James M. Mathew

A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring.

Background. New trends in immunosuppression in clinical transplantation include the use of antibody induction agents in protocols that emphasize reduction or avoidance of steroids and calcineurin inhibitors. Methods. In a randomized trial using three different antibody induction agents in 90 first renal transplant recipients from cadaver donors, group A received Thymoglobulin, group B received Alemtuzumab, and group C received Daclizumab. Maintenance immunosuppression included tacrolimus and mycophenolate in all three arms, and methylprednisolone in groups A and C only (standard clinical institutional practice). The targeted trough level of tacrolimus was between 8 and 10 ng/mL for groups A and C, respectively, with a targeted mycophenolate dose of 1 g twice daily. However, in group B, the target tacrolimus trough level was 4 to 7 ng/mL to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of mycophenolate, without steroid maintenance. Results. In this 15-month median postoperative interval report, there were no notable differences in demographics and patient and graft survivals. Acute rejection rates at 1 year were equivalent, that is, 5 of 30 in all three groups (16.6%). In group B, there was slightly lower renal function at 1 month, but no difference at 1 year. There was also significantly more leukopenia, but a greater percentage of T-regulatory cells and number of Fox-P3 mRNA copies by flow cytometry and semiquantitative polymerase chain reaction analysis, respectively, in group B. Conclusions. This preliminary analysis indicates that 80% of the patients in group B remained steroid-free 1 year postoperatively, with lower tacrolimus trough levels and no difference in other adverse events.

The effects of chimeric cells following donor bone marrow infusions as detected by PCR-flow assays in kidney transplant recipients.

40 recipients of first cadaver kidney transplants were given perioperative donor vertebral bone marrow infusions (DBMC), compared with 100 controls who did not receive donor bone marrow. The immunosuppressive regimen included OKT3, Tacrolimus, and steroid maintenance therapy, and, in some patients, newly introduced mycophenolate mofetil. This report describes the 24-mo actuarial follow-up and several immunological monitoring studies including sequential measurements of donor bone marrow lineage subset chimerism by the recently reported PCR-flow assay. This is a sensitive in situ PCR detection system for donor versus recipient histocompatibility genes as well as cell surface CD epitope markers using flow cytometry. The results indicate (a) the stabilization of the donor CD3+ and CD34+ cells in recipient peripheral blood at levels below 1% between 6 mo and 1 yr postoperatively, with a 10-fold higher level of donor cell chimerism of these lineages in recipient iliac crest marrow; (b) significantly lower levels of chimerism in peripheral blood up to 6 mo postoperatively in patients who had early acute (reversible) rejection episodes compared with those who did not; (c) a higher degree of chimerism seen in patients who were class II MHC HLA DR identical with their donors; (d) the identification of a high proportion of the donor bone marrow derived CD3 dimly staining subset of T cells (to which regulatory functions have been ascribed) in recipient peripheral blood and especially in recipient bone marrow; and (e) an unexpectedly increased susceptibility to clinically significant infections (primarily viral), and even death in the DBMC-infused group, compared with controls, but no graft losses because of rejection in the DBMC-infused group. Mixed lymphocyte culture assays showed a trend toward a greater number of nonspecifically low reactors in the DBMC group, as well as a greater number of nonspecifically high reactors in the controls (P = 0.058). The autologous mixed lymphocyte reaction also indicated a trend towards nonspecific immune activation in the DBMC group. Finally, anti-cytomegaloviral IgG antibody reactivity was significantly inhibited in the DBMC group 4-6 mo postoperatively (P = < 0.05). In the controls, there were no donor cell lineages detected by PCR-flow in the peripheral blood. These rather unexpected findings, indicating a more depressed cellular and humoral immune capacity in the DBMC cadaver kidney transplant recipients in this relatively early follow-up period, are discussed relevant to chimerism, MHC restriction, and suppressor activity brought about by specialized DBMC subsets, which still need to be defined.

Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants.

Nineteen subjects have more than 18 months’ follow-up in a phase IIb tolerance protocol in HLA–mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4+ and CD8+ T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.

Interleukin-10 Delivery via Mesenchymal Stem Cells: A Novel Gene Therapy Approach to Prevent Lung Ischemia–Reperfusion Injury

