Studies on the induction of tolerance of the H-Y antigen in mice with neonatal skin grafts.

In contrast to the uniform rejection of adult male skin isografts by C57BL/6 females, neonatal male skin isografts are frequently accepted. Moreover, 50% of all females which accept a neonatal male skin graft for 50 days accept a subsequent adult male skin graft as well. This ability of neonatal skin to produce tolerance has been investigated under a variety of experimental conditions. The results indicate: (a) Even when a newborn male skin graft is transplanted concomitantly with an adult graft, it can produce tolerance of the latter although it is less effective in this regard than when transplanted beforehand. (b) The continued exposure of the host to the newborn graft is vitally important in maintaining the unresponsive state; and most females deprived of these grafts for 50 days manifest an immune response when challenged with adult male skin. (c) Newborn male skin isografts raised on adult females are not as antigenic as normal male skin grafts. (d) Occasionally, even a presensitized female can be rendered tolerant by grafting with neonatal male skin. (e) Neonatal male skin grafts are not accepted when transplanted to the spleens of adult females although they may occasionally induce tolerance of a subsequent orthotopic adult male skin graft. The failure of these intrasplenic grafts to survive can be attributed at least partly to their small size since orthotopic grafts of comparable size usually do not survive. (f) Females bearing neonatal male skin grafts are not perceptible cellular chimeras. Because the unresponsive condition induced with neonatal skin is similar to that which results from multiparity, this latter condition has also received attention. In this regard it has been established that unlike the removal of a neonatal male skin isograft, the delayed grafting of isolated females with a previous history of multiparity does not result in many of them manifesting what may be considered an immune response. However, this delay in grafting does seem to impair the tolerance multiparity produces. The results are discussed in relation to other methods of producing tolerance in adult animals.

Immunogenicity and crossreactivity of specificity-associated markers on alloreactive T cells. Confirmation based on the model of tolerance abolition by adoptive transfer.

Syngeneic or parental strain T cells adoptively transferred into hybrid rats tolerant of third party alloantigens (L/DA tolerant of BN), in numbers insufficient to abolish tolerance, induce instead an active resistance to tolerance abolition with larger, usually effective dosages of donor cells. Of particular interest is the finding that immunization with T cells from one parental strain donor (e.g., DA) inhibited the tolerance-abolishing alloreactivity (anti-BN) of subsequently transferred T cells from the same (DA) and the other (L) parental strain donor. We conclude that anti-MHC receptors on T cells from different genetic backgrounds reactive to the same third party alloantigens share the same conserved immunogenic specificity-associated markers (SAM). The nonpolymorphism of anti-MHC receptors shown here in the transplantation tolerance model is a confirmation of the same conclusion drawn from earlier studies with the GVHD-resistance model, and it therefore suggests that these two models of T cell MHC interactions involve very similar mechanisms of T cell idiotypic regulation.

Influence of size and gene dosage on the survival of skin allografts on rats rendered tolerant at birth.

The attributes of the test grafts with which putatively tolerant rats are challenged influence their immune response. Lewis (Lew) rats inoculated at birth with Lew/BN F1 hybrid bone marrow cells accept large skin allografts more readily than small allografts, and F1 hybrid skin grafts survive better than BN transplants. The results indicate that the survivals of these major histocompatibility complex-incompatible grafts are determined by the same factors that operate when only weak histoincompatibilities prevail.

Studies on the conservation of epidermal specificies of skin and certain mucosas in adult mammals.

To determine whether the factor(s) responsible for the conservation of epidermal specificities in adult guinea pigs and hamsters resides in the germinal layer of the epidermis or in the dermis, thin grafts of skin, possessing qualitatively distinct regional characteristics, were separated into their superficial epidermal and dermal components with the aid of trypsin. Dermis of one type was combined with epidermis of another to produce "recombinant" grafts which were then transplanted to small, full thickness cutaneous sites on the thorax of geneticaily compatible hosts. A variant of this procedure involved transplanting sheets of superficial epidermis of various types to shallow split thickness recipient areas in the skin of the thorax. All grafts were maintained for 100 days before they were excised and examined histologically. The results indicate that, whereas the dermis determines the kind of epidermis produced in recombinant grafts involving the ear, the sole of the foot, and the trunk, this is not the case in recombinants which include tongue, esophageal, or cheek pouch epithelia. The one exception to this occurred when tongue or esophagus epithelia were transplanted to split thickness beds in trunk skin. Here they appeared to produce an epidermis characteristic of their new location. It is believed that this exception is probably due to the fact that the native follicular epidermis present in trunk dermis made such a substantial contribution to the new superficial epidermis that it behaved overtly as body skin epidermis. Taken together, these results suggest that basal layer cells of the superficial epidermis of sole of foot skin, ear skin, and the hair-bearing skin of the general integument behave as if they are equipotential, and that in adult life maintenance of these particular epidermal specificities is the outcome of persistent specific inductive stimuli from the underlying dermis. The results of subsidiary experiments are reported which indicate that the epithelial component of mammary gland tissue is also pluripotential, being capable of producing, under appropriate conditions, a normal-looking, fully stratified superficial epidermis.

