Host Thymus Research Articles

Single Agent CD45-Targeted Antibody Drug Conjugate Enables Full Mismatch Allogeneic Hematopoietic Stem Cell Transplantation in a Murine HSCT Model

Introduction Allogeneic hematopoietic stem cell transplant (Allo-HSCT) is a potentially curative treatment for malignant and non-malignant blood disorders. However, current conditioning regimens limit the use of this curative procedure in many eligible patients due to regimen-related mortality and morbidities, including organ toxicity, infertility, and secondary malignancies. We are developing novel antibody drug conjugates (ADC) as conditioning agents that can achieve full myeloablation as a single agent that may reduce toxicity associated with current conditioning regimens. We have generated an anti-murine ADC targeting CD45 and assessed its effectiveness as single agent conditioning regimen in a fully allogeneic murine HSCT model. Methods Our tool CD45 ADC is engineered for rapid clearance (t1/2=1.7hr) to enable HSCT after conditioning. A single dose of 3 mg/kg is fully myeloablative in C57BL/6 mice. To determine if the tool CD45-ADC could successfully condition recipients for fully mismatched allo-HSCT, we evaluated the ability of a single dose of 5 mg/kg of the tool CD45-ADC to condition C57BL/6 hosts (H-2b, CD45.2+) for transplant with cells from CByJ.SJL(B6) donors (H-2d, CD45.1+). A matched dose of an isotype ADC (Iso-ADC) was used as a negative control, while 9 Gy TBI was used as a conventional conditioning positive control. Conditioned mice were transplanted with 4x107 whole BM cells, and peripheral blood chimerism was assessed over 22 weeks. At 22 weeks, donor hematopoietic cell chimerism was evaluated in the spleen, bone marrow, and thymus of recipients. Results In the fully mismatched Balb/c → C57Bl/6 allo-HSCT model, conditioning with a single dose of 5 mg/kg of CD45-ADC as a single agent was well tolerated and enabled full allogeneic donor chimerism (n=2 separate experiments). Peripheral blood chimerism was observed in mice conditioned with CD45-ADC at week 4 and maintained through week 22 (Figure 1). Multilineage reconstitution was observed in the T-, B-, and myeloid cell compartments with >90% donor chimerism seen in each compartment, indicative of HSC engraftment. These results were comparable to chimerism seen in the 9 Gy TBI positive control. Treatment with a non-targeting isotype ADC at a matched dose was not effective (Figure 1). For all groups, stem cell chimerism in the bone marrow matched that in the periphery. Splenic and thymic donor immune cell reconstitution was similar between CD45-ADC and TBI conditioning at week 22 (Figure 1), demonstrating that CD45-ADC efficiently depletes host lymphocytes in secondary lymphoid organs while preserving the capacity of the host thymus to support de novo generation of donor-derived T cells after transplantation. Conclusion Conditioning with CD45-ADC was well-tolerated, fully myeloablative, and enabled complete chimerism in a full mismatch allo-HSCT model as a single agent. This targeted, readily translatable approach for safer conditioning could improve the risk-benefit profile for allogenic and haploidentical HSCT and may extend the curative potential of HSCT to more patients suffering from blood cancers and other diseases that may benefit from HSCT. Disclosures Hyzy: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Hammond:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sarma:Magenta Therapeutics: Ended employment in the past 24 months. Clark:Magenta Therapeutics: Current Employment. Bhat:Magenta Therapeutics: Current Employment. Lamothe:Magenta Therapeutics: Current Employment. Palchaudhuri:Magenta Therapeutics: Current Employment. Pearse:Magenta Therapeutics: Ended employment in the past 24 months. McDonagh:Magenta Therapeutics: Ended employment in the past 24 months. Kiem:Magenta Therapeutics: Consultancy; CSL: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees. Wagner:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BlueRock: Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees. Blazar:Magenta Therapeutics: Consultancy; Fate Therapeutics Inc.: Research Funding; BlueRock Therapeutics: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder; BlueRock Therapeuetic: Consultancy. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.

