High Levels Of Donor Chimerism Research Articles

Treosulfan-based conditioning for inborn errors of immunity.

Inborn errors of immunity (IEI) are inherited disorders that lead to defects in the development and/or function of the immune system. The number of disorders that can be treated by haematopoietic stem-cell transplantation (HSCT) has increased rapidly with the advent of next-generation sequencing. The methods used to transplant children with IEI have improved dramatically over the last 20 years. The introduction of reduced-toxicity conditioning is an important factor in the improved outcome of HSCT. Treosulfan has myeloablative and immunosuppressive properties, enabling engraftment with less toxicity than traditionally used doses of busulfan. It is firmly incorporated into the conditioning guidelines of the Inborn Errors Working Party of the European Society for Blood and Marrow Transplantation. Unlike busulfan, pharmacokinetically guided dosing of treosulfan is not part of routine practice, but data are emerging which indicate that further improvements in outcome may be possible, particularly in infants who have a decreased clearance of treosulfan. It is likely that individualized dosing, not just of treosulfan, but of all agents used in conditioning regimens, will be developed and implemented in the future. This will lead to a reduction in unwanted variability in drug exposure, leading to more predictable and adjustable exposure, and improved outcome of HSCT, with fewer late adverse effects and improved quality of life. Such conditioning regimens can be used as the basis to study the need for additional agents in certain disorders which are difficult to engraft or require high levels of donor chimerism, the dosing of individual cellular components within grafts, and effects of adjuvant cellular or immunotherapy post-transplant. This review documents the establishment of treosulfan worldwide, as a safe and effective agent for conditioning children with IEI prior to HSCT.

Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p<0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p<0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

CD45-Targeted Antibody Drug Conjugate Plus Post Transplant Cytoxan Is Sufficient to Enable Allogeneic Bone Marrow Transplant in a Minor Mismatch Mouse Model

Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting. CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models.

Use of a Processed Hematopoietic Stem Cell Product (FCRx) in Unmatched Related and Unrelated Donor — Recipient Pairs Is Associated with High Levels of Donor Chimerism and Donor-Specific Tolerance to Kidney Allografts

The prospect of eliminating the long term costs and side effects of drug-based immunosuppression (IS) is a compelling reason to pursue the establishment of transplant tolerance. Tolerance in kidney transplant (KTx) recipients has been achieved through the infusion of donor HSC under some form of conditioning. The increased upfront cost and intensive treatment required by tolerance induction protocols demands that the long term outcomes be superior to that achieved with SOC IS regimens. Since 2009 we have conducted a Phase 2 trial of combined stem cell/living donor kidney transplantation in mismatched and unrelated subjects where the stated goal has been the establishment of durable donor macrochimerism. Our protocol is based upon tolerogenic CD8+/TCR-facilitating cells (FCRx) and 200 cGy TBI-based nonmyeloablative conditioning. Recipients were conditioned with fludarabine (30mg/m2/dose, days -5,-4,-3), cyclophosphamide (50mg/kg/dose, day-3 and+3), 200 cGy TBI (day-1) followed by a living donor KTx (day0). A G-CSF mobilized peripheral blood mononuclear cell product was apheresed from the donor >2 weeks pre-KTx, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 + cells and FC, and cryopreserved until administration day+1 post-KTx. MMF and tacrolimus-based immunosuppression (IS) was weaned and discontinued at 1 year if chimerism, normal renal function and normal KTx biopsy were noted. 37 subjects were transplanted between 2009 and 2016. 17 of 37 subjects were transplanted from 0-2 of 6 HLA matched unrelated donors. 26 of 37 subjects achieved high levels of durable donor chimerism and have been removed from all immunosuppression (7-95 months). Durably chimeric subjects show normal yearly protocol biopsies at 6 months, 12 months, and yearly thereafter, while standard of care patients begin to show abnormal biopsies and deterioration of renal function as early as 24 months post-transplant. Among the 26 subjects who were removed from immunosuppression, none have experienced biopsy proven acute rejection, none have lost chimerism, and none have had to have immunosuppression resumed. Patient and graft survival at 5 years post-transplant are similar to standard of care (SOC) subjects. Two cases of graft-versus-host disease (GVHD) have occurred (6% incidence). One subject experienced grade 2 GVHD that responded to therapy and was successfully removed from all immunosuppression. One subject experienced steroid unresponsive grade 3-4 GVHD resulting in death. Among 7 durably chimeric subjects who had an underlying autoimmune disease that caused their renal failure, none have experienced recurrence of the autoimmune disease (expected rate of recurrence 40-60%). In summary, patient and graft survival at five years was comparable between FCRx and SOC pts. Tolerant FCRx subjects had significantly better renal function than SOC. Medical therapy for hypertension and hyperlipidemia was more common in SOC than tolerant FCRx pts. We conclude there are significant long-term medical benefits to establishing tolerance in KTx recipients using the FCRx approach that become apparent within < 5 years. DisclosuresHorwitz:Gamida Cell: Research Funding. Ildstad:Regenerex, LLC: Equity Ownership, Other: CEO.

Induction of Major Histocompatibility Complex-mismatched Mouse Lung Allograft Acceptance With Combined Donor Bone Marrow: Lung Transplant Using a 12-Hour Nonmyeloablative Conditioning Regimen.

