tolerance induced by male skin grafts in female cba ...

Aust. J. Exp. Biol. Med. Sci., 63 (Pt. 3) 319-321 (1985)

Brief Communication:

© TOLERANCE INDUCED BY MALE SKIN GRAFTS IN FEMALE CBA MICE CAN SUPPRESS THE GENERATION OF H-Y SPECIFIC CYTOTOXIC T CELLS by R. B. ASHMAN (From the Microbiology Department, John Curtin School of Medical Research. P.O. Box 334. Canberra. ACT, Australia, 2601.)

(Accepted for publication February 27, 1985.) Cell-mediated immune responses to the male antigen (H-Y) are subject to complex genetic and cellular regulation (reviewed by Simpson, 1982). Mice carrying the H-2'' haplotype reject primary grafts of male skin, and are thus primed for the subsequent generation of H-Y specific cytotoxic T cells (Tc cells) in vitro. With one exception (Ashman. 1983). mice of other haplotypes do not reject primary male skin grafts. However, if females of these strains are immunised with male cells via the footpad, rather than intravenously or intraperitoneally, they develop memory Tc cell precursors that differentiate into cytotoxic effector cells after in vitro challenge with male spleen cells (Mullbacher and Brenan, 1980; Fierz et ai, 1982). This mode of priming is also sufficient to enable a proportion of CBA female mice to reject male skin grafts (Simpson, Chandler and Pole. 1981); however, when a male graft is accepted, in either naive or primed mice, H-Y specific tolerance is induced in the recipient female (Ashman, 1982; 1984). This situation seems analogous to the induction of specific unresponsiveness to H-Y in C57 Bl/6 female mice after multiple pregnancies (Billingham, Silvers and \Viison, 1%5). This is known to be mediated, and can be transferred, by a thymus-dependent cell population (Smith and Powell, 1977), but the mode of action of these putative suppressor cells has not yet been analysed. Although the role of the Tc cells in tissue graft rejection is still the subject of considerable controversy, spleen cells from multiparous female mice, when challenged with male cells in vitro, show a variable impairment in their ability to generate H-Y specific effector Tc cells (Simpson, Benjamin and Chandler, 1981). It was thus of interest to determine whether the tolerance induced by male skin grafts in female CBA mice also affected the ability of the animals to make H-Y specific Tc cell responses. The analysis was undertaken as follows. Thirty CBA female mice were immunised with 1 x 10' male spleen cells in the hind footpad. Four weeks later 20 were grafted with syngeneic male tail skin by standard methods (Billingham and Medawar. 1955) under Nembutal anaesthesia. The plaster bandages were removed after 10 days and the grafts scored for a total of 80 days. Rejection was defined as total graft necrosis. Two grafts were technical failures—of the remainder, 10 were rejected. Subsequently, spleen cells from mice primed but not grafted, and from mice that had either accepted or rejected male skin grafts, were challenged in vitro with irradiated male cells. The results are shown in the Table. All controls showed male-specific lysis, whereas there was substantial, though variable, impairment of the ability to generate effector Tc cells from spleen cells of the tolerant mice. Three out of 5 mice in the group that rejected male skin grafts showed responses comparable with controls, while 2 showed much enhanced killing. All groups gave strong responses against BALB/c (H-2'') alloantigens, demonstrating that the decreased responsiveness was specific for the H-Y antigen. This impairment of Tc cell responses in H-Y tolerant mice admits of two possible interpretations. First, Tc cells might be directly involved in skin graft rejection; thus, their suppression would be essential for the maintenance of tolerance. Culture may lead to a variable weakening of the suppression, and the consequent detection of low levels of cytotoxicity by some activated cells. Alternatively, if graft rejection is mediated predominantly by DTH-like cells (Loveland et al., 1981), the reduced expression of Tc cell activity against H-Y, but not against BALB/c, can only be due to cross-reactivity of suppression of the idiotypes recognising H-Y in association with Class II MHC antigens, on idiotypes recognising H-Y in association with Class I restriction antigens. There is a precedent for this, in that cross-reactivity, at the clonal level, of an H-2

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restricted receptor idiotype on a different antigen in association with a 'non-self Class I restriction antigen has previously been reported (Hunig and Bevan, 1981). It is also interesting that rejection of male skin did not invariably enhance the Tc cell response. Neither footpad priming with male cells nor prior rejection of male skin results in solid immunity, as defined by rejection of skin grafts by all CBA females (Ashman, 1984); that is, in this experimental system, a relatively small antigenic challenge, presented as a skin graft, can overcome a pre-existing state of specific immunity and establish specific tolerance to the selfsame antigen. These data, together with the failure of skin grafts consistently to boost Tc cell responsiveness, further suggest that the mechanisms regulating the response to H-Y in CBA mice are inherently biased towards unresponsiveness, but that this can be modulated, albeit within limits, by variations in antigen dose and/or route of administration. Acknowledgements:

My thanks to Jane Bell for excellent technical assistance.

TABLE I H-Y specific Tc ceil responses of footpad-primed CBA female mice that had either accepted or rejected syngeneic male skin grafts. Percent cytotoxicity Treatment on Male macrophages P815 Primed, no graft 21-1 68-9 26-8 66 2 19-0 58-0 Primed, skin graft rejected

21-1 21-6 26-7 496 40-3

49-9 54-4 48-7 48-8 44-1

Primed, skin graft accepted

12-6 4-9 8-2 12;2

45-5 53-1 70-0 64-5

Mice were primed with male spleen cells via the footpad, and grafted with syngeneic male skin as described in the text. After 80 days spleen cell suspensions were prepared from individual mice and set up at 2xlO''/ml in 5 ml of Eagle's Minimum Essential Medium supplemented with 10 •* M 2-merceptoethanol and 5% foetal calf serum. One culture from each individual was stimulated with 5 x 10' 2000 R CBA male spleen cells and a second with 2-5 x lO' 2000 R BALB/c spleen cells as a third party control. After 5 days the cultures were harvested, resuspended in 1 ml and threefold dilutions tested, as appropriate, on "Cr-labelled CBA male and female thioglycollate-induced macrophage target cells, or on "Cr-labelled P8I5 (H-2'') tumour target cells in a final volume of 0-2 ml. After 4 h incubation, 0 1 ml aliquots of the supernatant were removed, "Cr release determined and percent lysis calculated. A straight line was fitted to the titration points by linear regression and the results calculated as percent lysis at a standard killer-totarget cell ratio (30:1 against H-Y; 4:1 against P815). Killing on female targets was negligible, and is not shown. The data presented are from one of two replicate experiments, both of which gave similar results.

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