T cell progenitor therapy-facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction.

Infusion of in vitro-derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell-deficient (Rag1-/-) marrow with WT in vitro-generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery.

Reprogramming and Recapitulating T Cells in the Immature Immune Environment of the Neonate to Induce Transplant Tolerance

Introduction: Semi-allogeneic bone marrow and spleen cells (BMC/SC) induce transplant tolerance in neonatal mice whereas allogeneic BMC/SC cause lethal graft-versus-host disease (GVHD). To prevent aGVHD and induce transplant tolerance with allo-SC/BMC we reprogrammed donor/host CD4 T cells by co-stimulation blockade to preserve regulatory CD4 T cells while recapitulating depleted donor/host CD8 T cells. Methods: C3H (H-2k) neonatal mice were injected iv with total or depleted B6 (H-2b) GFP+ SC/BMC. CD8 T and CD49b NK cells were depleted from donor inocula using StemCell Technologies kits. In vivo neonatal host CD8 T cells were depleted (Mab 53–6.7) and donor/host CD4 T cells reprogrammed (CD154 Mab MR1). Trafficking and interactions of injected cells were monitored by microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Neonatal mice injected with GFP+ allo-SC/BMC developed aGVHD, with diarrhea, reduced growth and early death. GFP+ cells proliferated and spread throughout injected mice indicating systemic inflammation. Depletion of donor/host CD8 T cells together with co-stimulation blockade of donor/host CD4 T cells (CD154) resulted in GFP signal being reduced and restricted to lymphoid organs (day 6). High resolution microscopy showed donor T and B cells positioned respectively in PALS and follicular regions of host spleen; lack of Ki67 immunostaining indicated many donor cells were not proliferating. Donor DC were detected in host thymus (day 6) suggesting a possible role in central tolerance. Donor-type B6 hearts transplanted into neonatally-treated adult C3H mice (n=5) (CD8 depleted/CD4 co-stimulation blockade) all continued to beat at 100 days post-transplant, 3 grafts with maximal strength and 2 grafts with diminished strength. In untreated control mice (n=3) donor-type hearts stopped beating by day 10. H&E staining of a strongly beating B6 heart graft at 100 days showed undamaged cardiomyocytes with little if any cellular infiltrate and absence of macrophages and T cells by immunostaining. Conclusion: Reprograming CD4 T cells while depleting/recapitulating CD8 T cells in allo-SC/BMC treated neonates leads to 100% cardiac allograft survival at 100days; low beat scores in 2/5 grafts suggests further optimization is required. These findings provide insight into robust tolerance induction in neonates. This project was supported with funding from the Canadian Institutes for Health Research and the Gordon English family.

The increase in thymopoiesis following T cell progenitor therapy is dependent upon the input population and continued interaction between developing T cells and the thymic microenvironment.

Abstract The inclusion of in vitro derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant (HSCT) aids in the recovery of the thymus damaged by total body irradiation and improves de novo thymopoiesis. To understand the interaction between proTs and the thymic microenvironment, wildtype (WT) mice were lethally irradiated and given T cell deficient donor (Rag1−/−) marrow along with in vitro generated proT from WT donors, limiting mature T cell development to infused proT. Donor proTs within the host thymus led to a significant increase in thymic epithelial cell (TEC) numbers by day 21 post-transplant, and increased actively cycling TECs as measured by Ki67 expression and BrdU uptake. However, that gain was temporary and lost by day 28, suggesting that continued signaling from proT cells is required to sustain TEC cycling and mass. We also find a significant improvement in total thymocyte number by day 21 followed by a significant increase in the total mature T cell number in the secondary lymphoid organs by day 28. This protective surge is also temporary, receding by day 60. In this time period, infused DN2 proTs selectively increased thymocyte number while DN3 proTs preferentially led to a greater TEC numbers. Interestingly, an exception in persistence occurs when DN3 proTs are used and the increase in mature T cells in the spleen persists at day 60. As a result of the lack of competition for thymic niches by cells from the Rag1−/− graft, a subpopulation of the infused proT persisted in the thymus in an immature state at day 60. These findings highlight the importance of the interaction between developing T cells and TECs in the proliferation and survival of these critical components of the thymic microenvironment.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1-Donor Cell Types

