Persistent Mixed Chimerism Research Articles

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.

Mixed Chimerism in T Cells after Allogeneic SCT Allows Early Diagnosis of Graft Rejection and Successful Treatment with Immunosuppression Withdrawal and/or Donor Leukocyte Infusion.

Background: Graft rejection constitutes a serious complication, infrequent after conventional (ablative) allogeneic SCT but with higher incidence after reduced intensity conditioning (RIC) or T-cell depleted (TCD) SCT.Objective: To evaluate the usefulness of chimerism monitoring for the early diagnosis of graft rejection in different SCT settings, as well as for the follow up of the response to treatment with immunosuppression withdrawal (ISW) and/or donor leukocyte infusions (DLI).Patients and Methods: The study includes 68 SCT (32 ablative, 19 RIC, 17 TCD -including 8 haploidentical-). 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: After initial engraftment in all patients, graft rejection was diagnosed in 8 (2 (6%) ablative, 2 (10%) RIC, 4 (23%) TCD), either established (severe pancytopenia and BM aplasia) or incipient (progressive decrease in PB and BM cell counts) a median of 52.5 days (range 23–110) after SCT. All patients showed mixed chimerism (MC) in BM and PB with higher percentages of recipient cells (%R) in PB. In 7/7 patients studied, T cells showed persistent MC with high %R (>50% in 6/7; <50% and >25% in 1/7). B cells showed MC in 5/6 patients studied, with lower %R (<15% in 3/5, >50% in 1/5). Only 2 patients showed MC, transient in one of them, in myeloid cells. 2 patients were not treated due to concurrent multiorgan failure and subsequently died. Reduction of IS in 5 patients obtained 1 response (normal PB and BM counts, and CC). The other 4 patients underwent ISW but no further response was obtained. One of them received a second SCT while the other 3 were treated with DLI, and all of them responded. The last patient (transplanted from a haploidentical family donor) who was not receiving IS, responded to treatment with DLI. Time from therapeutic intervention to response was variable with a median of 2 months (range 1–7). 4 patients developed GVHD>I, which was the cause of death in one and was controlled in the other three. One patient died from sepsis in complete remission (CR) 9 months after the transplant. 4 patients are alive in CR a median of 50 months after SCT (range 16–72).Conclusions: The observation of MC, mainly in T lymphocytes, together with a decrease in PB and BM cell counts, allowed early diagnosis and successful treatment (6/6 patients) of graft rejection.

Classical Tolerance Induction in Renal Transplantation — A Study of 11 Patients

Transplantation biology has accumulated evidence of persistent mixed lymphohematopoietic chimerism (MLHC) after allogeneic hematopoietic stem cell (HSC) grafting, resulting in donor-specific tolerance to alloantigens in solid organ transplantation. We aimed to design a safe, non-toxic, non-myeloablative, high-dose allogeneic HSC transplantation (HSCT) protocol to achieve grafting with persistent low-level MLHC-associated, donor-specific tolerance across major histocompatibility complex barriers in our living related donor (LRD) renal allograft recipients. HSC infusion into the portal, marrow and systemic circulation was performed in 11 LRD renal allograft recipients. All patients underwent a tolerance induction protocol (TIP), but two patients also had HSC infusion ( n = 1) or donor renal tissue inoculation ( n = 1) into the recipient thymus. All patients received low-dose immunosuppressive therapy for a mean of 1 year post-transplantation. Such therapy was discontinued for the following reasons: poor compliance; drug toxicity; and low-level chimerism, as tested across gendermismatched donors by a fluorescent in situ hybridization technique. All patients had prope tolerance with early, stable allograft function without any episodes of rejection, infection, or graft versus host disease. MLHC could be tested in seven patients, all of whom showed persistent MLHC (average 0.54%) in peripheral blood. All patients had stable allograft function for a mean of 32.6 months after the withdrawal of immunosuppressive therapy, and all had a normal clinical profile as tested by laboratory parameters. Of 483 patients who underwent TIP using HSCT, weaning of immunosuppressive therapy was implemented in 165 (34.2%), and 11 patients developed successful MLHC-associated donor-specific tolerance.

