Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts" Two types of host reactivities not requiring immunization in the mouse and not mediated by T lymphocytes were compared: resistance of irradiated and nonirradiated F1 hybrids to accept parental grafts of normal or malignant hemopoietic cells (Hh system), and the natural killer cell activity against mouse lymphomas (NK system). The effects of six independent variables known to influence resistance t o marrow grafts were investigated in the NK system using YAC-I lymphoma cells as targets. The following properties were shared: (a) maturation during the fourth week of life; (b) low sensitivity to acute total body irradiation; (c) dependence on the integrity of bone marrow as demonstrated by reduced reactivity in *gSr-treated mice; (d) suppression by a single injection of rabbit anti-mouse bone marrow serum; (e) suppression by a single injection of the anti-macrophage agents silica and r-carrageenan; and (f) suppression by multiple injections of parental spleen cells into F1 mice. These positive correlations are particularly significant because most of the variables have either opposing or no effect on conventional immunity. F1 mice rendered specifically unresponsive to parental marrow grafts, could retain NK cell activity, and genetically susceptible mice could be rendered hyporeactive in terms of NK cells, indicating that the specificities of YAC-1 and Hh-1 incompatible targets were different.

Host reactivities not requiring immunization in the mouse, especially natural resistance of irradiated animals to accept grafts of normal or malignant hemopoietic cells, were compared with NK activity against the YAC-1 lymphoma. The effects of several independent variables known to influence natural resistance in vivo had a similar effect on the NK system. Figure 12 lists an impressive array of shared properties and positive correlations. In contrast, the distinctions were few and minor. Many of the positive correlations were of particular significance since the experimental variables either have opposing or no effects on conventional induced immunity. The multiplicity and pervasiveness of these correlations suggest that the cellular mechanisms underlying natural reactivities are similar or common. Cytotoxic effectors mediating natural resistance to normal cells, tumors, and cells infected with intracellular pathogens may be distinct in terms of target selectivity, yet belong to a single cell lineage subject to common regulatory influences for differentiation and function. Regulation of reactivity via suppressor cells was studied in the NK system only. The spleens of mice selected for low levels of NK activity (resulting from young age, irradiation, and treatment with the macrophage-active agents l-carrageenan or hydrocortisone acetate) contained cells capable of inhibiting the lytic function of NK effectors taken from untreated adult donors. All the suppressor cells studied were thymus-independent, as judged by their occurrence in spleens of genetically athymic mice; the suppressive function was resistant to 2000 rads of gamma-rays administered in vitro and was not restricted by the major histocompatibility complex, without exception. However, two major classes of suppressors were identified: (a) macrophagelike cells inducible by l-carrageenan or hydrocortisone acetate, and (b) nonadherent cells found in spleens of untreated infants and of irradiated adult mice. It is proposed that the suppression of NK cytolysis demonstrated in vitro was a manifestation of regulatory mechanisms modulating the level of NK activity in vivo. Macrophagelike cells that are induced, activated, or inactivated by bacteria, viruses, hormones, and other agents may act as regulators of differentiation, maturation, and function of cells belonging to the NK lineage. Nonadherent cells could be either a distinct class of suppressors or immature NK cells capable of binding but not lysing target cells. In the latter case, regulation would be achieved via competitive binding of targets by pre-NK cells presumably in dynamic equilibrium with functional (i.e. matured) NK effectors.

F1 hybrid mice are capable of rejecting inbred parental strain bone marrow grafts after a single lethal exposure to X-rays. The incompatibility is genetically controlled by the Hybrid-histocompatibility-1 (Hh-1) locus in or near the D end of the Histocompatibility-2 (H-2) region. The onset of parental graft rejection begins 9–12 hr after transplantation and is completed by 24 hr. Maturation of hybrid resistance does not occur until the 22nd day of life. In adults, the resistance to parental marrow grafts can be temporarily abrogated or weakened by administration of cyclophosphamide or dead cultures of Corynebacterium parvum, acute supralethal exposures to radiation, or by split-dose irradiation with 6–37-day intervals. Parental marrow grafts elicit a transplantation reaction in irradiated F1 mice which is indistinguishable from that elicited in irradiated allogeneic (H-2-incompatible) hosts. Because of this immunogenetic similarity, the following question is raised: are the same or different alloantigens responsible for rejection of parental and allogeneic marrow grafts? In the first case, Hh-1 alleles would be recessive determinants of tissue-specific transplantation antigens, whereas in the second case they would be the determinants of parental- and tissue-specific antigens subject to genetic suppression in Hh-1 heterozygotes. Although the available evidence is not conclusive in excluding one of the two possibilities, it favors the concept that allograft reactivity to hemopoietic cells is elicited by recessive tissue-specific antigens.

