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Alloreactive T-effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Ex vivo nonspecific depletion of T cells from the donor graft impedes stem cell engraftment and posttransplant immune reconstitution. Teffs upregulate Fas after activation and undergo Fas ligand (FasL)–mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs in vivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes, taking advantage of the high-affinity interaction between biotin and streptavidin. SA-FasL–engineered mouse and human T cells underwent apoptosis after activation in response to alloantigens in vitro and in vivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients after cotransplantation with bone marrow cells, whereas all controls that underwent transplantation with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated the protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL–engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represents a practical approach with significant translation potential for the prevention of aGVHD.

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA-FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long-term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA-FasL-engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor-matched, but not third party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft-draining lymph nodes, and required phagocytes and TGF-β. At the maintenance phase, immune protection evolved into graft site-restricted immune privilege as the destruction of long-surviving SA-FasL-islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4(+)CD25(+)Foxp3(+) Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA-FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.

Immune imbalance in autoimmune disorders such as type 1 diabetes may originate from aberrant activities of effector cells or dysfunction of suppressor cells. All possible defective mechanisms have been proposed for diabetes-prone species: (i) quantitative dominance of diabetogenic cells and decreased numbers of regulatory T cells, (ii) excessive aggression of effectors and defective function of suppressors, (iii) perturbed interaction between effector and suppressor cells, and (iv) variations in sensitivity to negative regulation. The experimental evidence available to date presents conflicting information on these mechanisms, with identification of perturbed equilibrium on the one hand and negation of critical role of each mechanism in propagation of diabetic autoimmunity on the other hand. In our analysis, there is no evidence that inherent abnormalities in numbers and function of effector and suppressor T cells are responsible for the immune imbalance responsible for propagation of type 1 diabetes as a chronic inflammatory process. Possibly, the experimental tools for investigation of these features of immune activity are still underdeveloped and lack sufficient resolution, in the presence of the extensive biological viability and functional versatility of effector and suppressor elements.

Adopting prior models of sleep-wake transitions, a flip-flop switch in synchronized neurotransmitter activity is proposed to underlie restless leg syndrome onset. In this model, leg quiescence homeostasis sustained through concerted activities of several neurotransmitters in basal ganglia is perturbed and produces striatal motor activity along sensory activity associated with thalamocortical circuits (conscious urge and discomfort). This model explains the association of restless leg syndrome with a wide variety of associated pathologies emphasizing that perturbed function and imbalance may occur under different steady states of neurotransmitter levels. Likewise, this concept links various central and peripheral etiologies and integrates the augmenting and transient effects of therapeutic neuromodulators.

The prevalent current approach to type 1 diabetes (T1D) is the abrogation of pathogenic potential by immunosuppressive therapy, an intuitive approach aiming to slow down disease progression by the reduction of pathogenic burden. In spite of promising initial results in rodent models, there has been little efficacy of most lymphoreductive strategies in human subjects. Our analysis suggests that lymphopenia is the common denominator of ineffective immunosuppressive therapies: Immune rebound from lymphopenia is associated per se with increased susceptibility to immune reactivity, including relapse of autoimmunity. In addition, immune homeostasis and self-tolerance are not restored. These considerations raise the following question: What is the allowed degree of immunosuppressive therapy that does not elicit recurrent autoimmunity. More effective therapeutic strategies include targeted deletion of pathogenic cells, preferably in the pancreatic islets and regional lymphatics using selective T cell activation markers, re-education and remodeling of effector responses.

Receptor/ligand interactions of the tumor necrosis factor (TNF) superfamily are associated with versatile apoptotic and regulatory signaling pathways in parenchymal tissues and the immunohematopoietic system. Apoptotic signaling mediated by Fas and TNF receptor-1 (TNF-R1) is one of the major cytotoxic mechanisms used by immune cells to kill endogenous cells and exogenous pathogens. In this capacity, these ubiquitous effector mechanisms of cell death, along with perforin/granzyme, are direct mediators of β-cell injury that is inflicted in autoimmune insulitis in type 1 diabetes (T1D). At the same time, the TNF family receptor/ligand interactions are prime constituents of immune homeostasis that enforce negative regulation of sensitized immunocytes (1–3). All immune cells upregulate TNF family receptors upon activation and are therefore submitted to negative regulation by apoptosis within the process of activation-induced cell death (AICD), which is also essential to termination of inflammation (4). Tumor necrosis factor family receptor/ligand interactions participate as universal cytotoxic mechanisms in physiological turnover of cells, removal of dysfunctional cellular elements, and clearance of debris following injury of parenchymal tissues. As ubiquitous injury factors, the TNF family ligands concomitantly activate precursors and synergize with other growth factors to induce generation of renewed functional parenchymal cells to replace the injured tissue. For example, the Fas receptor and the cognate Fas-ligand (FasL) are constitutively expressed and transduce trophic regulatory signals in tissues such as glia, neurons, hepatocytes, and endothelium (5–7). Likewise, trophic signals are transduced by TNF superfamily receptors in hematopoietic stem and progenitor cells, which synergize with other growth factors to foster hematopoiesis under stress conditions (8). Therefore, common injury factors such as TNFα and interferons (IFN) couple hematopoietic activity to inflammation and injury in order to fuel immune reactions and support initial resolution of injury (9). The Fas/FasL interaction is a common effector mechanism of β-cell apoptosis and islet injury under inflammatory conditions, and physiological modulation of immune homeostasis, including control of aberrant autoimmune reactions. In the absence of specific characteristics of the toxic and anti-inflammatory pathways, it is questioned whether neutralization or reinforcement of the Fas/FasL interaction is of therapeutic value in autoimmune disorders. We examine a wide array of evidence arguing in favor of and against therapeutic neutralization of Fas and/or FasL and conversely, consider the feasibility of implementation of this molecular interaction as approaches to abrogate autoimmune diabetes. Despite focus on a particular signaling pathway, the Fas/FasL interaction, in a particular autoimmune disorder, T1D, the debate is rather relevant to multiple receptor/ligand interactions of the TNF superfamily and to the entire range of inflammatory and autoimmune disorders.

