Stable Mixed Chimerism Research Articles

T-Cell Chimerism Analysis by Mutiplex STR PCR after Non-Myeloablative Allogeneic Stem Cell Transplantation

Background: The ability of non-myeloablative allogeneic stem cell transplants to eradicate host neoplastic cells is based on the accumulating evidence of a graft-versus-malignancy (GVM) effect. Stable mixed chimerism (MC) is associated to the lower risk for the development of graft-versus-host diseases (GVHD), but this possibly occurs at the expense of the GVM effect. Therefore, assessment of the chimerism status is critical to allow immune intervention to maintain a state of donor-host tolerance and to prevent loss of the graft. Methods: Serial post-transplant peripheral blood samples were collected from 17 patients with various malignant diseases following non-myeloablative allogeneic stem cell transplantation. DNA was amplified from the T-cells, and the polymerase chain reaction (PCR) products were quantified by an automated fluorescent DNA analyzer. Results: All 17 patients showed T-cell MC at post-transplant, but this varied in degree and duration, and then 3 patterns emerged. Group 1: 5 patients experienced a short interval of T-cell MC prior to conversion to complete donor chimerism (CC) (median: 25 days). Group 2: 5 patients showed a rapid increase of host cells after a brief MC at a median of 21 days. They never achieved CC, and they relapsed or showed progressive diseases. Group 3: 7 patients showed persistent T-cell MC for 40-50 days, and they subsequently gradually converted to CC after a median of 112 days. Conclusion: All the patients achieved T-cell MC in post-transplant, but the CC development differed in frequency and speed. GVHD preceded the onset of T-cell CC in the majority of the patients. Serial engraftment monitoring of the T-cell chimerism status during the first 100 days after non-myeloablative stem cell transplantation is important in aiding the clinical management of such patients. (Korean J Hematol 2006;41:28-35.)

Renal Allograft Tolerance in DLA-Identical and Haploidentical Dogs After Nonmyeloablative Conditioning and Transient Immunosuppression With Cyclosporine and Mycophenolate Mofetil

Background Canine models of bone marrow and renal transplantation have provided important preclinical data relevant to developing novel therapeutic protocols for hematopoietic and solid organ transplantation in human beings. Nonmyeloablative transplantation has been shown to induce stable mixed hematopoietic chimerism in normal dogs and correct the phenotype of canine pyruvate kinase deficiency and Glanzman’s thrombasthenia. In this study, we investigated the potential for inducing renal allograft tolerance using a nonmyeloablative bone marrow transplantation strategy that induces mixed chimerism in DLA-identical dogs. Methods Reciprocal renal allografts were performed in 4 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy total body irradiation [TBI]) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with and without simultaneous bone marrow transplantation. Two DLA-identical control dogs received reciprocal renal allografts without TBI or immunosuppression with CSP and MMF. Serum creatinine (Cr) concentration was monitored to assess renal allograft function. Results The renal allografts were acutely rejected in the 2 DLA-identical dogs without TBI or immunosuppression. There was long-term (>1 year) renal allograft survival as evidenced by a normal (<2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous bone marrow transplantation. Conclusions Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal allograft tolerance in DLA-identical and DLA-haploidentical dogs without donor/host mixed hematopoietic chimerism. These findings suggest it may be possible to induce tolerance to solid organ transplants without the need for chronic immunosuppressive therapy or stable hematopoietic chimerism in the setting of both DLA-matched and haploidentical transplants.

Peripheral CD4+CD25+ Regulatory T Cells (Treg) Block Alloreactive Host Anti-Donor T Cells in Canine Mixed Hematopoietic Chimeras.

CD4+CD25+ Treg cells may be important immune regulators for the maintenance of immune tolerance after allogeneic hematopoietic cell transplantation (HCT). In the well-established canine model of nonmyeloablative HCT, stable mixed donor/host hematopoietic chimerism persists for the lifespan of the recipient following 2 Gray total body irradiation, dog leukocyte antigen (DLA)-identical bone marrow transplantation and combined postgrafting immunosuppression with mycophenolate mofetil and cyclosporine for 4 and 5 weeks, respectively. We hypothesized that CD4+CD25+ Treg are an important component of the active cellular mechanism of peripheral tolerance that maintains stable mixed chimerism in this HCT model. Previously, we showed that depletion of CD4+CD25+ Treg cells from mixed lymphocyte culture (MLC) significantly increased allo-specific proliferation in the DLA-mismatched setting. In the current study, we asked if depletion of CD4+CD25+ Treg from peripheral blood mononuclear cells (PBMC) obtained from dogs with stable mixed chimerism 1 to 4 years after nonmyeloablative HCT could generate minor histocompatibility antigen (mHAg)-specific, host anti-donor cytotoxic T lymphocytes (CTL). Responder PBMC from mixed chimeric dogs were cultured in a modified MLC with irradiated, CD34+ derived dendritic cells (DC) from the respective DLA-identical HCT donor at a 10:1 responder: stimulator ratio. On day 4 of MLC, CD4+CD25+ T cells were depleted by either immunomagnetic selection (Miltenyi) using the anti-CD25 monoclonal antibody (ACT-1) or the addition of 10−11M denileukin diftitox (DAB389IL-2, Ontak) for 18 hours. DAB389IL-2 is a fusion protein consisting of the ADP-ribosyltransferase domain of diphtheria toxin and IL-2. By flow cytometry analysis, depletion of CD4+CD25bright+ was 60%–66% with immunomagnetic selection and 70%–77% with DAB389IL-2. On day 5, culture media was changed to remove residual DAB389IL-2 in the respective flasks. A secondary MLC was established with CD4+CD25+ depleted responder cells and irradiated DC from the respective DLA-identical HCT donor. Peak proliferation of responder cells measured by 3H-thymidine incorporation was on day 4 of secondary MLC. There was a 54%–95% increase in proliferation of cells depleted of CD4+CD25+ T cells with immunomagnetic selection and a 121%–133% increase in proliferation of DAB389IL-2 treated cells compared with non-depleted responder cells. We evaluated mHAg-specific cytotoxic activity of the CD4+CD25+ depleted responder T cells by 51Cr release assay following 3rd MLC stimulation with respective DC. At a 10:1 effector: target ratio, there was 74%–82% specific lysis of the respective HCT donor cells by the CD4+CD25+ depleted responder T cells, while the non-depleted control responder cells had 4%–6% specific lysis. PCR analysis with informative microsatellite markers confirmed the alloreactive CTL were of host origin. In summary, depletion of CD4+CD25+ Treg from PBMC of mixed chimeras permitted the emergence of host anti-donor mHAg-specific CTL. These data suggest that mHAg-specific host anti-donor T cells persist in the peripheral blood of stable mixed chimeric dogs and that peripheral CD4+CD25+ Treg prevent alloreactive immune responses and maintain mixed chimerism. These results support the hypothesis that mixed hematopoietic chimerism established without T cell depletion relies on peripheral immune tolerance mechanisms.