Despite broad and intense conventional immunosuppression, long-term survival after lung transplantation lags behind that for other solid organ transplants, primarily because of allograft rejection. Therefore, new strategies to promote lung allograft acceptance are urgently needed. The purpose of the present study was to induce allograft tolerance with a protocol compatible with deceased donor organ utilization. Using the major histocompatibility complex-mismatched mouse orthotopic lung transplant model, we investigated a conditioning regimen consisting of pretransplant T cell depletion, low-dose total body irradiation and posttransplant (donor) bone marrow, and splenocyte infusion followed by posttransplantation cyclophosphamide. Our results show that C57BL/6 recipients of BALB/c lung allografts undergoing this complete short-duration nonmyeloablative conditioning regimen had durable lung allograft acceptance. Mice that lacked 1 or more components of this regimen exhibited significant graft loss. Mechanistically, animals with lung allograft acceptance had established higher levels of donor chimerism, lymphocyte responses which were attenuated to donor antigens but maintained to third-party antigens, and clonal deletion of donor-reactive host Vβ T cells. Frequencies of Foxp3 T regulatory cells were comparable in both surviving and rejected allografts implying that their perturbation was not a dominant cell-regulatory mechanism. Donor chimerism was indispensable for sustained tolerance, as evidenced by acute rejection of allografts in established chimeric recipients of posttransplantation cyclophosphamide after a chimerism-ablating secondary recipient lymphocyte infusion. Together, these data provide proof-of-concept for establishing lung allograft tolerance with tandem donor bone marrow transplantation using a short-duration nonmyeloablative conditioning regimen and posttransplant cyclophosphamide.

The Best of Both? Megadose Stem Cells with CD34 Selection and T-Cell Addback Enables Engraftment from Mismatched Unrelated Donors Using Reduced Toxicity Conditioning in Primary Immunodeficiency

▪Background: The optimal approach for transplanting patients with primary immunodeficiency from a mismatched unrelated donor (MMUD) remains unclear. With reduced intensity conditioning, when bone marrow (BM) was used as the stem cell source in such patients we have previously observed a high rate of very low level donor chimerism (4 of 12 patients) requiring a second transplant procedure. The use of peripheral blood stem cells (PBSC) can overcome this but results in a high incidence of acute (≥ Gde II 10/21, Gde III-IV 5/21) and chronic (10/21, extensive 7/21) graft-versus-host disease (GVHD) (Rao et al in press). We hypothesized that the use of megadose CD34 selected stem cells from PB with addback of a T-cell dose akin to BM might facilitate engraftment through improved competition for the stem cell niche without severe GVHD.Methods: We prospectively analysed outcomes on 20 consecutive patients with primary immunodeficiency (SCID n=5, HLH n=3, Other PID n=12) transplanted from 8/10 (n=4) or 9/10 (n=16) HLA MMUD at our institution between 2011-2015. The mean age at transplant was 5.1 years. All patients received reduced toxicity conditioning regimens (12/20 Fludarabine/Melphalan/Alemtuzumab; 8/20 Fludarabine/Treosulphan/Alemtuzumab) with Cyclosporin (CsA) and mycophenolate mofetil (MMF) GvHD prophylaxis. CD34 selection of donor PBSC was performed using CliniMACs and the mean CD34+ dose was 20.1x106/kg. At day 0 either 108 CD3/kg (cohort 1, n=6) or 3x108 CD3/kg (cohort 2, n=14) T-cells from the CD34- fraction were infused with the graft. Mean follow up was 31.9 months.Results: In cohort 1, all patients engrafted and 2 developed high level donor mixed chimerism (both curative) in the myeloid and lymphoid lineages at last follow up. Two patients had Grade II acute GvHD and 1 had moderate chronic GvHD. In view of the slow immune reconstitution in this cohort, the T-cell addback dose was increased to 3x108/Kg in subsequent patients. In cohort 2, all patients achieved full donor haemopoiesis initially, 6/14 developed high levels of donor chimerism in both myeloid and lymphoid lineages later and 1/14 progressed with 10% donor T-cell engraftment but remains disease-free. The incidence of significant aGVHD was low (grade II n=4, no grade III or IV) and no patient developed cGvHD. Overall across both cohorts, 10 patients had viral reactivations and there were 5 deaths (3 viral complications, 1 pulmonary vasculopathy, 1 cGVHD lungs). The disease free survival at 2 years 7 months follow up was 70% which compares favorably with a previous cohort transplanted with unmanipulated BM as the stem cell source (Fig 1). Immune reconstitution was delayed (mean CD3 and CD19 counts at day 100 post-transplant was 245 x 109/L and 315 x 109/L) with similar kinetics in both cohorts (Fig 2), comparable to RIC transplant using unmanipulated BM. By 1 year post transplant 11/14 evaluable patients achieved normal CD3+T-cell numbers and 8/14 normal CD19+ B-cell counts.Conclusion: The use of megadose peripheral blood stem cells with T-cell addback in the mismatched unrelated donor transplant setting results in high rates of curative engraftment and a low incidence of acute and chronic GvHD following reduced toxicity conditioning. However, T-cell reconstitution remains delayed (presumably reflecting the effect of Alemtuzumab on the infused T-cells) with a high incidence of viral complications. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immune Reconstitution in Recipients of Living Donor Kidney/Hematopoietic Stem + Facilitating Cell Transplants