Ischemia-reperfusion (IR) injury is an important cause of primary graft failure in lung transplantation. In this study, viral interleukin-10 (vIL-10)-engineered mesenchymal stem cells (MSCs) were tested for their ability to prevent lung IR injury. Bone marrow-derived MSCs were transduced with rvIL-10-retrovirus. After 120 min of warm left lung ischemia, rats received approximately 15 x 10(6) vIL-10-engineered MSCs (MSC-vIL-10), empty vector-engineered MSCs (MSC-vec), or saline intravenously. Mean blood oxygenation (PaO(2)/FiO(2) ratio, mmHg) was measured at 4 hr, 24 hr, 72 hr, and 7 days. As early as 4 hr post-IR injury with MSC-vIL-10 treatment, blood oxygenation was significantly (p < 0.05) improved (319 +/- 94; n = 7) compared with untreated (saline) controls (63 +/- 19; n = 6). At 24 hr post-IR injury, in the MSC-vIL-10-treated group there was a further increase in blood oxygenation (353 +/- 105; n = 10) compared with the MSC-vec group (138 +/- 86; n = 9) and saline group (87 +/- 39; n = 10). By 72 hr, oxygenation reached normal (475 +/- 55; n = 9) in the MSC-vIL-10-treated group but not in the saline-treated and MSC-vec-treated groups. At 4 hr after IR injury, lungs with MSC-vIL10 treatment had a lower (p < 0.05) injury score (0.9 +/- 0.4) compared with lungs of the untreated (saline) group (2.5 +/- 1.4) or MSC-vec-treated group (2 +/- 0.4). Lung microvascular permeability and wet-to-dry weight ratios were markedly lower in the MSC-vIL10 group compared with untreated (saline) controls. ISOL (in situ oligonucleotide ligation for DNA fragmentation detection) and caspase-3 staining demonstrated significantly (p < 0.05) fewer apoptotic cells in MSC-vIL10-treated lungs. Animals that received MSC-vIL10 therapy had fewer (p < 0.05) CD4(+) and CD8(+) T cells in bronchoalveolar lavage fluid compared with untreated control animals. A therapeutic strategy using vIL-10-engineered MSCs to prevent IR injury in lung transplantation seems promising.

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses.

Background. Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. Methods. Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. Results. The yield of RdD cells ranged from 0.1 to 0.9% of the starting material with the majority being TcR&agr;&bgr;, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 2–3 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated allogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. Conclusions. These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

The human bone marrow as an immunoregulatory organ.

It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1–3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define “acquired specific immunological tolerance” as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11–15), to date, specific immunological tolerance in organ transplantation in humans has not been possible.

Modulatory effects of human donor bone marrow cells on allogeneic cellular immune responses.

BACKGROUND In order to evaluate whether immunoregulatory mechanisms are brought about by human donor bone marrow cell infusions accompanying organ transplantation, we established in vitro culture systems analogous to the transplant model. METHODS Cell-mediated lympholysis (CML) and mixed lymphocyte culture (MLC) responses of peripheral blood lymphocytes or spleen cells stimulated with irradiated cadaver donor spleen cells in the presence of specific donor vertebral-body bone marrow cell (DBMC) modulators were tested. RESULTS When compared with spleen cells as modulator controls, DBMC inhibited both the proliferative and cytotoxic responses in a dose-dependent manner. Use of unirradiated and T cell-depleted DBMC was required for detection of the inhibitory activity. Furthermore, DBMC had to be added within the first 2 days after the initiation of the cultures for the down-regulation of CML (MLC) to occur. The down-regulation of MLC responses could not be shown to be antigen (donor) specific. Physical separation of DBMC from the responder-stimulator cells using the transwell system abrogated modulation of the CML (and MLC) reactions, suggesting the requirement of cell-cell contact for modulatory effect. The inhibitory activity by DBMC could not be overcome by addition of up to 200 U/ml of exogenous recombinant interleukin 2 to the cultures. However, it could be abrogated by restimulation with donor spleen cells, indicating that donor reactive cells were not deleted by DBMC in short-term cultures. CONCLUSIONS These results showed a regulatory role for DBMC in cellular immune responses against donor cell alloantigens, supporting the rationale for DBMC for facilitating clinical allograft acceptance.

Genomic Biomarkers Correlate with HLA-Identical Renal Transplant Tolerance

The ability to achieve immunologic tolerance after transplantation is a therapeutic goal. Here, we report interim results from an ongoing trial of tolerance in HLA-identical sibling renal transplantation. The immunosuppressive regimen included alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, and complete drug withdrawal by 24 months post-transplantation. Recipients were considered tolerant if they had normal biopsies and renal function after an additional 12 months without immunosuppression. Of the 20 recipients enrolled, 10 had at least 36 months of follow-up after transplantation. Five of these 10 recipients had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence, and 3 had subclinical rejection in protocol biopsies (nontolerant). Microchimerism disappeared after 1 year, and CD4(+)CD25(high)CD127(-)FOXP3(+) regulatory T cells and CD19(+)IgD/M(+)CD27(-) B cells were increased through 5 years post-transplantation in both tolerant and nontolerant recipients. Immune/inflammatory gene expression pathways in the peripheral blood and urine, however, were differentially downregulated between tolerant and nontolerant recipients. In summary, interim results from this trial of tolerance in HLA-identical renal transplantation suggest that predictive genomic biomarkers, but not immunoregulatory phenotyping, may be able to discriminate tolerant from nontolerant patients.

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.

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.