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi-allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1-donor cell types selectively alter neonatal immunity. In host thymus, F1-donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1-donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1-donor cells give rise to albumin-containing hepatocyte-like cells. The neonatal immune system is lymphopenic, Th-2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1-donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.

Impaired thymic expression of tissue-restricted antigens licenses the de novo generation of autoreactive CD4+ T cells in acute GVHD

AbstractDuring acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEChigh) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEChigh cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4+ T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEChigh cells. Our data, therefore, indicate that a functional compromise of the medullary mTEChigh compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.

Small-molecule inhibitor of glycogen synthase kinase 3β 6-Bromoindirubin-3-oxime inhibits hematopoietic regeneration in stem cell recipient mice.

Small-molecule inhibitors of glycogen synthase kinase 3β (GSK3β) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3β inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3β inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant.

Trafficking of Intra-Graft Donor T Cells After Skin Transplantation in Mice.

It is firmly established that the skin contains T lymphocytes including significant numbers of FoxP3+ regulatory T cells (Tregs). However, their role in skin transplantation is still unknown. Recent evidence has been provided by W. Burlingham's laboratory suggesting that intra-graft T cells may influence the alloresponse and rejection process (bi-directional alloimmunity). In this study, we investigated the trafficking of intra-graft donor T cells to the recipient's lymphoid organs after skin transplantation in mice. To test this C57BL/6 (B6) mice received a skin transplant from a double transgenic B6 donor whose T cells expressed DSred while FoxP3 expression was associated with GFP (Tregs being double positive). At different time points after transplantation, cell suspensions of recipients' lymph nodes, spleen and thymus were prepared. The presence and characterization of fluorescent donor cells was investigated using Image Stream (Amnis) technology. First, the presence of T cells (DSred+ FoxP3-) was detected only at d4-d7 posttransplantation i.e. after skin graft revascularization and essentially in the host's thymus. On the other hand, double positive (DSred+ FoxP3+) Tregs were found in all recipient's lymphoid organs. Surprisingly, high frequencies of single positive GFP+ cells were found in both ipsilateral lymph nodes and thymus. The nature of these cells is being investigated. In summary, our results show that most T cells leaving the graft are Tregs spreading through all recipient's lymphoid organs while intra-graft effector T cells migrate primarily to the host's thymus.

Administration Of Anti-CD20 Mab Is Highly Effective In Preventing But Ineffective In Treating Chronic GVHD While Preserving A Strong GVL Effect

