Host-versus-graft disease in mice after the induction of neonatal transplantation tolerance by two different methods

Newborn A mice were injected either with a single i.v. dose (Group A) or with repeated doses of (B10 x A)F1 semiallogeneic spleen cells (SSC) (Group B). A similar degree of partial transplantation tolerance (TT) to B10 skin allografts was revealed in both groups. No signs of acute, rapidly fatal host-versus-graft disease (HVGD) (anemia, leukocytosis, severe thrombocytopenia, hepatic infarcts, gastrointestinal bleedings) were found, rather a chronic HVGD developed [moderate thrombocytopenia, autoimmune antithymocyte antibodies (ATA)] in both groups. The mortality due to lymphoproliferative disorders (LPD) was significantly higher in Group A. Thus, repeated perinatal injections of (B10 x A)F1 SSC into A mice did not increase the tolerogenic and the LPD-inducing effect either, and they did not elicit acute HVGD in contrast to observations in other F1 donor-recipient combinations [1]. Consequently, the development of acute HVGD depends on immunogenetic factors and not on the repeated administration of SSC.     

CD40 ligation prevents neonatal induction of transplantation tolerance

To investigate the consequences of CD40 engagement on the neonatal induction of transplantation tolerance, BALB/c mice were injected at birth with (A/J x BALB/c) F1 spleen cells together with activating anti-CD40 mAb and grafted 4 wk later with A/J skin. Whereas A/J allografts were accepted in mice neonatally injected with F1 cells and control Ab, they were acutely rejected in mice injected with F1 cells and anti-CD40 mAb. Neonatal administration of anti-CD40 mAb resulted in enhanced anti-A/J CTL activity, increased IFN-gamma, and decreased IL-4 production by donor-specific T cells in vitro. Experiments using anti-cytokine mAb and IFN-gamma-deficient mice demonstrated that CD40 ligation prevents neonatal allotolerance through an IFN-gamma- and IL-12-dependent pathway. Finally, we found that newborn T cells express less CD40L than adult T cells upon TCR engagement. Taken together these data indicate that insufficiency of CD40/CD40L interactions contribute to neonatal transplantation tolerance.     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J x BALB/c)F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-gamma in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. III. Inhibition of the IgG Response to noncongenic cells by preimmunization with congenic cells

When B10.A (5R) mice (H-12i5) are immunized with spleen cells from congenic B10 mice (H-12b), they respond to alloantigens of the H-2Db region by producing antibodies of only IgM type. In contrast, they produce both IgM and IgG antibodies when immunized with A.BY cells (H-2b) that carry other foreign cell surface antigens (non-H-2) in addition to H-2Db. Preimmunization of 5R mice with two injections of congenic cells leads to an H-2Db specific inhibition of the IgG response to a subsequent immunization with A.BY cells. It is concluded that congenic B10 cells fail to activate helper T cells which are necessary to induce the switch from IgM to IgG production. Instead T- or B-cell tolerance may be induced with prohibits the subsequent IgG response to A.BY cells, possibly by way of suppressor T cells which may act either on B cells directly or via helper T cells.     

Coupling of palmitate to ovalbumin inhibits the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of A1(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.     

Prevention of marrow graft rejection without induction of graft-versus-host disease by a cytotoxic T-cell clone that recognizes recipient alloantigens

In allogeneic marrow transplantation, donor T cells that recognize recipient alloantigens prevent rejection but also cause graft-versus-host disease (GVHD). To evaluate whether the ability to prevent marrow graft rejection could be dissociated from the ability to cause GVHD, we generated a panel of four different CD8 cytotoxic T-lymphocyte clones specific for H2(d) alloantigens. Three of the clones caused no overt toxicity when as many as 20 x 10(6) cells were infused intravenously into irradiated H2(d)-positive recipients, and one clone caused acute lethal toxicity within 1 to 3 days after transferring 10 x 10(6) cells into H2(d)-positive recipients. One clone that did not cause toxicity was able to prevent rejection of (C57BL/6J x C3H/HeJ)F1 marrow in 800 cGy-irradiated (BALB/cJ x C57BL/6J)F1 recipients without causing GVHD. Large numbers of cells and exogenously administered interleukin-2 were required to prevent rejection. These results with different CD8 clones suggest that GVHD and prevention of rejection could be separable effects mediated by distinct populations of donor T cells that recognize recipient alloantigens.     

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.     

The role of cell migration and microchimerism in the induction of tolerance after solid organ transplantation

A new hypothesis has been proposed which states that microchimerism is the basis for the clinical tolerance seen in long-term survivors of solid organ transplants. Efforts to enhance microchimerism include simultaneous infusion of bone marrow of donor origin and transplantation of a solid organ. Studies are in progress to verify the phenomenon of microchimerism and its role in clinical tolerance.     

Control of neonatal tolerance to tissue antigens by peripheral T cell trafficking

Self tolerance is acquired by the developing immune system. As reported here, particular properties of the neonatal tissue contribute to this process. Neonatal skin, but not adult skin, was accessible for na?ve CD8 T cells. In mouse bone marrow chimeras generated at different ages, recent thymic emigrants were tolerized to a skin-expressed major histocompatibility complex class I antigen only during a neonatal period but not during adulthood. Blockade of T cell migration neonatally prevented tolerance induction. Thus, T cell trafficking through nonlymphoid tissues in the neonate is crucial for the establishment of self tolerance to sessile, skin-expressed antigens.     

