Pathological changes in F1 hybrid mice following transplantation of spleen cells from donors of the parental strains

It has already been reported by one of us (Boyse, 1959) that intraperitoneal transplants of spleen cells from A strain donor mice that had been immunized against C57BL strain tissues were invariably lethal to hosts of the F₁ (C57BL×A) constitution.

In this report an account is given of the pathological changes in F₁ hybrid hosts that have received intravenous or intraperitoneal transplants of spleen cells from parental strain donors, with particular reference to lethal transplants. Here the outstanding features are splenomegaly, wasting, progressive histiocytosis in the spleen and lymph nodes, disappearance of lymphoid tissue, focal necrosis of the spleen and cellular infiltrations round the hepatic vessels. Following transplantation by the intraperitoneal route there are also severe pancreatic lesions associated with fat necrosis and ascites.

Differential (donor/host) cell counts of host spleen cell suspensions indicate that the spleen may be virtually replaced by donor cells, the majority of which are histiocytes. Erythropoietic function is progressively supplanted by donor tissue.

Similar, but reversible and less extensive changes occur with other (non-lethal) donor/F₁-host combinations. These hosts do not waste and the proportion of donor cells included in the host spleens falls until their detection, by present methods, is no longer possible.

     

Life-long tolerance and chimerism in parental mice induced with F 1 hybrid cells

We have induced specific immunological tolerance towards (C57BL × CBA-T6T6) F₁ skin grafts in newborn recipients of the parental CBA strain by one injection of 20 × 10⁶ or 40 × 10⁶ F₁ spleen cells. Approximately 80 % of the CBA recipients were highly tolerant (the grafts survived for more than 50 days). Many of the highly tolerant mice retained fully preserved grafts until natural death. The highly tolerant CBA mice had dividing (C57BL × CBA-T6T6) F₁ cells in their spleen (12–13 %) and lymph nodes (12–15 %). The proportion of these cells decreased with time, but we found a few of them even at 567 days of life (0.7 and 4.2 %, respectively). We have concluded that specific immunological tolerance to tissue grafts induced at birth can last throughout the entire life-span of a mouse. The tolerance persists in spite of a very low proportion of dividing donor cells within the lymphoid tissue of tolerant hosts.     

CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice

Spleen cells from F₁ hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F₁ donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F₁ mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c × C57BL/6) F₁ hybrid (CB6F₁) nulnu mice or from euthymic CB6F₁ mice depleted from birth of CD4⁺ T cells, but not of CD8⁺ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F¹ nulnu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F₁ nulnu mice to induce neonatal tolerance to alloantigens is directly related to the long-term absence of mature CD4⁺ T cells. Interestingly, a new increase in the titers of anti-DNA Ab was observed when spleen cells from athymic CB6F₁ nulnu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F₁ spleen cells. This finding indicates that B cells from CB6F₁ nulnu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4⁺ T cells. These data indicate that the continuous absence of CD4⁺ T cells causes a reversible functional defect in F₁ spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.     

The fate of mouse spleen cells transplanted into homologous and F1 hybrid hosts

The fate of antibody-producing mouse spleen cells transplanted intraperitoneally into isologous, homologous and F₁ hybrid mice has been investigated by the use of donors hyperimmunized against sheep or human red cells. Survival of the transplanted cells was indicated by a rising titre of haemagglutinins in the host or by a secondary response to challenge with a critical dose of red cells. This dose was sufficient to evoke a vigorous secondary response in immunized mice but only a weak primary response in unimmunized mice.

Homotransplants between the C57BL and A strains did not survive in a functional state for longer than three days. It is suggested that antibody-producing cells are particularly susceptible to immune attack and may therefore be eliminated sooner than other types of cell.

In genetically-compatible (C57BL×A)F₁ hosts, transplants of C57BL cells survived for about two weeks: cells from isoimmune donors (immunized against the other parental strain) probably died out more rapidly. Transplants of A strain cells survived in some instances for four to five weeks: transplants from isoimmune donors invariably killed their hosts.

It is suggested that the law which states that the tissues of an inbred strain are compatible with hybrid hosts derived by crossing that strain with another may require qualification when applied to cells which produce antibody. In such circumstances the outcome may depend upon the innate resistance of the transplanted cells to continued antigenic stimulation. Where this is high, as in the A strain, the host may die: where it is low, as in the C57BL strain, the transplant may die.

     

Critical role of NK but not NKT cells in acute rejection of parental bone marrow cells in F1 hybrid mice.

F1 hybrid mice vigorously reject transplanted parental bone marrow (BM) cells, which is a phenomenon called "hybrid resistance (HR)". Since NK1.1(+) cells play crucial role in HR, both NK1.1(+)CD3(+) NKT cells and NK1.1(+)CD3(-) NK cells have been possible candidates of effector cells. To elucidate the major effector cells in HR, we employed Rag-2(-/-) mice devoid of T, B, and NKT cells and cytokine receptor common gamma subunit and Rag-2 double-deficient (gamma(c)(-/-(y))-Rag-2(-/-)) mice lacking all lymphoid cells including NK cells. Rag-2(-/-) F1 hybrid mice rejected parental BM cells to the extent similar to wild-type (WT) F1 hybrids. In contrast, male gamma(c)(-/y)-Rag-2(-/-) F1 hybrid mice were unable to reject parental BM cells. After reconstitution with NK but not NKT cells, male gamma(c)(-/y)-Rag-2(-/-) F1 hybrid mice restored the ability to reject parental BM cells. Collectively, it is concluded that NKT cells play little role, if any, and NK cells are the only cells involved in HR.     