Monitoring Graft Function after First Allogeneic Transplant for Mucopolysaccharidosis-IH: Patterns of Rejection and Outcome of Second Allograft Procedure.

Mucopolysaccharidosis type I (MPS-I [Hurler's syndrome]) is an autosomal recessive condition caused by a deficiency of alpha-L-iduronidase. We report single centre 20 year experience of outcomes of BMT in MPS-IH. As there is no consensus about graft monitoring and the indication for second transplant, we describe the monitoring of graft function, indications for and management of second BMT in this institution.Data was collected from 43 patients receiving 61 transplants who underwent allogeneic transplantation at the Royal Manchester Children's Hospital between 1985 and 2005. The median age at first transplant was 15 months (range 7– 44), and of these 23 were male and 20 were female. 24 of the first transplants were from an unrelated donor. Graft function was monitored prospectively in each transplant using enzyme levels, urinary GAGs and, for the last 33 transplants, VNTR analysis.Almost half of the 61 transplants carried out since 1985 have been done in the last five years. Of the 43 patients transplanted, 19 survived with stable engraftment, 16 patients rejected their first grafts and all 16 of these patients went on to receive a second transplant. GVHD occurred in 16 of 28 patients who received only 1 transplant. Of the 16 who had 2 procedures, 6 had GVHD after their first transplant and 8 after their second. 8 patients died and all deaths were due to transplant-related complications (1 EBV/LPD, 1 CMV, 4 Adenovirus, 2 GVHD). 24 patients received unrelated donor grafts. 16 of these MUD transplants were carried out before 2002 and 8 since 2002. 6 of the 8 deaths were in patients who received unrelated donor grafts and all 6 of these were pre 2002. This improvement in mortality is significant (Fisher's exact test 2-sided p= 0.04).45 of the 61 transplants were carried out after 1995 and these patients all had regular VNTR monitoring of their graft function. From these results 4 groups of graft status are identified: full engraftment, stable mixed chimerism, immediate rejection, early autologous reconstitution. 23 patients remain fully engrafted at 100% VNTR levels. 16 of these were after first transplant and 7 following their second transplant. 4 patients have stable mixed chimerism (2 after 1st transplant and 2 after 2nd). Although the VNTR levels are now not 100% in this group, they remained at 100% until at least 70 days post transplant. They reached a state of stable mixed chimerism at a mean of 203 days post transplant (range 133–280). The final level of mixed chimerism reached was a mean of 59% donor. 3 patients immediately rejected their graft with VNTR levels falling to 0% within 28 days of transplantation. 8 patients had early autologous reconstitution requiring a second transplant procedure once their VNTR levels fell to 20%, and in these there was early mixed chimerism (within 60 days after graft).All 16 patients who rejected their first graft went on to have a second transplant when their VNTR levels fell to<20%. 13 of these survived with stable engraftment and 3 died. The donor was the same in all but one case, and conditioning was usually of reduced intensity.SummaryGraft rejection is not uncommon after allogeneic BMT for MPS-1H and can be detected using VNTR, enzyme and urinary GAGs. Where chimerism is detected earlier than 60 days the graft is subsequently lost, whereas later mixed chimerism usually stabilises at adequate levels. Second transplant is effective and safe using the same donor.

Effect of Intravenous Busulfan Versus Total Body Irradiation on Donor Chimerism and Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Stem Cell Transplantation (AlloSCT) in Mice.

The goals of administering transplantation conditioning to leukemia patients undergoing alloSCT are to kill tumor cells, prevent rejection of the donor graft, create “space” for donor hematopoiesis, and provide an overall milieu that favors a donor T cell-mediated “graft-versus-leukemia” effect. However, transplantation conditioning also increases the incidence and severity of acute GVHD by damaging non-hematopoietic tissues and by inducing lymphopenia, which augments the antigen-driven expansion of alloreactive T cells. The purpose of this study was to compare the relative safety and efficacy of transplantation conditioning with intravenous busulfan (Busulfex; ESP Pharma, Edison, NJ) versus total body irradiation (TBI) by administering titrated doses of either agent to mice and comparing their effects on: 1) donor chimerism after syngeneic and semi-allogeneic (parent into F1) SCT; 2) graft rejection after MHC-matched allogeneic SCT; and 3) acute GVHD after semi-allogeneic SCT. Both agents induced titratable donor chimerism after syngeneic BMT, but TBI was more immunosuppressive, since allogeneic marrow engrafted after 700 cGy TBI but not after an equivalently myelotoxic dose of Busulfex (60 mg/kg). When given in equivalently myelotoxic doses, TBI always induced more GVHD than did IV busulfan, as measured by weight loss and the augmentation of donor T cell and myeloid chimerism. As a result, stable mixed hematopoietic chimerism was generated more readily in mice conditioned with nonmyeloablative doses of busulfan than in mice conditioned with equitoxic doses of TBI. These results demonstrate that IV busulfan promotes GVHD to a lesser extent than does an equivalently myelotoxic dose of TBI. IV busulfan may be the preferred agent if stable mixed hematopoietic chimerism is the desired outcome. The relative roles of lymphopenia versus tissue damage in the differential outcomes of TBI versus Busulfex conditioning are being investigated.

