Proceedings of the National Academy of Sciences Vol. 65, No. 1, pp. 70-73, January 1970

Cells Involved in Cell-Mediated and Transplantation Immunity in the Rabbit, I. The Noninvolvement of the Bone Marrow Reactive Cell in the Transplant Rejection Reaction* Maxwell Richter,t Nabih I. Abdou, and Robert D. Midgley

THE HARRY WEBSTER THORP LABORATORIES, DIVISION OF IMMUNOCHEMISTRY AND , AND THE DEPARTMENT OF SURGERY, ROYAL VICTORIA HOSPITAL, MONTREAL, QUEBEC, CANADA Communicated by Wilder Penfield, October 28, 1969 Abstract. Rabbits were subjected to 800 r to render them immunoincompetent with respect to the humoral immune response. These rabbits were, however, only slightly less capable of rejecting skin allografts than nonirradiated rabbits. Furthermore, the administration of normal or specifi- cally "primed" bone marrow cells (bone marrow obtained from a rabbit injected one to two days previously with the spleen cells obtained from the prospective skin donor) did not enhance or inhibit the rejection reaction. It is con- cluded that the cells mediating the humoral immune response are different from those mediating the cellular immune response.

Introduction. Previous investigations from this laboratory have established that at least two cell-types are required for the mediation of the humoral immune response in the rabbit, the antigen-reactive cell and the -forming cell.'-3 The antigen-reactive cell has been localized to the bone marrow.4 By subjecting normal rabbits to varying doses of irradiation, coupled with cell transfer experi- ments, we have previously demonstrated that a dependent relationship exists between the capacity of the rabbit to synthesize humoral antibody and the presence of functionally active antigen-reactive cells in the bone marrow.5 Evidence has also been presented strongly suggesting that these cells vacate the bone marrow after contact with the antigen, leaving behind antigen-reactive cells capable of transferring immunocompetence to a recipient 800 r irradiated rabbit with respect to all except the one used to immunize the cell donor." 2 Since the bone marrow can be depleted of antigen-reactive cells pre- committed to interact with an antigen as complex as the ,7 it was proposed to utilize the cell transfer system to determine the role of antigen- reactive cells in transplant rejection reactions. As will be seen below, the bone marrow antigen-reactive cell does not play a role in the mediation of this reaction. Materials and Methods. Black outbred rabbits were selected as both the spleen cell and skin donors and New Zealand white rabbits were used as the bone marrow donors and as the irradiated bone marrow recipients (Fig. 1). The spleen was excised from a rabbit intravenously anesthetized with nembutal (30 mg/kg body weight). The spleen 70 Downloaded by guest on September 26, 2021 VOL. 65, 1970 MICROBIOLOGY: RICHTER ET AL. 71

NORMAL BLACK RABBIT NORMAL WHITE RABBIT (donor B-I)7 (donor W-I)

CELLS /,- (5x 10) z /

SKIN PRIMED (day 1) ALLOGRAFT BONE MARROW CELLS (5x 108)

TIME (in days) FOR REJECTION OF SKIN ALLOGRAFT FROM DONOR B-I FIG. 1.-Protocol for the demonstration of different cell pathways resulting in and transplant rejection. cell suspension was prepared by application of slight pressure on spleen fragments through a wire mesh (50 mesh) into Medium 199 (Med 199) (Microbiological Assoc., Bethesda, Md.). The spleen cells were centrifuged and resuspended in Med 199 to a cell concen- tration of 108 splenic lymphoid cells per milliliter. The spleen cells (5 X 108) were then intravenously injected into a normal white rabbit. Twenty-four or 48 hr later, the rabbit was sacrificed and bone-marrow cell suspensions (referred to as primed bone marrow) were prepared as previously described." 2 These bone-marrow cells were then injected into 800 r irradiated white rabbits which served as the skin graft recipients. Skin grafts were performed as follows: Under intravenous nembutal anesthesia, the backs of both donor and recipient rabbits were shaved and prepped with Tincture of Merthiolate, using sterile technique with appropriate draping. The full thickness donor skin was removed, defatted, and placed on sterile saline-soaked gauzes. The donor site was closed primarily with 4-0 nylon sutures. A full thickness defect 2 cm in diameter was created on the backs of recipient rabbits and, using sterile technique, the donor skin was tailored to fit the recipient bed and held in place by means of interrupted 4-0 nylon sutures with a tie-over bolus dressing. The entire area was immobilized by means of collodion preparation of the surrounding nonoperated area and circumferential Kling dressing. The rabbits were placed in single cages. The dressings were removed and changed at 5- to 7-day intervals to allow for observation of take or rejection of the skin graft. The criteria for rejection of the skin grafts were those described by Billingham.6 The recipients of bone marrow primed with allogeneic splenic were bled at intervals of time and tested for the presence of circulating cytotoxic directed to leukocytes of the lymphocyte donor. The procedures used to detect these antibodies have been presented in detail previously.7 Results. As can be seen in Table 1, irradiated (800 r) rabbits were capable of rejecting skin allografts to the same extent as nonirradiated control rabbits. The transfer of normal or "primed" allogeneic bone marrow cells to irradiated Downloaded by guest on September 26, 2021 72 MICROBIOLOGY: RICHTER ET AL. PROC. N. A. S.