Secretion of ligands of the tumor necrosis factor (TNF) superfamily is a conserved response of parenchymal tissues to injury and inflammation that commonly perpetuates elimination of dysfunctional cellular components by apoptosis. The same signals of tissue injury that induce apoptosis in somatic cells activate stem cells and initiate the process of tissue regeneration as a coupling mechanism of injury and recovery. Hematopoietic stem and progenitor cells upregulate the TNF family receptors under stress conditions and are transduced with trophic signals. The progeny gradually acquires sensitivity to receptor-mediated apoptosis along the differentiation process, which becomes the major mechanism of negative regulation of mature proliferating hematopoietic lineages and immune homeostasis. Receptor/ligand interactions of the TNF family are physiological mechanisms transducing the need for repair, which may be harnessed in pathological conditions and transplantation. Because these interactions are physiological mechanisms of injury, neutralization of these pathways has to be carefully considered in disorders that do not involve intrinsic aberrations of excessive susceptibility to apoptosis.

The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34(+) progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34(+) progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

One of the therapeutic approaches to type 1 diabetes (T1D) focuses on enhancement of regulatory T cell (Treg) activity, either by adoptive transfer or supplementation of supporting cytokines such as interleukin-2 (IL-2). In principle, this therapeutic design would greatly benefit of concomitant reduction in pathogenic cell burden. Experimental evidence indicates that physiological recovery from lymphopenia is dominated by evolution of effector and cytotoxic cells, which abolishes the therapeutic efficacy of Treg cells. Targeted and selective depletion of effector T cells has been achieved with killer Treg using Fas ligand protein and a fusion protein composed of IL-2 and caspase-3, which showed remarkable efficacy in modulating the course of inflammatory insulitis in NOD mice. We emphasize a critical consideration in design of therapeutic approaches to T1D, immunomodulation without lymphoreduction to avoid the detrimental consequences of rebound recovery from lymphopenia.

Immune profiling of non-obese diabetic (NOD) is a widely employed tool to assess the mechanisms of inflammatory insulitis. Our analysis of the female NOD colony revealed similar distribution of lymphoid lineages to wild type mice, and at various ages of prediabetic and diabetic mice. The profiles of mesenteric and pancreatic lymph nodes differ and often change reciprocally due to directed migration of T cells towards the site of inflammation. Significant events in our colony include early decline in CD4(+)CD25(+)CD62L(+) Treg, accompanied by gradual increase in CD4(+)CD25(+)FoxP3(+) Treg in peripheral lymphoid organs and pancreatic infiltrates. Impressively, aged euglycemic mice display significant transient rise in CD4(+)CD25(-)FoxP3(+) Treg in the thymus, pancreas and draining lymph nodes. A significant difference was superior viability of effector and suppressor cells from new onset diabetics in the presence of high interleukin-2 (IL-2) concentrations in vitro as compared to cells of prediabetic mice. Overall, we found no correlation between FoxP3(+) Treg in the pancreatic lymph nodes and the inflammatory scores of individual NOD mice. CD25(-)FoxP3(+) Treg are markedly increased in the pancreatic infiltrates in late stages of inflammation, possibly an effort to counteract destructive insulitis. Considering extensive evidence that Treg in aged NOD mice are functionally sufficient, quantitative profiling evolves as an unreliable tool to assess mechanism and causes of inflammation under baseline conditions. Immune profiles are modulated by thymic output, cell migration, shedding of markers, proliferation, survival and in-situ evolution of regulatory cells.

Treg cells endowed with enhanced killing activity through decoration with Fas-ligand (FasL) protein (killer Treg) have been effective in delay of hyperglycemia in prediabetic non-obese diabetic (NOD) mice. In this study, we assessed the therapeutic efficacy of these cells, harvested from age-matched euglycemic NOD donors, on the course of disease in new-onset diabetics. One dose of 4 × 106 killer Treg cells stabilized blood glucose associated with increased insulin levels in 5 of 9 mice and partially reversed the severity of islet inflammation, whereas naive Treg cells did not modulate the course of disease significantly. Killer Treg cells were shown to operate through induction of cell apoptosis within the pancreatic lymph nodes, resulting in reduced efficiency of adoptive disease transfer to NOD/SCID recipients. A second mechanism of action consisted of increased fractions of CD4+CD25−FoxP3+ T cells in the pancreas and all lymphoid organs. Immunomodulation with FasL rather than Treg cells enhanced the expression of CD25 and FoxP3 in the thymus, suggesting a possible contribution of thymic output to prolonged stabilization of the glucose levels. Autologous Treg cells evolve as excellent vehicles for targeted delivery of FasL as an immunomodulatory protein, which delete pathogenic cells at the site of inflammation and induce systemic dominance of suppressor subsets.

