Persistence Of Donor Cells Research Articles

Photoreceptor nanotubes mediate the in vivo exchange of intracellular material

Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor‐derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule‐containing protrusions, termed photoreceptor nanotubes (PhNTs), that connect donor and host cells in vivo and in vitro. PhNTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with PhNT outgrowth by targeting Rho GTPase‐dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube‐like connections between neurons in vivo.

Clinical Outcomes of Microtransplantation for Older Adults with Acute Myeloid Leukemia

Introduction: Outcomes of acute myeloid leukemia (AML) in older patients remain unsatisfactory with low rates of complete remission (CR), disease-free survival (DFS), and overall survival (OS). Microtransplantation - in which allogeneic cells are infused without substantial engraftment (<2% donor cell engraftment) - has the potential to improve anti-tumor immunity without the risks of allogeneic hematopoietic stem cell transplant and graft-vs-host disease (GVHD).Methods: Adapting the protocol of Ai et al. (Blood 2011;117(3):936-41), newly diagnosed high-risk AML patients ≥ 55 years received induction with cytarabine100 mg/m2, idarubicin 12 mg/m2, and microtransplantation (7+3+MST). Microtransplantation was given 24-48 hours after 7+3 and consisted of a G-CSF mobilized leukapheresis product from a HLA-haploidentical family donor; a minimum of 1x108 CD3+ cells/kg were infused. Subjects in CR received 2 cycles of consolidation with high dose cytarabine (HiDAC); the second cycle was followed by another MST. Subjects not in CR were allowed to undergo a 2nd repeat induction cycle (7+3+MST).Results:We enrolled seventeen patients (Table 1) with a median of 248 days of follow up (range 37-887). The median age was 73 years (range 61-78); 5 (29%) had an antecedent hematologic disorder, and 10 (59%) had adverse-risk cytogenetics. Overall, 12 (71%) had poor-risk AML defined as either therapy-related AML, antecedent hematologic disorder, or adverse-risk cytogenetics; 2 (12%) had intermediate-risk AML, and 3 (18%) had favorable-risk AML (all three NPM1 positive).One patient developed sepsis during induction and did not receive microtransplantation; of the other sixteen patients that received at least one round of 7+3+MST, 10 (62.5%) achieved a complete remission, including 4 of 4 patients with intermediate or favorable risk AML (100%) and 6 of 12 with poor-risk AML (50%). Of the 10 in CR, 9 achieved CR after one round of induction and one required a second induction. Nine completed consolidation while one stopped after the first cycle of consolidation due to deconditioning. Three (19%) remain disease-free and alive at 100, 274, and 737 days. Interestingly, all three of these patients had fevers/chills during or immediately after MST. There were no further complications with MST, no cases of treatment-related mortality, and no cases of GVHD, consistent with absence of substantial engraftment. Estimated survival at six months was 67% and at one year was 34% (Figure 1). Correlative studies of immunomodulatory effects, including T-cell exhaustion and NK cell function, depth of response (minimal residual disease), and persistence of donor cells (microchimerism) are pending to better understand the potential mechanisms by which microtransplantation may improve disease responses.Conclusion: Microtransplantation is a potentially safe and effective therapy for older patients with AML. High rates of complete remission were observed in both those with favorable/intermediate risk disease as well as those with poor-risk disease. Further studies are warranted into the efficacy and mechanisms of microtransplantation. [Display omitted] DisclosuresSung:Novartis: Research Funding; Merck: Research Funding; Cellective: Research Funding. LeBlanc:Seattle Genetics: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Pfizer: Consultancy; AstraZeneca: Research Funding; American Cancer Society: Research Funding; Celgene: Honoraria; Cambia Health Foundation: Research Funding; Flatiron Health: Consultancy. Brander:Teva Pharmaceuticals, Genentech, AbbVie, Pharmacyclics: Consultancy; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. McKinney:Kite Pharma: Other: advisory comittee. Sipkins:Gilead Sciences, Inc.: Research Funding. Rizzieri:Shire: Research Funding; Erytech: Research Funding.