Neonatal disease profile in Larkana before and after establishment of neonatal ward

This is a study of 2050 neonatal admissions excluding neonatal tetanus in children hospital CMC Larkana from December, 1988 to July, 1993. Six hundred and eighty-nine cases were admitted in General Paediatrics Ward before establishment of neonatal unit and 1361 cases were admitted in neonatal ward. The aime of study was to compare the disease pattern and mortality rates before and after establishment of a neonatal unit in the same hospital. Number of admissions significantly increased after the establishment of neonatal unit but there was no decline in the mortality. Changes in disease pattern were observed particularly for Gastroenteritis and miscellaneous (undetermined) category. Seventy percent of admissions were males in Ist week of their life. Commonest cause of admission and mortality was birth anoxia.     

Association between the Rfp-Y haplotype and the incidence of Marek's disease in chickens

Certain haplotypes at the major histocompatibility (B) complex (Mhc) of the chicken provide an easily demonstrated influence on tumor formation following infections with Marek's disease virus (MDV). Recognition that there is a second histocompatibility complex of genes in the chicken, Rfp-Y, comprised of Mhc class I and class II genes, some of which are at least transcribed, evokes the question of whether this gene complex might also influence the outcome of MDV infections. To test this hypothesis, pedigree-hatched chicks in families from the original Rfp-Y-defining stock in which three Rfp-Y and two B system haplotypes are segregating were challenged with the RB1B strain of MDV. Birds with the Y3/Y3 genotype were found to have 2.3 times the risk of developing a tumor compared with birds with other Rfp-Y genotypes combined (P <0.02). Additionally, birds carrying the BR9/B11 genotype had 2.3 times the risk of tumor formation, relative to birds with the B11/B11 genotype (P <0.02). We found no evidence for an interaction between genotypes within the B and Rfp-Y systems. These data provide evidence that Rfp-Y haplotypes, as well as B haplotypes, can significantly influence the outcome of infection with MDV.     

Prevention of graft-versus-host disease and the induction of transplant tolerance by low-dose UV-B irradiation of BM cells combined with cyclosporine immunosuppression

GVHD is prevented and stable chimerism is induced in the rat BMT model by 700 J/m2 but not 100-500 J/m2 UV-B irradiation of allogeneic BM cells. Paradoxically, CsA which prevents GVHD in clinical BMT causes an aggressive autoimmune disease termed syngeneic GVHD in irradiated syngeneic BMT recipients after its withdrawal. Recently, we have shown that while 500-700 J/m2 UV-B irradiation of syngeneic BM cells combined with a 30-day course of CsA recipient immunosuppression impairs hemopoiesis due to lack of hemopoietic factors, a low dose of 100-300 J/m2 UV-B is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment. This study extends these findings to the P-to-F1 hybrid and fully allogeneic rat BMT models and examines the effectiveness of low-dose UV-B irradiation of BM cells combined with a short course of CsA treatment in the prevention of GVHD and induction of transplant tolerance. Lethally gamma-irradiated (10.5 Gy) LBNF1 recipients of naive or UV-B irradiated (100-700 J/m2) BMT were treated with CsA (12.5 mg/kg/day) for 30 consecutive days after BMT. All lethally irradiated LBNF1 that did not receive BMT died in < 16 days, while animals transplanted with UV-B (700 J/m2) BMT survived > 1 year without GVHD. In contrast, all recipients of naive BMT died of lethal GVHD in < 50 days. Similarly, all recipients of naive BMT that received a 30-day course of CsA therapy developed severe GVHD with 60% mortality after cessation of CsA therapy. CsA-treated recipients of BMT irradiated with 700 J/m2 died between 12 and 25 days from failure of hemopoiesis. In contrast, CsA-treated recipients of 100-200 J/m2 UV-B irradiated BMT showed full BM engraftment without GVHD after cessation of CsA and survived > 1 year. These results were reproducible in the fully allogeneic UV-B BMT model. To test for donor-specific tolerance, the animals challenged 100 days after BMT with cardiac allografts accepted permanently (> 100 days) Lewis but not BN (non-BMT parental donor) cardiac allografts. Our results confirm that 700 J/m2 UV-B irradiation of BM cells combined with CsA recipient immunosuppression impairs the recovery capacity of stem cells while the use of lower UV-B (100-200 J/m2) is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment and leads to induction of transplant tolerance.     

Restoration of T cell-independent type 2 induction of Ig secretion by neonatal B cells in vitro

The humoral immune response of neonates to T cell-independent type 2 (TI-2) Ags is markedly defective. We previously demonstrated that multivalent membrane Ig cross-linking, using dextran-conjugated anti-Ig Abs (anti-Ig-dextran), is an in vitro model for membrane Ig-dependent TI-2 induction of Ig secretion. In this work, we demonstrate that highly purified neonatal B cells are intrinsically defective in IgM secretion in response to anti-Ig-dextran and cytokines in vitro, as well as other modes of B cell activation, relative to adult B cells. However, costimulation of anti-Ig-dextran-activated neonatal B cells with either CD40-ligand, a recombinant bacterial lipoprotein, or LPS restores the IgM secretory response of neonatal B cells to adult levels. Analysis of Ig isotype secretion indicates that neonatal B cells have an enhanced capacity to secrete IgE and IgA relative to other Ig isotypes. These data suggest that neonatal B cells are competent to secrete Ig in response to TI-2 Ags if adequate costimuli are provided, and thus may have particular relevance for the design of vaccine strategies in the immunodeficient host. The data also suggest that neonatal B cells are programmed to secrete relatively enhanced amounts of IgE and IgA, which may be relevant for antimicrobial resistance at mucosal surfaces.