PMC

Figure 3a: Comparison of whole blood chimerism and T cell chimerism. For whole blood, the percentage of donor chimerism was determined by quantifying the ratio of peak heights for donor-or recipient-specific microsatellite amplicons after purification of DNA from the unfractionated whole blood. For T cell chimerism, CD3+/CD20- cells were first sorted using a FACSAria flow cytometric cell sorter, and then DNA was prepared from these sorted cells. The percentage donor chimerism was then determined by determining the ratio of peak heights for donor-or recipient-specific microsatellite amplicons. Y-axis: Peak T cell chimerism measured while on immunosuppression. X-axis: Whole blood chimerism measured at the same time-point as the T cell chimerism. Each animal is depicted once, as a color-coded point.
Figure 3b: CD14+ myeloid chimerism closely resembles whole blood chimerism. Shown is a representative result from RDe9 (day 56 post-transplant), in which donor chimerism was measured in whole blood, and in flow-cytometrically sorted CD3+ T cells and CD14+ myeloid cells.

(A) Whole blood, bone marrow, T cell, B cell, and granulocyte chimerism of recipient animals at day 30 post-transplant shown as individual points. R.53 is shown as a red point, all other recipients shown as black points. Mean and standard deviation are shown as blue boxes and whiskers.
(B) Longitudinal analysis of whole blood chimerism, bone marrow chimerism, T cell chimerism, B cell chimerism, and granulocyte chimerism for all transplant recipients. Color-coded bars below each graph indicate duration of immunosuppression (black bar: Sirolimus, “Sir”; red bar: anti-CD154, “5C8”; blue bar: Belatacept, “Bela”).
(C) Thymic chimerism at time of necropsy for 4 recipients for which thymic tissue was obtainable at necropsy (R.51, R.54, R.55, R59). Individual chimerism values shown as black points. Mean and standard deviation are shown as blue boxes and whiskers.
(D) Long-term follow up of whole blood chimerism, bone marrow chimerism, T cell chimerism, B cell chimerism, and granulocyte chimerism for a previously reported () recipient (‘RDe9’) with stable multilineage mixed-chimerism.

An historical scheme of chimerism. Since Owen discovered persistent mixed chimerism in Freemartin cattle, 3 strategies have been pursued in the attempt to induce allograft tolerance. These include 1) persistent mixed chimerism, 2) transient mixed chimerism and 3) full donor chimerism. Allograft tolerance via mixed chimerism has been achieved in various murine models (Slavin et al. in 1977 and Sharabi et al. in 1989). However, persistent mixed chimerism has been extremely difficult to achieve in primates and has only been achieved in HLA-matched transplant recipients by the Stanford group. of note, the neonatal tolerance achieved by Medawar in 1953 was not a complete reproduction of the Freemartin cattle observation. The chimerism achieved in neonatal tolerance was transient and the mechanism of tolerance may be different from that in Freemartin cattle. In 1964, Balner reported the possibility of inducing allograft tolerance via transient chimerism. However, his finding remained unheeded until we (MGH) found that transient mixed chimerism is sufficient to induce renal allograft tolerance in nonhuman primates. Our strategy with transient mixed chimerism has been successfully translated to both HLA-matched and HLA-mismatched human kidney transplantation. Full donor chimerism was until recently considered too risky to apply clinically for tolerance induction of transplanted organs. However, full donor chimerism and renal allograft tolerance have been achieved by the Northwestern group in HLA-mismatched kidney transplant recipients without an unacceptable incidence of GVHD.

PBMC and T-cell donor chimerism following HSCT. (A) Bubble chart demonstrating PBMC and T-cell donor chimerism levels at day 50 posttransplant. The area of each bubble represents the number of events. The first group (n = 37, red), displayed FC, in which both PBMC and T-cell donor chimerism was ≥99%. The second group (MC, n = 32, blue) demonstrated MC in both PBMC and T-cell fractions (PBMC and T-cell chimerism ≤98%). The pattern of PBMC donor chimerism up to day 365 according to donor chimerism at day 50 posttransplant. PBMC chimerism remained stable at complete (or near complete) donor chimerism in patients who demonstrated FC by day 50 posttransplant. In contrast, PBMC donor chimerism remained significantly lower in those found to have MC at day 50, even beyond the initiation of donor lymphocyte infusion (DLI) at day 180 (****P < .0001; 2-way ANOVA, with post hoc Tukey test, mean, and standard error shown for all data points; FC, n = 37; MC, n = 32). (B) The pattern of T-cell donor chimerism up to day 365 according to donor chimerism at day 50 posttransplant. (C) T-cell chimerism remained stable at complete (or near complete) donor chimerism for the first 100 days in patients who demonstrated FC by day 50 posttransplant, but displayed an average drop of 10% by day 180. In contrast, T-cell donor chimerism remained significantly lower in those found to have MC at day 50 with a trend toward partial correction following instigation of DLI at day 180 (****P < .0001, 2-way ANOVA, with post hoc Tukey test, mean, and standard error shown for all data points; FC, n = 37; MC, n = 32).