Regenerative engineered vascularized bone mediated by calcium peroxide.

One of the main challenges hindering the clinical translation of bone tissue engineering scaffolds is the lack of establishment of functional vasculature. Insufficient vascularization and poor oxygen supply limit cell survival within the constructs resulting in poor osseointegration with the host tissue and eventually leading to inadequate bone regeneration. Inspired by cues from developmental biology, we regenerative engineered a composite matrix by incorporating calcium peroxide (CaO2 ) into poly(lactide-co-glycolide) (PLGA) microsphere-based matrices and sought to assess whether the delivery of the byproducts of CaO2 decomposition, namely O2 , Ca2+ , and H2 O2 could enhance the regeneration of vascularized bone tissue. The composite microspheres were successfully fabricated via the oil-in-water emulsion method. The presence and encapsulation of CaO2 was confirmed using scanning electron microscopy, energy dispersive x-ray spectroscopy, thermogravimetric analysis, and X-ray powder diffraction. The microspheres were further heat sintered into three-dimensional porous scaffolds and characterized for their degradation and release of byproducts. The in vitro cytocompatibility of the matrices and their ability to support osteogenic differentiation was confirmed using human adipose-derived stem cells. Lastly, an in vivo study was performed in a mouse critical-sized calvarial defect model to evaluate the capacity of these matrices in supporting vascularized bone regeneration. Results demonstrated that the presence of CaO2 increased cellularization and biological activity throughout the matrices. There was greater migration of host cells to the interior of the matrices and greater survival and persistence of donor cells after 8 weeks, which in synergy with the composite matrices led to enhanced vascularized bone regeneration.

Clinical Studies in Hematologic Microtransplantation.

The anti-tumor effects of allogeneic hematopoietic stem cell transplantation depend upon engraftment of donor cells followed by a graft-versus-tumor (GVT) effect. However, pre-clinical and clinical studies have established that under certain circumstances, anti-tumor responses can occur despite the absence of high levels of durable donor cell engraftment. Tumor response with little or no donor engraftment has been termed "microtransplantation." It has been hard to define conditions leading to tumor responses without donor cell persistence in humans because the degree of engraftment depends very heavily upon many patient-specific factors, including immune status and degree of prior therapy. Likewise, it is unknown to what degree donor chimerism in the blood or tissue is required for an anti-tumor effect under conditions of microtransplantation. In this review, we summarize some key studies supporting the concept of microtransplantation and emphasize the importance of recent large studies of microtransplantation in patients with acute myelogenous leukemia (AML). These AML studies provide the first evidence of the efficacy of microtransplantation as a therapeutic strategy and lay the foundation for additional pre-clinical studies and clinical trials that will refine the understanding of the mechanisms involved and guide its further development as a treatment modality.

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction

Although c-kit(pos) cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose-effect relationship is unknown. Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×10(6), 0.75×10(6), 1.5×10(6), 3.0×10(6), and 6.0×10(6) cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×10(6)) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×10(6) significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×10(6) CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2'-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. Surprisingly, in this rat model of acute myocardial infarction, the dose-response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×10(6) is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

Cellular Immunotherapy: Using Alloreactivity to Induce Anti-Leukemic Responses without Prolonged Persistence of Donor Cells

A goal of cancer immunologists is to harness cellular immune responses to achieve anti-cancer responses. One of the strongest activating stimuli for the immune system is the encounter with cells expressing allogeneic HLA molecules. While alloreactive responses can negatively impact of the outcome of hematopoietic stem cell transplant because of graft-versus-host disease (GVHD), these same responses can have anti-leukemic effects. Donor lymphocyte infusions have been used in an attempt to harness alloreactive responses to achieve anti-leukemic responses. Because this protocol is usually carried out in the absence of recipient anti-donor responses, this protocol often induces GVHD as well as anti-leukemic responses. A recent study indicated the infusion of large number of haploidentical donor cells (1–2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies (100 cGy total body irradiation) resulted in 14 (7 major) responses/26 patients. A rapidly developing cytokine storm was observed, while no persisting donor cells could be detected at two weeks after infusion eliminating the possibility of GVHD. Characterization of the effector mechanisms responsible for the anti-leukemic responses in this protocol, should guide new approaches for achieving enhanced anti-leukemic responses using this protocol.

