Allochimeric therapy induces unique regulatory T cells that mitigate chronic rejection.

Induction of tolerance to an allogeneic graft without the need for nonspecific immunosuppression is a major goal of transplantation therapy. We have shown that treatment with molecularly engineered, allochimeric [alpha(1h)(1/u)]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.A(u)) or Lewis (LEW, RT1A(1)) amino acid substitutions for host-type ACI (RTI.A(a)) sequences in the alpha(1)-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. The mechanisms involved in the establishment and maintenance of specific allograft tolerance are still not fully understood. It is now clear that acquisition of transplantation tolerance is an active, highly regulated, multistep process. According to the pool size model of allograft tolerance, the allograft outcome, rejection, or tolerance often depend on the balance between cytopathic and regulatory T cells (T-regs). This study examined mechanisms of chronic rejection (CR) development on a model of cardiac transplant tolerance after adoptive transfer of T-regs followed by allochimeric therapy. Generation of T-regs was demonstrated in vitro by MLR coculture and confirmed by adoptive transfer of T cells from primary recipients to secondary hosts. To confirm the true nature of regulatory cells, we performed a second transfer into tertiary recipients. Unlike T-regs from tolerant hosts, T cells from naive rats did not prolong graft survival. Histological evaluation of T-regs-transfected groups showed absence of visible CR. In contrast, T-regs generated in recipients after high-dose cyclosporine treatment failed to inhibit CR in transferred singeneic recipients. Allochimeric therapy triggers generation of unique regulatory lymphocytes that mitigate development of chronic rejection through regulation of anti-inflammatory mechanisms and down-regulation of alloantibody response.

Donor MHC class I peptides in conjunction with self-epitopes induce donor-specific tolerance in a dose-dependent manner but unable to abrogate chronic rejection.

Understanding specific tolerance mechanisms is a primary goal of transplantation science. We have previously shown that hosts treated with MHC class I protein have donor sequences in the alpha1-helix of the alpha1 domain on a background of self-epitopes, resulting in the development of donor-specific tolerance. However, the nature of class I alloantigenic determinants that regulate the alloimmune response remains unclear. The alpha1-helical sequence of RT1.A,1 which shares RT1.A(u) sequences, was substituted in the RT1.A(a) molecule to produce the composite [alpha1h(l/u)]-RT1.A(a) MHC class I allochimeric molecule. Immunodominant epitopes were identified within the hypervariable region of the alpha1 domain of RT1.A(a) (ACI), RT1.Al (Lewis, LEW), and RT1.A(u) (Wistar Furth [WF]). To clarify the mechanisms of tolerance development through presentation of donor-type immunogenic epitopes and cryptic self-epitopes we used synthetic peptides corresponding to donor immunogenic determinants with peptides derived from recipient self-sequences (RT1.A(a)--aa 10 to 49 P1 and 91 to 120 P3; and P2 RT1.A(l/u) 50 to 90). ACI recipients of LEW and WF cardiac allografts were injected through the portal vein (PV) at day 0 with four doses (2, 0.5, 0.25, and 0.125 mg/rat) of three peptide mixtures in conjunction with subtherapeutic CsA (10 mg/kg for 3 days). Allograft survival was strongly dose-dependent. Only low-dose regimens were consistent in tolerance induction, but such therapy did not abrogate development of chronic rejection (CR), unlike allochimeric therapy with soluble MHC class I protein. Different effects of protein or synthetic peptide therapies on development of CR suggest that development of specific tolerance is an active immunologic process and it depends on the form of allogeneic epitopes presented.

Identification of early tolerance regulator genes induced by allochimeric therapy using microarray-based genomewide scan.

We have demonstrated that peri- or postoperative delivery of allochimeric [a1h(u)]-RT1.A(a) class I major histocompatibility complex molecules with donor-type (RT1A(u)) immunogenic epitopes presented in recipient-type (RT1A(a)) sequences induced donor-specific tolerance in ACI (RT1a) recipients of WF (RT1u) heart allografts. A genomic scan during the early posttransplant period was performed to elucidate the underlying operative mechanisms. A rat genome study after transplantation was carefully designed using Affymetrix Rat Genome 230 2.0 Array. The allochimeric treatment group is 3-day cyclosporine (CsA)-treated ACI recipients that accepted Wistar Furth RT1u cardiac allografts with postoperative dosage of allochimeric molecules, while the control is 3-day CsA-treated ACI recipients of WF cardiac allografts. All the samples were harvested 5 days after heart transplant as the early stage of tolerance detection. Following array data normalization and modeling, we compared the above two treatment groups and identified a total of 250 tolerance regulator genes induced by allochimeric molecules only.