Allogeneic Hematopoietic Stem-Cell Transplantation, Mixed Chimerism, and Tolerance in Living Related Donor Renal Allograft Recipients

Objective We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts. Patients and Methods Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad × 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW × 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4 +/CD 8 + T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 × 10 8 cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy. Results Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 ± 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients. Conclusion We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.

Favorable Impact of T Cell Depleted PBSCT on Transplant Outcomes Despite Delayed Donor T Cell Engraftment.

Although T cell depleted stem cell transplantation can reduce risks from GVHD, it is associated with incomplete engraftment and higher relapse rates. To mitigate these negative effects we evaluated a T cell depleted PBSCT protocol followed by delayed add-back of donor T cells. Between 2001–2004, 56 patients with hematological malignancies received a preparative regimen of total body irradiation 12Gy, fludarabine 125mg/m2, and cyclophosphamide 120mg/kg followed by a T cell depleted PBSCT from an HLA identical sibling. The T cell dose was adjusted to 2 x 104/kg in all patients. Patients not developing grade <II GVHD received 107CD3 cells/kg between days 45–100. Lineage-specific T cell and myeloid chimerism was monitored day 14, 30, 45, 60 and 90 post-transplant. Patients were aged between 11–56 (median 30y). Twenty six with CML in CP, acute leukemia in first remission, or MDS RA were designated as standard risk (SR) for transplant outcome. Thirty with more advanced or refractory disease were designated high risk (HR). Overall survival was 65±7% at a median of 1 year. Disease risk was the major factor affecting outcome with lower relapse and transplant-related mortality (TRM) and higher disease-free survival (DFS) in SR versus HR patients (3.5±3.5 vs. 42±11%, 7.5±4 vs. 17.5±8% and 85±7 vs 36±9% respectively, p< 0.0001). One patient rejected the transplant and died following two further transplant attempts. The remaining patients achieved rapid 100% donor myeloid engraftment, sustained unless relapse occurred. Donor T cell engraftment was incomplete. 0nly 12.5% of patients had full donor T cell chimerism (>95% donor T cells) on day 14, rising to 50% of patients by day 90, with persistent mixed chimerism in some patients beyond 6 months. Predictably, acute GVHD grade I–IV by d60 was significantly correlated with full T cell chimerism (p< 0.0001). There was no significant difference in day 30 lymphocyte count, CMV reactivation or transplant CD34 dose between fully or partially T cell chimeric recipients at any time point. Full T cell chimerism was significantly more frequent in HR vs SR patients (on day 90: 82 vs 24%, p= 0.002). Kaplan-Meier outcome estimates for mixed vs full T cell chimerism were therefore analyzed separately for SR and HR patients. All outcomes (relapse, TRM and DFS) were more favorable (but not significantly so) for patients who had mixed chimerism at any time point between d14–90: of 13 evaluable HR patients fully chimeric at d30 there was a 74% probability of relapse vs a 58% relapse for15 mixed chimeric patients (p=NS). No patient with SR disease relapsed. There was a non-significant DFS advantage for d30 mixed vs full chimeric patients: 95% vs 85%, and 40% vs 20% for SR and HR patients respectively. These results indicate 1) full T cell chimerism is more frequent in HR patients (who have received large cumulative doses of prior chemotherapy) 2) T cell depleted PBSCT have a high frequency of prolonged mixed T cell chimerism. This does not predispose to late graft rejection, is favorable for reducing GVHD and does not negatively affect CMV reactivation, relapse, TRM and DFS.