Ankle Bipolar Fresh Osteochondral Allograft Survivorship and Integration

Fresh osteochondral allografts represent a treatment option for early ankle posttraumatic arthritis. Transplanted cartilage survivorship, integration, and colonization by recipient cells have not been fully investigated. The aim of this study was to evaluate the ability of recipient cells to colonize the allograft cartilage and to assess allograft cell phenotype. Seventeen ankle allograft samples were studied. Retrieved allograft cartilage DNA from fifteen cases was compared with recipient and donor constitutional DNA by genotyping. In addition, gene expression was evaluated on six allograft cartilage samples by means of real-time reverse transcription-polymerase chain reaction. Histology and immunohistochemistry were performed to support molecular observations. Of fifteen genotyped allografts, ten completely matched to the host, three matched to the donor, and two showed a mixed profile. Gene expression analysis showed that grafted cartilage expressed cartilage-specific markers. The rare persistence of donor cells and the prevailing presence of host DNA in retrieved ankle allografts suggest the ingrowth of recipient cells into the allograft cartilage, presumably migrating from the subchondral bone, in accordance with morphological findings. The expression of chondrogenic markers in some of the samples argues for the acquisition of a chondrocyte-like phenotype by these cells. To our knowledge, this is the first report describing the colonization of ankle allograft cartilage by host cells showing the acquisition of a chondrocyte-like phenotype.

Use of alloreactivity to generate in vivo anti-leukemic responses (46.48)

Abstract A previous clinical trial demonstrated that infusion of haploidentical G-CSF mobilized WBC (1 - 2 x 10^8 cells/kg) into patients with refractory hematological malignancies (100 cGy irradiation) resulted in a high frequency of anti-leukemic responses including some complete responses despite a lack of detectable donor cell persistence beyond two weeks. Beyond a cytokine storm, no effector mechanisms were defined. It is possible that alloreactive responses were either capable of directly attacking the leukemic cells or reactivating the patient’s anti-leukemic effector cells that were already present via cross-reactivity. PBMNC were obtained from the blood of newly diagnosed leukemic patients, and separated into CD3- and CD3+ populations. The patient’s CD3+ cells were used as responder cells in a mixed lymphocyte culture and stimulated with mitomycin C treated patient’s CD3- cells or PBMNC from allogeneic normal donors chosen at random. On day 7, the effector cells were collected and tested for their ability to lyse 51Cr labeled patient CD3- cells. The results showed that the patient’s CD3+ cells stimulated with allogeneic PBMNC were able to generate anti-leukemic responses in a stimulator dependent fashion. Thus, this assay may be useful for predicting which donor would be best for patients receiving cellular immunotherapy and may also be useful for the defining the anti-leukemic effector cells that are present with this form of cellular immunotherapy.

506 HYMOVIS™, A HEXADECYLAMIDE HYALURONAN DERIVATIVE (HYADD®4-G), INHIBITS GENE EXPRESSION CHANGES INDUCED BY INTERLEUKIN-lp IN CHONDROCYTES AND SYNOVIAL FIBROBLASTS DERIVED FROM OSTEOARTHRHIS PATIENTS

data on mRNA expression showed that allograft cartilage tissues express cartilage specific markers. Conclusions: The colonization of human viable knee and ankle allografts by recipient cells was investigated by genetic typing and mRNA expression. There is an evidence of persistence of donor cells particularly in knee allografts. This event is rare in ankle allografts, where the prevailing presence of host DNA suggests the ingrowth of recipient cells into the allograft, presumably migrating from the subchondral bone, in accordance with histological findings. The observed synthesis of cartilage specific RNAs in some of the analysed samples argues for the acquisition of a chondrocyte-like phenotype by some of these cells.