Synergy between Nonmyeloablative Doses of Intravenous Busulfan and Post-Transplantation Cyclophosphamide for Induction of Tolerance to MHC-Compatible Stem Cell Allografts.

The goal of conditioning for nonmyeloablative allogeneic stem cell transplantation, or NST, is to permit the stable engraftment of donor cells with a minimum of toxicity to the patient. We have previously developed a minimal conditioning regimen, consisting of low dose total body irradiation (TBI; 100–200 cGy) and post-transplantation cyclophosphamide (Cy), that permits the induction of stable mixed hematopoietic chimerism following the transplantation of modest doses of MHC-identical allogeneic stem cells in mice. The goal of the current study was to determine whether intravenous (IV) busulfan (Busulfex; ESP Pharma, Edison, NJ), an agent with myelotoxic but minimal immunosuppressive activity, could be used in place of TBI in this regimen and still promote allograft tolerance. TBI was more immunosuppressive than Busulfex, because engraftment of allogeneic stem cells could be achieved in mice conditioned with 700 cGy TBI alone but not in mice conditioned with an equivalently myelotoxic dose of 60 mg/kg Busulfex IV. However, when post-transplantation Cy was added, stable mixed hematopoietic chimerism was achieved in mice conditioned with doses as low as 100 cGy TBI or 20 mg/kg Busulfex. Busulfex and post-transplantation Cy were both required for the induction of allograft tolerance, because graft rejection occurred in transplanted mice treated with either agent alone. The capacity of Busulfex to promote allograft tolerance in this model could not be explained by effects of the drug on the host immune system, because the graft rejection response was not restored in Busulfex-conditioned mice reconstituted with mature host lymphocytes at the time of transplantation. These results demonstrate that Busulfex effectively substitutes for TBI in promoting allograft tolerance, and suggests that the creation of marrow “space” promotes alloengraftment in conditions of suboptimal host immunosuppression.

Stable Mixed Hematopoietic Chimerism in Dogs Conditioned with Donor Antigen, Anti-CD154 Antibody and 100cGy TBI.

Stable mixed chimerism can be established in dogs given a sublethal dose of 200cGy total body irradiation (TBI) before and immunosupression with mycophenolate mofetil (MMF) and Cyclosporine (CSP). When the TBI dose is reduced to 100 cGy, only transient engraftment is observed. Here we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with the help of an anti-CD154 antibody which blocks the CD40-CD154 pathway.Accordingly, recipients were given a single i.v. injection of 5 mg/kg anti-CD154 antibody (day -5) followed one day later by combined i.v./sc. injections of 107 peripheral white blood cells/kg from their intended DLA-identical littermate marrow donors. TBI, 100cGy (delivered at 7 cGy/min), was given on day 0 followed by infusion of a median of 4.22x108 marrow cells/kg. Postgrafting immunosuppression consisted of MMF (10mg/kg, BID, days 0 to 28) and CSP (15 mg/kg BID, days -1 to 35). Six dogs have been transplanted. Two dogs are too early after transplant for evaluation, while four dogs have been followed for >9, 12, >18 and >26 weeks. All four dogs had initial engraftment. Three of four dogs have continued to show stable mixed donor/host hematopoietic chimerism among lymphocytes and granulocytes from the peripheral blood. One dog rejected the graft 12 weeks after transplantation. All six transplanted dogs are alive and well. Besides mild allergic reactions to the antibody in five of the six dogs (redness of the eyes, skin rashes and dry mucous membranes), no additional side effects attributed to the pretransplant treatment were observed.Data were consistent with previous findings in dogs, in which the costimulatory signal between B7 and CD28 was blocked, and support the hypothesis, that stable marrow allografts would be established by combining non-myeloablative pretransplant host immunosuppression and posttransplant immunosupression of both host and donor cells using MMF and CSP.

Reduced Intensity Conditioned Allogeneic Stem Cell Transplantation Is as Effective in Poor Risk as Standard Risk Acute Myeloid Leukaemia (AML).