Studies on the immunotherapy of runt disease in rats.

Using rats of the Lewis and BN (Ag-B locus incompatible) isogenic strains, a comparative study has been made of the capacity to prevent or mitigate the development of runt disease with: (a) lymph node cell suspensions from normal adult BN rats, (b) node cells, or (c) serum from donors sensitized against Lewis tissue antigens, or (d) heterologous anti-lymphocyte serum (ALS) raised in rabbits against rat thymocytes. Following a standard intravenous or intraperitoneal inoculation of 20 x 10(6) Lewis node cells into neonatal BN hosts, there are cutaneous manifestations of runt disease within 11-15 days and death invariably takes place within 20 days. However, complete protection is afforded by administration of a similar number of normal BN node cells via a different vein, or admixed with the otherwise harmful Lewis node cells. However, timing of the administration was crucially important-precedence or delay by as little as 4 hr resulted in a great impairment of protection. When the inoculations of the two cell suspensions were separated by 24 hr, no protection was afforded. These and other observations suggested that a necessary condition for protection of the hosts by unsensitized isologous cells requires that they establish a prompt and intimate confrontation with the homologous target cells. At the same dosage level, suspensions of node cells from sensitized isologous donors were much more effective therapeutically, saving the lives of 92% of treated subjects when administered after a delay of 3 days, and of 19% when the delay was 4 or 5 days. Of the various immunotherapeutic agents studied, daily injections of 0.2 ml of isoantiserum gave the best results, and could totally reverse the course of the disease even when initiated at age 10-13 days and subjects already presented symptoms. ALS, although inferior to isoantiserum at the dosage levels tested, proved to be superior to sensitized isologous cells as a protective agent, since the initiation of daily injections after delays of 6 or 8 days were still effective. The observations that delayed treatments of infant rats with isoantisera or ALS resulted in complete recoveries sustain the thesis that the lesions responsible for the fatal outcome of runt diseases are not inflicted at a very early stage. The efficacy of both isoantisera and ALS as a means of inhibiting the progression of homologous disease also suggests that they may have therapeutic value in situations where this condition is encountered.

Studies on the migratory behavior of melanocytes in guinea pig skin.

Pigment spread is the natural or experimentally procured (through grafting) progressive encroachment of pigmentation from black or red skin areas into juxtaposed white skin areas, or from black skin areas into red skin areas in spotted guinea pigs and other mammals. So far as spread from black into white skin is concerned, it had previously been shown that migration of epidermal melanocytes into skin lacking homologues of these cells was responsible. However, since red skin already has its own complement of phenotypically "red" melanocytes, the intriguing possibility remained that when black pigment encroaches upon red, rather than melanocyte migration being responsible, phaeomelanin (red)-producing melanocytes are transformed into eumelanin (black)-producing cells by some kind of serially transmissible factor derived from contiguous eumelanotic melanocytes. By utilizing two isogenic strains (Nos. 2 and 13) of spotted guinea pigs and their F(1) hybrids, the mechanism underlying the spread of pigment from black into red skin has been analyzed, employing cellular transplantation antigens as melanocyte "markers." The findings demonstrate unequivocally that a physical migration of pigment cells is responsible. By comparing the extents of pigment spread from black ear skin grafts, or from epidermal cell suspensions prepared therefrom, from parental strain or from F(1) hybrid donors in white host skin areas of F(1) hybrid guinea pigs, it has been possible to evaluate the influence of the intimate contact of melanocytes with alien transplantation antigens on their survival and migratory behavior. No evidence was forthcoming that pigment spread takes place less readily when the cells responsible are confronted by epidermal cells bearing foreign antigens than when they are confronted by cells of their own antigenic constitution. These findings are contrary to expectation if the phenomena of allogeneic inhibition or contact-induced cytotoxicity apply to normal cells in in vivo situations.