Allogeneic natural killer cells for refractory lymphoma

We reported that IL-2 activated autologous NK cells can induce, but not maintain durable remissions in lymphoma patients. We hypothesized that allogeneic NK cells may overcome class I MHC-mediated inhibition of NK cell killing. In a pilot study, we evaluated infusion of haploidentical donor NK cells for antitumor efficacy. Six patients with advanced B cell non-Hodgkin lymphoma (NHL) received rituximab, cyclophosphamide, and fludarabine as immunosupression to permit homeostatic NK cell expansion, followed by CD3-depleted NK cell-enriched cell products followed by subcutaneous IL-2 administration (10 x 10(6) units every other day x 6 doses). At 2 months, four patients showed an objective clinical response. We observed early donor cell persistence in two patients (blood and in tumor-bearing node), but this was not detectable beyond 7 days. All patients demonstrated substantial increases in host-regulatory T cells (Treg) after NK cell and IL-2 therapy (180 +/- 80 cells/microl vs. baseline: 58 +/- 24 cells/microl, p = 0.04) which may have limited donor cell expansion in vivo. These findings suggest safety and feasibility of allogeneic NK cell therapy in patients with lymphoma; however host Treg and inadequate immunodepletion may contribute to a hostile milieu for NK cell survival and expansion. Cell therapy trials should incorporate novel strategies to limit Treg expansion.

In vivo multimodal imaging of stem cell transplantation in a rodent model of Parkinson's disease

Stem cell therapy in the nervous system aims to replace the lost neurons and provide functional recovery. However, it is imperative that we understand the in vivo behaviour of these cells post-implantation. We report visualisation of iron oxide labelled bone marrow-derived stem cells (BMSCs) implanted into the striatum of hemi-parkinsonian rats by magnetic resonance imaging (MRI). Functional efficacy of the donor cells was monitored in vivo using the positron emission tomography (PET) radioligand [11C]raclopride. The cells were visible for 28 days by in vivo MRI. BMSCs provided functional recovery demonstrated by a decreased binding of [11C]raclopride. Although, histology confirmed the persistence of donor cells, no tyrosine hydroxylase positive cells were present. This suggests that BMSCs may have a limited paracrine effect and influence functional recovery. We demonstrate, using multimodal imaging, that we can not only track BMSCs but also establish their effects in a pre-clinical model of Parkinson's disease.

Comment on ‘Tolerance Versus Immunosuppression: A Perspective’

Comment on ‘Tolerance Versus Immunosuppression: A Perspective’

Pre-Transplant Infusion of Donor CD4+ CD25+ T Cells Suppresses Host Anti-Donor MiHA-Specific CD8 T Cells and Facilitates Stable Mixed Chimerism Following MHC-Matched Allogeneic Marrow Transplant.