Background: Perspectives of immunotherapy to cancer mediated by bone marrow transplantation (BMT) in conjunction with dendritic cell (DC)-mediated immune sensitisation have yielded modest success so far. In this study, we assessed the impact of DC on graft vs tumour (GvT) reactions triggered by allogeneic BMT. Methods: H2Ka mice implanted with congenic subcutaneous Neuro-2a neuroblastoma (NB, H2Ka) tumours were irradiated and grafted with allogeneic H2Kb bone marrow cells (BMC) followed by immunisation with tumour-inexperienced or tumour-pulsed DC. Results: Immunisation with tumour-pulsed donor DC after allogeneic BMT suppressed tumour growth through induction of T cell-mediated NB cell lysis. Early post-transplant administration of DC was more effective than delayed immunisation, with similar efficacy of DC inoculated into the tumour and intravenously. In addition, tumour inexperienced DC were equally effective as tumour-pulsed DC in suppression of tumour growth. Immunisation of DC did not impact quantitative immune reconstitution, however, it enhanced T-cell maturation as evident from interferon-γ (IFN-γ) secretion, proliferation in response to mitogenic stimulation and tumour cell lysis in vitro. Dendritic cells potentiate GvT reactivity both through activation of T cells and specific sensitisation against tumour antigens. We found that during pulsing with tumour lysate DC also elaborate a factor that selectively inhibits lymphocyte proliferation, which is however abolished by humoral and DC-mediated lymphocyte activation. Conclusion: These data reveal complex involvement of antigen-presenting cells in GvT reactions, suggesting that the limited success in clinical application is not a result of limited efficacy but suboptimal implementation. Although DC can amplify soluble signals from NB lysates that inhibit lymphocyte proliferation, early administration of DC is a dominant factor in suppression of tumour growth.

Graft-versus-host disease (GVHD) can be prevented by Fas-mediated selective depletion of host-sensitized donor lymphocytes ex vivo. We tested the hypothesis that Fas-mediated depletion of lymphocytes in the absence of host-specific antigenic stimulation can alleviate GVHD. Brief exposure (24 hours) of unstimulated donor lymphocytes to Fas ligand (FasL) ex vivo results in balanced apoptosis of CD8+ and CD4+ subsets with preferential depletion of CD62L and CD69, increased T regulatory fractions, and sustained responses to stimulation. This procedure ameliorates weight loss and improves the clinical and histologic score of skin and gastrointestinal GVHD with and without concurrent transplantation of hematopoietic progenitors and irrespective of conditioning-induced tissue injury. Although FasL-resistant donor T cells are less potent effectors of GVHD, they facilitate hematopoietic progenitor engraftment when infused with or after the graft and retain the potential to elaborate graft-versus-tumor reactions. These findings in a preclinical model together with the known trophic effects of FasL on primitive hematopoietic progenitors suggest that brief ex vivo incubation of hematopoietic grafts with FasL may improve the outcome and safety of clinical T cell–replete allogeneic and haploidentical transplants.

Tumor necrosis factor-α (TNF-α) has been suggested to exert detrimental effects on hematopoietic progenitor function that might limit the success of transplants. In this study, we assessed the influences of TNF-α and its two cognate receptors on the function of fresh umbilical cord blood (UCB) and cryopreserved mobilized peripheral blood (mPB). CD34+ progenitors from both sources are less susceptible to spontaneous apoptosis than lineage-committed cells and are not induced into apoptosis by TNF-α. Consequently, the activity of UCB-derived severe combined immune deficiency (SCID) reconstituting cells and long-term culture-initiating cells is unaffected by this cytokine. On the contrary, transient exposure of cells from both sources to TNF-α stimulates the activity of myeloid progenitors, which persists in vivo in UCB cell transplants. Progenitor stimulation is selectively mediated by TNF-R1 and involves activation of caspase-8, without redundant activity of TNF-R2. Despite significant differences between fresh UCB cells and cryopreserved mPB cells in susceptibility to apoptosis and time to activation, TNF-α is primarily involved in tropic signaling in hematopoietic progenitors from both sources. Cytokine-mediated tropism cautions against TNF-α neutralization under conditions of stress hematopoiesis and may be particularly beneficial in overcoming the limitations of UCB cell transplants.

As physiological pathways of intercellular communication produced by all cells, cytokines are involved in the pathogenesis of inflammatory insulitis as well as pivotal mediators of immune homeostasis. Proinflammatory cytokines including interleukins, interferons, transforming growth factor-β, tumor necrosis factor-α, and nitric oxide promote destructive insulitis in type 1 diabetes through amplification of the autoimmune reaction, direct toxicity to β-cells, and sensitization of islets to apoptosis. The concept that neutralization of cytokines may be of therapeutic benefit has been tested in few clinical studies, which fell short of inducing sustained remission or achieving disease arrest. Therapeutic failure is explained by the redundant activities of individual cytokines and their combinations, which are rather dispensable in the process of destructive insulitis because other cytolytic pathways efficiently compensate their deficiency. Proinflammatory cytokines are less redundant in regulation of the inflammatory reaction, displaying protective effects through restriction of effector cell activity, reinforcement of suppressor cell function, and participation in islet recovery from injury. Our analysis suggests that the role of cytokines in immune homeostasis overrides their contribution to β-cell death and may be used as potent immunomodulatory agents for therapeutic purposes rather than neutralized.