Rapid Cyclosporine Tapering and Isotretinoin Usage after Unrelated Donor Hematopoietic Stem Cell Transplantation in Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare type of childhood leukemias, and allogeneic hematopoietic stem cell transplantation (HSCT) is known to be the only way to cure the disease. Unfortunately, relapse is still the most frequent cause of treatment failure after transplant in JMML. We investigated the feasibility of inducing graft versus leukemia (GVL) effect and the use of a differentiating agent even after unrelated HSCT in children with JMML. Seven consecutive patients with JMML underwent unrelated HSCT at a median age of 17 months. The sources of grafts were bone marrows (n=3) or HLA 1- or 2-antigen mismatched cord bloods (n=4). Only 3 of the 7 patients were in complete remission before transplantation. Intravenous busulfan, cyclophosphamide, and etoposide were used as preparative agents except in one who was conditioned with TBI-based regimen. Cyclosporine was used universally for GVHD prophylaxis with additional use of short-term methotrexate in bone marrow transplants and of methyl-prednisolone in cord blood transplants. Cyclosporine was tapered rapidly from around 1 month post-HSCT and isotretinoin (75–100 mg/m2/day) was used in selected patients who have any risk factors of relapse. Cyclosporine blood levels were 247.8±91.1, 146.6±104.2, and 88.8±52.6 ng/mL at 1, 2, and 3 months post-transplant, respectively. There was no grade 3 or 4 acute GVHD and only 2 patients developed grade 2 acute GVHD which was improved without additional treatment. Chronic GVHD was developed in 3 (1 limited, 2 extensive) of the evaluable 5 patents, which was all resolved after combined use of immune suppressive agents. Initial chimeric status analysis at 1 month revealed complete donor chimerism (CC) in 4 patients, mixed chimerism (MC) in 2 and autologous recovery (AR) in one. One of the patients with MC and the one with AR were in disease-free status. One patient whose chimeric status changed from CC to MC eventually relapsed. One patient with initial MC with residual disease turned to CC with complete remission. Another patient with initial MC but with no evidence of disease is on treatment with isotretinoin without relapse for 3 months even with persistent MC. The patient with AR relapsed early after transplant. Five patients are alive relapse-free and disease-free with a median follow-up of 16 months after transplant. The Kaplan-Meier probability of event-free survival was 66.7%. We suggest that GVL induction strategy with concomittant use of a differentiating agent might have a role to suppress leukemic relapse in JMML.

Analysis of Chimerism Induction Following Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) with a Reduced-Intensity Regimen.

<Background> Although the analysis of chimerism induction has become an important diagnostic tool for providing better clinical management of patients undergoing allogeneic HSCT, the techniques have not been fully standardized. Moreover, it is currently unknown whether the onset of graft-versus-host disease (GVHD) is related to the status of mixed chimerism (MC) or complete donor-type chimerism (CDC).<Methods> First, to validate our chimerism analysis system, we performed experiments with artificially mixed cell samples from healthy volunteers to examine the reliability of short tandem repeat (STR) determination by quantitative polymerase chain reaction (PCR). We confirmed a linear correlation between the proportion of mixed cells and the calculated ratio, with a correlation coefficient of 0.99, which enables the detection of target cells at 3% (median standard deviation, 1.6%). Next, using this validated system, we prospectively evaluated the kinetics of chimerism in CD3+, CD19+, and peripheral blood mononuclear cells (PBMC), for correlation with the occurrence of GVHD in 19 patients with various hematological diseases (median age, 53y) who had received allogeneic HSCT from an HLA-identical sibling donor between July 2003 and February 2004. The preparative regimen was fludarabine/busulfan (BU) (n=12) or cladribine/BU (n=7). GVHD prophylaxis consisted of cyclosporin alone (n=8), cyclosporin plus short-term methotrexate (n=7), or tacrolimus (n=4). Chimerism analysis was repeated weekly after transplantation.<Results> We evaluated 405 consecutive blood samples from these 19 recipients, 12 of whom developed acute GVHD. Six of these 12 patients showed MC, i.e. 91% (83–94%; MC) donor cells in the CD3+ fraction at the onset of GVHD, but all except one subsequently achieved CDC within a median of 15 (7–33) days without additional DLI. The remaining patient showed persistent MC and relapsed 158 days after transplantation.<Conclusion> We found that the presence of MC in the CD3+ fraction is rather common at the onset of acute GVHD, but GVHD subsequently eradicates residual host hematopoietic cells. Alternatively, GVHD is part of a clinical manifestation of an immune reaction that is related to the induction of CDC.

