Donor Cell Infusion Research Articles

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

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. 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. The yield of RdD cells ranged from 0.1 to O.9% of the starting material with the majority being TcRalphabeta, 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 23 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 alogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

In Vivo Persistence of Donor Cells following Adoptive Transfer of Allogeneic Dendritic Cells in HIV-Infected Patients.

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

Allograft tolerance induced by cyclophosphamide without prior inoculation of donor cells—immune suppression and redirection

Objectives: To determine the possibility and cellular mechanism of inducing allograft tolerance by multiple injection of a lower dose of cyclophosphamide without prior infusion of donor cells. Methods and results: Heterotopic heart grafts were performed in MHC mismatched strain combinations (C57/B6 vs. BALB/c). Cyclophosphamide (40 mg/kg) was given intravenously on days 0, 2, 4 and 7 without prior infusion of donor cells. Long-term (>100 days) allograft survival with normal histology was achieved. The long-term survivors accepted the donor skin grafts, but rejected the third-party skin grafts. Cyclophosphamide treatment initially led to profound lymphocytopenia, inhibition of spontaneous blastogenesis and low levels of lymphocyte proliferation response to both donor and third-party antigens. Ultimately, donor-specific tolerance occurred demonstrated by normal levels of peripheral lymphocytes, spontaneous blastogenesis and lymphocyte proliferation response to third-party antigens, and low levels of lymphocyte proliferation response to donor antigen. A switch of cytokines from IFNγ dominant to IL-4 dominant, a low level of IgM and a high level of IgG1 were found in tolerant mice. Conclusions: Allograft tolerance can be induced by a short course of cyclophosphamide without prior donor cell inoculation. Tolerance induced is characterized initially by non-specific immunosuppression, which progresses to donor-specific hyporesponsiveness associated with the development of a Th2 dominant cytokine response.

Bone marrow cell trafficking following intravenous administration

To address trafficking of transplanted marrow cells immediately after intravenous infusion, we examined the early fate of infused non-adherent, low-density donor bone marrow cells in a syngeneic mouse model. The presence of infused donor cells, marked with indium-111 oxine (111In), with the fluorescent dye PKH26, or by a detectable transgene marker, was evaluated at 3-48 h in a variety of tissues, including peripheral blood. All three cell-marking methods indicated a rapid (< 4 h) influx of cells into the bone marrow, liver, spleen, muscle and other tissues. Moreover, these tissues remained positive for the 48 h observation period. Interestingly, analysis of PKH26-positive cells in non-myeloablated animals demonstrated that approximately 17% of infused donor marrow cells localized to the marrow space within 15 h, whereas a smaller proportion of donor cells (approximately 1-2%) localized to the marrow in recipients preconditioned by irradiation. In an effort to enrich for cells that specifically home to the bone marrow, PKH26-labelled donor marrow cells were recovered from the first host and infused into a secondary recipient. Although this was a phenotypically undefined population of cells, no increase was observed in the relative fraction of PKH26-labelled cells returning or 'homing' to the marrow of the second recipient. Taken together, these data suggest both that marrow engraftment may be mediated by non-specific 'seeding' rather than a specific homing signal, and that efficient targeting of transplanted cells to the marrow is a complex multifaceted process.

Donor blood monocytes but not T or B cells facilitate long-term allograft survival after total lymphoid irradiation.

Previous studies showed that a combination of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte globulin (ATG), and a single donor blood transfusion induced tolerance to ACI heart allografts in Lewis rats. All three modalities were required to achieve tolerance. The objective of the current study was to determine the subset(s) of cells in the donor blood that facilitated long-term allograft survival. Lewis hosts received TLI, ATG, and donor cell infusion after heart transplantation. Graft survival, mixed leukocyte reaction (MLR), and intragraft cytokine mRNA were studied. The intravenous injection of 25 x 10(6) ACI peripheral blood mononuclear cells (PBMC) significantly prolonged graft survival as compared with that of Lewis hosts given TLI and ATG alone. Injection of highly enriched blood T cells or splenic B cells adjusted for the number contained in 25 x 10(6) PBMC failed to induce significant graft prolongation. Unexpectedly, depletion of monocytes (CD11b+ cells) from PBMC resulted in the loss of graft prolongation activity. Enriched populations of monocytes obtained by plastic adherence were more efficient in prolonging graft survival than PBMC on a per cell basis. Hosts with long-term grafts (>100-day survival) showed evidence of immune deviation, because the MLR to ACI stimulator cells was vigorous, but secretion of interferon-gamma in the MLR was markedly reduced. In situ hybridization studies of long-term grafts showed markedly reduced levels of interferon-gamma mRNA as compared with rejecting grafts. Infusion of donor monocytes facilitated graft prolongation via immune deviation.