Surmounting the barrier mediated by host T cells against successful engraftment of MHC-matched allogeneic marrow is a crucial objective of hematopoietic stem cell transplant (HCT). We proposed that the ability of unmanipulated donor CD4+ CD25+ regulatory T cells (Tregs) to suppress activation of host effector CD8+ T cells would enhance donor HC engraftment between strains disparate for multiple minor HA (MiHA), and promote stable chimerism. C57BL/6 (H-2b, Ly9.1−) mice conditioned (5.5 Gy TBI) 24 hrs earlier, were transplanted with Tregs and TCD-BM from 129P3/J (H-2, Ly9.1+) mice. By four weeks post-HCT, the mean frequency of circulating donor-derived (total Ly9.1+) cells was 15.7 ± SE 5.9% in recipients of 4 × 106 TCD-BM + Tregs (4.5 × 105) compared to the absence of donor chimerism in recipients of allogeneic HCT only (0.3 ± 0.09%) - a finding also evident from the analysis of circulating donor B220+ cells (8.7 ± 3.0 vs. 0.2 ± 0.06%). Based on these findings, we hypothesized that pre-HCT infusion of Tregs into a lymphopenic, allogeneic host might enhance their facilitating function. Accordingly, Tregs (4 or 1.5 × 105) were transplanted 24hr. post-conditioning either 3 days prior to HCT or co-transplanted with (day 0) donor TCD-BM. The transplant was rejected in recipients not administered donor Tregs. The higher dose of Tregs administered pre-transplant or co-transplanted facilitated equivalent donor cell chimerism. In contrast, administration of 1.5 × 105 Tregs resulted in chimerism only when this donor population was infused 3 days prior to HCT (1.6±0.9%). To test the hypothesis that donor Tregs suppressed host anti-donor specific CD8 T cells, we monitored host CD8+ T cell responses to the immunodominant donor epitope H60 by tetramer staining (LTFNYRNL/Kb). One month post-HCT of TCD-BM alone, the frequency of circulating tetramer+ CD8+ cells in these recipients rejecting the marrow graft was clearly greater compared to the frequency in recipients of TCD-BM + Tregs which engrafted (p<0.0008). In contrast, the host anti-donor CD8 levels were not different between recipients of TCD-BM alone and TCD-BM + D.0 'low dose' Treg administration which failed to engraft (p>0.05). Notably, transplants using unmanipulated host Tregs failed to support engraftment. These findings illustrate that donor Tregs support chimerism via suppression of host anti-donor antigen-specific T cells. Finally, the persistence of donor cells in circulation as well as in lymphoid tissues 3 months post-HCT indicated that long-term, stable chimerism was established by this Treg administration regimen. In total, our results demonstrate that donor Tregs promote MHC-matched allogeneic marrow engraftment under conditions of reduced intensity conditioning and that the strategy of pre-HCT Treg infusion supports the notion that antigen driven expansion of donor anti-host reactive Treg cells prior to stem cell transplants can increase their efficacy to facilitate hematopoietic engraftment. Experiments to study transient vs. long-term engraftment and expansion of donor Treg cells are being examined.

Immune modulation and microchimerism after unmodified versus leukoreduced allogeneic red blood cell transfusion in cancer patients: results of a randomized study

Transfusion of red blood cells (RBCs) has been associated with immunomodulatory effects. Persistence of donor cells in the recipient may be contributive. A randomized single-center trial was conducted to compare microchimerism and immune responses in 35 patients undergoing cancer surgery and transfused perioperatively with either unmodified RBCs (UN-RBCs, n = 18) or leukoreduced RBCs (LR-RBCs, n = 17). Biologic parameters included microchimerism assessment peripheral blood mononuclear cell (PBMNC) phenotyping, cytokine production by stimulated PBMNCs, FoxP3 gene expression, and T-cell repertoire (TCR) analysis. Microchimerism was documented in 8 of 18 patients after UN-RBC transfusion while absent after LR-RBC transfusion (0/17; p = 0.001). After UN-RBC transfusion, microchimerism was associated with increased interleukin (IL)-10 production (p = 0.02), reduced TCR alteration (p = 0.04), and reduced CD56+ cell counts (p = 0.02) when compared to recipients without evidence for microchimerism. FoxP3 gene expression did not differ significantly between both treatment groups nor with the presence or absence of microchimerism in the UN-RBC group. Finally, after an initial early decrease after surgery and transfusion, IL-12 production increased and more significantly so after UN-RBC transfusion versus LR-RBC transfusion (p = 0.05). UN-RBC-induced microchimerism is associated with specific immunomodulatory effects in cancer patients who received transfusions during surgery.

Microchimerism maintains deletion of the donor cell-specific CD8+ T cell repertoire.