Skin homografts: tolerogenic versus immunogenic influences in mice.

In strain combinations involving multiple non-H-2 disparities, neonatal skin grafts may survive significantly longer than adult grafts of similar genotype on normal adult hosts, and repeatedly outlive grafts of adult origin on immunosuppressed recipients. Moreover, newborn grafts of long-standing may render their hosts unresponsive to adult skin grafts from the same donor strain. With some H-2-compatible strain combinations in which homozygous neonatal grafts are rejected, F(1) hybrid (heterozygous) grafts of similar age not only may survive indefinitely, but also may induce tolerance of subsequent adult parental strain homografts. These tolerogenic and gene dosage effects, although much weaker, can likewise be revealed with H-2-incompatible neonatal skin grafts.

The role of passenger leukocytes in the anomalous survival of neonatal skin grafts in mice.

The anomalous survival of neonatal C3H skin grafts on CBA mice is correlated with the emigration of passenger leukocytes from the graft vasculature. Thus, newborn homografts whose leukocyte populations are eliminated by X-irradiation or by transient sojourn on an intermediate adult C3H host, do not display prolonged survival. Moreover, the continued presence of the newborn grafts is not requisite to the maintenance of the unresponsive state, an observation consonant with the demonstration that CBA mice bearing long-term neonatal C3H skin grafts are leukocyte chimeras. In contrast, neonatal male C57 skin grafts may persist on C57 females after heavy irradiation of the donor, or after passage on an intermediate adult male host. In addition, tolerance is broken by removal of long-persistant newborn grafts from hitherto unresponsive females, and chimerism is not detectable in female C57 mice tolerant of infant male isografts. Finally, leukocytes of neonatal C3H origin, inoculated subcutaneously into CBA males, may occasionally render these animals unresponsive to subsequent adult C3H skin homografts, whereas those taken from infant C57 males usually sensitize their adult female hosts. Thus, passenger leukocytes are implicated in the extended survival of C3H neonatal homografts on CBA recipients, but not in the persistence of H-Y-incompatible neonatal skin isografts on C57 females.

Major histocompatibility complex (MHC) restriction of foreign transplantation antigens in rats rendered tolerant at birth.

Evidence is presented that MHC restriction of foreign transplantation antigens occurs when tolerance is induced. Whereas PVG and F344 rats rendered tolerant at birth with (DA X PVG)F1 and (DA X F344)F1 hybrid bone marrow cells (BMC), respectively, accept ACI skin grafts, presumably because the foreign transplantation antigens of these third party grafts, which are MHC-compatible with DA, are recognized only in association with the MHC of the hosts, DA rats rendered tolerant with (DA X PVG)F1 or (DA X F344)F1 hybrid BMC usually reject ACI skin. Further support that MHC restriction accompanies the induction of tolerance is provided by the observation that Lewis.1N rats rendered tolerant at birth with athymic (nude) Wag BMC are much more likely to accept BN.B2 (MHC-compatible with Wag) skin grafts, than BN (MHC-compatible with Lewis.1N) grafts.

Quantitative studies on the mixed lymphocyte interaction in rats. II. Relationship of the proliferative response to the immunologic status of the donors.

The influence of the immunologic status of the cell donors on the proliferative behavior of rat lymphocytes in the mixed lymphocyte interaction has been studied. Mixed cultures of cells from various parental and F(1) combinations having morphologically distinguishable sex chromosomes exhibited unidirectional proliferative reactivity. The mitotic figures were predominately of parental origin. Lymphocytes from donors made tolerant at birth to homologous transplantation isoantigens were specifically unreactive against cells bearing antigens of the tolerance inducing strain, but not to indifferent third party homologous lymphocytes. Cells from animals that had been surgically thymectomized at birth exhibited a markedly and sometimes totally diminished reactivity against homologous lymphocytes. Presensitization of the cell donors resulted in a curtailment of proliferative reactivity in cultures with cells bearing the immunizing antigens. This may reflect the destructive properties that lymphocytes from sensitized animals are known to possess. The results of these experiments show that the proliferative activity of lymphocytes in the mixed lymphocyte interaction accurately reflects the immunologic status of the cell donors, and these findings provide further support for the premise that the mixed lymphocyte interaction represents a primary immunologic response by cells in culture against homologous cells bearing histocompatibility antigens.

Cellular basis of tolerance in neonatally induced mouse chimeras.

In mice, thymectomized as young adults, neonatally induced tolerance persists in the putative absence of cell chimerism. The finding provides evidence that a selective deficiency of specific clones of lymphocytes exists in transplantation tolerance when induced under the conditions of these experiments.