Poor risk cytogenetics and multi-lineage dysplasia are commonly found in older patients with acute myeloid leukaemia (AML) and despite intensive chemotherapy prognosis remains grim. We compared the outcome of standard risk to poor risk AML (both according to cytogenetic risk group and presence of underlying dysplasia) following reduced intensity conditioned (RIC) allogeneic haematopoietic stem cell transplantation (allo-HSCT) in 65 patients (32 males, 33 females) with a median age of 53years (range 22–65years). Conditioning regimen incorporated fludarabine(150mg), Alemtuzumab(100mg) and busulphan(8mg–16mg). 35/65 had a background of multi-lineage dysplasia (MLD-AML), 30/65 de-novo AML. According to cytogenetics, 48 were standard risk, 15 poor risk and 2 good risk (both good risk cases were in second remission). Median number of chemotherapy regimens administered prior to RIC HSCT was 3 (range 1–8), with 33/65 receiving FLAG chemotherapy and 2/65 a previous autologous HSCT. 57/65 were in complete remission (CR) at time of HSCT, 6/65 partial remission (PR) and 2/65 refractory disease. 51/65 were in first remission, 14/65 second remission. Sibling donor was used in 25/65, volunteer unrelated donor (VUD) in 40/65 (1 antigen mismatch 13/40, 2 antigen mismatch 3/40). Bone marrow (BM) was administered to 17/65 and peripheral blood stem cell (PBSC) to 48/65.Median follow-up was 389days (range 36–1731) for all patients and for those alive 633days (range 71–1731). Failure to engraft occurred in 1 patient and late graft rejection in 3 patients. The day+100 transplant related mortality for all patients was 11%. The overall survival (OS) at 1year for siblings versus VUD was 77% v 58% (p=0.46) and the disease free survival (DFS) 50% versus 53% (p=0.98). The OS for de-novo AML versus TLD-AML at 1year 73% versus 59% (p=0.34) and DFS 59% versus 46% (p=0.06). Comparison of the OS at 1year for the standard versus poor risk cytogenetic groups was 63% versus 72% (p=0.33), and DFS 50% versus 57% (p=0.68) respectively. 15/65 developed acute GvHD grade II–III. The cumulative incidence of chronic GvHD was 19% in those surviving after day+100. 46% of patients achieved full donor engraftment on lineage specific chimerism at day+100. Incremental donor leukocyte infusions (DLI) were administered to 25 patients. 16/25 received DLI for mixed chimerism, 9/16 reverted to 100% donor, 4/16 continued with stable mixed chimerism and 3/16 developed relapsed disease. 9/25 received DLI for disease relapse resulting in complete remission in 3/9 cases. 10/23 patients administered DLI developed GvHD grade II–III. In summary, RIC allo-HSCT incorporating Alemtuzumab was equally well tolerated in both sibling and VUD allografts, with a low incidence of acute and chronic GvHD. The results show favourable outcome in a poor risk cohort of patients with AML, with comparable OS compared to standard risk/de-novo AML. DFS was not affected by cytogenetic risk however comparison of de-novo AML to MLD-AML did show a trend (p=0.06) in favour of de-novo AML.

Use of Pre-Emptive Donor Lymphocyte Infusions To Achieve Durable Remission Following Alemtuzumab Based Reduced Intensity Conditioning (RIC) Transplantation for AML or MDS.

Use of alemtuzumab in RIC HSCT reduces the incidence of graft rejection and graft vs host disease(GvHD). However, there can be a delay in full T-cell donor engraftment. As a dominant donor T-cell chimerism may be important to achieve a strong graft vs leukaemia effect(GvL), we examined the impact of pre-emptive DLI (pDLI) on patients with falling donor chimerism. 76 patients with AML or MDS were treated with RIC HSCT (fludarabine 150mg/m2, busulphan 8mg/kg, alemtuzumab 100mg). Complete sublineage chimerism data up to day +100 was available on all patients. The underlying diagnoses were AML n=27, MDS n=49. 33 patients had early disease vs 44 advanced disease (advanced disease as defined by AML >CR1, MDS RAEB or AML with multilineage dysplasia). The median recipient age was 51.6 years (range:19–72), with median follow-up of 526 days (range:137–1256). There were 30 sibling and 50 VUD allografts. Stem cell source was 61 PBSC vs 15 BM. 62 patients were fully HLA matched and 14 patients were HLA mismatched.CD15 engraftment occurred rapidly with 95% of patients achieving full donor chimerism(FDC) at day 30 and 96% at day 100. In contrast, CD3 engraftment was significantly delayed, with only 50% of patients FDC at day 30, 47% at day 100. Incremental doses of pDLI were considered for patients with falling donor chimerism (<50% donor) after day 100. Patients had immunosuppresion withdrawn, and had to have no GvHD. 20 patients received a total of 55 doses of pDLI. 10/20 had advanced disease, and 6/20 had unfavourable cytogenetics. Median donor CD3 chimerism at time of pDLI was 31.5%(range:7–59). The median CD3 dose of pDLI was 8.4x106/kg, with the first dose given at a median of day +176 (range:104–494). The median interval between pDLI was 8 weeks(range:4–22). 15 patients had FDC restored at median of 130 days following first doses of pDLI (range:36–523). 8/20 developed acute Gd II-IV GvHD following pDLI, with 2 patients dying of GvHD related complications. 2 patients relapsed with AML following treatment: with 1 death, and 1 patient currently undergoing treatment. 2 patients had not reached FDC at follow-up. A further 9 patients received DLI for cytogenetic or morphological relapse. Time to first dose of DLI was 257 days (range:76–837). The median CD3 dose was 1.67 x 107/kg. 3 patients were FDC and 6 patients MDC at time of relapse. All 3 patients with FDC failed to respond to DLI. Complete remission was seen in 3/6 patients with MDC. 4/9 patients developed acute Gd II-IV GvHD. 5/9 patients have died(all of underlying AML).The outcome of patients receiving pDLI was compared with patients with FDC(n=28), and stable mixed chimerism(defined as donor CD3 chimerism >70%) who did not receive DLI(n=18). There was no significant difference in recipient age, disease, disease stage, HLA type, cell source or cell dose between groups. However, there were more sibling donors in the group receiving pDLI(p=0.02). The 2 year DFS, OS and relapse rate was comparable between patients with FDC, stable chimerism and those receiving pDLI (59% vs 83% vs 67% p=0.22), (62% vs 88% vs 75% p=0.13), (12% vs 17% vs 15% p=0.74) respectively. In summary, pre-emptive DLI is effective in reversing falling donor chimerism, and can induce prolonged remission, even in a sub-group of patients with high risk disease. A dominant donor CD3 chimerism(>70%) may be sufficient to acheive an allo-immune effect in majority of patients.

The Different Dynamics of Chimerism Evolution Determines the Success of Myeloablative, Reduced Intensity Conditioning and T-Cell Depleted Allogeneic Stem Cell Transplantation.