Donor T cell-mediated graft versus leukemia/lymphoma (GVL) effect plays a critical role in preventing tumor relapse in leukemia/lymphoma patients treated with allogeneic hematopoietic cell transplantation (HCT). However, these same donor T cells also induce acute and chronic graft versus host disease (GVHD), which remains a major obstacle for allogeneic HCT as a curative therapy for hematological malignancies. Chronic GVHD is a systemic lupus- and scleroderma-like autoimmune syndrome. We and others reported that donor B cells play important roles in augmenting pathogenesis of chronic GVHD in mouse models and humans. Rituxan (an anti-CD20 mAb) has been used in the clinic for the treatment of ongoing chronic GVHD, but the effect is variable and minimum in some reports, and the mechanisms for this ineffectiveness remains unclear. In the current studies, we evaluated the effect of in vivo administration of a depleting anti-CD20 mAb in preventing and treating autoimmune-like chronic GVHD as well as the impact on GVL effect. With the mouse chronic GVHD model of DBA/2 donor to MHC-matched BALB/c recipient established in our lab (Blood, 2006, J. Immunol 2012), we found that one intravenous injection of anti-CD20 (50mg/kg) immediately after HCT effectively prevented induction of autoimmune-like chronic GVHD. While all (12/12) recipients treated with control IgG developed chronic GVHD with proteinuria and hair-loss and died by 30 days, none (0/12) of the anti-CD20-treated recipients developed proteinuria or hair-loss and all survived for more than 50 days after HCT without signs of chronic GVHD. In addition, the anti-CD20-treated recipients eliminated BCL1 leukemia/lymphoma cells without signs of chronic GVHD. The preventative anti-CD20 treatment had little impact on donor CD8+ T cell activation and expansion in the periphery and allowed for the strong CD8+ T cell-mediated GVL effect. The effective prevention of chronic GVHD by anti-CD20 was associated with significant changes in donor B and CD4+ T cells as well as associated with protection of host thymus. Preventive anti-CD20 treatment depleted IL-6-producing donor B cells and increased IL-10-producing B cells. In addition, the treatment also significantly reduced donor CD4+ T cell expansion, the percentage of IFN-g-producing CD4+ T as well as CD4+ T cells expressing CD8αα, the latter of which has recently been reported to represent over-activated pathogenic CD4+ T cells, such that the host thymus was protected from donor T-cell-mediated damage. Anti-CD20 or control IgG treatment of recipients with ongoing chronic GVHD 15-20 days after HCT did not show any difference in disease progress, and all (8/8) in each group died within 30 days after HCT. The ineffectiveness of anti-CD20 therapy was associated with little reduction of CD19+ B or CD4+ T cells, as the majority of CD19+ B cells became CD20- in the recipients with ongoing chronic GVHD. These results indication that anti-CD20 can effectively prevent induction of autoimmune-like chronic GVHD while preserving the GVL effect, but anti-CD20 is ineffective in treating ongoing chronic GVHD. (This study is supported by the Nesvig Lymphoma Foundation). Disclosures:Chan:Genentech Inc.: Employment.

Prolonged survival in mice with advanced tumors treated with intra-bone marrow-BMT plus fetal thymus transplantation with restoration of T cell functions (P2177)

Abstract Thymic function decreases in line with tumor progression in patients with cancer, resulting in immunodeficiency and a poor prognosis. In the present study, we attempted to restore thymic function by BALB/c (H-2d) syngeneic (Syn) or B6 (H-2b) allogeneic (Allo) bone marrow transplantation (BMT) using intra-bone marrow-bone marrow transplantation (IBM-BMT) plus Syn-, Allo- or C3H (H-2k) 3rd-party fetal thymus transplantation (TT). Although the BALB/c mice with advanced tumors (Meth-A sarcoma; H-2d, >4 cm2) treated with either Syn- or Allo-BMT alone showed a slight improvement in survival compared with non-treated controls, the mice treated with BMT + TT showed a longer survival. The mice treated with Allo-BMT + Allo-TT or 3rd-party TT showed the longest survival. Interestingly, although there was no difference in main tumor size among the BMT groups, lung metastasis was significantly inhibited by Allo-BMT + Allo-TT or 3rd-party TT. Although host thymus was still atrophied, transplanted thymus was well-engrafted. T cell functions increased significantly, whereas number of myeloid suppressor cells and regulatory T cells significantly decreased in these mice. These results suggest that BMT + TT, particularly Allo-BMT + Allo-TT or 3rd-party TT, is most effective in prolonging survival as a result of the restoration of T cell function in hosts with advanced tumors.