Summary Hematopoietic chimerism is one of the effective approaches to induce tolerance to donor-derived tissue and organ grafts without administration of life-long immunosuppressive therapy. Although experimental efforts to develop such regimens have been ongoing for decades, substantial cumulative toxicity of combined hematopoietic and tissue transplants precludes wide clinical implementation. Tolerance is an active immunological process that includes both peripheral and central mechanisms of mutual education of coresident donor and host immune systems. The major stages include sequential suppression of early alloreactivity, establishment of hematopoietic chimerism and suppressor cells that sustain the state of tolerance, with significant mechanistic and temporal overlap along the tolerization process. Efforts to devise less toxic transplant strategies by reduction of preparatory conditioning focus on modulation rather than deletion of residual host immunity and early reinstitution of regulatory subsets at the central and peripheral levels.

Ischemic or mechanical injury to the optic nerve is an irreversible cause of vision loss, associated with limited regeneration and poor response to neuroprotective agents. The aim of this study was to assess the capacity of adult bone marrow cells to participate in retinal regeneration following the induction of anterior ischemic optic neuropathy (AION) and optic nerve crush (ONC) in a rodent model. The small-sized subset of cells isolated by elutriation and lineage depletion (Fr25lin(-)) was found to be negative for the neuroglial markers nestin and glial fibrillary acidic protein (GFAP). Syngeneic donor cells, identified by genomic marker in sex-mismatched transplants and green fluorescent protein, incorporated into the injured retina (AION and ONC) at a frequency of 0.35%-0.45% after intravenous infusion and 1.8%-2% after intravitreous implantation. Perivascular cells with astrocytic morphology expressing GFAP and vimentin were of the predominant lineage that engrafted after AION injury; 10%-18% of the donor cells incorporated in the retinal ganglion cell layer and expressed NeuN, Thy-1, neurofilament, and beta-tubulin III. The Fr25lin(-) cells displayed an excellent capacity to migrate to sites of tissue disruption and developed coordinated site-specific morphological and phenotypic neural and glial markers. In addition to cellular reconstitution of the injured retinal layers, these cells contributed to endothelial revascularization and apparently supported remodeling by secretion of insulin-like growth factor-1. These results suggest that elutriated autologous adult bone marrow-derived stem cells may serve as an accessible source for cellular reconstitution of the retina following injury.

T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.

The mechanisms of autoimmune reactivity onset in type 1 diabetes (T1D) remain elusive despite extensive experimentation and discussion. We reconsider several key aspects of the early stages of autoimmunity at four levels: islets, pancreatic lymph nodes, thymic function and peripheral immune homeostasis. Antigen presentation is the islets and has the capacity to provoke immune sensitization, either in the process of physiological neonatal β cell apoptosis or as a consequence of cytolytic activity of self-reactive thymocytes that escaped negative regulation. Diabetogenic effectors are efficiently expanded in both the islets and the lymph nodes under conditions of empty lymphoid niches during a period of time coinciding with a synchronized wave of β cell apoptosis surrounding weaning. A major drive of effector cell activation and expansion is inherent peripheral lymphopenia characteristic of neonates, though it remains unclear when is autoimmunity triggered in subjects displaying hyperglycemia in late adolescence. Our analysis suggests that T1D evolves through coordinated activity of multiple physiological mechanisms of stimulation within specific characteristics of the neonate immune system.

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL–engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL–engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+Foxp3+ regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

Background: Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT. Methods: Mice bearing congenic (H2Ka) Neuro-2a tumours were grafted with allogeneic (H2Kb) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DCNeuro2a) were inoculated (on day +7) in conjunction with donor (H2Kb) and haploidentical (H2Ka/b) lymphocytes. Results: Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DCNeuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2Ka/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT. Conclsions: The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours.

Camptothecins (CPTs) are topoisomerase I inhibitors chemotherapeutic agents used in combination chemotherapy. We showed previously that combination of moxifloxacin (MXF) and CPT induced inhibitory effects on topoisomerase I activity, on proliferation of HT-29 cells in vitro and enhanced apoptosis, compared to CPT alone. Analysis of secretion of the pro-angiogenic factors IL-8 and VEGF showed significant reduction by MXF. Using a murine model of human colon carcinoma xenograft, we compared the effects of MXF/CPT in vitro to MXF/irinotecan combination in vivo. We show that the MXF/CPT inhibitory effects observed in vitro are reflected in the inhibition of the progressive growth of HT-29 cells implanted in SCID mice. Using caliper measurements, Doppler ultrasonography, image analyses and immunohistochemistry of nuclear proteins (Ki-67) and vascular endothelial cells (CD-31) we show that addition of MXF (45mg/kg) to a relatively ineffective dose of irinotecan (20mg/kg), results in a 50% and 30% decrease, respectively, in tumor size and a decrease in Ki-67 staining. Power Doppler Ultrasound showed a significant, pronounced decrease in the number of blood vessels, as did CD-31 staining, indicating decreased blood flow in tumors in mice treated with MXF alone or MXF/irinotecan compared to irinotecan. These results suggest that the combination of MXF/irinotecan may result in enhanced anti-neoplastic/anti-angiogenic activity.