Background: The dynamics of chimerism evolution determines the success of allogeneic stem cell transplantation (SCT). Several factors such as the intensity of the conditioning regimen, the T-cell content of the graft or the GVHD prophylaxis, influence the degree of chimerism after SCT. Objective: To evaluate the dynamics of chimerism after different SCT settings (ablative, reduced intensity conditioning -RIC- and T-cell depleted -TCD-) and its influence in the success of the procedure.Patients and Methods: The study includes 68 SCT: 32 ablative (including 7 from HLA matched unrelated donors -UD-), 19 RIC and 17 TCD (including 8 from haploidentical donors and 2 from UD with 1 HLA disparity). Chimerism analysis was performed by FISH for the sex chromosomes or STR-PCR (sensitivity 1 Samples obtained on days +30, +100, +180, +365 and once a year thereafter, included bone marrow (BM) and peripheral blood (PB). Moreover, chimerism was analyzed in PB and leukocyte lineages (T lymphocytes CD3+, B lymphocytes CD19+ and myeloid cells CD15+ isolated (purity >95%) by immunomagnetic means, AutoMACS, Miltenyi Biotec), every 2 weeks, starting on day +15 (except in ablative), and until complete chimerism (CC) was achieved. Results of chimerism follow-up were censored once the diagnosis of relapse or rejection was established.Results: The incidence (% patients) of mixed chimerism (MC) on day +30 (ablative: BM 27%, PB 15%; RIC: BM 40%, PB 41%, CD3 40%; TCD: BM 31%, PB 33%, CD3 54%), as well as its dynamics (MC on day +100: ablative BM 8%, PB 4%; RIC BM 8%, PB 15%, CD3 22%; TCD BM 20%, PB 40%, CD3 37%) were different in the three SCT settings. All except 2 CD15 samples analyzed showed CC. Moreover, the percentage of recipient cells (%R) was significantly higher after RIC and TCD than after ablative SCT, as well as in T lymphocytes than in BM or PB (7/8 cases with simultaneous studies showed MC in T lymphocytes and CC in PB). All RIC SCT evolved to CC by day +180 while TCD SCT showed persistent MC (2 patients with stable MC after one year). The incidence of rejection was greater after RIC (2/19) and TCD (4/17) than after ablative SCT (2/32). All these patients showed MC, mainly in T lymphocytes, which allowed early diagnosis and successful treatment with immunosuppression withdrawal and donor leukocyte infusion. Patients with CC in PB/T lymphocytes on day +30 had a higher incidence of GVHD>I than those with MC. In the present series, however, a relationship between chimerism and relapse, disease free survival or overall survival, was not observed.Conclusions: SCT with greater incidence of MC (RIC and TCD) favor immune tolerance between donor and recipient which reduces the risk/severity of GVHD at the expense of a higher incidence of graft rejection.

Donor-Specific Tolerance Is Induced after Nonmyeloablative Conditioning and Allogeneic Hematopoietic Cell Transplantation and May Persist after Graft Rejection.

The association between stable hematopoietic chimerism and donor specific tolerance (DST) for solid organ grafts was demonstrated clinically and in different experimental models. However, the existing data also demonstrate that the establishment of hematopoietic chimerism does not always correlate with the development of DST, especially for skin grafts. As these issues could have major implications for organ transplantation in general, the present study was undertaken to assess DST in recipients with transient or stable hematopoietic cell (HC) chimerism established after nonmyeloablative conditioning regimens. In a canine model of allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning, DST to skin grafts was evaluated in DLA-identical recipients with stable mixed hematopoietic chimerism (MC) (n=11), or following transient HC engraftment (n=22). There was a significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with stable MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in 4 recipients with MC developed an inflammatory reaction without skin graft loss consistent with a chronic rejection process. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged in recipients after transient HC engraftment compared to normal recipients (P=0.002). However, a chronic inflammatory process without graft loss developed in all of the HC donor-derived skin grafts from this group. An increased time to rejection of the hematopoietic graft was significantly associated with an improved survival of the subsequent skin graft (P=0.02). The time to rejection of third-party (DLA-nonidentical) skin grafts (n=40) was the same in recipients with stable MC and previous HC graft as well as normals (median- 8 days). Autologous skin grafts (n=40) survived for the duration of follow-up without developing chronic inflammation. Extended HC donor-specific skin graft survival (>15 days) was predictive of subsequent stable engraftment at second HCT in recipients with previous graft rejection after first HCT. Five of 6 dogs with extended survival of the skin graft beyond day 15 became stable MC after second HCT compared to 0/9 dogs with skin grafts surviving less than 11 days (P=0.0008). In this model, DST to skin grafts in recipients with stable MC after nonmyeloablative HCT may not be complete, and chronic inflammation may develop without loss of that graft. Partial DST may persist after rejection of HC grafts established with nonmyeloablative conditioning. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.

Influence of Hematopoietic Cell Lysate Vaccinations and Dendritic Cell Enriched Marrow Grafts on Engraftment after Nonmyeloablative Hematopoietic Stem Cell Transplantation.