Cell Fate Modulation by Microvesicles: Transcriptionally-Mediated and Long Term in Nature

Abstract 4801Cell-derived membrane enclosed vesicles containing mRNA, protein, microRNA, and DNA, can enter cells and effect a phenotype change. We have shown that lung-derived microvesicles enter marrow cells inducing them to express pulmonary epithelial cell-specific protein and mRNA, a variety of microRNA and to enhance their capacity to engraft in irradiated mice and express the phenotype of type II pneumocytes (Aliotta et al, Exp Hematol 38,2010). In the present studies using rat/mouse hybrid cultures and measuring species-specific mRNA, we have shown that immediately after co-culture of rat lung across from mouse marrow, mouse marrow cells express both rat and mouse specific surfactants B and C mRNA. However, when these cells are cultured in steel factor supported long-term culture, rat-specific mRNA disappears rapidly, while mouse-specific mRNA persists out to 12 weeks in liquid culture. Identical studies with rat liver cultured across from mouse marrow have shown early expression in mouse marrow of both rat and mouse albumin mRNA, but in long term in vitro culture, expression of albumin mRNA was mouse-specific. Thus, the major long-term persistent event is an alteration of transcription in the target marrow cells. In a similar fashion, marrow modulated by lung microvesicles in vitro and engrafted into lethally irradiated (950 cGy split dose) mice, evidences expression of pulmonary epithelial cell-specific mRNA or protein (surfactants) in host lung, marrow, thymus, spleen and liver 6 weeks after engraftment – the furthest time tested. These results indicate that microvesicle cell fate modulation is biologically meaningful and represents an important new mechanism for cell phenotype determination. Disclosures:No relevant conflicts of interest to declare.

Transplantation Tolerance Induced in Humans at the Fetal or the Neonatal Stage

Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens. Engraftment needs no conditioning regimen prior to transplantation in neonates with severe combined immunodeficiency disease or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.

Towards Clinically Feasible Co-Stimulatory Blockade for Tolerance Induction: Transient Treatment with CTLA4-Ig, Anti-CD48 and FTY720 Enables Durable Engraftment of T Cell Depleted BM Allografts Following Non-Myeloablative Conditioning.

AbstractAbstract 3726Induction of specific durable immune tolerance towards transplanted donor cells and tissues, without chronic administration of toxic immunosuppressive drugs, represents a most desirable goal in transplantation medicine. Two major approaches addressing this challenge have shown promise in rodent studies. The first is the use of co-stimulatory blockade to inhibit T cells with anti-donor specificity, and the second uses bone marrow (BM) transplantation to achieve a tolerizing chimeric state in which donor antigens are continuously presented in the host thymus. The exceptional clinical potential of the latter approach has been recently demonstrated in a pilot study of combined renal and bone-marrow transplantation from a single donor. However, the presence of alloreactive T cells in the BM graft imposes a significant risk for GVHD which is unacceptable in patients with non-malignant conditions. While T cell depleted BMT could prevent GVHD, the high rejection rate in the absence of aggressive conditioning treatment, represents a significant obstacle. More than a decade ago a co-stimulatory blockade based on anti CD40L was successfully used to induce chimerism following T depleted BMT. However, anti CD40L was later found to be thrombotic in humans, leading to a search for a safer protocol. In previous studies attempting embryonic pancreas xeno-transplantation (Tchorsh-Yutsis et al. Diabetes 2009), we were able to achieve optimal maintenance of the embryonic graft upon transient treatment with CTLA4-Ig and anti CD48, in conjunction with continuous immune suppression with FTY720. However, durable tolerance was not achieved and rejection ensued upon cessation of immune suppression. Therefore, in the present study we sought to investigate the potential of transient treatment with CTLA4-Ig, anti CD48 and FTY720, the equivalents of which are available for clinical use, combined with transplantation of ‘mega dose’ T cell depleted BMT. If successful, such a protocol could achieve the desired durable tolerizing chimeric state. Considering the importance of providing empty niches for successful BM engraftment, we initially determined the minimal myeloablation with busulphan which can induce durable chimerism following infusion of congenic B6-SJL(Ly-5.1) TDBM (25×106) into B6 (Ly-5.2) mice. Testing doses ranging from 10mg/Kg to 100mg/Kg busulphan showed that donor type chimerism above 50% was attained at doses >50mg/Kg (40±26%, 66±7% and 75±2% chimerism at 50, 60 and 100 mg/Kg). Consequently, the sublethal dose of 60mg/Kg was selected for further use in all attempts to induce allogeneic chimerism, in conjunction with transient debulking of host lymphocytes by a single infusion of anti CD4 and anti CD8 depleting antibodies. The well tolerated combined sublethal conditioning presented a formidable barrier for engraftment of allogeneic ‘megadose’ T cell depleted BM, and no chimerism was achieved. However, addition of transient post transplant treatment with CTLA4-Ig, anti CD48 and FTY720 (Fig 1A), led in two independent experiments to marked donor type chimerism with a median follow up of 116 days (range: 70 to 163 days) beyond cessation of immune suppression (Fig.1B). Thus, while no chimerism could be detected in mice treated post transplant with FTY720 alone (0/7), transient post transplant immune suppression with CTLA4-Ig, anti CD48 and FTY720 resulted in more than 80% donor type chimerism in 8 of 11 mice. As can be seen in Fig.1C, significant chimerism was attained in both the myeloid and lymphoid lineages. Since agents such as Belatacept (CTLA4-Ig) and Alefacept (blocking the interaction of CD48) are available for clinical use, our results suggest a potentially feasible co-stimulatory blockade approach for the induction of durable hematopoietic chimerism under non myeloablative conditioning, as a platform for cell therapy and organ transplantation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Presence of donor-derived thymic epithelial cells in [B6→MRL/lpr] mice after allogeneic intra-bone marrow–bone marrow transplantation (IBM–BMT)