Immune functions of spleen lymphocytes of rats subjected to chronic irradiation and antioxidant (ubiquinone Q-9) diet.

The dynamics of T and B cell immunity in spleens from rat exposed to whole-body chronic irradiation with dose rates of 12.9 cGy/day (range 1-10 Gy) and 3.0 cGy/day (range 0.57-2.04 Gy) were investigated. gamma-irradiation with a dose-rate of 12.9 cGy/day was shown to produce a wave-like suppression of the T lymphocyte mitogenic response. Irradiation with a dose-rate of 3.0 cGy/day caused a decrease in immune response of T lymphocytes 48 days after onset of exposure (total dose 1.4 Gy). It was also shown that chronic irradiation with a dose-rate of 3.0 cGy/day produced significant changes in the DNA of T lymphocytes. Our results show that the radiation-induced suppression of immune functions and damage to DNA structure were partially eliminated when animals were fed a daily diet supplemented with a natural antioxidant, ubiquinone Q-9. The inhibiting effect of chronic irradiation was more pronounced in B lymphocytes because of their higher radiosensitivity.

Early and long-term effects of chronic low-dose radiation and coenzyme Q diet on the proliferation of rat spleen T cells.

Chronic whole-body irradiation (0.43 cGy/day; total doses 11, 23, and 35 cGy) caused non-monotonous long-term disturbances in rat splenic T lymphocytes proliferation. Immunosuppression observed after chronic exposure had no correlation with the splenic cell number and the decrease in the fluorescence intensity of Hoechst 33258-DNA complex in T lymphocytes. Small, but significant radioprotection was observed with Coenzyme Q diet immediately after irradiation. These results indicate that the changes in T cell immunity, T cell viability, and T cell DNA state after exposure to low dose radiation are not interrelated.

[Production of tumor necrosis factor (TNF) by macrophages and T-lymphocytes of rodents as affected by acute gamma-irradiation]. / Produktsiia faktora nekroza opukholei (FNO) makrofagami i T-limfotsitami gryzunov v usloviiakh ostrogo gamma-oblucheniia.

TNF production in peritoneal macrophages and in splenic T cell from mice and ground-squirrels were performed after acute in vivo and in vitro doses of ionizing radiation ranging from 0.1-6 Gy. It was observed that doses in the range 0.5-6 Gy induced the increase of TNF production along with the decrease of mitogen-induced proliferation in T lymphocytes.

[Effect of an antioxidant (ubiquinone Q-9) and chronic low-dose irradiation on rat chromatin structure and T-lymphocyte proliferation]. / Vliianie antioksidanta (ubikhinona Q-9) i khornicheskogo oblucheniia v malykh dozakh na strukturu khromatina i proliferatsiiu T-limfotsitov krys.

We have demonstrated the protective effect of natural antioxidant coenzyme Q-9 on the T-cell proliferative capacity and chromosomal DNA state under low dose chronic irradiation. The DNA-staining fluorescent dye Hoechst-33258 and acridine orange were to observed the nuclear morphology changes in T-lymphocytes. Ubiquinone diet partially restored the decreasing in immune response and the luminescence intensity of DNA-Hoechst-33258 complex in T-cells of low dose irradiated rats.

[Stimulation of murine natural killer cells by weak electromagnetic waves in the centimeter range]. / Stimuliatsiia estestvennykh killernykh kletok myshei, podvergnutykh deistviiu slabykh élektromagnitnykh voln santimetrovogo diapazona.

Irradiation with electromagnetic waves (8.15-18 GHz, 1 Hz within, 1 microW/cm2) in vivo increases the cytotoxic activity of natural killer cells of rat spleen. In mice exposed for 24-72 h, the activity of natural killer cells increased by 130-150%, the increased level of activity persisting within 24 h after the cessation of treatment. Microwave irradiation of animals in vivo for 3.5 and 5 h, and a short exposure of splenic cells in vitro did not affect the activity of natural killer cells.