Recipients of hemopoietic transplants are usually preexposed to whole body irradiation to deplete the blood-forming system, and so provide the transferred cells with a "graft bed" and stimuli for maximal proliferation and differentiation. It is generally assumed that irradiation in the lethal dose range would also be immunosuppressive and enable allogeneic cells to engraft. This assumption, however, is not correct. In the course of studies on the genetics of hybrid resistance to parental bone marrow grafts, it was noted that allografts did not establish themselves in irradiated hosts of certain mouse strains while succeeding in hosts of other strains (1). The mice in which marrow grafts grew and those in which the same cells failed sometimes belonged to inbred strains sharing the same//-2 alleles. This suggested that genes other than those specifying major transplantation antigens influenced the outcome of marrow allografts in irradiated mice. Additional evidence is now presented in support of this view. The data also indicate that a peculiar type of incompatibility for hemopoietic allografts is common in irradiated mice, and that it results from destructive host anti-graft reactions. Since the processes of cellular proliferation, antibody formation, and skin graft rejection are impaired after whole body irradiation, bone marrow allografts are presumably rejected by a mechanism previously unknown. This type of aUograft reaction is peculiar because it does not require proliferation of lymphoid cells and is tissue specific, thymus independent, and regulated by genetic factors which apparently do not affect the fate of other solid grafts. Materials and MethodsMice.--Most inbred and F1 strains were raised in our animal colony, and were derived from pedigreed breeders supplied by G. D. Snell, Jackson Laboratory, Bar Harbor, Maine (C57BL/ 10ScSn [abbreviated B10], C3H.SW, B10.D2, A

Spleen cell suspensions of unprimed donor mice containing precursors of immunocytes have been transplanted into X-irradiated recipient mice. In the presence of antigen (sheep erythrocytes) these precursors, called antigen-sensitive units, gave rise to progeny cells secreting specific antibody. We studied quantitatively the production of cells releasing IgM hemolysins (direct plaque-forming cells), IgG hemolysins (indirect plaque-forming cells), and hemagglutinins (cluster-forming cells). We found that each of these immunocyte populations was distinct, i.e., that cells releasing agglutinins did not, as a rule, release hemolysins, and vice versa. We also found that cell populations secreting IgM hemolysins did not shift, under certain experimental conditions, to the production of IgG hemolysins during the primary immune response. By transplanting graded numbers of spleen cells, we succeeded in limiting to one or a few the number of antigen-sensitive units that reached the recipient spleen. We estimated thereby the frequency of antigen-sensitive units in donor cell suspensions and tested their potential for production of immunocytes of more than one type. Our results indicated that antigen-sensitive units were unipotent for they displayed in the spleens of unprimed donors the same restrictions of function and heterogeneity (antibody-specificity differentiation, antibody-class differentiation) found among antibody-forming cells. Furthermore, antigen-sensitive precursors for direct plaque-forming cells, indirect plaque-forming cells, and cluster-forming cells were detected in the spleens of unprimed mice in different frequencies, i.e., 1 in ∼ 106, 1 in ∼ 7 x 106, and 1 in ∼ 19 x 106 spleen cells, respectively. We concluded that relatively advanced differentiation of potentially competent cells occurs before sheep erythrocyte administration. The relevance of this finding for the broad spectrum of immunologic reactivities and for the heterogeneity of antibody responses to given antigens was discussed.

Spleen cells of (C57BL/6 X C3H/He)F1 mice were assayed for natural killer (NK) cell activity against YAC-1 and FBL-3 lymphoma targets at several intervals after total-body exposure to a high sublethal dose of 137Cs or 60Co gamma-rays. NK cell activity did not decline for the first 12 days but decreased sharply thereafter and remained low until day 24. The recovery of splenic NK cell activity was delayed. Beginning on day 28, the activity was slowly increased, reaching near-normal levels (80% of controls) 41-59 days after irradiation. Suppressor cells detectable during the period of lowest NK cell activity, i.e., on days 17 and 19, may have been responsible for the delayed and slow recovery. These studies indicated that a) mature effectors of natural cytotoxicity are relatively radioresistant renewable cells with a lifespan of about 2 weeks whose progenitors are radiosensitive cells b) the kinetics of decline and especially of recovery of NK cell activity may be influenced by suppressor cells. Should NK cell activity confer resistance to autochthonous lymphomas in vivo, it may be a significant consideration for strategies of tumor therapy by cytotoxic agents that reconstitution of the NK cell pool is a slow process and that suppressor cell function must be overcome for full recovery.

The proliferation and differentiation of hemopoietic cells from genetically anemic Wv/Wx,W/Wv, and Wv/Wv mice, and from nonanemic carrier W/+, Wb/+, and Wv/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method. Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice. Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis. The severity of the hemopoietic impairment was gene-dose dependent. The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis. The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in uitro were neither decreased in number nor impaired in proliferation and differentiation. Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells.The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in nitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line. Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series. W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation.

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