We have previously shown that allogeneic intra-bone marrow–bone marrow transplantation (IBM–BMT) can be used to treat autoimmune diseases in MRL/lpr (H-2 K) mice with replacing not only hematolymphoid cells but also stromal cells by normal C57BL/6 (B6: H-2 b) mouse cells. In the present study, we examined for existence of donor-derived thymic epithelial cells (TECs) in the host thymus using green fluorescent protein (GFP)-B6 (H-2 b) mice. In [GFP-B6→MRL/lpr] chimeric mice, splenocytes and thymocytes were completely replaced by donor-type cells, and levels of serum autoantibodies and proteinuria were significantly – reduced to those levels of normal donors. Interestingly, GFP-expressing TECs – not only medullary TECs, which express mouse thymus stromal (MTS)-10, but also cortical TECs, which express cytokeratin 18 – were found. Also, the number of autoimmune regulator (AIRE) expressing TECs, which regulates tissue-specific antigens to delete autoreactive cells, was reduced in the chimeric mice to that of the donor, whereas the number of forkhead box N1 (FOXN1) expressing TECs, which are crucial in the terminal differentiation of TECs, remained unchanged. These findings suggest that BMCs contain the precursors of functional TECs, and that they can differentiate into TECs, thereby correcting thymic function.

Adoptive Immunotherapy for CMV Infection Complicating Haploidentical Transplants: Challenges Posed by the HLA Restriction of Immuno- Dominant Donor T Cells