Interruption of IL-2 signalling is an attractive therapeutic target in autoimmune disorders. In this study we evaluated the effect of a fusion protein composed of IL-2 and caspase-3 (IL2-cas) on NOD mice, as compared with disease induction by cyclophosphamide.IL2-cas was assessed in NOD mice at various ages and in conjunction with cyclophosphamide administration. The effect of IL2-cas on diabetogenic cells was evaluated in adoptive transfer experiments and in cell suspension in vitro.IL2-cas induced apoptosis in T cells expressing the alpha chain of the IL-2 receptor (cluster of differentiation [CD]25) in vitro, with superior survival of T cells expressing CD4 and forkhead box P3 (FOXP3). The fusion protein decreased mixed lymphocyte reactivity, and pretreatment with IL2-cas decreased the efficacy of adoptive transfer of diabetes into NOD severe combined immunodeficiency mice. Administration of one dose of IL2-cas decreased the incidence of diabetes in NOD mice, showing a superior beneficial effect when administered at young age, and effectively blocked induction of hyperglycaemia by cyclophosphamide, reducing the severity of islet inflammation. Administration of IL2-cas caused an acute increase in CD25(-)FOXP3(+) T cells in the lymph nodes, pancreas and thymus in NOD mice, with similar effects in wild-type mice. Administration of IL2-cas after onset of hyperglycaemia resulted in superior survival.Targeted elimination of cells expressing the IL-2 receptor by this fusion protein disrupts the autoimmune pathogenesis in prediabetic and diabetic NOD mice, despite depletion of CD25(+) regulatory T cells. Furthermore, this particular fusion protein is permissive to the development of FOXP3(+) T cells that might contribute to protracted protection from the progression of insulitis and overt hyperglycaemia.

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.

Targeted depletion of immune cells expressing the interleukin-2 (IL-2) receptor can exacerbate inflammatory bowel disease (IBD) through elimination of regulatory T (Treg) cells, or ameliorate its course by depletion of cytotoxic cells. To answer this question we used a fusion protein composed of IL-2 and caspase-3 (IL2-cas) in an experimental model of DSS-induced toxic colitis. In a preventive setting, co-administration of DSS with a daily therapeutic dose of IL2-cas for seven days improved all disease parameters. Although CD4(+)CD25(+) T cells were depleted in the mesenteric lymph nodes, a fractional increase in CD4(+)FoxP3(+) T cells was observed in the spleen. Likewise, IL2-cas therapy improved the outcome of established disease in a chronic model of colitis. These data demonstrate that therapies that use IL-2 as a targeting moiety exert a protective effect over the colon under conditions of inflammation. The efficacy of IL-2-targeted therapy is attributed to reduced activity of reactive T cells, which ameliorates the secondary inflammatory infiltration. IL2-cas evolves as a potential therapeutic tool in IBD.

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.

Activation-induced cell death is a general mechanism of immune homeostasis through negative regulation of clonal expansion of activated immune cells. This mechanism is involved in the maintenance of self- and transplant tolerance through polarization of the immune responses. The Fas/Fas-ligand interaction is a major common executioner of apoptosis in lymphocytes, with a dual role in regulatory T cell (Treg) function: Treg cell homeostasis and Treg cell-mediated suppression. Sensitivity to apoptosis and the patterns of Treg-cell death are of outmost importance in immune homeostasis that affects the equilibrium between cytolytic and suppressor forces in activation and termination of immune activity. Naive innate (naturally occurring) Treg cells present variable sensitivities to apoptosis, related to their turnover rates in tissue under steady state conditions. Following activation, Treg cells are less sensitive to apoptosis than cytotoxic effector subsets. Their susceptibility to apoptosis is influenced by cytokines within the inflammatory environment (primarily interleukin-2), the mode of antigenic stimulation and the proliferation rates. Here, we attempt to resolve some controversies surrounding the sensitivity of Treg cells to apoptosis under various experimental conditions, to delineate the function of cell death in regulation of immunity.

A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause–result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.©2007 Wiley Periodicals, Inc BioEssays 30:875–888, 2008. © 2008 Wiley Periodicals, Inc.

The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4+CD25+FoxP3+ T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.

Regulatory T cells (Treg) play a pivotal role in tolerance to self-antigens and tissue grafts, and suppression of autoimmune reactions. These cells modulate the intensity and quality of immune reactions through attenuation of the cytolytic activities of reactive immune cells. Treg cells operate primarily at the site of inflammation where they modulate the immune reaction through three major mechanisms: a) direct killing of cytotoxic cells through cell-to-cell contact, b) inhibition of cytokine production by cytotoxic cells, in particular interleukin-2, c) direct secretion of immunomodulatory cytokines, in particular TGF-β and interleukin-10. In addition to differential contributions of these mechanisms under variable inflammatory conditions, mechanistic complexity and diversity evolves from the diverse tasks performed by various Treg cell subsets in different stages of the immune reaction. Here we attempt to integrate the current experimental evidence to delineate the major suppressive pathways of Treg cells.

Regenerative medicine opens new avenues and promises towards more effective therapies for autoimmune disorders. Current therapeutic strategies for type I diabetes focus on three major directions, with distinct advantages and disadvantages: arrest of autoimmunity, islet transplantation and generation of neoislets. There is mounting evidence that candidate stem cells residing in the hematopoietic compartments participate in regeneration of pancreatic islets following chemical and autoimmune injury in vivo. The apparent major mechanisms include immunomodulation, revascularization, support of endogenous beta-cell regeneration and differentiation into insulin-producing cells. Review of the current evidence suggests that some divergent observations depend primarily on the experimental design, which both limits and accentuates developmental events. The flood of publications reporting negative results appears to reflect primarily suboptimal experimental conditions for differentiation of putative stem cells, rather than limited developmental plasticity. Stem cells modulate the course of autoimmune diabetes through multiple mechanisms, including de novo generation of units capable to sense, produce and secrete insulin. Therefore, the charged debate over controversies surrounding developmental plasticity should not impede attempts to design curative therapies for this disease.