DISTINCT TOLERANCE PATHWAYS IN SENSITIZED GRAFT RECIPIENTS

506 Allograft (Tx) rejection in sensitized patients represents one of the most challenging problems in clinical transplantation. In our model of accelerated rejection, LEW rats sensitized with BN skin Tx (d-7) reject LBNF1 cardiac Tx in <36 h. Recently, we have shown striking efficacy of two therapeutic approaches to induce tolerance in this stringent model. In the first, CD4-targeted therapy with RIB-5/2, a nondepleting mAb (10x 5 mg/rat/d; d-7→+21) results in permanent cardiac Tx survival in 99% of sensitized rats. In the second, blocking CD28-B7 T cell costimulation by treatment with CTLA4Ig (0.5 mg/rat d -5 & +2) + infusion of donor cells (20×106; d-7 & 0), produces indefinite Tx survival in ca. 40% of rats. Thus, selective blockade of activation signal 1 (CD4 mAb) or signal 2 (CTLA4Ig) may independently induce Tx tolerance. We now analyzed host immune events associated with these distinct tolerance pathways, and studied as to whether long-term cardiac Tx in presensitized hosts are free of chronic rejection, and hence in the strictest sense may be considered truly tolerant. Spleen cells (100×106) harvested in the maintenance (>120 d) phase of CD4 mAb-induced tolerance readily conferred donor-specific tolerance after i.v. transfer into new sets of cardiac or renal Tx hosts. This organ-nonspecific tolerant state could be then transferred with regulatory T cells in an "infectious" manner into new cohorts of test rat recipients. In contrast, the same number of spleen cells(100×106) from CTLA4Ig-conditioned hosts failed to transfer tolerance. However, features of "infectious" tolerance pathway could be detected after infusion of >300×106 of spleen cells from CTLA4Ig-treated rats. Competitive RT-PCR was used to study cytokine gene expression at the Tx site. CD4 mAb therapy abolished transcription of both Th1 (IL-2/IFNγ) and Th2 (IL-4/IL-10) cytokine genes in the early and late post-Tx phases, as compared to rejecting controls. In contrast, CTLA4Ig treatment resulted in a progressive and selective sparing of Th2-type cytokines at the Tx site. Cardiac Tx harvested at >100 d from tolerant rats showed little evidence of arteriosclerosis. Indeed, both RIB-5/2 mAb and CTLA4Ig treated hosts had very similar mean neointimal vessel occlusion scores at 0.80±0.36 and 0.82±0.30 (mean±SD; n=4/group), respectively. The number of diseased vessels in the two groups was not different either, with 48.7%±18.3 in the RIB-5/2 mAb group and 54.3%±30.8 in the CTLA4Ig group being affected. Conclusion: (i) CD4 mAb-induced blockage of activation signal 1 or CTLA4Ig-mediated blockage of co-stimulatory signal 2 may induce a true transplantation tolerance in presensitized rat recipients; (ii) the"infectious" pathway operates in a strictly cell dose-dependent manner, it may thus represent the natural host tolerogenic mechanism independent of the exogenous modulation; (iii) immune deviation towards Th2-type cytokine programs in the graft itself does not necessarily result in chronic rejection.

Allogeneic peripheral blood progenitor cells for treatment of relapse after bone marrow transplantation.