T-cell depleted hematopoietic stem cell transplants (HSCT) from HLA haplotypedisparate related donors are being increasingly used for the treatment of patients lacking an HLA-compatible related or unrelated donor. Acute and chronic graft versus host disease can be prevented without added prophylaxis if techniques achieving extensive T-cell depletion are employed. Improved conditioning regimens and transplants providing high doses of CD34+ cells have also insured consistent engraftment. However, these patients have a higher and more prolonged risk of serious viral infections than recipients of matched unmodified or T-cell depleted grafts.Adoptive immunotherapy with donor-derived pathogen-specific T-cells can restore effective resistance in these patients. However, T-cells sensitized and propagated in vitro usually respond to only 1–2 immunodominant epitopes from any antigeneic protein presented by different donor HLA alleles. We questioned how often T-cells generated from an HLA-haplotype disparate donor would be restricted by donor-unique, rather than shared HLA alleles since such T-cells would be inactive against virus-infected cells of a haplotype disparate host. Accordingly, we tested 8 cytomegalovirus (CMV) seropositive HLA haplotype disparate parent or sibling donors, as well as 4 patients, each transplanted from one of these donors 1 or more years previously. In each case, T-cells were sensitized with autologous DCs loaded with a pool of overlapping 15-mers spanning the sequence of CMVpp65, an immunodominant protein of cytomegalovirus, for 21–28 days. Using a matrix of peptide subpools, we then mapped the epitopes eliciting IFNγ+ T-cell responses and then determined their HLA-restriction by assaying their specific cytotoxicity against a panel of EBV B-cell lines loaded with the immunogenic epitope each sharing a single HLA allele with the donor. Of the 8 donors tested, 6 responded to CMV pp65 epitopes presented by HLA alleles shared with the haplotype matched recipient, while 2 donors responded exclusively to epitopes presented by donor unique HLA alleles. Recipients of transplants from the latter two donors experienced prolonged CMV viremia, one with CMV chorioretinitis, requiring months of antiviral treatment. CMV-specific T-cells isolated from each of the four transplanted patients over 1 year post transplant were specific for epitopes presented by HLA alleles shared by their donors. In the patients transplanted from donors whose CMV-pp65 specific T-cells were restricted by donor-unique HLA alleles, the epitope targeted and HLA restriction of the engrafted T-cell differed from that of the transplant donors own T-cells, suggesting that these T-cells may have developed from donor precursors differentiating in the host thymus.Thus, in a significant proportion of cases (2/8 in this study), virus-specific T-cells generated in vitro from HLA haplotype disparate donors may be restricted by donor-unique HLA alleles and, as a consequence, inactive against virus-infected host cells. For such patients, techniques insuring selective generation of T-cells specific for epitopes presented by shared alleles should be employed. Our findings may also in part explain the prolonged susceptibility of these patients to viral infection. Since immunodominant T-cells reactive against the same epitopes are also those detected at highest frequency in the donor's blood, they would likely be the only virus-reactive T-cells transferred in a T-cell depleted graft. As a consequence, effective CMV-specific responses might be delayed until new T cells are generated from precursors developing in the host thymus.

Functional characterization of human T cells generated in vitro from hematopoietic stem cells

T lymphocytes are a class of white blood cells that play a major role in the adaptive immune response. T cells are generated in the unique microenvironment of the thymus; however, the precise molecular signals enabling their differentiation are still being elucidated. Using the OP9‐DL1 stromal system we have demonstrated the generation of human T cells from umbilical cord‐blood (UCB) hematopoietic stem cells (HSCs). Here, we characterize T cell specification and commitment events induced at both the phenotypic and molecular level examined by the acquisition of T‐lineage cell surface markers and the induction of T cell associated genes, respectively. In addition, in vitro derived T cell subsets are mature and functional as they proliferate and secrete IFNγ following stimulation. Furthermore, we demonstrate the ability of in vitro‐derived human T cell progenitors to engraft and differentiate into a mouse host thymus in vitro. Finally, HSCs transduced to express an HIV (Human Immunodeficiency virus) inhibitory construct have been generated, and will be evaluated for decreased susceptibility to infection when challenged with HIV. This system has important clinical implications as it provides the foundation for a large supply of human T lymphocyte progenitors, which could be used for the immune‐reconstitution of T cell‐depleted individuals.Research supported by the Ontario HIV Treatment Network (OHTN) and the Canadian Institutes of Health Research (CIHR)

Involvement of Dendritic Cells in Allograft Rejection New implications of Dendritic Cell-Endothelial Cell Interactions