Solid organ transplant recipients generally require life-long immunosuppressive agents to maintain graft acceptance. This is associated with a number of toxicities, including renal dysfunction, metabolic abnormalities, opportunistic infection and malignancies. Calcineurin inhibitors, the most frequently used agents for immunosuppression, induce nephrotoxicity, with gradual renal decline in many transplant recipients. As a result, approaches to induce tolerance remain a major focus of research. We report here the long-term results from a reduced-intensity nonmyeloablative conditioning approach to establish high levels of donor chimerism without graft-versus-host disease (GVHD) or engraftment syndrome in mismatched related and unrelated recipients of combined living donor kidney/hematopoietic stem cell transplant (HSCT). Recipients were conditioned with 200 cGy total body irradiation plus fludarabine (30 mg/kg day -5, -4, -3) and cyclophosphamide (50 mg/kg days -3 and +3) and administered donor granulocyte colony-stimulating factor mobilized stem cells, enriched for hematopoietic stem cells (HSC) and graft facilitating cells (FCs) and depleted of GVHD-producing cells (FCRx). Subjects ranged in age from 18 to 65 years. All subjects were HLA-disparate from their living kidney/HSCT donors, ranging from 5 of 6 related to 0 of 6 unrelated. Eight were unrelated and 11 related to their donors. Subjects were maintained on tacrolimus and mycophenolate mofetil (MMF) for 6 months. At 6 months, if protocol biopsy is normal and chimerism present, the MMF is discontinued. Similarly, the tacrolimus is discontinued at 12 months if stable renal function, normal protocol biopsy and chimerism are present. 30 subjects have been enrolled and 25 transplanted. Nineteen subjects have > 1 year follow up. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression. Time off IS ranges from 8 months to 48 months. None have developed GVHD. We have herein prospectively evaluated immune reconstitution and immunocompetence. Return of CD4+ and CD8+ T central and effector memory cell populations was rapid. Examination for recent thymic emigrants (T cell staining for CD31+ CDRA+, TCR excision circle analysis) indicated that the majority (>97%) of these chimeric cells are being produced de novo. The TCR repertoires post-transplant in chimeric subjects were as diverse as pre-transplant donors and recipients but were distinct from donor or recipient pre-transplant. An increase in the CD4+ regulatory T cell/CD4+ effector T cell ratio was seen early in durably and transiently chimeric patients, suggesting active immunoregulation. Patients have been successfully vaccinated per American Society of Bone and Marrow Transplantation guidelines without loss of chimerism or development of allograft rejection. Of four subjects immunized for hepatitis B and whose donors were not, three retained immunologic memory after transplantation. Most chimeric subjects retained memory for measles, mumps, rubella and varicella. Chimeric subjects generated immune responses to pneumococcal vaccine. Opportunistic viral infections such as BK viremia and cytomegalovirus activation were absent after cessation of immunosuppression. These findings suggest that immunologic recovery is robust in chimeric subjects who receive kidney/FCRx and that durable chimerism is associated with donor-specific tolerance to renal allografts. DisclosuresBozulic:Regenerex, LLC: Employment. Tollerud:Regenerex, LLC: Chief Operating Officer Other. Ildstad:Regenerex, LLC: Chief Executive Officer Other.

Conventional CD4+CD25- and Regulatory CDCD4+25+ t Cells Have Opposite Effects on Progenitor Cells and Hematopoietic Reconstitution Following Stem Cell Transplantation

The bone marrow (BM) is a complex microsystem to support lifelong blood production. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines. Following hematopoietic cell transplantation (HCT) in the phase of hematopoietic reconstitution maximal blood production is needed. In an HCT donor HSC are given together with immune cells in the belief that T cells support HSC engraftment and regeneration of the blood system. Yet, states of immune mediated BM insufficiency, hypoplasia, and cytopenias are often observed. Moreover, with increased use of reduced intensity conditioning (RIC) engraftment failure has reemerged as a serious problem.Here, we studied the effects of distinct T cell subsets on hematopoietic reconstitution following HCT; specifically, we examined how conventional CD4+CD25- T cells (CD4conv) vs regulatory T cells (CD4+CD25+, Treg) modify the BM environment and influence donor-HSC activity and engraftment. We used minor-mismatched mouse models, non-myeloablative total body irradiation (TBI) conditioning and transplantations of purified HSC (KTLS; cKit+Thy1.1loLin–Sca-1+) plus selected T cell subsets. Recipients of HSC, HSC+Treg or HSC+CD8+ - but not HSC+CD4conv- demonstrated prompt donor engraftment with mixed chimerism in all lineages. Transplantation of HSC+Treg resulted in significantly faster lymphocyte recovery and higher levels of donor chimerism compared with recipients of HSC, HSC+CD8+ or HSC+CD4conv. Particularly B-cell regeneration was markedly higher in HSC+Treg-recipients compared with all other groups. In contrast, (B-) lymphopoiesis was severely impaired in recipients of HSC+CD4conv; when lymphocytes recovered eventually they were of host type. Moreover, in BM and spleens of HSC+CD4conv recipients pronounced hypocellularity was observed. This suppression of hematopoiesis was due to IFNg secretion of donor CD4conv cells, which were activated by dendritic cells via IL-12. High cytokine levels (of both IL-12 and IFNg) were only detectable in the BM (and not the spleen) of HSC+CD4conv recipients, where they resulted in an arrest of early hematopoiesis at the stage of short-term HSC and decreased cell-cycle activity within the progenitor compartment. As a consequence more mature multipotent progenitors were lacking. The key role of IFNg in halting hematopoietic maturation was confirmed by using CD4conv cells from IFNg-/- mice, which had no suppressive effects on BM cellularity and maturation of blood cells; rather, recipients of HSC+IFNg-/-CD4conv cells had equivalent cell numbers and subset distributions as mice given HSC alone. We hypothesized that differences of hematopoietic regeneration and donor engraftment relate to cell cycle activity of HSC in presence of CD4conv vs Tregs. To study the influence of these CD4-subsets on HSC cycling in more detail FACS-purified Treg vs CD4conv cells were infused into congenic mice following low-dose TBI-stimulation. In fact, on d+8 post-infusion HSC in the BM of Treg-recipients had increased cell-cycling activity in long-term-HSC and multipotent-progenitor fractions compared with mice given CD4conv cells or radiation only. These data lead us to speculate that Tregs promote, directly or indirectly, HSC proliferation; in the context of an allogeneic HCT this increased cycling of host HSC my open-up the niche space required to allow donor HSC to engraft. In contrast, CD4convinhibited HSC cycling activity, resulting in BM hypoplasia and cytopenias; at the same time donor HSC engraftment was impaired due to HSC-niche occupation by quiescent host HSC.Our findings underscore the critical role of T cells in regulating hematopoiesis under physiologic conditions and even more following allogeneic HCT. While donor T cells are generally believed to improve regeneration of the blood post-HCT and to be required to overcome host barriers, our data suggest Treg facilitate engraftment and hematopoiesis by increasing HSC cycling-activity and thereby making marrow sites available. CD4conv appear to have the opposite effect, resulting in decreased HSC proliferation and maturation - thus occupation of HSC niches. Our studies are of particular relevance to allogeneic HCT settings using RIC, where host HSC persist and grafts can be rejected by residual host immune cells. DisclosuresNo relevant conflicts of interest to declare.