Donor leukocyte infusions (DLI) are an effective therapy for patients who relapse with leukemia after bone marrow transplantation (BMT). Severe graft-versus-host disease and prolonged periods of pancytopenia compromise the success of this treatment in a substantial number of patients. We used filgrastim-mobilized peripheral blood progenitor cells (PBPCs), in some cases preceded by cytoreductive therapy, to circumvent some of the problems associated with DLI. Eleven patients (median age 41 years) received a total of 20 donor cell infusions. Their diagnosis was CML in hematological (two patients) or cytogenetic relapse (two patients), six patients suffered from acute myeloid leukemia (AM; n = 5) or Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL Ph+). One patient had multiple myeloma (MM). All six patients with acute leukemias received cytoreductive therapy prior to PBPC infusions; three patients with CML were pretreated with IFN alpha. Four of four patients with CML responded to PBPC infusions and currently are in complete clinical and molecular remission for time periods between 1 and 12 months. Six of six patients with acute leukemias achieved a complete remission. All of them relapsed after a median remission duration of 24 weeks (range 11-49 weeks). Three patients relapsed at extramedullary sites (CNS, testes, skin). Four of six acute leukemia patients received further cytoreductive therapy. All patients responded again and are in complete remission for time periods between 14 and 615 days. Two patients with acute leukemias have died due to dissemination of the disease. The patient with MM did not respond and is alive with disease. Severe (grade III) acute GVHD developed in two of 11 patients, three patients developed grade II disease, six patients did not show any signs of GVHD. Extensive chronic GVHD has developed in two cases to date. Patients with chemotherapy prior to PBPC infusion developed neutropenia and thrombocytopenia with a maximum duration of 20 and 14 days, respectively; prolonged periods of neutropenia did not occur. Two patients developed long-lasting thrombocytopenia in spite of PBPC infusion, in one case followed by leukemic relapse. Repeated courses of chemotherapy and PBPC infusion were generally tolerated well; no early deaths due to treatment-related toxicity or GVHD were observed. We conclude that the use of allogeneic PBPC instead of DLI in patients with relapse after BMT is technically feasible and safe. The efficacy of PBPC infusions seems comparable to DLI in patients with CML. Patients with acute leukemias also achieved complete albeit transient remissions. Aggressive chemotherapy followed by PBPC infusions resulted in only limited duration of cytopenia. The usage of PBPC infusion instead of non G-CSF-mobilized donor cells for treatment of relapse after BMT may reduce pancytopenia-related complications and merits further investigation.

Delayed Infusion of Immunocompetent Donor Cells After Bone Marrow Transplantation Breaks Graft-Host Tolerance and Allows for Persistent Antileukemic Reactivity Without Severe Graft-Versus-Host Disease

The development of graft-host tolerance after bone marrow transplantation (BMT) is crucial to avoid the problems of graft-versus-host disease (GVHD) and graft rejection. GVHD can be eliminated by depleting mature donor T cells from the BM inoculum, thereby facilitating the development of graft-host tolerance. However, T-cell depletion often results in an increased incidence of graft rejection and an increased frequency of leukemia relapse. Thus, although graft-host tolerance is a desirable outcome, it can pose a significant threat to leukemia-bearing hosts. Using a major histocompatability complex (MHC)-matched allogeneic model of BMT (B10.BR into AKR), we found that irradiated recipients given donor BM alone displayed mixed T-cell chimerism and did not develop GVHD. Graft-host tolerance developed by 8 weeks after BMT in these chimeras, and they were susceptible to low-dose leukemia challenge. When sufficient numbers of donor spleen cells, as a source of T-cells, were added to the BM graft, AKR hosts developed severe and lethal GVHD. Antihost reactive donor T cells persisted in chimeras undergoing GVHD, indicating that graft-host tolerance did not develop. When administration of the spleen cells was delayed for 7 to 21 days after BMT, there was significantly less mortality because of GVHD. Day 21 was the optimal time for infusion of cells without development of GVHD. Graft-host tolerance was broken by the delayed infusion of donor cells, as indicated by the persistence of antihost-reactive donor T cells in these chimeras in T-cell receptor cross-linking and mixed lymphocyte reaction assays. Importantly, the persistence of antihost-reactive donor T cells correlated with along-term antileukemic effect that was still present at 100 days after transplant. Multiple infusions of immunocompetent donor cells could be administered without increasing the risk for GVHD if delayed until 21 days post-BMT. Delayed infusions of donor spleen cells also resulted in a long-term antileukemic effect in the absence of GVHD in an MHC-haplotype-mismatched model of BMT (SJL into [SJL x AKR]F1). Although delayed infusion of normal donor cells did not induce GVHD, spleen cells from donors previously sensitized to host alloantigens induced GVHD when infused 21 days after BMT. Thus, the ability of previously activated cells to induce GVHD was not inhibited in the same manner as naive cells. Results from limiting dilution analysis assays indicated that alloactivated interleukin-2-secreting CD4+ T cells were preferentially inhibited over cytolytic T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Donor leucocyte imaging in patients with AIDS: a preliminary report

