Induction and mechanism of tolerance to bovine serum albumin in mice given total lymphoid irradiation (TLI).

BALB/c mice given total lymphoid irradiations (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.     

Late somatic effects in mice after total lymphoid irradiation

Late somatic effects of total lymphoid irradiation have been investigated in BC3F1 mice. A total X-ray dose of 34 Gy was distributed in 17 daily fractions. The cumulative mortality curve is shifted in time because all the irradiated mice died earlier than the unirradiated controls. There was a 24% shortening of life span. A marked increase of solid tumor incidence, mostly due to skin cancers, was observed (66% vs 30%). In contrast, the incidence of malignant lymphomas was greatly reduced in irradiated mice (6% vs 49%). Furthermore, nephrosclerosis was a common finding in the irradiated group (38% vs 8%). Death-rate analysis revealed an association between life shortening and the presence of solid tumors and nephrosclerosis at death.     

Strong and weak immune responses across the same major histocompatibility barrier in rats

Inbred rat strains, Fischer 344 (F-344) and Lewis (LEW), share the serologicalAg-Bl allele and react very weakly in mixed lymphocyte culture (MLC). Despite this apparent identity atAg-B, these strains differ markedly in their immune responses to anAg-B disparate third strain Marshall 520 (M-520) (Ag-B6). F-344 recipients allowed M-520 heart grafts an extended survival, whereas LEW recipients rejected them rapidly. F-344 and M-520 showed a weak response in MLC in contrast to a strong response for LEW and M-520. F-344 produced antisera in response to injection of M-520 cells that had a relatively high antibody titer but low cytotoxic activity. F-344 responded to another strain, Buffalo (BUF) (alsoAg-B6), in a similar fashion. F-344 apparently can produce a strong allogeneic response, as it was able to rapidly reject heart grafts from (LEW x Brown-Norway) F₁ donors (LBN) (Ag-B 1/3). The low response of F-344 to M-520 probably was not due to shared antigens between the two strains because M-520 heart grafts underwent rapid rejection in LEW hosts highly tolerant to F-344. To explain the contrasting response of F-344 and LEW to theAg-B6 disparity, we propose that it is controlled by an immune-response gene(s); that F-344 has a low-responding allele and LEW has a high-responding allele. The data do not reveal a location for this proposed gene. The high-responding allele appears to be dominant, as M-520 hearts were rejected rapidly by (F-344 x LEW) F₁ recipients.     

Split tolerance in nude mice transplanted with 2'-deoxyguanosine-treated allogeneic thymus lobes

To elucidate the acquisition of self tolerance in the thymus, full-allogeneic thymic chimeras were constructed. Athymic C3H and BALB/c nude mice were reconstituted with the thymic lobes of BALB/c and B10.BR fetuses, respectively, that were organ cultured for 5 days in the presence of 2'-deoxyguanosine. T cells in these chimeras were tolerized to the host MHC in both MLR and CTL assays. In contrast, T cells in the chimeras exhibited split tolerance for the thymic MHC haplotype. CTL specific for class I MHC of the thymic haplotype were generated not only from the peripheral T cells of the chimeras but also from thymocytes re-populated in the engrafted thymic lobes. However, T cells in these chimeras responded poorly to the class II MHC of the thymic haplotype in a standard MLR assay. In a syngeneic MLR culture upon stimulation with enriched APC of the thymic haplotype, only 22 to 48% of the responses were mediated by CD4+ cells, and proliferations of CD4- cells were prominent. There were no haplotype-specific suppressor cells detected which would cause the unresponsiveness to the thymic class II MHC. These results indicated that the thymic lobes treated with 2'-deoxyguanosine were defective in the ability to induce the transplantation tolerance for the class I MHC expressed on the thymus, although the same thymic lobes were able to induce the transplantation tolerance for the thymic class II MHC.     

Pathways analysis of differential gene expression induced by engrafting doses of total body irradiation for allogeneic bone marrow transplantation in mice

A major challenge in allogeneic bone marrow (BM) transplantation is overcoming engraftment resistance to avoid the clinical problem of graft rejection. Identifying gene pathways that regulate BM engraftment may reveal molecular targets for overcoming engraftment barriers. Previously, we developed a mouse model of BM transplantation that utilizes recipient conditioning with non-myeloablative total body irradiation (TBI). We defined TBI doses that lead to graft rejection, that conversely are permissive for engraftment, and mouse strain variation with regards to the permissive TBI dose. We now report gene expression analysis, using Agilent Mouse 8x60K microarrays, in spleens of mice conditioned with varied TBI doses for correlation to the expected engraftment phenotype. The spleens of mice given engrafting doses of TBI, compared with non-engrafting TBI doses, demonstrated substantially broader gene expression changes, significant at the multiple testing-corrected P <0.05 level and with fold change ≥2. Functional analysis revealed significant enrichment for a down-regulated canonical pathway involving B-cell development. Genes enriched in this pathway suggest that suppressing donor antigen processing and presentation may be pivotal effects conferred by TBI to enable engraftment. Regardless of TBI dose and recipient mouse strain, pervasive genomic changes related to inflammation was observed and reflected by significant enrichment for canonical pathways and association with upstream regulators. These gene expression changes suggest that macrophage and complement pathways may be targeted to overcome engraftment barriers. These exploratory results highlight gene pathways that may be important in mediating BM engraftment resistance.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vbeta11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).     