Early after transplantation, donor lineage-negative bone marrow cells (lin− BMC) constitutively upregulated their expression of Fas ligand (FasL), suggesting an involvement of the Fas/FasL axis in engraftment. Following the observation of impaired engraftment in the presence of a dysfunctional Fas/FasL axis in FasL-defective (gld) donors or Fas-defective (lpr) recipients, we expressed a noncleavable FasL chimeric protein on the surface of donor lin− BMC. Despite a short life span of the protein in vivo, expression of FasL on the surface of all the donor lin− BMC improved the efficiency of engraftment twofold. The FasL-coated donor cells efficiently blunted the host alloimmune responses in primary recipients and retained their hematopoietic reconstituting potential in secondary transplants. Surprisingly, FasL protein improved the efficiency of engraftment in syngeneic transplants. The deficient engraftment in lpr recipients was not reversed in chimeric mice with Fas− stroma and Fas+ BMC, demonstrating that the host marrow stroma was also a target of donor cell FasL. Hematopoietic stem and progenitor cells are insensitive to Fas-mediated apoptosis and thus can exploit the constitutive expression of FasL to exert potent veto activities in the early stages of engraftment. Manipulation of the donor cells using ectopic FasL protein accentuated the immunogenic and nonimmunogenic interactions between the donor cells and the host, alleviating the requirement for a megadose of transplanted cells to achieve a potent veto effect. Disclosure of potential conflicts of interest is found at the end of this article.

Abundant information is available on the involvement of various cellular and molecular mechanisms in beta cell apoptosis. The experimental evidence is controversial and difficult to reconcile, and the mechanisms of evasion of the autoreactive clones from immune surveillance are poorly understood. Multiple apoptotic pathways play a role in destructive insulitis, including perforin/granzyme, Fas/Fas-ligand (FasL), and other members of the necrosis factor superfamily. These pathways present redundant behaviors in both the initial and late stages of beta cell injury, and at the same time, each molecular mechanism is dispensable in the evolution of autoimmune diabetes. There may be a preferential use of perforin/granzyme in CD8(+) T cell-mediated lysis, which participates in onset of autoimmunity, and a predominance of FasL in CD4(+) T cell-mediated insulitis. Several cytokines released in the inflammatory infiltrate induce Fas expression in beta cells, priming them to FasL-mediated apoptosis. In this review, we focus on the possible participation of multiple cell subsets and molecular mechanisms in the pathogenesis of diabetes to the point where inflammation incites an irreversible vicious cycle that perpetuates beta cell death.

Two competing hypotheses aiming to explain the onset of autoimmune reactions are discussed in the context of genetic and environmental predisposition to type 1 diabetes (T1D). The first hypothesis has evolved along characterization of the mechanisms of self-discrimination and attributes diabetic autoimmunity to escape of reactive T cells from central regulation in the thymus. The second considers frequent occurrence of autoimmune reactions within the immune homunculus, which are adequately suppressed by regulatory T cells originating from the thymus, and occasionally, insufficient suppression results in autoimmunity. Besides thymic dysfunction, deregulation of both effector and suppressor cells can in fact result from homeostatic aberrations at the peripheral level during initial stages of evolution of adaptive immunity. Pathogenic cells sensitized in the islets are efficiently expanded in the target tissue and pancreatic lymph nodes of lymphopenic neonates. In parallel, the same mechanisms of peripheral sensitization contribute to tolerization through education of naive/effector T cells and expansion of regulatory T cells. Experimental evidence presented for each individual mechanism implies that T1D may result from a primary effector or suppressor immune abnormality. Disturbed self-tolerance leading to T1D may well result from peripheral deregulation of innate and adaptive immunity, with variable contribution of central thymic dysfunction.

Small-sized adult bone marrow cells isolated by counterflow centrifugal elutriation and depleted of lineage markers (Fr25lin(-)) have the capacity to differentiate into insulin-producing cells and stabilize glycemic control. This study assessed competitive migration of syngeneic stem cells to the bone marrow and islets in a murine model of chemical diabetes. VLA-4 is expressed in ∼ 25% of these cells, whereas CXCR4 is not detected, however, it is transcriptionally upregulated (6-fold). The possibility to enrich stem cells by a bone marrow homing (BM-H) functional assay was assessed in sequential transplants. Fr25lin(-) cells labeled with PKH26 were grafted into primary myeloablated recipients, and mitotically quiescent Fr25lin(-)PKH(bright) cells were sorted from the bone marrow after 2 days. The contribution of bone marrow-homed stem cells was remarkably higher in secondary recipients compared to freshly elutriated cells. The therapeutic efficacy was further increased by omission of irradiation in the secondary recipients, showing a 25-fold enrichment of islet-reconstituting cells by the bone marrow homing assay. Donor cells identified by the green fluorescent protein (GFP) and a genomic marker in sex-mismatched transplants upregulated PDX-1 and produced proinsulin, affirming the capacity of BM-H cells to convert in the injured islets. There was no evidence of transcriptional priming of freshly elutriated subsets to express PDX-1, insulin, and other markers of endocrine progenitors, indicating that the bone marrow harbors stem cells with versatile differentiation capacity. Affinity to the bone marrow can be used to enrich stem cells for pancreatic regeneration, and reciprocally, conditioning reduces the competitive incorporation in the injured islets.