Rare cases of stable allograft acceptance after discontinuation of immunosuppression are often accompanied by macrochimerism (> 1% donor cells in blood) or microchimerism (< 1% donor cells in blood). Here, we have investigated whether persistence of donor cells is the cause or the consequence of long-lasting CTL unresponsiveness. We found that engraftment of splenocytes bearing a single foreign MHC class I-restricted epitope resulted in lifelong donor cell microchimerism and specific CTL unresponsiveness. This status was reversed in a strictly time- and thymus-dependent fashion when the engrafted cells were experimentally removed. The results presented herein show that microchimerism actively maintains CTL unresponsiveness toward a minor histocompatibility antigen by deleting the specific repertoire and thus excluding dominant, T cell extrinsic mechanisms of CTL unresponsiveness independent of systemically persisting donor cell antigen.

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo

Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.

Successful Remission of Poor Prognosis AML after Adoptive Transfer and In Vivo Expansion of Human Haploidentical NK Cells.

We previously demonstrated that although autologous NK cell therapy after hematopoietic cell transplantation (HCT) is safe, it does not provide an anti-tumor effect. We hypothesize that the lack of NK cell inhibitory receptor (KIR) mismatching with autologous tumor cells limits anti-tumor efficacy. Allogeneic NK cell infusions may overcome this restriction. Here we tested haploidentical, related donor NK cell infusions in a non-transplant setting to establish safety with a goal of in vivo NK cell expansion. A low intensity outpatient immune suppressive regimen of fludarabine (Flu) alone was tested in 8 renal cell carcinoma patients. A higher intensity inpatient regimen of high-dose cyclophosphamide and Flu (Hi-Cy/Flu) was tested in 12 patients with poor prognosis AML, all with active leukemia and most who failed 1 or more standard theries. NK cells were enriched from donor apheresis products using CD3 depletion by the CliniMacs system. All patients received subcutaneous IL-2 after infusions. Patients who received the lower intensity Flu regimen showed transient persistence of donor cells, but no in vivo expansion (<0.1%) using a PCR based chimerism assay of unique MHC class I alleles in the donor but not present in the recipient. There were no objective responses in renal cell carcinoma patients. In marked contrast, 6 of 7 AML patients who were evaluable for engraftment receiving Hi-Cy/Flu showed engraftment of donor cells, some for > 28 days. Three of 6 patients had peak engraftment levels of 10%, 100% and 100% of PBMC in the first 2 weeks after the haploidentical infusion. The remaining three patients had lower levels of approximately 1%. Five of 12 patients have achieved a remission with this therapy. Compared to patients who failed to clear their leukemia, the remission patients had significantly higher proportions of circulating NK cells (35±8% vs. 1.5±0.4%, p=.001) and cytotoxicity against K562 targets (50±10% vs. 10±5% at E:T 20:1, p=0.01) suggesting that in vivo expansion was required to achieve efficacy. We next tested whether endogenous cytokines play a role in the in vivo expansion. Based on its established role in NK cell differentiation and on recent mouse studies, IL-15 was a likely candidate to drive homeostatic NK cell expansion. Flu patients showed only a slight increase in IL-15 after several days compared to pre-treatment levels. In marked contrast, AML patients had significant increases in plasma IL-15 after Hi-Cy/Flu therapy, which appeared before haploidentical cell infusions and was sustained for several weeks. There was an inverse correlation between the absolute lymphocyte count and the IL-15 concentration (r = −.62, p-value < .0001). We conclude that Hi-Cy/Flu therapy allows for expansion of allogeneic NK cells in vivo in part through increased endogenous IL-15 concentrations, which can induce remissions in poor prognosis AML patients. For the first time, these findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of AML used alone or as an adjunct to HCT. Future studies will address NK cell donor-recipient mismatch at KIR ligands, patient selection and other variables that impact on response.

