Mixed Hematopoietic Chimerism Research Articles

Improvement in Hemolytic Parameters and Tricuspid Regurgitant Jet Velocity (TRV) Following Non-Myeloablative Allogeneic Stem Cell Transplantation (allo-SCT) in Adults with Severe Sickle Cell Disease (SCD).

Although SCD has a variable clinical course, many individuals develop end-organ complications that are associated with significant morbidity and early mortality. SCD-related pulmonary hypertension is a frequently recognized complication (up to 30%), is associated with significant mortality, and is resistant to hydroxyurea or chronic exchange transfusions. Currently myeloablative allo-SCT in children under age 16 is curative in the majority, preventing or ameliorating many of the complications of the disease. However, it is unclear whether reduced intensity allo-SCT in adults that allows mixed hematopoietic chimerism could offer the same benefit. Furthermore, reversal of pulmonary hypertension has not been previously demonstrated in either setting. Beginning in 2003, we initiated an IRB approved clinical trial testing a nonmyeloablative approach in adults with severe SCD. Protocol entry criteria include end-organ complications, (stroke, pulmonary hypertension TRV≥ 2.5 m/s, or frequent vaso-occlusive crises or acute chest syndrome not ameliorated by hydroxyurea. Conditioning was achieved with low dose radiation, alemtuzumab, and sirolimus (rapamycin). All patients receive unmanipulated G-CSF mobilized peripheral blood progenitors obtained from an HLA-matched sibling. Assessment of donor chimerism is measured by microsatellite PCR of CD3 and CD14/15 positive white cells. Hemolysis is monitored by serial measurements of total bilirubin, lactate dehydrogenase, hemoglobin and absolute reticulocyte counts. Transthoracic echocardiography is performed in all patients, and serially in those with TRV ≥ 2.5 m/s. Transmitral flow, Doppler determinations of valvular regurgitant velocity, and left ventricular stroke volume are assessed and graded. A total of 8 patients have accrued. 4 of 8 patients met criteria for pulmonary hypertension. Post transplant follow-up for these 4 patients ranged from 7 to 36 months and stable donor lymphoid (13–39%) and myeloid (53–100%) chimerism was observed in all 4. This pattern of mixed chimerism was sufficient for full donor erythroid engraftment as all demonstrated normal hemoglobin, allowing for therapeutic phlebotomy to correct transfusional iron overload. Improvement in TRV and hemolytic parameters were observed (see table) as early as 3 months post transplant. The average pre- and post allo-SCT values were: TRV 3.1 → 2.5 m/s (p=0.13, Wilcoxon rank sum), total bilirubin 4.0 → 0.6 (p=0.1), lactate dehydrogenase 487 →178 units/L (p=0.2), hemoglobin (hgb) 8.6 → 12.6 g/dL (p=0.002), and reticulocyte (retic) count 326 → 68 k/uL (p=0.01). Our results indicate that reduced intensity allo-SCT in adults patients with SCD allows for full donor erythroid engraftment, normalization of blood counts, reversal of hemolysis, and improvement in TRV.TRV and hemolytic parametersPatient 1Patient 2Patient 3Patient 4AverageTRVPre3.72.63.42.63.08(m/s)BMT2.62.42.52.32.45T biliPre8.34.01.91.94.0(0.1–1.1 mg/dL)BMT0.80.50.50.40.6LDHPre1065330338215487113–226 units/L)BMT23417820990178HgbPre8.18.58.88.98.6(g/dL)BMT12.913.911.911.612.6ReticPre344213417331326(31–105 k/uL)BMT7632768768

Everolimus and Cyclosporine in Combination with 2Gy TBI as Pre- and Posttransplant Immunosuppression in a Canine Transplantation Model.