Tolerance to parenchymal self. Regulatory role of major histocompatibility complex-restricted, OX8+ suppressor T cells specific for autologous renal tubular antigen in experimental interstitial nephritis.

BN rats develop interstitial nephritis after immunization with rabbit, but not rat renal tubular antigen. Using RT1n rat strains that differentially express tubular antigen, we investigated the unresponsiveness of BN rats to BN tubular antigen (BN-TBM) using delayed-type hypersensitivity (DTH) responses to BN-TBM as a measure of cell-mediated immunity. Our results indicate that rat strains expressing tubular antigen respond to immunization with BN-TBM with the clonal expansion of antigen-specific, cyclophosphamide-sensitive, OX8+, MHC-restricted suppressor T cells. Such suppression appears to be relevant to the maintenance of tolerance to parenchymal self, since chronic cyclophosphamide therapy abrogates suppression and results in significant interstitial nephritis.

The behavior of skin grafts incompatible with respect to skin alloantigens on mice rendered tolerant at birth with lymphoid cells.

It has been reported that lethally irradiated adult B6 mice restored with (A X B6) hemopoietic cells subsequently reject adult A-strain skin grafts because they are not tolerant of A-strain skin-specific (Sk) antigens. This thesis has been confirmed by a series of experiments conducted on neonatally treated animals. Thus sublethally irradiated neonatal B6 mice, inoculated with (A X B6) lymphoid cells, permanently accept these cells while subsequently rejecting adult strain A skin grafts. Further evidence that the destruction of these grafts results from the reaction of the host to Sk antigens, is provided by the fact that similarly treated recipients often permanently accept neonatal A-strain skin. Such grafts usually induce tolerance of adult A-strain skin grafts.

Influence of culturing on the survival of major histocompatibility complex-compatible and -incompatible thyroid grafts in rats.

Culturing Fischer thyroid fragments promotes their survival in major histocompatibility complex (MHC) -incompatible ACI rats but not in MHC- compatible Lewis animals.

"Intrinsic" immunological tolerance in allophenic mice.

Mice experimentally derived from pairs of conjoined, undifferentiated, cleavage-stage embryos of different histocompatibility genotypes can retain cells of each strain, which still produce their characteristic antigenic products. The animals are permanently tolerant of cells of both original types, remain free of runt disease, and display a normal and specific immune response to introduction of a foreign antigen. Absence of autoimmunity in development of ordinary animals is explainable by the "intrinsic" kind of tolerance found here.

Mixed leukocyte reactions and histocompatibility in rats.

Mixed leukocyte culture tests have been carried out on two hetero geneous but genetically defined backcross populations of rats to determine whether the reactions observed can provide the basis for histocompatibility matching. The experiments were designed so that only-one-way reactions could occur. Only when the donors of individual leukocyte mixtures differed at the important Ag-B histocompatibility locus was there any in vitro reactivity, and differences at this locus were invariably associated with the prompt rejection of skin homografts. Determination of compatibility at this locus proved to be important in that it facilitated the prolongation of survival of skin homografts by immunosuppressive therapy.

Genetic background and expressivity of histocompatibility genes.

A difference in the reactivity of F(1) hybrid female mice to skin grafts from male donors of each of their parental strains suggests that the genetic background can influence the efficacy of the Y antigen to elicit rejection of the graft.

Male specific antigen: homology in mice and rats.

Male lymphoid cells from some strains of rats can sensitize C57BL/6 female mice against C57BL/6 male skin grafts; this indicates that the male specific antigen of these two species is homologous.

H-Y antigen: behavior and function.

The factors are reviewed which affect the expression of H-Y antigen, a cell surface component that has been extensively analyzed in mice but which may be ubiquitous in all vertebrates. The phylogenetic stability of this antigen and its association with the Y chromosome indicate an important role in primary sex determination.

Spontaneous diabetes in rats: destruction of islets is prevented by immunological tolerance.

Spontaneous diabetes occurring in "BB" rats (derived from a colony of outbred Wistar rats) is the result of destruction of pancreatic islets by infiltrating mononuclear cells (insulitis) and may be a disease very similar to human juvenile onset diabetes. Both diseases probably have an autoimmune etiology. Evidence is presented that islets transplanted to diabetic BB rats are destroyed by the original disease process. Inoculation of bone marrow from normal (nondiabetes-susceptible) rat donors into neonatal BB recipients usually prevented the development of hyperglycemia.