For almost half a century immunologists have tried to tear down the MHC barrier, which separates two unrelated individuals during transplantation. Latest experimental data suggest that a breakthrough in vitro is imminent. Dendritic cells (DCs), which activate naïve allo-reactive T-cells (TCs), play a central role in the establishment of allo-antigen-specific immunity. Allograft solid organ rejection is initiated at the foreign endothelial cell (EC) layer, which forms an immunogenic barrier for migrating DCs. Thus, DC/EC interactions might play a crucial role in antigen-specific allograft rejection. Organ rejection is mediated by host allo-reactive TCs, which are activated by donor DCs (direct activation) or host DCs (indirect activation). Direct allo-antigen presentation by regulatory dendritic cells (DCreg) can play an instructive role towards tolerance induction. Several groups established that, DCregs, if transplanted beforehand, enter host thymus, spleen, or bone marrow where they might eventually establish allo-antigen-specific tolerance. A fundamental aspect of DC function is migration throughout the entire organism. After solid organ transplantation, host DCs bind to ECs, invade allograft tissues, and finally transmigrate into lymphoid vessels and secondary lymphoid organs, where they present allo-antigens to naïve host TCs. Recent data suggest that in vitro manipulated DCregs may mediate allo-transplantation tolerance induction. However, the fundamental mechanisms on how such DCregs cause host TCs in the periphery towards tolerance remain unclear. One very promising experimental concept is the simultaneous manipulation of DC direct and indirect TC activation/suppression, towards donor antigen-specific allo-transplantation tolerance. The allo-antigen-specific long-term tolerance induction mediated by DCreg pre-transplantation (with simultaneous short-term immunosuppression) has become reproducible in the laboratory animal setting. Despite the shortcomings of laboratory animal studies, strong promises are deriving from these studies for clinical kidney, heart, and liver transplantation.

Donor T-Cell Development in Host Thymus After Heterotopic Limb Transplantation in Mice

We developed a mouse model of heterotopic limb transplantation in which we took advantage of Thy1.1 and Thy1.2 congenic strains to track and characterize donor T cells, to determine the role of recipient's thymus in mixed T-cell chimerism induction as well as transplant immunocompetence. The vascularized Thy1.1 limb graft composed of femur, muscle, and skin (VBT) survived long-term in more than 87.5% of Thy1.2 recipients. Percentages of donor-type Thy1.1 T cells increased from day 30 to 90 in thymus and spleen of recipients. Most peripheral donor T cells displayed a naïve phenotype and a few others were regulatory T cells. Thymectomy prevented peripheral T-cell chimerism. Congenic VBT in immunodeficient RAG mice restored their ability to reject skin allografts. These observations suggest that donor T cells differentiated in host thymus might contribute to maintenance of mixed chimerism after transplantation of tissue composite grafts that include vascularized bone.

Role of Thymus in Operational Tolerance Induction in Limb Allograft Transplant Model

In this study, we evaluated the role of host thymus in tolerance induction in composite tissue allografts (CTA) across major histocompatibility complex (MHC) barrier during a 7-day alphabeta- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1(1)) received vascularized hind-limb allografts from hybrid Lewis x Brown-Norway (F1), (LBN, RT1(1+n)) donors. Mixed lymphocyte reaction (MLR) and skin grafting assessed donor-specific tolerance in vitro and in vivo, respectively. Flow cytometry determined the efficacy of immunosuppressive protocols and the presence of donor-specific chimerism. Immunocytochemistry revealed the presence of donor-specific cells in the lymphoid organs of recipients. Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the alphabeta-TCR/CsA protocol, the MST of allografts was 51 days. For euthymic rats, the MST of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with alphabeta-TCR/CsA resulted in indefinite allografts survival (MST=370 days). MLR and skin grafting confirmed donor-specific tolerance in euthymic recipients. Flow cytometry showed stable chimerism in the euthymic rats and transient chimerism in thymectomized limb recipients. Immunoperoxidase staining revealed the persistence of donor-derived cells in the lymphoid tissues of euthymic recipients. We found that the presence of thymus was imperative for the induction of donor-specific tolerance in rat hind-limb composite tissue allografts using a alphabeta-TCR/CsA protocol.