Induction of allograft tolerance after total lymphoid irradiation (TLI): development of suppressor cells of the mixed leukocyte reaction (MLR).

We searched for the presence of suppressor cells of the MLR in C57BL/Ka leads to BALB/c chimeras. The chimeras were made with total lymphoid irradiation (TLI) and marrow transplantation. Spleen cells from the old chimeras inhibited the MLR of BALB/c responder cells against C57BL/Ka stimulator cells. Inhibition was specific for the stimulator cells, since no effect on the MLR was observed with C3H or BALB.C3H stimulator cells. Maximal inhibition was achieved when the responder cells in the MLR shared the H-2 haplotype of the chimeric recipient. Spleen cells obtained from chimeras young 30 to 40 days after BM transplantation inhibited the MLR nonspecifically, since similar marked inhibition was observed regardless of the H-2 haplotype of the responder or stimulator cells. The finding of antigen-specific and nonspecific suppressor cells is similar to that observed in mice rendered tolerant to bovine serum albumin after treatment with TLI.     

The development of humoral immunity to tissue-specific tubular basement membrane alloantigens after renal transplantation across the major histocompatibility barrier in rats immunomodulated with blood transfusions and cyclosporin

The development of a humoral immune response to the tubular basement membrane (TBM) alloantigen of Brown-Norway (BN) rat kidneys was studied after transplantation of BN rat kidneys into bilaterally nephrectomized Lewis (LEW) rats. The LEW rat recipients consisted of four groups receiving no form of immunosuppression, pretransplantation cyclosporin alone, or pretransplantation donor-specific or donor-nonspecific transfusions combined with cyclosporin. The latter two regimens induce indefinite allograft survival in the majority of recipients. Circulating antibody to collagenase-solubilized BN rat renal basement membrane (CS-BN-RBM) was present in all four groups of transplant recipients within 1 week after transplantation, and no significant differences in antibody levels were noted between rats receiving no immunosuppression (survival of 1-2 weeks) and the groups of rats who received various immunosuppressive regimens and survived longer. Circulating antibody to BN-CS-RBM continued to increase in quantity in the cyclosporin-treated group until the time of death (2-10 weeks post-transplantation). In the much longer lived combined transfusion and cyclosporin-treated groups, circulating antibody to BN-CS-RBM generally attained a maximum at approximately 2 to 4 months post-transplantation and then plateaued or decreased somewhat before the time of death (3-16 months post-transplantation). No correlation was found between quantity of circulating anti-BN-CS-RBM antibody and post-transplantation survival. Comparative study of the quantity of circulating antibody to BN-CS-RBM (the presumed nephritogenic antigen of experimental tubulointerstitial nephritis in the BN rat) in serum from transplant recipients as compared to serum from BN rats with severe experimental tubulointerstitial nephritis (TIN) (as induced by immunization with heterologous TBM antigens) demonstrated a greater quantity of potentially nephritogenic antibody circulating in transplant recipients than in BN rats with experimental TIN. Histologically, the transplanted kidneys in immunomodulated recipients demonstrated focal chronic interstitial inflammatory infiltrates with tubular atrophy and relative sparing of the glomeruli. The development of immune responses to tissue-specific alloantigens may become of clinical significance as graft-survival times are increased.     

Transplantation of metanephroi across the major histocompatibility complex in rats

To determine whether transplanted metanephroi grow, differentiate, and function in hosts that differ in major histocompatibility complex loci (RT1 loci in rats) from donors in a defined way, we implanted metanephroi from embryonic day (E) 15 PVG (RT1(c)) rat embryos into the omentum of nonimmunosupressed uninephrectomized PVG-RT1(avl) (host) rats. By 4 wk posttransplantation, metanephroi had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts had normal structure and ultrastructure. At 12 wk posttransplantation, weights of metanephroi were 54 +/- 8 mg. Inulin clearances were 0.9 +/- 0.3 microl. min(-1). 100 g rat wt(-1). In vitro, splenocytes from PVG rats stimulated the proliferation of cells originating from both PVG-RT1(avl) rats in which a transplant had been performed and PVG-RT1(avl) rats with no transplant. Full-thickness PVG-RT1(avl) skin engrafted normally on PVG-RT1(avl) rats in which PVG metanephroi had been previously implanted and metanephroi retained a normal appearance. In contrast, skin from PVG rats sloughed, and the tubular architecture of metanephroi was obliterated by a mononuclear cell infiltrate consistent with acute rejection. Here we show for the first time that functional chimeric kidneys develop from metanephroi transplanted across the MHC into nonimmunosupressed hosts and provide evidence that a state of peripheral immune tolerance secondary to T cell "ignorance" permits their survival.     