Superior long-term repopulating capacity of G-CSF+plerixafor-mobilized blood: implications for stem cell gene therapy by studies in the Hbb(th-3) mouse model.

High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.

Human CD8+/TCR−/CD56dim/- Facilitating Cells Facilitate Homing and Engraftment of Human HSC in Vivo

Abstract 4093Hematopoietic stem cell (HSC) transplantation has become a common procedure for treatment of hematopoietic malignancies and autoimmune disease. Despite significant advances in HSC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain limitations to application in the clinic. However, these risks can be overcome through less toxic nonmyeloablative conditioning and cell depletion strategies to remove GVHD causing-cells while retaining engraftment enhancing-tolerogeneic cells. We were the first to discover CD8+/TCR− graft facilitating cells (FC) in mouse bone marrow. The addition of as few as 30,000 FC to 10,000 HSC significantly enhances engraftment of HSC in allogeneic recipients without causing GVHD. FC also potently enhance engraftment of limiting numbers of syngeneic HSC. Human CD8+/TCR- FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (mPBMC). In the CD8+/TCR- FC, 48% of cells expressed CD3ε+, 43% were FoxP3+, 43% were CD11c+, 19% were CD19+, and 30% were HLA-DR+. Approximately 55% of FC are also CD56dim/-, and the remaining population is CD56bright. The morphology of human CD8+/TCR− FC with Wright-Giemsa staining under light microscopy suggested that the human FC population is heterogeneous. Here we evaluated if human FC enhance human HSC or progenitor homing to bone marrow of NOD/SCID/IL-2rγnull (NSG) mouse recipients. CD45+CD34+ HSC and CD8+/TCR−/CD56dim/-FC were sorted from mPBMC. NSG recipients were conditioned with 1100 cGy of total body irradiation (TBI). 24 hours after TBI, 100,000 HSC with or without 300,000 CD8+/TCR−/CD56dim/- FC were transplanted into conditioned NSG recipients. Recipients were euthanized 16 hours after transplantation. Bone marrow was harvested from femurs and tibias of recipients and plated in Colony Forming Culture (CFC) Assays. Recipients of HSC plus FC generated significantly more colony formation (colonies = 110) compared with HSC alone (colonies = 65) (P = 0.011), suggesting that CD8+/TCR−/CD56dim/- FC enhanced homing of HSC or progenitors to bone marrow. To test if human CD8+/TCR−/CD56dim/- FC facilitate engraftment of human HSC in NSG mice, 300,000 CD8+/TCR−/CD56dim/- FC were mixed with 100,000 HSC and transplanted into NSG recipient mice conditioned with 325 cGy TBI. Mice that received HSC alone served as controls. At 30 days after transplantation, PBL typing showed that 34% (10 of 29) recipients of HSC alone engrafted. In contrast, 78% of recipients (n = 23) of HSC plus CD8+/TCR−/CD56dim/- FC engrafted, and donor chimerism in PB was 1.1% ± 0.8% and 4.1% ± 1.3% (P <0.05), respectively. At 6 months after transplantation, NSG recipients of HSC + CD8+/TCR−CD56dim/- FC exhibited persistent donor chimerism in PB (9.1% ± 6% vs. 3.8% ± 3.5%) (P <0.05) and significantly higher levels of donor chimerism in spleen (26.3% ± 11.8% vs. 12.3% ± 9.8%) (P <0.05) and BM (11.6% ± 4.8% vs. 2.9% ± 1.3%) (P <0.05) compared to recipients of HSC alone. Our data indicate that CD8+/TCR−/CD56dim/- FC facilitate homing of human HSC or progenitors and enhance engraftment of human HSC in NSG recipient mice. Disclosures:Bozulic:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Human CD8+/TCR−/CD56dim/− facilitating Cells Enhance Engraftment of HSC In NOD/SCID/IL2rγnull Recipients.