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION, MIXED CHIMERISM AND TOLERANCE IN LIVING RELATED DONOR RENAL ALLOGRAFT RECIPIENTS

P911 Aims: We designed a prospective, randomized control clinical trial to evaluate the efficacy and safety of achieving mixed chimerism associated tolerance protocol in living related donor (LRD) renal allograft recipients. Methods: Sixty six consecutive patients divided in two equal groups of 33 patients with end stage renal disease, were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups {treated (Tn) and control (Cn)} were well-balanced in their clinical, lab parameters and donor HLA match profile. Tn underwent thymic transplantation of donor renal tissue, 2 donor specific transfusions, low intensity conditioning and high dose hematopoietic stem cell transplantation (HSCT) before renal transplantation. Conditioning regimen included low dose target specific irradiation (to abdominal and inguinal lymph nodes, bone marrow(BM) of thoracolumbar vertebrae and part of pelvis on alternate days, 100 rads x 4), anti-T cell antibodies (15 mg/kg body weight (BW)), cyclophosphamide (10 mg/kg BW x 2 consecutive days) and cyclosporine (CsA) (3 mg/kg BW/day). Unfractionated HSCT procured from donor marrow was administered in recipient’s BM, portal and peripheral circulation within 24 hours of achieving CD 4+/ CD 8 + T- cell count < 10% of normal. This was supplemented by peripherally mobilized stem cells, with mean total dose of 20 x 10 8 cells/kg BW of recipient administered peripherally. Renal transplantation was performed after negative LCM. Donor specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by FISH technique. Both groups received CsA and prednisolone for immunosuppression, Cn received mycofenolate mofetil/ Azathioprine in addition. Rejection was treated with standard anti-rejection treatment. Immunosuppression was withdrawn 6 months after renal transplantation in patients with consistent positive chimerism. Clinical tolerance was defined by achieving stable allograft function for more than 100 days without immunosuppression confirmed by allograft biopsy. Results: Over a mean follow-up of 210 days, all Tn patients had stable allograft function with mean serum creatinine (SCr) of 1.20 mg/dl, no rejection/ CMV infections/ graft and patient loss. Low level donor-specific cytotoxic antibodies were observed in all of them. CsA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was noted in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5 % before and 1 ± 0.3 % after transplantation). All 4 patients on drug withdrawal have developed donor-specific tolerance with a mean follow up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of Cn was 1.45 mg/dl over a mean follow up of 216 days. Acute rejection was observed in 17 (51.5 %) patients, no CMV infection / patient loss was noted and 1(3.03%) graft was lost in Cn. No graft Vs host disease was observed in Tn. Conclusions: We have achieved mixed hematopoietic chimerism associated tolerance with high dose HSCT, intrathmic donor renal tissue transplantation and minimum conditioning without any adverse effects.

Feasibility of a cost-effective approach to evaluate short tandem repeat markers suitable for chimerism follow-up

Precise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients. To set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT. Six dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients. Four markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67-0.88) under our experimental system. A sensitivity of 0.8-1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH. To our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage of Taq polymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient. The data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.

Reduced intensity conditioning and prophylactic DLI can cure patients with high-risk acute leukaemias if complete donor chimerism can be achieved.