Hypothalamo-pituitary-adrenocortical dysregulation in aging F344/Brown-Norway F1 hybrid rats

Hypothalamo-pituitary-adrenocortical (HPA) axis aging was studied in young (3 mo), middle aged (15 mo) and aged (30 mo) F344/Brown Norway hybrid rats. This strain was selected to obviate HPA-relevant pathologies found in other aging models. Aged, unstressed rats showed enhanced central HPA drive, marked by elevated ACTH release and decreased pituitary proopiomelanocortin and corticotropin-releasing factor receptor 1 (CRH-R1) mRNAs. Acute corticosterone responses to spatial novelty were exacerbated in aged rats; however, responses to restraint or hypoxia were not affected. Chronic stress exposure also differentially increased HPA drive in aged animals, marked by elevated paraventricular nucleus CRH peptide levels and pituitary proopiomelanocortin mRNA. Plasma ACTH and pituitary POMC and CRH-R1 mRNA expression in middle-aged rats were intermediate those of young and aged animals. Middle-aged animals responded to chronic stress with disproportionate increases in CRH mRNA levels, and increased corticosterone secretion following hypoxia but not novelty. The results suggest a gradual increase in HPA tone across the aging process, culminating in marked hyperresponsivity to both acute and chronic stress in senescence.     

Depletion of asialo-GM1+ cells from the F1 recipient mice prior to irradiation and transfusion of parental spleen cells prevents mortality to acute graft-versus-host disease and induction of anti-host specific cytotoxic T cells.

Graft-versus-host disease (GVHD) was induced in irradiated (750 rad) (CBA x C57BL/6)F1 hybrid mice by an intravenous injection of 30 x 10(6) CBA spleen cells and 5 x 10(6) syngeneic F1 bone marrow cells. The GVHD resulted in the death of 80% of recipients within 9 days. However, when radioresistant Asialo-GM1+ cells were depleted from the recipients with a single injection of anti-Asialo-GM1 antibody 2 days before irradiation and transplantation, mortality decreased significantly (to 11%). During the GVHD, anti-host specific cytotoxic T cell (CTL) activity could be shown in vitro in the spleens of mice suffering from the GVHD if suppressor activity was first abolished by in vitro culture procedures. This CTL activity, however, was not detectable in the spleens of anti-ASGM1 antibody pretreated hosts. The results indicate that radioresistant ASGM1+ cells of host origin are necessary for the induction of both anti-host CTL and lethal GVHD.     

Schedule-induced polydipsia in F1 hybrid rats of wild-caught and domestic Norway rats

Five F1 hybrid rats of a wild-caught Norway male rat and a domestic, albino Norway female rat and five domestic, albino Norways rats were tested on fixed-time 1-min food schedules for the development of schedule-induced polydipsia. All five F1 hybrid rats and three domestic rats developed polydipsia. These data generalized schedule-induced polydipsia to the F1 hybrid generation of wild-caught and domestic rats. The implications of these results for Symons and Sprott's [12] suggestion of a genetic component for schedule-induced polydipsia are discussed.     

Experimental myositis in rats. II. The sensitivity of spleen cells to syngeneic muscle antigen.

The transformation responses to a syngeneic muscle homogenate of spleen cells, lymph node cells and blood lymphocytes have been studied in rats with allergic myositis. Significantly increased responses to muscle were obtained only for spleen cells from rats which had received two or more immunizing injections of heterologous muscle with Freund's complete adjuvant. Other populations of lymphoid cells which had previously been shown to be capable of transferring the disease did not show significant transformation responses to muscle. Increased responses in spleen cells were obtained using a supernatant ultracentrifugal fraction as well as a whole muscle homogenate. Fractionation of the antigen showed blast stimulating activity to be present in fractions of differing molecular size, suggesting that the antigen(s) responsible may be present in polymeric form or may be bound to molecules of varying molecular size.     

Radiosensitive nature of paravascular infiltrate-producing potential of parental spleen cells

The injection of spleen cells from donors of parental strain into F₁ hybrid mice results in paravascular infiltration (PVI) in the portal tracts of the recipients' livers. The infiltrates can be enumerated. Whole-body irradiation (802 r) of the recipients before the injection of parental spleen cells does not reduce the number of PVI formed. Irradiation (1500 r) of the donors eradicates the capacity of their spleen cells to produce PVI. In vivo radiation inactivation experiments indicate that the LD₃₇ for donor PVI-producing cells is approximately 47 r and the D₀ is 102 r. These data support the contention that the formation of PVI is a function of donor cells and depends upon cell division.