Depletion of donor-reactive cells as a new concept for improvement of mismatched bone marrow engraftment using reduced-intensity conditioning

New nonmyeloablative strategies to improve acceptance of mismatched bone marrow (BM) may compensate for the inadequate supply of compatible grafts. Recently we proposed to facilitate engraftment of mismatched BM by selective depletion of activated donor-reactive host cells with cyclophosphamide (CY). Here we have compared engraftment of allogeneic BM after depletion of antigen-activated host lymphocytes by CY, with BM engraftment following general immunosuppression by the same CY dose. Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY in order to deplete activated T cells (day 1), and transplanted with a second C57BL/6 BM inoculum (day 2) in order to achieve BM engraftment. Alternatively, mice received an equal dose of donor BM cells in a single injection one day after the same CY dose. Treated animals were repeatedly tested for persistence of donor cells in the blood. Depletion of alloantigen-primed lymphocytes by 200 mg/kg CY provided stable GVHD-free engraftment of allogeneic BM in nonirradiated mice, while immunosuppressive treatment with the same CY dose alone resulted in BM rejection. Low-dose irradiation before priming with donor BM allowed the tolerance-inducing CY dose to be reduced to 100 mg/kg. Alloantigen-primed lymphocyte depletion (APLD) by a reduced CY dose resulted in engraftment of donor BM after a significantly lower irradiation dose than treatment with irradiation and CY alone. Our results demonstrate that conditioning that focuses on APLD has a definite advantage over general immunosuppression with CY and radiation therapy.

Quantification of donor microchimerism in sex-mismatched porcine allotransplantation by competitive PCR

The persistence of donor cells in recipient circulation and peripheral tissues post-transplantation has been demonstrated in solid organ allotransplantation and xenotransplantation models. Although this state of microchimerism has been postulated as the basis for graft acceptance, chimerism has not been directly linked to the maintenance of peripheral tolerance or prevention of rejection. Studies have demonstrated that the qualitative presence or absence of donor microchimerism bears no association with graft acceptance. Our preliminary work suggests that there is a threshold chimerism necessary for the induction of donor-specific hyporesponsiveness. Because the kinetics of donor cell accumulation and distribution in allograft recipients are largely unknown, quantitative analyses are needed to evaluate chimerism's significance to donor-specific tolerance. We developed a quantitative, competitive PCR assay to precisely measure the amount of chimerism in male to female transplant pairs by targeting the sex-determining region of the Y chromosome (SRY gene). Traditionally, this technique requires that serial known amounts of an SRY-specific competitive template (CT) be coamplified with a constant amount of sample DNA to determine the equivalence point of the relative band intensities of the PCR products. However running a panel of PCRs with CT amounts above and below the equivalence point to generate a standard curve for ever' sample is laborious. Here we describe the generation of a single standard curve that permits the rapid and reliable quantification of microchimerism after coamplification of sample DNA with a single amount of CT.

Effect of KRN7000 on induced graft-vs-host disease

Objective Graft-vs-host disease (GVHD) is a serious complication of allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), for which no effective therapy exists. In our study, KRN7000, a synthetic analog of α-galactosylceramide, known for its ability to activate natural killer T cells, was tested for its ability to prevent onset of GVHD in a murine model of haploidentical major histocompatible (MHC) mismatched hematopoietic cells. Methods Irradiated (BALB/cXC57BL/6)F 1 mice were inoculated with parental C57BL/6 splenocytes with or without SCT. KRN7000 was given intraperitoneally as single or multiple doses at 100 μg/kg/dose and mice were followed up for GVHD clinical symptoms and for survival. The effect of KRN7000 treatment was also tested in vitro for the induction of suppression of alloreactivity in mixed lymphocyte reaction (MLR). Results KRN7000 prevented development of GVHD symptoms in almost all mice and 52/53 mice maintained a healthy profile for more than 235 days. Most vehicle-treated mice or untreated controls died of GVHD within a median of 3 weeks. KRN7000 treatment did not prevent engraftment of donor cells following sublethal total-body irradiation (TBI) and allowed durable persistence of donor cells following lethal TBI and SCT. Splenocytes derived from KRN7000-treated mice suppressed efficiently mixed lymphocyte reaction (MLR) in vitro. Conclusion GVHD induced by alloreactive lymphocytes can be prevented by KRN7000. GVHD prevention may be accomplished by regulation of T cell function and might thus provide a new modality for safer SCT and DLI.