23 patients with ALL (n=9) and AML (n=14) underwent nonmyeloablative stem cell transplantation (NST) from an HLA-identical donor after conditioning with fludarabine (180 mg/m(2)), busulfan (8 mg/kg) and anti-T-lymphocyte globulin (40 mg/kg). After NST, 20/23 patients engrafted. Ten out of 14 patients with uncontrolled disease reached complete remission. A multiplex-PCR using short tandem repeats was used for chimerism analysis and detected mixed chimerism (MC) in 14/22 evaluable patients (64%) after NST. Prophylactic donor lymphocyte infusions (DLI) were given to 11/14 patients with MC; MC converted to complete donor chimerism (CC) in 6/11 patients within 2-6 weeks. All patients with persistent MC with or without DLI relapsed during further follow-up. MC predicted impending relapse 4-52 weeks before clinical diagnosis. Ten of 23 patients (43%) are alive 2-34 months after stem cell transplantation. 12 of 23 patients (52%), have died from leukaemia after NST. One out of 23 patients has died from severe sepsis. In conclusion, NST leads to stable engraftment and complete remission in patients with advanced acute leukaemias. NST can cure a substantial proportion of these patients, but the relapse rate is still high. Repeated chimerism analysis is a useful tool to detect recipient cells, especially in patients without molecular markers of disease and can be used to monitor immunomodulatory therapies. MC is unstable in these patients and predicts impending relapse. Prophylactic DLI can convert MC to CC, which seemed to lower relapse risk.

T cell and B cell tolerance to GALalpha1,3GAL-expressing heart xenografts is achieved in alpha1,3-galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism.

We have previously demonstrated that mixed xenogeneic chimerism and donor-specific T-cell tolerance can be induced in the rat-to-mouse species combination by using a relatively nontoxic, nonmyeloablative conditioning regimen. However, natural antibodies (NAbs) against Galalpha1,3Gal (Gal) pose an additional major barrier to pig-to-human vascularized xenograft acceptance. To determine whether the mixed chimerism approach could also overcome this humoral barrier, T cell-depleted rat (GalT+/+) bone marrow cells (BMC) were transplanted to alpha1,3-galactosyltransferase deficient (GalT-/-) mice conditioned with a nonmyeloablative regimen, consisting of transient T cell and natural killer (NK) cell depletion, 3 Gy whole body irradiation, and 7 Gy thymic irradiation. By giving a high dose (180x106) of rat BMC, persistent mixed chimerism could be induced in GalT-/- mice, although the level of donor-type hematopoietic repopulation declined over time. Induction of mixed chimerism was associated with a rapid disappearance of anti-Gal and anti-rat NAb in the sera. Both anti-Gal Ab-producing cells and B cells with receptors recognizing Gal were undetectable in mixed chimeras, even when the chimerism levels declined, suggesting that a very low level of chimerism could effectively maintain B-cell tolerance to Gal, probably by clonal deletion and/or receptor editing. Mixed chimeras accepted subsequently transplanted donor-type rat hearts (>100 days) without immunosuppressive therapy, whereas delayed vascular and even hyperacute rejection of rat hearts occurred in conditioned control GalT-/- mice. Cellular rejection occurred by 5-6 days in conditioned control wild-type mice. These findings demonstrate that induction of mixed chimerism with a nonmyeloablative regimen can prevent vascularized xenograft rejection by cellular and anti-Gal Ab-dependent pathways in GalT+/+-to-GalT-/- species combinations.

Effects of mixed chimerism and immune modulation on GVHD, disease recurrence and survival after HLA-identical marrow transplantation for hematologic malignancies.

