Immune "tolerance profiles" in donor bone marrow infused kidney transplant patients using multiple ex vivo functional assays.

Ex vivo identification of donor-specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living, related-donor kidney transplant patients up to 11 years postoperatively, i.e., 20 haploidenticals with donor bone marrow cell (DBMC) infusions, eight noninfused haploidentical controls (haplo controls), and 11 HLA-identical controls (HLA-id), using multiple ex vivo immune assays. We observed that no patients developed donor-specific antibodies. The majority showed donor-specific CTL unresponsiveness from year 1 onward. Thirteen of 20 DBMC recipients became specifically donor MLR nonreactive. Depletion of donor cells in DBMC recipients still MLR reactive increased donor-specific reactivity by 75% +/- 36% (p = 0.04). Adding them back in low concentration caused antigen specific inhibition. The frequencies of ELISPOT granzyme-B and interferon-gamma-producing cells somewhat paralleled the CTL and MLR responses. In the trans vivo DTH, 14 of 19 DBMC recipients demonstrated donor-specific unresponsiveness and 16 of 19 showed "linked suppression," vs none of eight and one of eight haplo controls and vs six of 10 and one of 10 HLA-ids, respectively. Most importantly, when all six assays were performed simultaneously, 10 of 18 DBMC, five of 10 HLA-ids, and no haplo controls were specifically donor unresponsive long term. We propose that a cluster analysis combining these assays will reveal tolerant recipients in whom IS minimization may safely be tested. This appears to have occurred in many DBMC-infused recipients.

Transplantation of the spleen: effect of splenic allograft in human multivisceral transplantation.

OBJECTIVES: To describe the effect of the splenic allograft in human multivisceral transplantation. SUMMARY BACKGROUND DATA: We performed transplants of the spleen as part of a multivisceral graft in an attempt to decrease both the risk of infection from an asplenic state and the risk of rejection by a possible tolerogenic effect. To our knowledge, this is the first report of human splenic transplantation in a large series. METHODS: All primary multivisceral recipients who received a donor spleen (N = 60) were compared with those who did not receive a spleen (N = 81). RESULTS: Thirty-five of 60 (58%) are alive in the spleen group, and 39 of 81 (48%) are alive in control group (P = 0.98). In univariate analysis, splenic recipients showed superiority in freedom-from-any rejection (P = 0.02) and freedom-from-moderate or severe rejection (P = 0.007). No significant differences were observed in analyses of infectious complications between the spleen and control groups. Both platelet and leukocyte counts became normal in splenic patients, whereas these counts were significantly increased in nonsplenic recipients. Observed incidence of graft versus host disease (GVHD) was 8.25% (5 of 60) in the spleen group and 6.2% (5 of 81) in the control group (P = 0.70). Increased incidence of autoimmune hemolysis was observed in the spleen group. CONCLUSIONS: Allograft spleen can be transplanted within a multivisceral graft without significantly increasing the risk of GVHD. The allogenic spleen seems to show a protective effect on small bowel rejection. Further investigation with longitudinal follow-up is required to precisely determine the immunologic and hematologic effects of the allograft spleen.

FoxP3 mRNA transcripts and regulatory cells in renal transplant recipients 10 years after donor marrow infusion.

BACKGROUND: We update more favorable 10-year deceased donor kidney transplant survival in 63 recipients infused perioperatively with donor vertebral body bone marrow (DBMC-i) vs. 219 noninfused controls having equivalent immunosuppression and demographics. We questioned if this was associated with putatively regulatory FoxP3 mRNA and cell phenotypes (CD4+CD25+high percentages and high DC2:DC1 ratios) in DBMC-i vs. noninfused controls. METHODS: Baseline studies were performed on peripheral blood lymphocytes (PBLs) vs. marrow in normal laboratory volunteers of CD4+CD25+high percentages and DC2:DC1 by flow cytometry, and FoxP3 mRNA in CD3+ cells by real-time polymerase chain reaction. Similar studies were performed on PBL of the majority of the 10-year patients remaining with graft function: 21 (of the remaining 37) DBMC-i vs. 55 (of the remaining 105) controls. RESULTS: In normal subjects, all parameters were significantly higher in marrow than in PBL, supporting our previous reports of ex vivo DBMC immunoregulation. At 9.8+/-.02 years posttransplant in DBMC-i vs. controls, death-censored percent graft failure was 17.5% vs. 32.9% (P=0.02) with 247.6+/-24 vs. 79.9+/-3.1 (mean+/-SE) FoxP3 copies/5,000 CD3+ cells (P=0.0001). PBL CD4+CD25+high percentages were lower, but DC2:DC1 values higher in both recipient groups than in end-stage renal disease patients who had lower FoxP3 levels (40.8+/-5.9, P<0.0001), consistent with non-CD4+CD25+high T regulatory cells generated long-term posttransplant. Individual higher FoxP3 values correlated with higher iliac crest chimerism in DBMC-i, but not in controls (with 50-fold lower chimerism). In chronically rejecting controls, FoxP3 was further decreased. CONCLUSIONS: Peritransplant DBMC-i has higher 10-year renal transplant acceptance, chimerism, and FoxP3 mRNA in thus-far unclarified regulatory cell phenotypes.

The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation.

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post-transplantation, with stable graft function. Forty-five (study group: G1) received DBMC, and 59 (control group: G2) did not. Immunosuppression was reduced by one third upon enrollment, by another third the second year of the study and was completely withdrawn the third year. Patient and graft survival were similar between the two groups. Although rejection episodes were significantly less in G1 the first 2 years of the study (35% vs. 57%, p = 0.016), there was no significant difference overall (74% vs. 81%, p = 0.14). Until February 2004, 20 patients, 10 in each group, were immunosuppression free for 1-3 years. Approximately 20% of long-term survivors of liver transplantation can successfully discontinue their immunosuppression. DBMC infusions, do not increase this likelihood.