Reduced adiposity in ob/ob mice following total body irradiation and bone marrow transplantation

Objective: The objective of this study was to assess long‐term metabolic consequences of total body irradiation (TBI) and bone marrow transplantation. Severe obesity develops due to both hypertrophy and hyperplasia of adipocytes. We hypothesized that TBI would arrest adipose tissue growth and would affect insulin resistance (IR). Research Methods and Procedures: We exposed 2‐month‐old female ob/ob mice to 8 Grays of TBI followed by bone marrow transplantation and tested the animals for body weight (BW) gain, body composition, blood glucose, and insulin sensitivity. Results: Two months after TBI, irradiated mice stopped gaining BW, whereas non‐treated mice continued to grow. At the age of 9.5 months, body mass of irradiated mice was 60.6 ± 1.4 grams, which was only 61% of that in non‐treated ob/ob controls (99.4 ± 1.6 grams). Body composition measurements by DXA showed that decreased BW was primarily due to an impaired fat accumulation. This could not result from the production of leptin by bone marrow‐derived adipocyte progenitors because inhibition of the obese phenotype was identical in recipients of both B6 and ob/ob bone marrow. Inability of the irradiated mice to accumulate fat was associated with hepatomegaly, lower levels of monocyte chemoattractant protein‐1 expression in adipose tissue, and increased IR. Discussion: Our data argue in favor of the hypothesis that inability of adipose tissue to expand may increase IR. This mouse model may be valuable for studies of late‐onset radiation‐induced IR in humans.     

Evidence for the control of Eosinophilia by the major histocompatibility complex in mice

The genetic control of eosinophilia has been studied in congenic strains of mice. Eosinophilia was induced with cyclophosphamide followed by keyhole limpet hemocyanin in complete Freund's adjuvant. After this treatment, BALB/c mice developed a high eosinophil response, whereas CBA, C57BL and A/J mice developed a low one. The major histocompatibility complex (M-HQ was found to exert a control on eosinophilia, as B 10.D2 mice developed a higher eosinophil response than B10, B10.A, or B10.BR. BALB/c-H-2 ^k mice had a lower response than BALB/c, and A.TL mice had a higher response than A/J or A.TH. If a single gene within the MHC is responsible for these effects, the most likely position for it is in the vicinity of the Tla locus. Splenectomy reduced eosinophilia in BALB/c and A.TL mice, but not in A/J mice,indicating that the spleen is a significant site of eosinophil production in high responder strains.     

Induction of transplantation tolerance in non-human primate preclinical models

Short-term outcomes following organ transplantation have improved considerably since the availability of cyclosporine ushered in the modern era of immunosuppression. In spite of this, many of the current limitations to progress in the field are directly related to the existing practice of relatively non-specific immunosuppression. These include increased risks of opportunistic infection and cancer, and toxicity associated with long-term immunosuppressive drug exposure. In addition, long-term graft loss continues to result in part from a failure to adequately control the anti-donor immune response. The development of a safe and reliable means of inducing tolerance would ameliorate these issues and improve the lives of transplant recipients, yet given the improving clinical standard of care, the translation of new therapies has become appropriately more cautious and dependent on increasingly predictive preclinical models. While convenient and easy to use, rodent tolerance models have not to date been reliably capable of predicting a therapy's potential efficacy in humans. Non-human primates possess an immune system that more closely approximates that found in humans, and have served as a more rigorous preclinical testing ground for novel therapies. Prior to clinical adaptation therefore, tolerance regimens should be vetted in non-human primates to ensure that there is sufficient potential for efficacy to justify the risk of its application.     

Induction of oral tolerance in mice by continuous feeding with beta-lactoglobulin and milk

We examined the immune response of mice to beta-lactoglobulin (BLG), a potent milk allergen, after continuous feeding of a BLG solution or milk instead of drinking-water. Strong suppression of the anti-BLG antibody response and antigen-specific T cell response were observed in mice fed BLG and milk. Although the profile of the antibody specificity to BLG peptides in mice fed BLG or milk was different from the control, the dominant determinants were still recognized and a limited number of new recognition sites appeared by BLG or milk feeding. These findings suggest that continuous feeding with BLG or milk not only induced tolerance in the periphery, but also priming. In terms of the antigen-specific IgG subclass response, the production of both IgG1 and IgG2a was significantly reduced. The cytokine secretion of interleukin (IL)-2, IFN-gamma and IL-10 was also reduced in the culture supernatants of lymph node cells from the mice fed BLG. The results indicate that the continuous feeding of BLG or milk induces suppression of both Th1- and Th2-dependent responses. This may reflect a state of oral tolerance induced by food ingestion.