BACKGROUND: Everolimus (RAD) is a potent immunosuppressant used for the prevention of acute allograft rejection in renal and cardiac transplantation. RAD in combination with cyclosporine A (CSA) demonstrates comparable efficacy to mycophenolate mofetil (MMF) and azathioprine, respectively, whereas it has a more feasible side effect profile. Studies that investigate RAD in hematopoietic stem cell transplantation (HSCT) are lacking. The aim of the present study was to evaluate the efficacy and safety of RAD in combination with CSA in a nonmyeloablative HSCT model.METHODS: Dogs were given conditioning with 2Gy total body irradiation (TBI) before allogeneic HSCT (d0). Dog leukocyte antigen-identical littermate marrow was infused i.v. at a median of 3.6x10E8 TNC/kg. Three dogs received unmodified marrow (group 1) whereas three other dogs were given sensitized marrow from a donor that had rejected the graft of a prior HSCT from the sibling that served as recipient in the current experiments (group 2). All dogs received an immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to 35) and 0.25mg BID PO (days 0 to 27) initially. Pharmacokinetics of RAD and CSA at day 5 were used for doses adaptation to reach targeted trough level of 3 8ng/ml for RAD and C0 and C2 level of 300 500ng/ml and 1500 2000ng/ml for CSA, respectively. Hematotoxicity, blood lipids, and creatinine were monitored.RESULTS: All dogs engrafted following HSCT and first hematopoietic mixed chimerism was detectable by day 7. Dogs in group 1 engrafted with a median maximum granulocyte chimerism of 82% (54–95%) at day 28. Median maximum PBMC chimerism was 52% (26–62%) at day 34. One dog rejected the graft after 10 weeks, 1 dog died from infection at week 9 but was a mixed hematopoietic chimera at the time of death (28% granulocytes, 36% PBMC), and 1 dog continues to be mixed chimeric through week 27 (0% granulocytes, 16% PBMC). Two of the 3 dogs in group 2 developed full donor chimerism among granulocytes and PBMC and remain stable full hematopoietic chimeras through weeks 15 an 26. The other dog rejected the graft after 10 weeks. Median RAD trough level at day 5 was 7.2ng/ml (2.3–12.0ng/ml). Synergism between RAD and CSA caused enhancement in CSA C0, C2, and AUC values by 112, 106 and 45%, respectively, compared to CSA levels in CSA/MMF treated dogs. CSA dose reduction was initiated in 3/6 dogs based on pharmacokinetics. Within all dogs a transient decrease in platelet and leukocyte counts with median nadirs of 0x10E9/l (0–21x10E9/l) at day 11 and 1.6x10E9/l (0.7–3.2x10E9/l) at day 8 was observed. Creatinine, cholesterol, triglyceride levels remained within normal limits.CONCLUSION: Immunosuppression with RAD/CSA as pre- and posttransplant conditioning is feasible in the canine HSCT model. RAD was well tolerated and allowed dose reduction of CSA. Using RAD and pre-sensitized bone marrow, full donor chimerism was achievable. Therefore, our data indicate that the combination of RAD/CSA might be used as alternative for CSA/MMF in the 2Gy nonmyeloablative HSCT setting.

INDUCTION OF MIXED CHIMERISM AND TRANSPLANTATION TOLERANCE IN A NON-HUMAN PRIMATE LUNG ALLOGRAFT MODEL: EARLY RESULTS

PURPOSE: Durable transplantation tolerance has been achieved by the induction of mixed hematopoietic chimerism in a variety of animal models, including a fully allogeneic kidney transplant model at our own institution. Here, we sought to determine whether a similar approach could be applied to a non-human-primate lung allograft model.

Recent Advances in Immune Modulation

Successful gene therapy protocols rely on the hypo-responsiveness of the immune system to transgene products generated from gene transfer vectors. In order to prevent cytotoxic lymphocyte or antibody formation induced by transgene expression, various strategies derived from recent advances in immune tolerance induction protocols have been tested in gene therapy model systems. Current immunosuppressive drugs were used to nonspecifically target T-cell activation, clonal expansion, and differentiation into effector cells. Central tolerance can be induced from intrathymic deletion of T cells with thymically expressed antigens or generation of hematopoietic mixed chimerism. Peripheral tolerance to transgenes may be achieved by several different pathways including deletion of activated/effector T cells by depleting antibodies, generation of T cell apoptosis or anergy by costimulation blockade, and active suppression by T regulatory cells. This review outlines the development of these strategies using various immune modulation regimens and protocols to induce long-term immune tolerance specific to the transgene product.