Background: Stable mixed hematopoietic chimerism can be established in a canine marrow transplantation model using nonmyeloablative conditioning consisting of total body irradiation (TBI, 2Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine A (CSA). Reduction of TBI to 1Gy resulted in rejection of marrow grafts in this model even if CD3 depleted donor PBMC were added to the graft. Here, we investigated whether addition of immature dendritic cells or postgrafting in vivo stimulation of donor T cells against recipient derived hematopoietic antigens are capable of enhancing targeted GVH reactions and allow engraftment.Methods: Dog leukocyte antigen-identical littermates received 2Gy TBI (group 1, control, n=7) and 1Gy TBI (group 2, n=7; group 3, n=6) before allogeneic stem cell transplantation (SCT). All recipients were given postgrafting immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to +35) and 10mg/kg MMF BID PO (days 0 to +27). In addition, group 2 was vaccinated ID and SQ with recipient blood cell lysates (3x/week w1-w5; 1x/week w6-w9) together with locally applied granulocyte macrophage-colony stimulating factor (125μg/vaccination). The vaccine consisted of PBMC and granulocytes at a ratio of 1:1 (105 cells/kg before lysis). Dogs of group 3 received ex-vivo generated immature monocyte-derived dendritic cells (MoDC) of donor origin in addition to marrow at day 0. MoDC doses ranged from 6.4–10.9x105 cells/kg (median 7.9x105). Littermate donor marrow was infused IV at a median of 3.4x108 (1.6–11.4x108) nucleated cells/kg.Results: The median platelet and leukocyte nadirs in group 1 were 30x109/l (median at day 11) and 2.4x109/l (median at day 8), respectively. In groups 2 and 3 the median platelet and leukocyte nadirs were 110x109/l (median at day 12) and 4.8x109/l (median at day 7). All dogs in group 1 engrafted and showed mixed hematopoietic chimerism among PBMC and granulocytes (after 4 weeks (w4): median 32% (5–44%) and 68% (10–74%), respectively). 4/7 animals remained stable mixed chimeras >25 weeks, 3/7 rejected their graft (w10, w14, w16). All dogs in group 2 showed initial engraftment and chimerism of donor PBMC and granulocytes (w4: median 12% (10–19%) and 19% (11–24%), respectively), but rejected their grafts a median of 10.3 weeks (w8-w11) after SCT. 4/6 dogs in group 3 had a median w4-chimerism in PBMC and granulocytes of 14% (11–15%) and 20% (16–27%), respectively and 2/6 dogs are too early after SCT to be evaluated. Currently 3/4 evaluable dogs in group 3 remain mixed chimeric. In comparison to group 1 engraftment kinetics of groups 2 and 3 indicate a delayed engraftment combined with significantly lower donor PBMC and granulocytes chimerisms and significantly reduced engraftment duration (group 2). No differences in w4-chimerism patterns between groups 2 and 3 could be observed.Conclusion: Our data show that vaccinations with recipient hematopoietic cell lysates failed to expedite long term bone marrow engraftment following a 1Gy TBI conditioning. Addition of donor-MoDC to the graft might favour sustained engraftment but a longer follow up is needed. Furthermore, equal dosed MMF applied orally seems to be inferior to SQ applications in postgrafting immunosuppression since compared to historical controls less mixed chimeras could be generated following 2Gy TBI.

Non-Myeloablative Allogeneic Transplantation (NMT) with T-Cell Replete Graft for Relapsed Chemosensitive Follicular Lymphoma (FL): Donor Lymphocyte Infusion (DLI) To Convert Stable Mixed Chimerism to Full Donor Chimerism Is Not Necessary in the Absence of Disease Progression.

DLI is commonly provided for mixed chimerism post NMT, even in the absence of measurable disease. This is with a high risk for graft-versus-host disease (GVHD) and a major cause of mortality and morbidity after NMT. We hypothesized that this practice may not be routinely needed in pts with indolent diseases, such as FL, and who continue to have stable mixed chimera (SMC) after NMT. We defined SMC as presence 50% to 99% donor cells by PCR analysis, and without any significant decrease of > 20% on two consecutive analysis. We treated prospectively 47 pts with relapsed FL with a NMT after conditioning with fludarabine, cyclophosphamide and high-dose rituximab as previously described (Blood 89:3595, 2001). ATG was added for 2 pts receiving unrelated transplants. Tacrolimus and methotrexate were used for GVHD prophylaxis. All pts received a non-manipulated graft, from peripheral blood (45 pts) or marrow (2 unrelated donors). At the time the first analysis was undertaken post NMT (median 30 days), 13 of 18 pts (72%) in clinical complete remission (CR) and 24 of 29 pts (83%) who had evidence of active disease at study entry had mixed chimera (P = 0.2). The % of donor cells were 93% and 75%, respectively. When the subsequent PCR analysis to assess engraftment was undertaken (median time, 89 days post NMT), median % donor cells were 100% and 95%, respectively, for the pts who were in CR or had active disease at study entry. All pts achieved CR post NST. Median time to achieve CR for pts who had active disease at study entry was 5.5 months after transplant. Twelve pts in CR continue to have SMC at the time of their last PCR analysis, undertaken at a median of 12 months (range, 6 to 55 months) post NMT. Nineteen pts were tested for the amplification of bcl-2 rearrangement from the marrow by PCR. They all had evidence of clonal disease prior study entry. All converted to PCR-negativity. Sixteen of these 19 pts had mixed chimera when they were first tested to be PCR (−) at a median time of 3 mos (range, 1–6 months) post NMT. At a median time follow-up time of 34 months, overall survival and disease-free survival of all 47 pts at 3-year were 88 and 85%, respectively. Only one relapse occurred. This occurred in a pt who had 60% donor at 3 months post NMT; it then decreased to 42% at 9 months. This was followed by graft failure and relapse. DLI was provided to only one pt with decreasing chimerism. This resulted to conversion to full donor after the infusion of to 1 x 105 CD3+ cells/kg, from the HLA-identical sibling donor. The incidence of acute grade II–IV and chronic (extensive and limited) GVHD in the study was 17% and 51%, respectively. These data suggest that DLI may not be needed for SMC after non-T cell depleted NMT for pts with FL who do not have evidence of progression. This strategy resulted in lower than expected acute and chronic GVHD and improved survival.

Durable Engraftment of Purified Allogeneic Hematopoietic Stem Cells Following Non-Myeloablative Conditioning.