BackgroundThe success of allogeneic bone marrow transplantation (allo-BMT) is affected by underlying disease relapse. Although mixed chimerism (MC) is not necessarily a poor prognostic factor, several groups have suggested that MC is associated with an increased risk of disease relapse. There is evidence that patients with MC benefit from additional immunotherapy if the treatment is started in minimal residual disease status (mixed chimerism status), not in frank hematological relapse. The purposes of this study are to evaluate 1) the risk for relapse or graft rejection in correlation to persistent MC status after allo-BMT, and 2) the possibility of preventing relapse by immune modulation treatments (withdrawal or rapid taper-off of post-transplant immuno-suppression, additional interferon treatment, or the administration of donor lymphocytes) in hematologic malignancies.Patients and MethodsOf 337 allogeneic donor-recipient pairs between March 1996 and August 1998, 12 patients who showed persistent or progressive MC and who received immune modulation treatments were evaluated. Twelve patients, median age 31 years (range 9 to 39 years), received an allo-BMT for: acute myelogenous leukemia (AML, n = 5), chronic myelogenous leukemia (CML, n = 4), acute lymphocytic leukemia (ALL, n = 3). Serial polymerase chain reaction (PCR) analysis of YNZ 22-, 33.6-minisatellites or Y chromosome-specific PCR analysis at short term intervals (pre-and post-transplant 1, 3, 6, 9, … months) was performed. Once MC was detected, immune modulation treatments on the basis of increasing MC in an early phase of recurrence of underlying disease were started.ResultsNine of 12 patients converted to complete chimerism (CC) (AML 5/5, CML 3/4, ALL 1/3). Four of 9 CC patients developed graft-versus-host disease (GVHD) grade ≤2 during immune modulation. All were treated successfully with steroids. Three patients who were not converted to CC showed relapse of underlying diseases or graft failure.ConclusionThe results demonstrate that, in patients with hematologic malignancies after allo-BMT, persistent MC is associated with relapse of underlying diseases or graft failure. Furthermore, when patients receive early immune modulation treatment, MC can be changed to complete donor pattern chimerism and ultimately prevent relapse.

Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies

Mixed lymphohematopoietic chimerism can be induced in mice with bone marrow transplantation (BMT) after a nonmyeloablative preparative regimen that includes cyclophosphamide, anti-T-cell antibody therapy, and thymic irradiation. These mixed chimeras are resistant to the induction of graft-versus-host disease (GVHD) after delayed donor leukocyte infusions (DLIs), despite a potent lymphohematopoietic graft-versus-host reaction that converts the mixed chimeric state to a full donor one. Based on this animal model, we initiated a trial of nonmyeloablative therapy with HLA-matched or -mismatched donor BMT and DLI for refractory hematologic malignancies. Twenty-one of 36 patients enrolled in this trial received a genotypically (n = 20) or phenotypically (n = 1) HLA-matched donor transplant; results reported here are for those patients only. Preparative therapy consisted of cyclophosphamide in doses of 150 to 200 mg/kg; peritransplant antithymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal radiation therapy); and cyclosporine. Eighteen of 20 evaluable patients developed persistent mixed lymphohematopoietic chimerism as defined by >1% donor peripheral white blood cells until at least day 35 posttransplantation. Ten patients received prophylactic DLI beginning 5 to 6 weeks after BMT for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-leukemia effect. Fourteen of 20 evaluable patients (70%) achieved an antitumor response; 8 of these responses were complete, and 6 were partial. Of the 8 evaluable patients who received prophylactic DLI, 6 showed conversion to full donor chimerism. Five of the 9 evaluable patients (56%) who received prophylactic DLI achieved a complete response, compared with 3 of 11 patients (27%) who did not receive prophylactic DLI. Currently 11 patients are alive, and 7 of these are free of disease progression at a median follow-up time of 445 days (range, 105-548 days) posttransplantation. Transplantation-related complications included cyclophosphamide-induced cardiac toxicity in 3 of 21 patients (14%) and grade II or greater GVHD in 6 patients (29%). One patient (5%) died from a complication of BMT, and 1 patient (5%) died from GVHD after 2 prophylactic DLIs were given for conversion of chimerism. In summary, mixed lymphohematopoietic chimerism was reproducibly induced after a novel nonmyeloablative preparative regimen incorporating chemotherapy, peritransplant antithymocyte globulin, and thymic irradiation, allowing for early administration of DLI in 10 of 21 patients. After treatment, striking antitumor responses were observed in the majority of patients with chemotherapy-refractory hematologic malignancies. Biol Blood Marrow Transplant 2000;6(3A):309-20.

Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.

Twenty-six transplanted thalassemic patients out of 295 analyzed, showed the presence of persistent mixed chimerism, over a period of time varying between 2 and 11 years after BMT. Despite the presence of large numbers of residual host cells, these transplanted thalassemic patients no longer require red blood cell transfusions and have a functional graft, producing sufficient levels of hemoglobin A ranging from 8.3-14.7 g/dl. These ex-thalassemic patients with persistent mixed chimerism, although they did not achieve complete donor engraftment are no longer exposed to the risk of graft rejection. The mechanisms underlying this apparent state of tolerance or education in these patients are at the present time unknown. However, these observations may be useful for physicians involved in defining optimal strategies for clinical gene therapy, in utero hematopoietic stem cell transplantation and adoption of less toxic conditioning regimens in mini-transplantation.

Coexistence of Two Functioning T-Cell Repertoires in Healthy Ex-Thalassemics Bearing a Persistent Mixed Chimerism Years After Bone Marrow Transplantation

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous beta-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.

Adoptive immunotherapy in canine chimeras.

Chimerism and tolerance after bone marrow transplantation provide excellent conditions for adoptive immunotherapy with T cells of the marrow donor. We studied adoptive immunotherapy in dog leukocyte antigen-identical canine littermate chimeras. Mixed chimeras were produced by conditioning treatment with total body irradiation of a dose of 10 Gy, a uniformly lethal dose in dogs, and infusion of between 1 x 10(8) and 2 x 10(8)/kg mononuclear marrow cells treated with absorbed antithymocyte globulin for inactivation of T cells. Donors were of opposite sex. Persistent mixed chimerism was induced in six of nine dogs, chimerism was complete in one dog, and only transient in two dogs. Tolerance to donor skin grafts was demonstrated in eight dogs, including a dog without cytogenetic evidence of chimerism. Lymphocytes of the marrow donor (between 3.2 x 10(8)/kg and 4.1 x 10(8)/kg) were transfused at various times after transplantation. Nontransfused dogs survived without graft-versus-host disease (GVHD), whereas dogs transfused on days 1 and 2 and dogs transfused on days 21 and 22 developed GVHD and died. In contrast, dogs transfused on days 61 and 62 or later survived without GVHD. Chimerism converted from mixed to complete in six of six transfused dogs and in one of eight nontransfused dogs (P<0.005). Donor lymphocyte transfusions 2 years and 4.5 years after transplantation induced split chimerism with lymphoid cells of donor origin and myeloid cells of host origin in one dog and complete chimerism in the other dog. Before lymphocyte collection, donors were immunized against tetanus toxin. Seven days after lymphocyte transfusion, recipients were given booster injections of tetanus toxoid and primary immunization against diphtheria toxin. In transfused animals, antibody titers against tetanus were demonstrated already before the booster injection. Transfused animals developed higher titers of antibody against tetanus and diphtheria toxin than nontransfused animals. Donor lymphocytes converted mixed chimerism into complete chimerism without producing GVHD, when the transfusion was delayed for 2 months or later after transplantation. Transfusion of donor lymphocytes transferred immune reactivity against tetanus toxin and improved reactivity against diphtheria toxin as a new antigen.