Mixed Chimerism Achieved by a Nonlethal Conditioning Regimen Induces Donor-Specific Tolerance to Lung Allografts

Graft rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem in lung transplantation (Tx). Mixed hematopoietic chimerism has been shown to produce long-lasting donor-specific transplant tolerance without immunosuppressive drugs in animal models; however, most conditioning regimens required to achieve mixed chimerism are too toxic for clinical use. The aim of this study was to develop a nonlethal conditioning regimen to induce tolerance to lung allografts. Four to 6-wk old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as organ donors and recipients, respectively. The recipient conditioning regimen included: 10 mg/animal antilymphocyte globulin (on day-5), 1 mg/kg/d tacrolimus (days 1 to 10), total body irradiation (500 cGy; day 0), and donor bone marrow (DBM) Tx (100 x 10(6) T-cell depleted cells on day 0 following irradiation). Six weeks after DBM Tx, chimeric animals received orthotopic left lung Tx. Graft survival was monitored by chest X-ray and histology. Long-term DBM engraftment was observed: hematopoietic chimerism in the peripheral blood was 12.4 +/- 3.4%, 36.7 +/- 14.1%, and 31.9 +/- 14.1% at 30 d, 6 mo, and 16 mo following DBM Tx, respectively. There was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>400 d) donor-specific lungs (RT1.A(a); n = 8), yet rapidly rejected (<8 d) third party hearts (RT1.A(l); n = 5). Graft (lung) tolerant (>150 d) chimeric recipients accepted secondary donor-specific heart grafts (>150 d; n = 4) but rejected third party heart grafts (<7 d; n = 3). Graft tolerant recipients demonstrated reduced (P < 0.05) in vitro donor-specific lymphoproliferative response and cytotoxicity, and no evidence of acute or chronic graft rejection. Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung allografts.

Perspectives en immunothérapie des allogreffes de tissus composites

Hand and composite tissue allotransplantation (CTA) holds great potential for reconstructive surgery but its development is currently limited by the side effects of the immunosuppressive drugs. Induction of specific tolerance, a situation where the recipient does not mount an immune response against the allograft but remains fully immunocompetent, holds exciting promise. Generation of mixed hematopoietic chimerism by infusing the recipient with donor bone marrow cells has been shown to induce tolerance without chronic immunosuppression. Genetic matching of the donor and the recipient is another option for transplanting composite tissues with only an initial course of immunosuppression. Experiments demonstrated long-term survival of musculoskeletal allografts between MHC-matched miniature swine. Finally, new immunosuppressive agents with a more targeted action will reduce side effects and may prevent the development of chronic rejection. Skin-specific immunosuppression is particularly useful for limb transplants since skin, regarded as the most antigenic component, is easily accessible to topical or irradiation therapies.

Different Mechanisms Control Peripheral and Central Tolerance in Hematopoietic Chimeric Mice

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor-specific transfusion and anti-CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNgamma-producing CD44(high)CD8+ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T-cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.

B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-αGAL–producing B-1 cells

AbstractB-1b cells produce IgM natural antibodies against α1-3Galβ1-4GlcNAc (αGal). These can be tolerized by nonmyeloablative induction of mixed chimerism using αGal-positive (αGal+) donor marrow. We assessed the role of CR1/2 in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in α1-3galactosyltransferase (GalT−/−) and GalT−/−Cr2−/− mice with αGal+ BALB/c marrow donors. Anti-αGal Ab and anti-αGal Ab–producing B cells became undetectable in GalT−/− chimeras, whereas they persisted in chimeric GalT−/−Cr2−/− mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1–cell tolerance, we generated GalT−/− radiation chimeras in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from αGal+ allogeneic bone marrow (BM) donors, anti-αGal–producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Our results suggest a possible role for follicular dendritic cells that pick up immune complexes via CR1/CR2 receptors in the tolerization of B-1b cells.

347: Intentional induction of mixed hematopoietic chimerism as platform for cellular therapy after HLA-matched allogeneic stem cell transplantation in childhood leukemia patients

To maximize graft-versus-leukemia (GvL) effect while minimizing or avoiding graft-versus-host disease (GvHD) we designed a prospective study of allogeneic PBSC transplantation aimed to achieve a mixed chimerism (MC) status. In order to ensure this objective we combined the use of reduced-intensity conditioning and graft manipulation by means of CD34+ selection. To improve immunological reconstitution early “low dose” (0.5 × 105/Kg CD3+ cells) DLI was scheduled once a month for six months. The PBSC CD34+ selection was performed by CliniMACS device.

CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation

AbstractIn utero hematopoietic-cell transplantation (IUHCT) can induce donor-specific tolerance to facilitate postnatal transplantation. Induction of tolerance requires a threshold level of mixed hematopoietic chimerism. CD26 is a peptidase whose inhibition increases homing and engraftment of hematopoietic cells in postnatal transplantation. We hypothesized that CD26 inhibition would increase donor-cell homing to the fetal liver (FL) and improve allogeneic engraftment following IUHCT. To evaluate this hypothesis, B6GFP bone marrow (BM) or enriched hematopoietic stem cells (HSCs) were transplanted into allogeneic fetal mice with or without CD26 inhibition. Recipients were analyzed for FL homing and peripheral-blood chimerism from 4 to 28 weeks of life. We found that CD26 inhibition of donor cells results in (1) increased homing of allogeneic BM and HSCs to the FL, (2) an increased number of injected animals with evidence of postnatal engraftment, (3) increased donor chimerism levels following IUHCT, and (4) a competitive engraftment advantage over noninhibited congenic donor cells. This study supports CD26 inhibition as a potential method to increase the level of FL homing and engraftment following IUHCT. The resulting increased donor chimerism suggests that CD26 inhibition may in the future be used as a method of increasing donor-specific tolerance following IUHCT.

Hematopoietic chimerism induces renal and skin allograft tolerance in DLA-identical dogs

Hematopoietic chimerism, a state where donor and recipient bone marrow cells coexist, is associated with donor-specific tolerance. Nonmyeloablative bone marrow transplantation (BMT) has been shown to induce stable mixed hematopoietic chimerism in dog leukocyte antigen (DLA)-matched dogs. The potential for inducing renal and skin allograft tolerance with nonmyeloablative BMT was investigated in DLA-identical and DLA-haploidentical dogs in this study. Renal allografts were performed in 8 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with (n = 8) and without (n = 4) simultaneous BMT. Skin allografts were performed in 2 DLA-identical and 4 DLA-haploidentical dogs after stopping CSP and MMF. Two DLA-identical control dogs received renal allografts without TBI, BMT, or immunosuppression with CSP and MMF. Molecular chimerism was determined with a PCR-based DNA microsatellite assay. Serum creatinine (Cr) concentration, urine specific gravity, and sequential renal biopsies were monitored to assess renal allograft function. Donor-type blood cells were first detected 4 weeks posttransplantation in both the myeloid and lymphoid lineages. Donor chimerism was present for at least 76 weeks in the DLA-identical dogs. Mixed chimerism was not observed in the DLA-haploidentical dogs or DLA-identical dogs that did not undergo BMT. The renal allografts were acutely rejected within 14 days in the 2 DLA-identical control dogs. There was long-term (> 5 yrs) 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 BMT. Renal allograft inflammation was severe in the control dogs, mild to moderate in the DLA-haploidentical dogs, and minimal in the DLA-identical dogs. Donor-specific skin grafts were accepted in the DLA-identical dogs but rejected in the DLA-haploidentical dogs. Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal and skin allograft tolerance in DLA-identical and permit long-term renal allograft survival in DLA-haploidentical dogs. These findings suggest it may possible to obtain long-term allograft survival in DLA-identical and -haploidentical dogs without the need for chronic immunosuppressive therapy.

A Novel Allogeneic Transplant Conditioning Regimen Designed for Tolerance Induction in Patients with Severe Sickle Cell Disease.

