b homogenized tissues emulsified into IFA. Responding spleen cells were restimulated with irradiated B/b spleen cells. The CTL obtained were incubated 4 h at an E : T = 50 : 1 with 51Cr-labelled RMA-S cells loaded with 20 CTL priming. This could apply especially to the differences observed when CTL priming was performed with GVHD tissues instead of normal tissues (Fig. 6). Altogether, these.