Donor bone marrow transplantation: chimerism and tolerance.

Infusion of donor bone marrow (DBM)-derived cells continue to be tested in clinical protocols intended to induce specific immunologic tolerance. Central clonal deletion of donor-specific alloreactive cells associated with mixed chimerism reliably produced long-term graft tolerance. In this setting, depletion of recipient T cells by antilymphocyte antibodies and subsequent repopulation by donor hematopoietic cells after donor bone marrow infusion (DBMI) are prerequisites for tolerance induction. Major advances have been made in animal models and in pilot clinical trials and the key questions with the future perspectives are presented in this article.

Donor bone marrow infusion in deceased and living donor renal transplantation.

The infusion and persistence in a transplant recipient of donor-derived bone marrow cells (DBMC) of multi-lineage can lead to a state of permanent chimerism. In solid vascular organ transplantation, the donor bone marrow lineage cells can even be derived from the transplant organ, and these cells can be detected in very small numbers in the recipient. This has been called microchimerism. Much controversy has developed with respect to the function of chimeric cells in organ transplantation. One idea is that the occurrence of these donor cells found in microchimerism in the recipient are coincidental and have no long-term beneficial effect on engraftment. A second and opposing view, is that these donor cells have immunoregulatory function that affect both the acute and chronic phases of the recipient anti-donor responses. It follows that detecting quantitative changes in chimerism might serve as an indication of the donor-specific alloimmune or regulatory response that could occur in concert with or independent of other adaptive immune responses. The latter, including autoimmune native disease, need to be controlled in the transplant organ. The safety and immune tolerance potential of DBMC infusion with deceased and living donor renal transplants was evaluated in a non-randomized trial at this center and compared with non-infused controls given identical immunosuppression. Overall DBMC infusions were well tolerated by the recipients. There were no complications from the infusion(s), no episodes of graft-vs-host disease (GVHD) and no increase infections or other complications. In the deceased DBMC-kidney trial, actuarial graft survival at 5 years was superior especially when graft survival was censored for recipient death. Acute rejections were significant reduced in patients given two DBMC infusions, and chronic rejection was dramatically reduced in all DBMC treated patients. The most interesting finding was that the degree of microchimerism slowly increased over the years the DBMC group that had exhibited no rejection episodes. In the DBMC-living related trial, the incidence of acute rejection did not differ between groups. However, DBMC chimerism in recipient iliac crest marrow had increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused, with the generation of T- regulartory cells in-vitro assays.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation.

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34(+) stem cells and the CD34(-) early progeny like CD38(+), CD2(+), CD5(+) and CD1(+) lymphoid cells as well as CD33(+) (but CD15(-)) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell-cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34(+) cells were found to give rise both to CD3(-) TCRalphabeta(+), as well as CD3(+) TCRalphabeta(+) cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.

Effect of living-related donor bone marrow infusion on chimerism and in vitro immunoregulatory activity in kidney transplant recipients.

BACKGROUND: In a previously reported series of donor-specific bone marrow cell (DBMC) infusions in cadaver kidney transplant recipients, there appeared to be an improvement in long-term graft survival (6 years) and fewer chronic rejections, which correlated with increasing DBMC chimerism (approximately 1.4% in the iliac crest bone marrow compartment now at 6 years). Prompted by this, we embarked on a study of DBMC infusion in living-related donor (LRD) kidney transplant recipients. METHODS: Between November 1996 and May 2000, 47 LRD kidney transplant recipients received donor iliac crest marrow (1.8 x 10(8)+/-1.9 x 10(8) cells/kg body weight+/-SD) in a single infusion 4 days postoperatively. Either OKT3 (n=26) or daclizumab (n=21) were used for induction therapy, with maintenance tacrolimus, mycophenolate mofetil, and methylprednisolone immunosuppression. These recipients were prospectively compared with 39 noninfused LRD kidney transplants (control group), which received equivalent immunosuppression in the same time period. Clinical follow-up ranged from 19.0 months to 61.6 months (mean 33.2 months). Polymerase chain reaction-flow chimerism analysis and in vitro assays of immunoregulatory activity of chimeric cells were performed. RESULTS: The incidence of acute rejection over this period of time was 10.6% and 10.3%, respectively (i.e., did not differ between groups). Immunosuppressive dosages were somewhat (but not statistically) lower over time in the DBMC group. Four-year actuarial patient and graft survival for the DBMC-infused group was 98% and 98%, and 98% and 95% for the control group, respectively ( =NS). DBMC infusion was well tolerated, with no increase in infectious episodes. DBMC chimerism in recipient iliac crest marrow has increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused. DBMCs and (donor) peripheral blood mononuclear cells purified by immunobeads from recipient blood or bone marrow (recipient-derived donor cells) inhibited mixed leukocyte responses of the recipient to the donor more strongly than freshly obtained peripheral blood cells drawn from the donors or even compared with bone marrow cells aspirated from the donors in a previously reported group of experiments. Additionally, similarly purified recipient-derived recipient cells from the same chimeric recipient more strongly inhibited the same mixed leukocyte response reactions autologously than a large group of nonchimeric (autologous) bone marrow modulating cells in similar reactions. CONCLUSIONS: These observations confirm that an immunoregulatory process appears to have been generated by DBMC infusion, encouraging a further decrease in immunosuppressive dosing using such assays in the future.