Allogeneic hematopoietic stem cell (HSC) transplantation remains the only curative approach for sickle cell disease (SCD) patients, yet high risk of procedural toxicities and graft-versus-host disease limits this approach. We chose a low-dose radiation approach utilizing rapamycin based upon its unique ability to promote T cell tolerance. We tested this approach in vivo in a murine bone marrow transplantation model comparing a short course of conventional immunosuppression with cyclosporine to that with rapamycin, with long-term, high-level chimerism attained only in mice treated with rapamycin. We have now begun accrual to an IRB approved clinical trial testing this approach in SCD adults and report the results in our first five subjects. Protocol entry criteria include irreversible complications from sickle cell disease (such as prior strokes or pulmonary hypertension defined as a tricuspid-regurgitant jet velocity [TRV] ≥2.5 m/s), or reversible complications (such as frequent vaso-occlusive crises or acute chest syndrome) not ameliorated by a 6-month course of hydroxyurea. Conditioning was achieved with a single radiation dose of 300cGy, alemtuzumab (1mg/kg total), and oral rapamycin targeting trough levels between 10–20 ng/ml. All patients received unmanipulated mobilized peripheral blood progenitors obtained from an HLA-matched sibling. Assessment of donor chimerism was measured by microsatellite PCR of CD3 and CD14/15 positive white cells. Conditioning was well tolerated in all patients. Two patients inadvertently received only 200 cGy. CD34+ cell doses ranged from 5.72–10 × 10e6/kg, and CD3+ cell doses ranged from 1.93–5.35 × 10e8/kg. In 4 of the 5 patients in whom follow-up is sufficient, an early peak in myeloid chimerism was seen at 95–100% at day 28, with stabilization at 48–100% long-term. Lower level lymphoid chimerism was seen early, ranging from 3–23%, with stabilization at 5–47% long-term. Hemoglobin electrophoresis revealed complete replacement by donor type hemoglobin in all 4 patients by day 120, with amelioration of the phenotype allowing for therapeutic phlebotomy. Unfortunately, two patients have experienced recurrent sickle cell disease, though T cell responses to donor assessed by the mixed lymphocyte reaction revealed continued tolerance to donor with preserved T cell responses to third party antigens. These results argue against rejection and suggest competition by surviving endogenous HSCs as a cause of graft loss. Additionally, retransplant of one of the patients in relapse thus far using 400 cGy, alemtuzumab, and rapamycin has re-established full donor erythroid chimerism. Stable mixed chimerism persists out to 23 months in one patient and 10 months in another. Finally, no patient to date has developed any symptoms or signs of acute or chronic GVHD. Our results demonstrate the ability of allogeneic HSC transplantation using a relatively simple conditioning regimen to achieve mixed hematopoietic chimerism without the development of GVHD, suggest the importance of adequate hematopoietic suppression, and may allow extension to alternative donor transplantation.

Ex Vivo Expanded T Regulatory (Treg) Cells Block Conversion of Mixed Chimeras To Complete Donor Chimerism.

A highly effective method to establish long-term, stable mixed hematopoietic chimerism was developed in the dog model. This involves nonmyeloablative allogeneic hematopoietic cell transplantation (HCT), consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen (DLA)-identical marrow, and short-term post-grafting immunosuppression. We hypothesized that CD4+CD25+ Treg cells may be important regulators for the maintenance of cellular immune tolerance after allogeneic HCT. Previously, we showed in 8 mixed chimeras that naive donor lymphocyte infusion (DLI) did not change the level of donor chimerism. However, reconditioning mixed chimeras with 2 Gy TBI followed by DLI “breaks” tolerance and increases the level of donor chimerism. Seven mixed chimeras were reconditioned with 2 Gy TBI followed by DLI. Within 4 weeks after DLI, conversion to 100% donor chimerism was seen in 5 of 7 dogs and 2 dogs had a > 50% sustained increase in donor chimerism. Four recipients developed graft-versus host disease (GVHD). A control group of 3 mixed chimeras reconditioned with 2 Gy TBI without DLI had no change in donor chimerism. These results suggest that reconditioning with 2 Gy TBI followed by DLI can break the tolerance mechanism established in mixed chimeras. Next we asked if CD4+CD25+ Treg cells obtained from mixed chimeras before reconditioning could block the increase in donor chimerism following 2 Gy TBI and DLI. Peripheral blood mononuclear cells (PBMC) from 8 mixed chimeras were obtained by leukapheresis and cultured in bulk mixed leukocyte culture (MLC) with 3rd party DLA-mismatched, unrelated and irradiated CD34+ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). On day 4 of MLC, CD25+ cells were isolated by positive immunomagnetic selection. Next, artificial antigen presenting cells (aAPC, KT32) were added to expand the CD4+CD25+ Treg cells. The aAPC expressed Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 and irradiated prior to stimulation of CD4+CD25+ Treg. After 7 days, Treg were expanded a median of 23 (range, 8–36)-fold. Expanded CD4+CD25+ Treg were assessed for phenotype and in vitro function. The Treg cells were generated from 8 mixed chimeras and were infused back into the respective dogs (median dose 1× 107/kg) after reconditioning with 2 Gy TBI and immediately prior to DLI. In 6 of 8 dogs there was no change in the level of donor chimerism at 16–20 weeks follow-up; 2 dogs converted to complete donor chimerism within 6 weeks. Treatment with expanded Treg cells blocked conversion to complete donor chimerism after 2 Gy TBI +DLI in 6 of 8 dogs, compared with significant increases in donor chimerism for all 7 dogs after 2 Gy TBI +DLI not given Treg (p=.007). None of the 8 Treg recipient dogs developed GVHD, compared with 4 of 7 not given Treg, (p=0.02). A control group of 4 mixed chimeras were infused with expanded, non-Treg CD25− T cells. To this end, CD25+ T cells were immunomagneticaly depleted on day 4 of MLC. CD25− T cells were expanded with aAPC. Three of 4 dogs converted to the complete donor chimerism within 7 weeks after 2 Gy TBI, non-Treg and DLI. These results suggest that ex vivo expanded CD4+CD25+ Treg cells have in vivo function in a large animal model and can restore the tolerance mechanism in mixed chimeras that is broken by 2 Gy TBI and DLI.

