Establishing hematopoietic mixed chimerism can lead to donor-specific tolerance to transplanted organs and may eliminate the need for long-term immunosuppressive therapy, while also preventing chronic rejection. In this review, we discuss central and peripheral mechanisms of chimerism induced tolerance. However, even in the long-lasting presence of a donor organ or donor hematopoietic cells, some allogeneic tissues from the same donor can be rejected; a phenomenon known as split tolerance. With the current goal of creating mixed chimeras using clinically feasible amounts of donor bone marrow and with minimal conditioning, split tolerance may become more prevalent and its mechanisms need to be explored. Some predisposing factors that may increase the likelihood of split tolerance are immunogenicity of the graft, certain donor-recipient combinations, prior sensitization, location and type of graft and minimal conditioning chimerism induction protocols. Additionally, split tolerance may occur due to a differential susceptibility of various types of tissues to rejection. The mechanisms involved in a tissue’s differential susceptibility to rejection include the presence of polymorphic tissue-specific antigens and variable sensitivity to indirect pathway effector mechanisms. Finally, we review the clinical attempts at allograft tolerance through the induction of chimerism; studies that are revealing the complex relationship between chimerism and tolerance. This relationship often displays split tolerance, and further research into its mechanisms is warranted.
A large body of literature has documented an inconsistent relationship of peripheral donor cell chimerism with alloimmune tolerance following kidney transplantation. We revisit this association with assays capable of quantifying cellular microchimerism with 150–1500-fold greater sensitivity than previously utilized allo-antibody based flow cytometric approaches. Forty renal transplant patients, 20 with concurrent donor bone marrow infusion (DBMI) and 20 control participants without infusion were prospectively monitored for peripheral blood microchimerism using donor polymorphism-specific quantitative real-time PCR. Thirty-eight patients were evaluated for microchimerism, 19 in each group. The frequency of testing positive for (95% vs. 58%, p = 0.02) and mean concentrations of microchimerism (115 ± 66 vs. 13 ± 3 donor genomes/million recipient genomes, p = 0.007), respectively, were higher in infused patients compared with controls. Thirty-one patients maintained stable graft function; 17 in the DBMI group vs. 14 in controls. Patients with stable graft function in the DBMI group compared with control patients harbored microchimerism more frequently (94 vs. 50%, p = 0.01) and at higher concentrations (123 ± 67 vs. 11 ± 4, p = 0.007), respectively. Significant correlation between dose of infused cells and microchimerism levels was found post-transplant (p = 0.01). Using very sensitive assays, our findings demonstrate associations between the presence and quantity of microchimerism with stable graft function in infused patients.
The pathways regulating immunological tolerance are complex and overlapping. Here, we comment on our findings that the PD-1, CTLA-4, LAG-3 and TGFβ inhibitory molecules are all involved in mediating peripheral CD8 T‑cell tolerance induced by anti-CD40L and allogeneic bone marrow transplantation in mice. These observations suggest the possibility of targeted manipulation of these pathways for induction of mixed hematopoietic chimerism for donor-specific transplantation tolerance.
Microchimerism refers to the presence of less than 1% of non-host cells in a person. Our group developed a reliable method for separating viable microchimeric cells from the host environment. Optimal separation of microchimeric cells at proportions as low as 0.01% could be established with two monoclonal antibodies directed against different HLA antigens, one targeting the microchimeric cells and the other the host cells. Purity of separated cell populations was validated by HLA-allele-specific and Y-chromosome directed real-time qPCR assays. The methodology was used successfully to separate microchimeric maternal cells from child umbilical cord mononuclear cells after pregnancy.
Cell sorting with HLA monoclonal antibodies targeting allelic differences enables reliable microchimeric cell detection and separation in blood specimens. With this approach, maximal enrichment of potentially viable microchimeric cells from a background cell population is reached, which opens the way to phenotypical and functional characterization of microchimeric cells.
The neonatal period of life is described as particularly susceptible to develop tolerance. This status of neonatal tolerance has been studied for decades since the discovery that semiallogeneic spleen cells inoculated at birth can induce donor-type graft acceptance. The host neonatal T‑cell compartment that may account for this propensity to develop tolerance is mostly characterized by a Th2-type polarized response and a default in the cytotoxic T lymphocyte (CTL) functions that allow the establishment of lymphoid chimerism and promote donor-type graft survival. We highlighted a new role of alloreactive Th17 cells as a critical barrier to neonatal tolerance that prevents this lymphoid chimerism and further demonstrated that the Th2 immune deviation is essential to control this Th17-type response. We discuss here the potential impact of breaking the tolerizing effects of exposure of the developing offspring to alloantigens in the induction of Th17-type immunity.
Liver transplant has become life-saving therapy for thousands of patients with end stage liver disease in the United States, but chronic rejection and the toxicities of immunosuppression remain significant obstacles to the further expansion of this modality and “transplant tolerance” remains a central goal in the field. So we and others are looking for alternative post-transplant strategies. We set out to ‘engineer’ repopulation after transplantation in a strain combination [dark agouti (DA) to Lewis green fluorescent protein+ (LEW GFP+)] which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Our central finding is purposeful manipulation of the immune response with low dose immunosuppression and liberation of stem cells for a very short period after transplantation results in long-term transplant acceptance by two mechanisms: transforming the liver (donor) to self (host) phenotype, and auto-suppression of the specific allograft response.