Human allograft acceptance is associated with immune regulation

Abstract

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

Figure 1

Trans vivo DTH responses: donor antigen–linked DTH nonresponsiveness to TT. (a) Eight million PBMCs from patient D.S. were injected into the footpads of SCID mice and the swelling measured after 24 hours. This background swelling was used to calculate the specific swelling when PBMCs plus the different antigens were included in the injection mixture. Injection of PBMCs plus self antigens (Ag) did not result in swelling, whereas injection of PBMCs plus self Ag plus TT resulted in swelling (not shown). These data are combined from six separate experiments performed over 8 months. (b) Eight million PBMCs from patient J.B. were injected into the footpads of SCID mice and the swelling measured after 24 hours. This background swelling was used to calculate the specific swelling when PBMCs plus donor alloantigens, TT, or donor alloantigens plus TT were included in the injection mixture. Data are combined from three separate experiments over 9 months.

Figure 2

Specificity analysis of donor antigen–linked DTH nonresponsiveness. (a) Eight million PBMCs from patient D.S. were injected into the footpads of Balb/c SCID mice either alone or mixed with 10 μg EBV lysate or 10⁵ autologous or donor-derived EBV-transformed LCL. Swelling was measured after 24 hours and is expressed as the change over the swelling induced by injection of PBMCs alone. Injection of EBV lysate or EBV-transformed LCL alone did not result in any increase in footpad thickness. The PBMCs also did not respond to control lysate prepared from Raji cells (not shown). (b) Eight million PBMCs from patient D.S. were injected into the footpads either alone or mixed with 10⁵ irradiated 721.221, A2-.221, or B62-.221 LCL. Swelling was measured as described above. (c) Eight million PBMCs from patient D.S. were mixed with TT or TT plus purified HLA-A2 or HLA-B62 and injected into the footpads of SCID mice. Swelling was measured after 24 hours as described above.

Figure 3

Effect of cytokine neutralization on donor-reactive DTH. (a) Eight million PBMCs from patient D.S. were mixed with 10 μg donor or self antigen or 10⁵ irradiated donor-derived EBV-transformed LCL and 25 μg of either control rabbit IgG or rabbit anti-human TGF-β IgG and injected into the footpads of Balb/c SCID mice. Swelling was measured after 24 hours. Results are expressed as the change over the swelling induced by injection of PBMCs alone. To determine the effect of neutralizing antibodies to IL-10, 8 million PBMCs from patient D.S. were mixed with 10 μg donor antigen or 10⁵ irradiated donor-derived EBV-transformed LCL and 25 μg of either control goat IgG or goat anti-human IL-10. Swelling was measured after 24 hours, as described above. (b) Eight million PBMCs from patient A.J. were mixed with 10 μg donor antigens and 25 μg of either control rabbit IgG or rabbit anti-human TGF-β IgG and injected into the footpads of SCID mice. Swelling was measured as described above. To assess the effect of neutralizing antibodies to IL-10, PBMCs from patient A.J. were mixed with donor or self antigens and 25 μg of either control Ig or anti–IL-10. The results shown are from three separate experiments. In two experiments, the anti–IL-10 was a mAb with a mouse IgG-1 control. In the third experiment, goat anti-human IL-10 and goat IgG were used as the specific antibody and control, respectively. The results were not significantly different whether the polyclonal antibody or mAb was used.