Ex Vivo Generated Cytotoxic T Cells (CTL) for Adoptive Immunotherapy across MHC Barriers Retain Anti-Leukemic Capacity (GVL) without Eliciting Graft Versus Host Disease (GVHD): Crucial Role of Recipient-Derived Antigen Presenting Cells (APC).

BACKGROUND: While recipient-derived APC have proven to be crucial in mediating GVL after allogeneic hematopoietic stem cell transplantation (HCT), this desired effect seemed to be inevitably linked to the risk of developing severe GVHD. It has been elegantly shown in murine models that delayed donor lymphocyte infusions (DLI) into mixed rather than full hematopoietic chimeras produce dramatically improved GVL effects and virtually no GVHD. However, avoidance of GVHD has been more difficult when this intelligent concept was transferred into the clinical setting. Here we demonstrate that donor-derived T cells of MHC-mismatched origin retain strong anti-leukemic activity and lose nonspecific alloreactivity upon specific in vitro priming and consecutive short term expansion.METHODS: We chose a fully MHC-mismatched allogeneic bone marrow transplantation model using B10.A mice (H-2a) as donors and C57BL/6 (H-2b) mice as recipients to produce either full (controls) or stable mixed hematopoietic chimeras. After full hematopoietic recovery mice were challenged with a lethal dose of a C57BL/6-derived leukemic cell line (C1498) and consecutively injected with either naive DLI, ex vivo generated CTL or PBS only. CTL were generated by specifically priming lymphocytes of MHC-mismatched donor origin on bone marrow-derived dendritic cells (DC) (either donor- or recipient-derived) that had been pulsed with recipient-derived leukemia cell lysates. This was followed by expansion using αCD3/αCD28 coated microspheres.RESULTS: We show thatex vivo generated CTL of allogeneic origin can mediate anti-leukemic effects only when primed on recipient-derived DC that had been pulsed with leukemia cell lysates (CTL versus controls, p < 0.05 ). In contrast, loading recipient-derived leukemia lysates on donor-derived DC (T cells and DC syngeneic to each other) abrogates the anti-leukemia effect of adoptively transferred CTL in vitro and in vivo.CTL exhibit a strong proliferative response in vitro when stimulated with irradiated leukemic cells of allogeneic origin (C1498), however, this response is significantly weaker (3-fold) when compared to naive DLI. Remarkably, the nonspecific proliferative response (alloreactivity) of CTL is completely abolished when irradiated C57BL/6 splenocytes are used as stimulators.CTL maintain their responsiveness to leukemia in vivo as demonstrated by cure rates from leukemia in up to 40% of mice. Importantly, upon adoptive transfer into leukemia bearing mice after MHC-mismatched HCT, CTL lose their unspecific alloreactivity in vivo as well as proven on microscopic sections of GVH target organs.Anti-leukemia effects of CTL across MHC-barriers are comparable to naive DLI in leukemia bearing mice (40% versus 50% cure rate).CTL are able to convert mixed hematopoietic chimerism into full hematopoietic chimerism (30% donor →100% donor). In contrast, mixed chimerism remains unchanged when CTL are transferred that had been primed ex vivo on donor-derived DC. This data is encouraging in that it provides further evidence that strong GVL effects can be obtained across MHC barriers without eliciting severe GVHD.