AbstractAbstract 3709CD8+/TCR− graft facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC). They are distinct from HSC since they do not generate multilineage engraftment when infused alone, nor do they form colonies in vitro. The function of human FC remains to be defined. Here, we phenotypically characterized the human CD8+/TCR− FC subpopulations and evaluated their function in vivo and in vitro. Human CD8+/TCR− FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (PBMC). Approximately 55% of FC express CD56dim/−, and the remaining population is CD56bright. In the CD8+/TCR−/CD56bright cell population, approximately 65% of cells express CD11c+ and 67% of cells express CD11b+. In CD8+/TCR−/CD56dim/− cell population, the majority of cells express CD3ε+ (80%), 17% were CD11c+, 16% were CD19+, 14% were CD11b+, 11% were CD123+, and 30% were HLA-DR+. To evaluate whether human CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo, we transplanted 100,000 HSC alone or plus 300,000 CD8+/TCR−/CD56dim/− FC into NOD/SCID/IL2rγnull (NSG) recipient mice conditioned with 325 cGy of total body irradiation. At 30 days after transplantation, 8 of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of recipients (n = 16) receiving HSC plus CD8+/TCR−/CD56dim/− FC engrafted, and donor lymphocyte and monocyte chimerism in PB was 0.53% ± 0.16% and 3.93% ± 1.28%, respectively. At 6 months after transplantation, NSG recipients of HSC alone lost donor chimerism in PB and no donor cells were detected in spleen and BM. In contrast, NSG recipients of HSC + CD8+/TCR−CD56dim/− FC exhibited durable donor chimerism in PB and showed significantly higher levels of donor chimerism in spleen (13%) and BM (9.43%) compared to recipients of HSC alone. To evaluate the function of CD8+/TCR−/CD56dim/− FC in vitro, HSC were incubated with CD8+/TCR−CD56dim/− FC for 18 hrs and then cultured in methylcellulose for 14 days in colony-forming cell assay. HSC plus CD8+/TCR−CD56dim/− FC generated significantly more colonies compared with HSC alone (P = 0.0038), suggesting that human CD8+/TCR−/CD56dim/− FC have a direct effect on the clonogenicity of HSC (Figure). In summary, our data indicate that (1) human CD8+/TCR− FC are a heterogeneous cell population; (2) CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo and promote HSC clonogenicity in vitro; and (3) recipients of CD8+/TCR−/CD56dim/− FC maintain a durable donor chimerism in BM and spleen. [Display omitted] Disclosures:Bozulic:Regenerex, LLC: Employment. King:Regenerex, LLC: Employment, Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.

Dissociation between peripheral blood chimerism and tolerance to hindlimb composite tissue transplants: preferential localization of chimerism in donor bone.

Mixed chimerism induces donor-specific tolerance to composite tissue allotransplants (CTAs). In the present studies, we used a nonmyeloablative conditioning approach to establish chimerism and promote CTA acceptance. Wistar Furth (RT1A(u)) rats were conditioned with 600 to 300 cGy total body irradiation (TBI, day-1), and 100 x 10(6) T-cell-depleted ACI (RT1A(abl)) bone marrow cells were transplanted on day 0, followed by a 11-day course of tacrolimus and one dose of antilymphocyte serum (day 10). Heterotopic osteomyocutaneous flap transplantation was performed 4 to 6 weeks after bone marrow transplantation. Mixed chimerism was initially achieved in almost all recipients, but long-term acceptance of CTA was only achieved in rats treated with 600 cGy TBI. When anti-alphabeta-T-cell receptor (TCR) monoclonal antibody (mAb) (day-3) was added into the regimens, donor chimerism was similar to recipients preconditioned without anti-alphabeta-TCR mAb. However, the long-term CTA survival was significantly improved in chimeras receiving more than or equal to 300 cGy TBI plus anti-alphabeta-TCR mAb. Higher levels of donor chimerism were associated with CTA acceptance. The majority of flap acceptors lost peripheral blood chimerism within 6 months. However, donor chimerism persisted in the transplanted bone at significantly higher levels compared with other hematopoietic compartments. The compartment donor chimerism may be responsible for the maintenance of tolerance to CTA. Long-term acceptors were tolerant to a donor skin graft challenge even in the absence of peripheral blood chimerism. Mixed chimerism established by nonmyeloablative conditioning induces long-term acceptance of CTA, which is associated with persistent chimerism preferentially in the transplanted donor bone.