Based on predictive, preventive and personalized medicine (PPPM) strategy for treatment of cancer, immunotherapy and additional targeted anti-cancer modalities should be considered for cure of cancer at an early stage of the disease following successful conventional treatment. Alternatively, PPPM strategy should be applied at the stage of minimal residual disease (MRD) induced following successful re-induction of second or subsequent remission. Fortunately, a stage of minimal residual disease can be accomplished in most patients with cancer following conventional treatment but in the absence of visible disease or lack of symptoms, additional treatment is usually denied. Considering PPPM using innovative and safe treatment focusing on immunotherapy should be considered for patients at risk because eradication of MRD can be accomplished whereas treatment of overt disease may no longer be possible. Similarly to the treatment of infectious diseases, the goal should be to recognize the high-risk cases upfront and treat MRD because residual malignant cells cannot be detected by available imaging technologies. The take home message is that at the stage of minimal residual disease focusing on immunotherapy and other targeted anti-cancer modalities may be safe, successful and possibly the only way to cure cancer, or at least to better control tumor progression.

The exact process that leads to the eruption of autoimmune reactions against beta cells and the evolution of diabetes is not fully understood. Macrophages and T cells may launch an initial immune reaction against the pancreatic islets of Langerhans, provoking inflammation and destructive insulitis. The information on the molecular mechanisms of the emergence of beta cell injury is controversial and points to possibly important roles for the perforin-granzyme, Fas-Fas-ligand (FasL) and tumor-necrosis-factor-mediated apoptotic pathways. FasL has several unique features that make it a potentially ideal immunomodulatory tool. Most important, FasL is selectively toxic to cytotoxic T cells and less harmful to regulatory T cells. This review discusses the intrinsic sensitivity of beta cells to FasL-mediated apoptosis, the conditions that underlie this beta cell sensitivity, and the feasibility of using FasL to arrest autoimmunity and prevent islet allograft rejection. In both the autoimmune and transplant settings, it is imperative to progress from the administration of nonspecific immunosuppressive therapy to the concept of beta-cell-specific immunomodulation. FasL evolves as a prime candidate for antigen-specific immunomodulation.

The continuing interest in the biology of stem cells is enhanced by new discoveries surrounding developmental plasticity of both embryonic and adult stem cells. Adoptive transfer of concepts and definitions from the hematopoietic system to other tissue stem cells suggests inclusion of characteristics such as ability to self-renew and differentiate to functionally reconstitute a tissue/organ of origin. How adequate and accurate are these definitions? Within the great unknown of how these cells function, modulate their gene expression patterns and respond to extrinsic signals, it is apparent that there are numerous levels of stemness. We may envision a scale of developmental flexibility. At one end of the scale are positioned the embryonic stem cells, and at the other end are positioned partially-differentiated, differentiation restricted (committed) tissue/organ stem cells. There is evidence that some stem cells in the adult are pluripotent, thus positioned close to the embryonic end of the stem scale. It is uncertain yet to what extent stem cells can move back and forth along the stem scale.

Gene therapy as an immunomodulatory approach has the potential to treat various inherited and acquired immune-based human diseases. However, its clinical application has several challenges, varying from the efficiency of gene transfer, control of gene expression, cell and tissue targeting, and safety concerns associated with the introduction of exogenous DNA into cells/ tissues. Gene therapy is also a time- and labor-intensive procedure. As an alternative, we recently developed a novel technology, ProtEx, that allows for rapid, efficient, and durable display of exogenous proteins on the surface of cells, tissues, and organs without detectable toxicity. This technology exploits the strong binding affinity (Kd = 10 - 1 5 M) of streptavidin with biotin and involves generation of chimeric molecules composed of the extracellular portions of immunological proteins of interest and a modified form of streptavidin, biotinylation of biological surfaces, and decoration of the modified surface with chimeric proteins. Biotin persists on the cell surface for weeks both in vitro and in vivo, thereby providing a platform to display exogenous proteins with extended cell surface kinetics. Two chimeric proteins, rat FasL (SA-FasL) and human CD80 (CD80-SA), were generated and tested for cell surface display and immunomodulatory functions. SA-FasL and CD80-SA molecules persisted on the surface of various cell types for extended periods, varying from days to weeks in vitro and in vivo. The cell surface kinetics, however, were protein and cell type dependent. SA-FasL showed potent apoptotic activity against Fas + cells as a soluble protein or displayed on the cell surface and effectively blocked alloreactive responses. The display of CD80-SA on the surface of tumor cells, however, converted them into antigen-presenting cells for effective stimulation of autologous and allogeneic T-cell responses. ProtEx technology, therefore, represents a practical and effective alternative to DNA-based gene therapy for immunomodulation.

We follow with interest, evidence published in this Journal concerning the sensitivities to apoptosis of effector and regulatory T cells (Treg) in inflammatory bowel disease (IBD), as a possible mechanism of breakdown in self-tolerance.1–4 The persistent and relapsing pattern of IBD might originate from outstanding resistance of human enteric cells to apoptosis5 or excessive apoptosis of Treg.3 Thus, therapeutic approaches aiming to restore enteric immune homeostasis should either delete effector cells or improve Treg survival, though it is unclear whether Treg apoptosis is causative or a consequence of enteric autoimmunity in IBD patients.3 Targeted deletion of effector cells has been achieved with a fusion protein composed of interleukin-2 (IL-2, internalisation moiety) and active caspase-2 (apoptotic molecule), bypassing the apparent defective caspase function in colitogenic cells.6 We reasoned that Treg endowed with enhanced killing activity might significantly attenuate the course of inflammatory colitis by shifting the effector/suppressor balance. In prior studies, we showed that administration of relatively small numbers of Treg decorated with Fas-ligand (FasL) protein (killer Treg), but not naive Treg, delays the onset of hyperglycaemia in prediabetic non-obese diabetic …