CTLA4-Ig-Based Conditioning Regimen to Induce Tolerance to Cardiac Allografts

Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.

Immune-Mediated Myelopathy following Allogeneic Stem Cell Transplantation

We report a case of immune-mediated myelopathy occurring 4 months after unrelated bone marrow transplantation for myelodysplastic syndrome. After the tapering of cyclosporine for graft-versus-host disease prophylaxis, the patient developed several neurological symptoms mimicking the clinical features of multiple sclerosis (MS). Although neurological exacerbation was well stabilized with a bulk dose of corticosteroid, sustained improvement of neurological deficits occurred after the patient developed hematopoietic mixed chimerism (HMC). This experience may provide clinical evidence supporting the current therapeutic concept, in which HMC induction can potentially cure several immune diseases, including MS.

Partial Donor-Specific Tolerance to Delayed Skin Grafts After Rejection of Hematopoietic Cell Graft

Donor-specific tolerance (DST) is induced after allogeneic hematopoietic cell transplantation (HCT) and is a potential strategy for prolonging survival of solid organ grafts. DST may persist in recipients with transient mixed hematopoietic chimerism (MC) when solid organ transplantation and HCT are done concomitantly. In a canine model of allogeneic HCT after nonmyeloablative conditioning, DST to skin grafts was evaluated in dog leukocyte antigen (DLA)-identical recipients with stable MC (n=11), or after rejection of the hematopoietic cell (HC) graft (n=19). There was significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in four recipients with MC developed an inflammatory reaction without skin graft loss. This may represent partial DST. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged compared to normal recipients even when skin grafting was delayed until after rejection of the HC graft (P=0.002). An inflammatory reaction developed in all nine of the surviving HC donor-derived skin grafts in this group, but there was no graft loss at last follow-up (median, 30 [range, 9-84] weeks). An increased time to rejection of the hematopoietic graft was associated with prolonged survival of the subsequent skin graft (P=0.02). In a model of stable MC, DST to skin grafts may be complete or partial. Partial DST can persist after HC graft rejection even if solid organ transplantation is delayed. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.

Chimerism and outcomes after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning

Allogeneic hematopoietic cell transplantation (HCT) following nonmyeloablative conditioning has been extensively evaluated in patients with hematologic malignancies who are ineligible for conventional HCT because of age or medical comorbidities. Nonmyeloablative regimens have led to an initial state of mixed hematopoietic chimerism defined as coexistence of donor- and host-derived hematopoiesis. While nonmyeloablative regimens have been associated with reduced regimen-related toxicities in comparison with conventional myeloablative conditioning, graft rejection, graft-versus-host disease (GVHD), and disease progression have remained significant challenges. In this article, after briefly introducing current techniques for chimerism assessment, we describe factors affecting donor chimerism levels after nonmyeloablative conditioning, and then review data suggesting that chimerism assessment early after HCT might help identify patients at risk for graft rejection, GVHD and relapse/progression. Finally, we discuss how these observations have opened the way to further research protocols evaluating manipulation of postgrafting immunosuppression, and/or infusion of donor immune cells.

The authors’ reply to the comments by Urban et al. on ‘Skin transplantation to monitor clinical donor‐related tolerance in mixed hematopoietic chimerism’ (Pediatr Transplant 2006; 10: 128–131)

The authors’ reply to the comments by Urban et al. on ‘Skin transplantation to monitor clinical donor‐related tolerance in mixed hematopoietic chimerism’ (Pediatr Transplant 2006; 10: 128–131)

Comments on ‘Skin transplantation to monitor clinical donor‐related tolerance in mixed hematopoietic chimerism’ by Mache et al. (Pediatr Transplant 2006;10:128–131)

Comments on ‘Skin transplantation to monitor clinical donor‐related tolerance in mixed hematopoietic chimerism’ by Mache et al. (Pediatr Transplant 2006;10:128–131)