Persistence of Mixed Chimerism in Patients Transplanted for the Treatment of Thalassemia

Molecular genetic techniques permit sensitive assessment of host hematopoiesis after marrow transplantation for thalassemia. Information on this persistence and the cell lines in which it occurs may permit therapeutic intervention in patients at high risk for rejection and/or relapse. The objective of this study, therefore, was to determine the evolution and cell line distribution of persistent mixed chimerism detected in 55 patients treated for beta thalassemia. Our findings indicated that rejection occurred in 20 patients, the host component disappeared in 20, and mixed chimerism without transfusion need persisted for 1 to 7 years in 15. In three patients with stable mixed chimerism for 4, 5, and 7 years, host hematopoiesis fluctuated between 25% and 75%. Despite this, donor pattern beta-globin chain synthesis maintained hemoglobin levels between 10 and 13.5 g/dL without transfusion. In these three patients, the polymerase chain reaction of the VNTR and the fluorescent in situ hybridization analysis revealed the coexistence of donor and host cells in the different peripheral blood cell subpopulations and precursors studied (CD2+, CD4+, CD8+, and CD19+ granulocytes; glycophorin-A+, erythroid burst-forming units, CD33+, granulocyte-macrophage colony-forming units). We found that rejection and disease recurrence occur in approximately one third of patients with early mixed chimerism. High levels of host type hematopoiesis can be present in patients not requiring transfusion.

Characterization of mixed allogeneic chimeras. Immunocompetence, in vitro reactivity, and genetic specificity of tolerance.

Mixed allogeneically reconstituted mice (B10 + B10.D2----B10) that specifically accept B10.D2 tail skin allografts were examined for in vivo and in vitro immunocompetence, patterns of hematopoietic repopulation, and in vitro reactivity. In vitro, mixed allogeneic chimeras (B10 + B10.D2----B10) manifested specific tolerance in mixed lymphocyte reactions and cell-mediated lympholysis to B10 and B10.D2 splenocytes, with normal responses to third-party (B10.BR) cells. Such chimeras were immunocompetent in B cell and helper T cell responses, as assessed by their primary plaque forming cell responses to in vivo sheep red blood cell immunization. This is in contrast to fully allogeneic chimeras, which responded less well. In addition, survival of the mixed allogeneic chimeras in a conventional animal facility was superior to that of fully allogeneic chimeras, and similar to syngeneically reconstituted (B10----B10) mice. Specific tolerance to skin grafts, degree of allogeneic engraftment, and persistence of chimerism was also assessed in a noncongenic mixed allogeneic combination (B10 + C3H----B10). Such animals manifested specific hyporeactivity to C3H skin allografts, but eventual chronic rejection of the grafts occurred in spite of stable and persistent mixed chimerism. MHC-congenic (B10.BR) skin grafts were accepted indefinitely in the same animals, suggesting that skin-specific non-major histocompatibility complex antigens were responsible for rejection of the C3H skin allografts.

Pretransplant conditioning with busulfan (Myleran) and cyclophosphamide for nonmalignant diseases. Assessment of engraftment following histocompatible allogeneic bone marrow transplantation.

Four pediatric patients with diseases potentially curable by bone marrow transplantation (BMT)--i.e., common variable immune deficiency, Wiskott-Aldrich Syndrome (WAS), mucopolysaccharidosis type VI, and mucopolysaccharidosis type I received a conditioning regimen consisting of busulfan and cyclophosphamide prior to BMT from HLA-identical, mixed leukocyte culture (MLC)-unreactive siblings. Only one of the four patients achieved full engraftment. Of the remaining three patients, one experienced failure of engraftment, and two had persistent mixed chimerism. Although no serious complications were directly related to the preparative therapy, the doses of busulfan and cyclophosphamide previously described to be adequate for conditioning children with WAS were completely effective in only one of three pediatric patients in this series. Despite a higher dose of busulfan (16 mg/kg), mixed chimerism was observed in a subsequent patient. Of 13 evaluable patients in the literature in whom busulfan and cyclophosphamide had been used as preconditioning regimens for a variety of nonmalignant conditions, eight demonstrated lack of complete and sustained engraftment. On the other hand, clinical improvement has accompanied partial engraftment in some cases. We conclude that additional immunosuppressive and/or myeloablative conditioning is necessary if complete engraftment is attempted in histocompatible allogeneic BMT for many of the nonmalignant disorders.