Transplantation of purified allogeneic hematopoietic stem cells (HSC) has the potential to be a curative treatment for autoimmune diseases. Before it becomes a viable therapy, however, the treatment-related mortality and difficulty of achieving engraftment must be addressed. Our research has focused on developing non-myeloablative regimens that lead to donor-derived engraftment of purified HSC in a murine model. Total lymphoid irradiation (TLI) consists of low-dose fractionated irradiation targeted to the thymus, abdomen, and peripheral nodes, while the skull, lungs, and long bones remain shielded. The non-myeloablative conditioning regimen of TLI and anti-thymocyte globulin (ATG) was followed by HSC transplantation. HSCs were isolated by the composite phenotype of Thy1.1+, c-kit+, Sca-1+, and lineage- (KTLS) or, in strains lacking the Thy1.1 marker, c-kit+, Sca-1+, and lineage- (KSL). We tested HSC transplantations across three major histocompatiblity complex (MHC)-matched strain combinations known through previous studies in our group to have significantly different barriers to engraftment. In all three strain combinations we observed stable mixed chimerism (approximately 50% donor-derived cells) when high doses of HSC (10,000/mouse) were administered. Chimerism was measured at thirty-day intervals, and initially sharply increased and then stabilized around day ninety post-transplantation. In prior studies from our laboratory in a spontaneously arising autoimmune diabetes model, we demonstrated that mixed allogeneic chimerism alone following low dose total body irradiation (TBI) and HSC transplantation was sufficient to block the autoimmune pathogenesis. In order to establish a second clinically relevant conditioning regimen, we attempted here to lower the dose of TBI by using cyclophosphamide and ATG in addition to low dose TBI. However, less robust engraftment was observed as compared to the TLI/ATG approach. To study how TLI/ATG allows engraftment, we have examined the marrow of TLI/ATG conditioned, untransplanted animals. Though TUNEL and Caspase-3 assays did not show a significant increase in apoptosis compared to controls, a 71% decrease in the quantitative number of HSCs isolated from these animals was observed. This depletion of HSCs in the marrow could provide a niche for donor HSCs to inhabit. Further histologic studies on lymphoid organs exposed to radiation through TLI, including the thymus and spleen, are ongoing and may further elucidate the mechanisms by which TLI reconditions the host immune system. The durable mixed chimerism observed following TLI/ATG conditioning and HCT will be applied to mice affected with the rodent form of multiple sclerosis (experimental autoimmune encephalomyelitis) and to tolerance induction of solid-organ grafts.SUMMARY: The combination of TLI/ATG non-myeloablative conditioning and transplantation of allogeneic HSC leads to a durable mixed chimeric state between donor and host and will next be applied to the induction of tolerance to autoantigens and alloantigens.

Development of Late over Early Full Donor Chimerism (FDC) Results in Improved Progression-Free and Overall Survival in Patients with Advanced Malignant Lymphomas Receiving Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).

Mixed chimerism (MC) can be induced in mice after non-myeloablative conditioning and allogeneic HSCT. Full donor hematopoiesis conversion from MC can occur when nontolerant donor lymphocyte infusions (DLI) are given weeks following the transplant as a result of lymphohematopoietic GVH reactions that eradicate leukemia/lymphoma without GVHD. Based on this murine model, we initiated a trial in patients with advanced hematologic malignancies of non-myeloablative therapy with HLA-matched related donor HSCT with intentional induction of MC followed by prophylactic DLI (pDLI) at day +35 post-HSCT in those without clinical GVHD. This analysis examines the influence of the kinetics of sustained donor lymphoid (CD3+ cells) chimerism on transplant outcomes. Fifty-four patients with advanced hematologic malignancies received nonmyeloablative preparative therapy (cyclophosphamide, equine antithymocyte globulin, thymic irradiation and a brief course of cyclosporine) and HLA-matched HSCT (bone marrow, BM, n=42; GCSF-mobilized peripheral blood stem cells, PBSC, n=12). Chimerism analyses were performed weekly until day +100 post-HSCT and then once monthly for a year. Twenty-nine patients with aggressive malignant lymphomas (NHL, n=21; HD, n=8) were selected out of 50 evaluable patients in an effort to analyze a relatively uniform group in terms of their disease and sustained chimerism; patients who lost the allograft were removed from this analysis. Twenty five patients attained stable FDC, defined as > or = 90% donor T cells, and 4 maintained stable MC. Using 42 days post-HSCT as the defining point for chimerism conversion 13 patients achieved early FDC and 16 patients achieved late FDC. Median time to achieve FDC in the early and late groups was 14 days (range, 7–42) and 70 days (range, 49–91) respectively. Separation produced fairly equal distribution by disease characteristics as follows (early to late): NHL, 10 to 11 and HD, 3 to 5. The early group included 10 PBSC recipients who did not receive pDLI because of the spontaneous conversion to FDC; in contrast, the majority of the late group received BMT and 13 of them received DLI (pDLI, n=10, therapeutic DLI, n=3). The incidence of acute GVHD (grades II–IV) was similar between the early and late groups with rates of 77% (n=10 of 13) and 69% (n=11 of 16) respectively; for chronic GVHD, there was a tendency toward an increased rate in the early group of 75% (3/4) compared to the late group rate of 63% (5/8). Overall disease response rates in the early and late groups were 62% and 69%, but there was a trend toward improved complete remission rates in the late group relative to the early group (50% vs 31%). Progression-free survival (PFS) and overall survival (OS) at 1 year were improved in the late compared to the early group [31% (5/16) vs 15% (2/13) and 50% (8/16) vs 38% (5/13), respectively]. At 5 years, there was a statistically significant improved PFS and OS rate in the late group over the early group [31% (5/16) vs 0% (0/13), p =0.037, for both PFS and OS]. In conclusion, these data support the observation from our mouse models that the most potent graft-vs-tumor effect occurs in mixed chimeras with late chimerism conversion, often after delayed pDLI.