Long-term follow-up of donor chimerism and tolerance after human liver transplantation

We aimed to quantify peripheral donor chimerism (DC) and to analyze its association with graft and recipient outcome. Forty-two liver transplant recipients and their respective donors were studied, providing a total of 148 posttransplantation serum samples. DC was assessed with real-time quantitative polymerase chain reaction (qPCR) to detect polymorphic markers. DC did not decrease with time post-transplantation and was higher in child recipients versus adults and in recipients of deceased donor liver transplants versus recipients of live donor liver transplants. Higher levels of DC were detected in Rh-positive blood group donors, in O blood group recipients versus A blood group recipients, and in recipients with hepatitis C virus versus recipients with alcoholic cirrhosis. High DC was associated with patients with organ damage due to recurrent disease and rejection. Stable, high levels of DC, in the absence of other major clinical events, may thus be a marker of transplantation tolerance, and this knowledge may help to tailor immunosuppressive treatment. In conclusion, qPCR is a useful technique for DC follow-up in liver transplantation, although the evolution of DC levels should be analyzed in accordance with the clinical outcome of the patient.

Sirolimus, Tacrolimus, and Low-Dose Methotrexate as Graft-versus-Host Disease Prophylaxis in Related and Unrelated Donor Reduced-Intensity Conditioning Allogeneic Peripheral Blood Stem Cell Transplantation

We assessed the combination of sirolimus, tacrolimus, and low-dose methotrexate as acute graft-versus-host disease (aGVHD) prophylaxis after reduced-intensity conditioning (RIC) allogeneic peripheral blood stem cell (PBSC) transplantation from matched related (MRD, n = 46) and unrelated (URD, n = 45) donors. All patients received fludarabine and intravenous busulfan conditioning followed by transplantation of mobilized PBSC. The median time to neutrophil engraftment was 13 days. The cumulative incidence of grade II-IV and III-IV aGVHD were 16% and 7%, respectively. There was no difference in the incidence of aGVHD between MRD and URD cohorts. Two-year cumulative incidence of extensive chronic GVHD (cGVHD) was 40%. Relapse-free survival (RFS) at 2 years was 34%: 21% in MRD and 45% in URD. Overall survival (OS) at 2 years was 59%: 47% in MRD and 67% in URD. High levels (>90%) of donor derived hematopoiesis were achieved in 59% of patients early after transplantation. The addition of sirolimus to tacrolimus and low-dose methotrexate as GVHD prophylaxis following RIC with fludarabine and low-dose intravenous busulfan is associated with rapid engraftment, low rates of aGVHD, and achievement of high levels of donor chimerism.

Tacrolimus and Sirolimus without Methotrexate as Acute GVHD Prophylaxis after Matched Related Donor Reduced Intensity Conditioning (RIC) Stem Cell Transplantation (SCT).

We have previously shown that the combination of tacrolimus and sirolimus (TAC/SIR) without methotrexate (MTX) is a safe and effective regimen for GVHD prophylaxis after myeloablative SCT. However, TAC/SIR has not been investigated in the RIC setting. We hereby report a prospective phase II trial testing TAC/SIR as GVHD prophylaxis after RIC SCT from matched related donors (MRD). All patients received fludarabine (FLU) 30 mg/m2 IV and intravenous busulfan (BU) 0.8 mg/kg IV daily × 4 days (day -5 thru day -2) as conditioning, followed by transplantation of filgrastim mobilized peripheral blood stem cells. Filgrastim 5 mcg/kg SC QD was started on day +1 until neutrophil engraftment. Tacrolimus and sirolimus were started on day-3, and doses were adjusted to maintain target serum trough levels 5–10 ng/ml and 3–12 ng/ml, respectively. Twenty-six patients have been transplanted, with a median age of 52 years (range 29–64 yrs). Diagnoses include NHL (8), AML (6), HD (5), CLL/SLL (3), CML (2), MDS (1), MM (1). Median CD34+ cells infused was 8.06 × 106 cells/kg (range: 2.96–38.0 × 106 cells/kg). All patients had sustained hematologic engraftment. Only 4 (15%) patients developed neutropenia below ANC 500, and 6 (23%) had platelet counts below 20K. One patient had late graft failure at 7 months post transplant. Grade II-IV acute GVHD incidence was 20%, with grade III-IV incidence of 12%. No hepatic VOD or thrombotic microangiopathy was observed. Day +100 transplant related mortality (TRM) was 0%. The cumulative incidences of TRM and relapse at 1 year were 4% and 38%, respectively. The cumulative incidence of chronic GVHD at 1 year was 74%. A high level (>90%) of donor-derived hematopoiesis was achieved in 68% by 1 month post transplant. Median donor chimerism at between day 20–50 post transplant was 93.5% (range 32%-99%), and 94% (range 22%-100%) at day 85–115. With a median follow-up of 13.5 months among survivors (range 5.5–19 months), progression-free survival (PFS) and overall survival (OS) at 1 year were 58% and 79%, respectively. Compared to a historical cohort of 47 MRD transplant recipients treated with the same FLU/BU conditioning regimen and using TAC/SIR ± mini-MTX (5 mg/m2 IV day +1,3,6) as GVHD prophylaxis, there was no statistical difference between TAC/SIR vs. TAC/SIR/MTX in the incidence of grade II-IV acute GVHD (20% vs 11%, p= 0.48), cumulative incidence of relapse at year (38% vs. 57%, p= 0.25), 1-yr PFS (58% vs. 41%, p= 0.33), or 1-yr OS (79% vs. 73%, p= 0.79). These results demonstrate that omission of mini-MTX is permissible, and that the combination of TAC/SIR alone as GVHD prophylaxis following reduced intensity conditioning with FLU/BU is associated with low rates of acute GVHD, TRM, and high levels of donor chimerism after MRD SCT.

Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment: role in homing efficiency in NOD/SCID mice

The use of mesenchymal stem cells (MSC) for cell therapy relies on the capacity of these cells to home and engraft long-term into the appropriate target tissue(s). Homing of MSC to bone marrow (BM) post-transplantation can occur, but does so with only poor efficiency. This study was designed to evaluate the role of the SDF-1/CXCR4 axis in the homing of Flk1+ MSC derived from human fetal BM. We investigated the expression of CXCR4 in Flk1+ MSC stimulated with a cytokine cocktail and explored their homing ability 24 hours after intravenous infusion into sublethally irradiated NOD/SCID mice. The peripheral blood was analyzed and human cells in recipients' BM were quantified from 2 to 6 months after transplantation. We found that Flk1+ MSC harbored intracellular CXCR4 which can be rapidly induced to the cell surface within a few hours. Short-term (24 hours) stimulation with the cocktail of cytokines resulted in up-regulation of both cell surface and intracellular CXCR4, increasing in vitro migration capacity to SDF-1 and homing to the BM of irradiated NOD/SCID mice. Moreover, compared to non-treated cells, transplantation of cytokine-treated Flk1+ MSC resulted in faster hematologic recovery and higher levels of donor chimerism in BM. Neutralization of CXCR4 significantly reduced homing and engraftment of Flk1+ MSCs in murine BM. These results suggest that the SDF-1/CXCR4 axis plays an important role in the regulation of motility of Flk1+ MSC. Increasing CXCR4 expression might be a potential strategy to improve engraftment of MSC in BM and accelerate the recovery of hematopoiesis.

High Levels of Early Donor Chimerism and Treatment-Responsive Disease Predict Improved Progression-Free Survival Following Non-Myeloablative Transplantation for Advanced CLL.

CLL remains an incurable disease with standard chemotherapy. Although recent data suggest that CLL can respond to graft vs leukemia effects, myeloablative allogeneic stem cell transplantation is associated with a high transplant-related mortality in this population of older, heavily pretreated patients. We report the outcomes of 50 patients with advanced CLL who underwent NST between January 2001 and August 2004. All patients received fludarabine 30 mg/m2 x 4 days and intravenous busulfan 0.8 mg/kg/d x 4 days. 94% received G-CSF mobilized peripheral blood stem cells while 6% received bone marrow. Graft vs host disease (GVHD) prophylaxis included tacrolimus plus low dose methotrexate (56%) or cyclosporine plus prednisone (30%) based regimens. Most patients had a HLA-matched unrelated donor (62%), 30% a HLA-matched related donor, and 8% a HLA-mismatched donor. The median age of the patients was 53 years (range 35–67), with a median time from diagnosis to NST of 6.4 years (range 0.2–14.7). The patients were heavily pretreated, with a median of 5 prior therapies; 98% of patients had received fludarabine, 96% alkylating agents, 80% rituximab, and 32% alemtuzumab. 22% of patients had relapsed after prior autologous stem cell transplant. Most patients had active disease at time of NST, with only 16% in complete remission and 26% in partial remission. 52% of patients were in active relapse and 6% had failed to respond to any attempted therapy (induction failures). 50% of patients developed grade 4 neutropenia and their median time to neutrophil engraftment was 11.5 days. The incidence of grade 2–4 acute GVHD was 36%, and of chronic extensive GVHD 33%. Eleven of 25 patients (44%) in active relapse or induction failure at time of NST achieved an objective response. The median follow-up of surviving patients is 12.4 months (range 5.6 mos-4.0 yrs), with one-and two-year PFS 38% (95% CI 24–52%) and 28% (13–42%). The one- and two-year OS in this highly refractory population is 59% (95% CI 44–75%) and 48% (31–65%). Patients achieving >75% donor-derived hematopoiesis 1–2 months post-NST had a 30% risk of relapse or death from disease, as compared to 72% for those with lower donor chimerism (p= 0.007). Relapse was the principal cause of treatment failure, resulting in 10 deaths; other deaths were due to infection (n = 5), GVHD (n = 3) and respiratory failure (n = 1). In Cox proportional hazards regression analysis considering pre-transplant parameters such as age, sex mismatch, disease status (CR/PR vs relapse/induction failure), donor type, aGVHD prophylactic regimen and Rai stage, only disease status was an independent risk factor for poor OS (HR 4.7, p= 0.02). Both older age and disease status (HR 2.7, p= 0.02) were associated with poor PFS. Although treatment-responsive disease was the primary predictor of outcome, nonetheless 44% of patients with refractory disease still achieved objective responses after NST. High levels of donor chimerism 1–2 months post-NST were associated with a reduced risk of relapse or death from disease. These results suggest that NST is a reasonable treatment option for patients with advanced CLL, and that strategies to augment donor chimerism early after NST may result in improved long-term outcomes.

A Two Step Nonmyeloablative Allogeneic Transplantation Protocol Using In Vitro T-Cell Depleted Mobilized Stem Cells Followed by Donor Lymphocyte Infusions in Patients with Advanced CLL.