rabbits did not interfere with or enhance their capacity to reject the skin grafts. On the other hand (not shown in Table 1), irradiated recipients of normal bone marrow cells could mount an immune response directed toward lymphocytes of the skin graft donor (cytotoxic titers of 30 to 100), whereas irradiated recipients of primed bone marrow (primed with respect to lymphoid cells of the skin donor) were unable to form detectable quantities of circulating antibodies toward the cells of the skin donor. TABLE 1. The rejection of skin allografts by irradiated (800r) rabbits given either normal or primed allogeneic bone marrow cells. Irradiated white recipient rabbit injected with primed* bone marrow cells obtained Skin allograft Time (in days) from donor injected with obtained from for rejection spleen cells obtained from following of skin following black rabbit black rabbit allograft B 1 16-18 B I B 1 15-17 B 1 B 2 14-16 B 1 B 3 16-18 B 2 18-21 B 2 B 1 17-19 B 2 B 2 15-18 B 2 B 3 14-17 Recipient irradiated only B 1 19-21 (no marrow cells transferred) B 2 16-19 B 3 15-19 Control, non-irradiated rabbit B 1 15-17 (no cells transferred) B 2 14-17 B 3 14-18 * Primed bone marrow is that obtained from a rabbit 1 to 2 days following immunization with the antigen. Discussion. Previous investigations conducted in this laboratory have unequivocally implicated an interaction between two cell types in the successful mediation of the humoral immune response-the bone marrow antigen-reactive cell (ARC) and the antibody-forming cell (AFC) of as yet undetermined origin.3 The ARC cell has been shown to be relatively radio-sensitive to 800 r in vivo irradiation2' 3 since the bone marrow cells of such a rabbit cannot transfer anti- body-forming capacity to other 800 r irradiated rabbits. The recovery of the capacity of the irradiated rabbit to synthesize humoral antibodies parallels the recovery of the ARC cells in the bone marrow,5 with complete recovery by 4 to 6 weeks. Bone-marrow cells obtained from rabbits one to two days following immunization with an antigen, i.e., sheep red blood cells (S-rbc), could not transfer immunocompetence to 800 r irradiated rabbits with respect to S-rbc whereas they could transfer immunocompetence with respect to all other antigens.2 These data suggest that the ARC cells vacate the bone marrow after contact with the antigen in vivo, leaving behind a bone marrow deficient in ARC cells directed toward the immunizing antigen. These findings were consistently obtained regardless of whether the antigen used was an inanimate protein,2 a nonreplicating ,2 or a viable, potentially proliferative lymphocyte.' On the basis of these findings, a protocol was devised to deter- Downloaded by guest on September 26, 2021 VOL. 65, 1970 MICROBIOLOGY: RICHTER ET AL. 73

mine the role of the ARC cell in the induction of cellular-type immunity, as represented by the transplant rejection reaction. If the ARC cell does have a specific function here, then the irradiated recipients of lymphocyte-primed bone marrow should not be able to reject skin transplants obtained from the rabbit which had originally donated lymphocytes used to prime the bone marrow donor until their own ARC cell had regenerated. Such was not the case, how- ever. The irradiated recipients of primed bone marrow cells were able to reject skin grafts as effectively as irradiated rabbits not given any bone marrow cells or given normal allogeneic bone marrow cells. It would therefore appear that the capacity to reject an allogeneic transplant does not involve the mediation of the ARC cell. Whether the capacity to evoke cellular immunity is entirely defined by a unique type of immunocompetent cell or by the antibody-forming cell identical to that mediating humoral immunity remains to be determined. However, if the latter is the case, then it would appear that the same cell capable of mediating cellular immunity (efferent limb of immune response) would also possess the capacity to "recognize" and interact with the original antigen (afferent limb of immune response), a property possessed by the memory cell mediating humoral immunity but not by the virgin ARC or AFC cell in the rabbit,2' 3 mouse, and rat.8-10 Experiments are currently in progress to better define the relationship of the cells mediating the humoral and cellular immune responses in the rabbit. * This investigation was supported by a grant from the Medical Research Council of Canada. t Medical Research Associate, Medical Research Council of Canada. I Singhal, S. K., and M. Richter, J. Exp. Med., 128, 1099 (1968). 2 Abdou, N. I., and M. Richter, J. Exp. Med., 129, 757 (1969). 3 Richter, M., and N. I. Abdou, J. Exp. Med., 129,1261 (1969). 4 Richter, M., B. Rose, and N. I. Abdou, Int. Arch. Alekrgy, 38, 269 (1970). 5 Abdou, N. I., B. Rose, and M. Richter, J. Exp. Med., 130, 867 (1969). 6 Billingham, R. E., in Transplantation of Tissues and Cells, ed. R. E. Billingham and W. K. Silvers (Philadelphia: The Wistar Institute Press, 1961), Chap. 1. 7 Abdou, N. I., and M. Richter, these PROCEEDINGS, 63, 1136 (1969). 8 Claman, H. N., E. A. Chaperon, and R. F. Triplett, J. Immunol., 97, 828 (1966). 9 Mitchell, G. F., and J. F. A. P., Miller, J. Exp. Med., 128, 821 (1968). 10 Davies, A. J. S., E. Leuchars, V. Wallis, R. Merchant, and E. V. Elliot, Transpl., 5, 222 (1967). Downloaded by guest on September 26, 2021