Evidence for ineffective erythropoiesis in severe sickle cell disease

AbstractPeripheral destruction of sickled erythrocytes is a cardinal feature of sickle cell disease (SCD). Less well established is the potential contribution of ineffective erythropoiesis to the pathophysiology of this hemoglobinopathy. Since patients with SCD frequently develop mixed hematopoietic chimerism after allogeneic nonmyeloablative stem cell transplantation, we used this opportunity to directly compare the differentiation and survival of SCD and donor-derived erythropoiesis in vivo. Donor and recipient erythropoiesis was compared in 4 patients with SCD and 4 without SCD who developed stable mixed hematopoietic chimerism following transplant. Molecular analysis of chimerism in peripheral blood and bone marrow demonstrated higher expression of donor-derived β-globin RNA relative to the level of donor-derived genomic DNA in patients with SCD. Analysis of chimerism in immature (glycophorin A–positive [GYPA+], CD71hi) and mature (GYPA+, CD71neg) erythroblasts confirmed the intramedullary loss of SS erythroblasts with progressive maturation. In patients with SCD, relative enrichment of donor erythroid precursors began to appear at the onset of hemoglobinization. Ineffective erythropoiesis of homozygous hemoglobin S (SS) progenitors thus provides a maturation advantage for homozygous hemoglobin A (AA) or heterozygous hemoglobin S/hemoglobin A (SA) donor erythroid precursor cells that results in greater donor contribution to overall erythropoiesis following stem-cell transplantation and improvement of clinical disease.

Murine and Math Models for the Level of Stable Mixed Chimerism to Cure β‐Thalassemia by Nonmyeloablative Bone Marrow Transplantation

Stable mixed chimeric stem cell transplantation in hemoglobinopathies exploits shorter erythroid survival in hemolytic anemias, providing normal donor red blood cells with a competitive survival advantage. This study examined the level of stable mixed chimerism necessary for complete hematological cure of the thalassemic phenotype, using a nonmyeloablative busulfan chemotherapeutic preparation. Thalassemic mice transplanted from congenic wild-type donors developed partial mixed chimerism. Hematologic cure required >80% donor red blood cells and only >13% donor white blood cells. Murine and human transplant results were compared with a math model for survival advantage of donor peripheral blood cells produced by steady-state chimeric marrow.

Sustained and stable hematopoietic donor‐recipient mixed chimerism after unrelated cord blood transplantation for adult patients with severe aplastic anemia

We evaluated the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT (cord blood stem cell transplantation). Nine patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60 mg/kg) and ALG (120 mg/kg). The prophylaxis of GVHD consisted of standard CsA and MTX. Patients have a media age of 25.3 yr (range: 15-37), and a median weight of 57.2 kg (range: 52.5-60) at the time of transplantation. Cord blood searches were all conducted at Guangzhou Cord Blood Bank. The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Engrafted evidence has been found in seven patients involved by biomolecular analyses showing donor-recipient mixed chimerism post-transplant which was stable and persistent. After a median follow up of 32.2 months (range: 4-69), seven patients were alive and disease free. This study shows that durable donor-recipient stable mixed chimerism can be achieved by unrelated CBSCT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation.

Nonmyeloablative Conditioning with Busulfan before Matched Littermate Bone Marrow Transplantation Results in Reversal of the Disease Phenotype in Canine Leukocyte Adhesion Deficiency

Leukocyte adhesion deficiency (LAD)–1, a primary immunodeficiency disease caused by molecular defects in the leukocyte integrin CD18 molecule, is characterized by recurrent, life-threatening bacterial infections. Myeloablative hematopoietic stem cell transplantation is the only curative treatment for LAD-1. Recently, canine LAD (CLAD) has been shown to be a valuable animal model for the preclinical testing of nonmyeloablative transplantation regimens for the treatment of children with LAD-1. To develop new allogeneic transplantation approaches for LAD-1, we assessed a nonmyeloablative conditioning regimen consisting of busulfan as a single agent before matched littermate allogeneic bone marrow transplantation in CLAD. Three CLAD dogs received busulfan 10 mg/kg intravenously before infusion of matched littermate bone marrow, and all dogs received posttransplantation immunosuppression with cyclosporin A and mycophenolate mofetil. Initially, all 3 dogs became mixed chimeras, and levels of donor chimerism sufficient to reverse the CLAD phenotype persisted in 2 animals. The third dog maintained donor microchimerism with an attenuated CLAD phenotype. These 3 dogs have all been followed up for at least 1 year after transplantation. These results indicate that a nonmyeloablative conditioning regimen with chemotherapy alone is capable of generating stable mixed chimerism and reversal of the disease phenotype in CLAD.

Efficacy and Limitations of Cyclophosphamide‐induced Tolerance against αGal Antigen

In the present study, we have elucidated the efficacy of two cyclophosphamide (CP)-induced tolerance protocols for the induction of B-cell tolerance against Galalpha1-3Galbeta1-4GlcNAc (alphaGal) antigens. alpha1,3-galactosyltransferase-deficient (GalT-/-; H-2(b/d)) mice received with 1 x 10(8) AKR (alphaGal+/+ H-2k) spleen cells (SC) followed by 200 mg/kg CP, or alternatively followed by 200 mg/kg CP, 30 mg/kg Busulfan (BU) and 1 x 10(8) T-cell-depleted AKR bone marrow cells (BMC). The generation of both anti-alphaGal and anti-donor antibodies were completely suppressed, but normal antibody production against third party antigens was observed after BALB/c skin grafting in both groups of GalT-/- mice. In GalT-/- mice, treated with SC and CP, mixed chimerism was not observed. Cellular rejection was observed in grafted donor AKR hearts with an absence of humoral rejection, whereas humoral rejection was observed in untreated GalT-/- mice. On the other hand, long-term mixed chimerism and permanent acceptance of donor AKR skin graft and heart graft were achieved in GalT-/- mice treated with SC, CP, BU and BMC. These results demonstrate the efficacy of classical drug-induced tolerance in the induction of B-cell tolerance against alphaGal antigens. However, induction of stable mixed chimerism was required for the suppression of cellular rejection.