Transplantation of stem cells that are T-cell depleted using Campath 1H incubation following non-myeloablative (NM-alloSCT) conditioning leads to durable donor engraftment with minimal GVHD using HLA-identical family sibling and unrelated donors. The high levels of donor chimerism allow the subsequent use of DLI to treat residual disease (Exp. Hematol. 2003; 31: 865–872). Patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) have a short life expectancy. We analyzed 12 patients with advanced CLL (9), PLL (1) or M. Waldenstrom (2) treated with NM-alloSCT. Median age was 56 years. Recipient conditioning consisted of fludarabine (30 mg/m2, 6 days), ATG (10 mg/kg, 4 days) and busulphan (3.2 mg/kg, 2 days). High numbers of G-CSF mobilized peripheral blood CD34+ cells from 11 related and 1 unrelated donors were collected (median 12 x 106/kg). The graft was T-cell depleted by 30 minutes incubation with 20 mg Campath 1H. No post-transplant GVHD prophylaxis was administered. The transplant procedure was very well tolerated. All patients engrafted rapidly. Only 1 patient experienced GVHD gr I (skin) after alloSCT. Sequential chimerism studies of bone marrow showed sustained mixed chimerism with a median donor percentage at 3 months after alloSCT of 86% (range 8–100%). At a fixed time period of 6 months after NM-alloSCT, low dose DLI was administered (5 x 106 CD3+ cells/kg) in 8 patients (3 pts too early after alloSCT and one pt died before DLI). The disease status pre-DLI was persistent disease in 4 pts and CR in 4 pts. After DLI, four patients experienced severe aGVHD (≥ grade 2) and 1 limited cGVHD. Importantly, the observed GVHD after low dose DLI was well managable. One patient received a second transplant from the original donor after myeloablative conditioning due to progressive disease with concommitant decreasing mixed chimerism. Donor chimerism increased after DLI with a median donor percentage of 98% (range, 3–100%) at 3 months after DLI. Presently at a median follow-up of 423 (range 46–1572) days, 9 patients are alive. Four patients are in complete remission, 3 with partial remission and 2 with progressive disease. Three patients have died, two due to infection and one by unknown cause.In conclusion, in-vitro T-cell depleted NM-alloSCT followed by low dose DLI is a well tolerated strategy in older patients with advanced CLL. The low incidence of GVHD and the high levels of donor chimerism after NM-alloSCT allow the use of DLI to treat residual CLL. Our results demonstrate a profound GVL effect in patients with advanced CLL. NM-alloSCT may prolong survival for this group of patients.

High Levels of Donor Chimerism Early after Non-Myeloablative Transplantation Predictive of Overall and Progression Free Survival but Not Risk of Acute Graft Versus Host Disease for Patients with AML or MDS.

Non-myeloablative (NST) transplantation is increasingly used in the treatment of patients with AML and MDS who are not candidates for myeloblative transplant. Relapse of disease remains a major cause of treatment failure after NST. Predictive factors to identify patients at high risk of relapse are needed to identify patients who would benefit from additional interventions. Attainment of a high degree of donor engraftment achieved early after transplantation may indicate the presence of a more significant allo-immune effect. We have performed a retrospective analysis of 64 patients with AML and MDS receiving NST, assessing the impact of donor chimerism when measured early after transplantation on outcome. Overall survival (OS), progression free survival (PFS) and risk of graft versus host disease (GVHD) were compared for patients achieving ≥90 % or <90% donor derived hematopoiesis when measured 1 month after transplant. All patients received fludarabine 30 mg/m2/day x 4 days and intravenous busulfan (Busulfex)0.8 mg/kg/day x 4 days for conditioning. All patients received calcineurin-inhibitor based GVHD prophylaxis. All patients received PBSC with G-CSF at 5 mcg/kg beginning day 1 after transplantation. Chimerism was measured using FISH for sex mismatched patient donor pairs or by STR analysis. 37 patients had ≥90% donor derived hematopoiesis, 27 patients had <90% donor derived hematopoiesis after transplantation. The two groups had similar characteristics with a median age of 57 yrs (range 21–70) for patients ≥90% and 58 yrs (range 32–69) for patients <90%. Of patients achieving ≥90%, 23 patients had AML and 14 MDS. Of patients <90%, 13 had AML and 14 with MDS. 7 of 16 (44%) patients with early stage disease(AML in CR1 or early stage MDS) achieved ≥90% donor hematopoiesis, while 30 of 48 (63%) patients with advanced disease achieved ≥90%. 17 of 29 (59%) patients with unrelated donors achieved ≥90% donor derived hematopoiesis, while 20 of 33 (61%) patients with matched related donors achieved ≥90% donor derived hematopoiesis. 21 of 32 (66%) patients with donor-recipeint sex mismatch achieved ≥90% while 16 of 32 (50%) patients with same sex donors were ≥90%. The median follow-up for surviving patients achieving ≥90% donor chimerism was 12 months and 15 months for those <90%. Patients achieving ≥90% donor chimerism had a significantly improved 1-year (71% versus 41%) and 2-year (39% versus 19%) OS (p=0.05). Similarly, for patients achieving ≥90% donor chimerism, there was a trend toward an improved PFS at 1-year (49% versus 30%) and 2-years (32% versus 19%) (p=0.08). There was no difference in the risk of developing stage 2–4 acute GVHD, 19% for both patients above and below 90%. Achieving high levels of donor chimerism when measured early after NST predicts for an improved overall survival and there is a trend toward an improved progression free survival. This may represent the presence of an enhanced graft versus leukemia effect in these patients. The degree of donor chimerism does not predict the development of acute GVHD. These results suggest that patients with <90% donor derived hematopoiesis may be candidates for strategies to enhance donor chimerism.