The early stages of homing, seeding, and engraftment of hematopoietic stem and progenitor cells are poorly characterized. We have developed an optical technique that allows in vivo tracking of transplanted, fluorescent-tagged cells in the host femurs. In this study we used fluorescence microscopy to monitor the topologic and chronologic patterns of hematopoietic cell seeding in the femoral bone marrow (BM) of mice. PKH-labeled cells homed to the femur within minutes after injection into a peripheral vein. Most cells drifted within the marrow space and gradually seeded in clusters close to the endosteal surface of the epiphyseal cortex. Three days after transplantation 85% to 94% (14%) of PKH-labeled cells in the femoral marrow were located within 100 μm of the epiphyseal bone surface (P < .001 versus the more central cells), whereas labeled cells were absent in the femoral diaphysis. Primary seeding of juxtaendosteal, epiphyseal marrow occurred independently of recipient conditioning (myeloablated and nonconditioned hosts), donor-recipient antigen disparity, or the phenotype of the injected cells (whole BM and lineage-negative cells) and was consistently observed in secondary recipients of BM-homed cells. Seeding in regions close to the epiphyseal bone was also observed in freshly excised femurs perfused ex vivo and in femurs assessed without prior placement of optical windows, indicating that the site of primary seeding was not affected by surgical placement of optical windows. Four to 5 days after transplantation, cellular clusters appeared in the more central regions of the epiphyses and in the diaphyses. Centrally located cells showed decreased PKH fluorescence, suggesting that they were progeny of the seeding cells, and brightly fluorescent cells (quiescent first-generation seeding cells) were observed close to the bone surface for as long as 24 days after transplantation. These data indicate that the periphery of the femoral marrow hosts primary seeding and that quiescent cells continue to reside in the periphery for weeks and do not divide. The site of proliferation of transplanted cells is the center of the marrow space.

Homing of transplanted bone marrow cells (BMC) to the host bone marrow (BM) is the first step of engraftment towards durable multilineage haematopoietic reconstitution. We used an in vivo assay to track PKH-labelled cells in the BM of mice after transplantation, using fluorescence microscopy through an optical window placed over the distal femoral epiphysis. Within hours after intravenous injection, the cells moved in and out the femur, and were mobile within the marrow space. One hour after injection of whole BMC into non-conditioned syngeneic and allogeneic recipients, the homing efficiencies (HE) were 1.23 +/- 0.14% and 0.12 +/- 0.02% respectively (P 80%) were quiescent in the BM during the first 3 d. HE were twofold higher in busulphan-myeloablated recipients (P < 0.001 vs non-conditioned), and allogeneic transplantation resulted in 84 +/- 9% donor chimaerism at 4 weeks. The HE of lin- cells was 16-fold higher than that of lin+ cells (P < 0.001), and the subset of lin- SCA-1+ cells was 4.6-fold higher in the BM-homed cell population (P < 0.001 vs lin- cells). Approximately 1,500 of the BM-homed cells rescued 62-71% secondary syngeneic and allogeneic myeloablated recipients. Strikingly, the HE could be predicted during the first 3 d after transplantation by correcting the measurements performed in vivo for the enrichment of progenitors in donor inoculum, donor-recipient antigen disparity and myeloablative conditioning.

Long-term display of exogenous proteins on the cell surface may have important research and therapeutic implications. We report a novel method for the cell-surface display of proteins that involves generation of a chimeric protein with core streptavidin, biotinylation of cells, and "decoration" with the protein. A chimeric protein with the extracellular portions of FasL (SA-FasL) was efficiently displayed on the cell surface within 2 hr without detectable cellular toxicity. Biotin and SA-FasL persisted on the cell surface for weeks in vitro and in vivo. Immunomodulation with SA-FasL-decorated splenocytes effectively blocked alloreactive responses in naive and presensitized rodents and prevented the rejection of allogeneic pancreatic islets. This approach may serve as an alternative to gene transfer-based expression with broad research and therapeutic applications.

This work describes an optical technique for characterization of the early stages of hematopoietic stem cell (HSC) engraftment under physiological conditions and in real time. Bone marrow cells (BMCs) labeled with PKH membrane linkers were injected into conditioned recipients (B10-->B10.BR mice) preoperated for placement of optical windows over femoral epiphyses. Labeled cells were tracked in vivo by fluorescence microscopy. Cellular adhesion to the BM stroma was tested with laser tweezers, and viability was assayed by the propidium iodide (PI) exclusion test, as determined from energy-transfer measurements of the pair PKH67-PI in freshly excised femurs in situ. At optimal concentrations for in vivo tracking, 1-4 micro M PKH dyes neither impaired the viability of BMCs nor the capacity of allogeneic HSCs to reconstitute hematopoiesis in myeloablated recipients. The optical window allowed in vivo visualization of 23%-26% of the PKH-labeled BMCs in the femur. The homing efficiencies at 16 hours posttransplantation were quantified as 1.77% +/- 0.15% and 0.21% +/- 0.02% for syngeneic and allogeneic BMCs, respectively. In femurs excised 16 hours after transplantation, 70% +/- 9% of the cells were adherent to the BM stroma, and two-thirds of the cells were PI negative (viable). In vivo tracking and in situ assessment of labeled HSCs in recipient BM provide important quantitative and qualitative insights into the early stages of engraftment. Correlation of early events and the efficiency of durable engraftment serve as the basis for a systematic approach toward optimization of the conditions for transplantation.

The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 +/- 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6-10 cells over an area of approximately 70 microm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen-disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non-HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.