Four patients with the acquired immunodeficiency syndrome (AIDS) and fever were investigated using donor leucocyte scans. The lung/liver and lung/spleen uptake ratios in these patients were compared with the uptake ratios in donor leucocyte scans in seven neutropenic (non-AIDS) patients and five patients who had autologous leucocyte scans performed over the same time period. For all scans indium-111-oxime-labelled leucocytes were used, except for one AIDS patient in whom technetium-99m hexamethyl-propylene amine oxide (HMPAD)-labelled donor leucocytes were used. There were no adverse reactions to the donor cell infusions. Two patients had repeat studies 8 weeks apart (from different donors) without ill effect. There were no differences in the 111In uptake ratios between the three groups. There were three positive studies in the patients with AIDS, and these elucidated the cause of the pyrexia in all three. The negative case is more difficult to confirm, but the clinical course and the absence of focal disease on post-mortem have been taken to support the scan findings. There was no difference in the acceptability of the technique or the distribution of the labelled leucocytes between the AIDS and non-AIDS patients. Donor leucocyte imaging of patients with AIDS is probably more effective and considerably less hazardous for technical staff than autologous leucocyte methods. This study demonstrates that the technique can be applied successfully to patients with AIDS.

APPARENT LACK OF H-Y-SPECIFIC SUPPRESSOR CELLS IN FEMALE MICE GIVEN MALE SPLEEN CELLS INTRAVENOUSLY

Mice given a pretransplant i.v. infusion of spleen cells from donors incompatible with the recipients with respect to individual minor H antigens exhibit a delayed rejection of skin grafts from these incompatible donors. Attempts were made to detect suppressor T cells, possibly induced by the i.v. infusion of donor cells, among spleen cells from female mice made hyporesponsive to H-Y antigen by an i.v. infusion of male spleen cells. When transferred to nonimmune immunocompetent mice, lymphoid cells from hyporesponsive females delayed the rejection of male skin grafts only slightly. Similar results were obtained when putative suppressor cells were transferred to immunodeficient hosts having had their potential to reject male skin grafts restored with either nonimmune or specifically immune female lymphoid cells. The observed suppression of graft rejection could be accounted for, to a significant extent, by male spleen cells carried over with the transferred female cells to the assay recipients. Though an active host-derived suppressor mechanisms was not found, transferred spleen and lymph-node cells obtained from females given a prior i.v. infusion of male spleen cells were poor at restoring the capacity to reject male skin grafts to immunodeficient female recipients, whereas equal numbers of lymphoid cells from untreated females were much better. Thus, i.v. infusion of male spleen cells appeared to deplete the peripheral lymphoid tissue of the recipients of responsivity to H-Y antigen.

Increased elevation of peripheral leukocyte counts by infusion of histocompatible granulocytes.

Abstract. The results of 38 HL‐A and other antigen matching studies revealed an inverse relationship between the percent of donor‐recipient serologic discordance and increases in recipient granulocyte levels 1 h after infusion of donor cells. Selected unrelated, parent, and sibling donors possessing mean percent serologic discordances of 33, 22, and 16, respectively, increased postinfusion granulocyte counts by 10, 46, and 117%, respectively. A mean of 1.58 × 1010 total granulocytes collected from sibling donors was associated with a significant elevation of recipient levels, as compared to unfavorable results with the infusion of 2.05 × 1010 and 2.31 × 1010 total granulocytes harvested from less compatible parent and selected unrelated donors, respectively.