Thymus Grafts Research Articles

IKZF1 Mutation Mediate Resistance to IMiDs in Human Hematopoietic Stem Cells

Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model.Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G.Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment.Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. DisclosuresLentzsch:Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Antithymocyte Globulin Treatment at the Time of Transplantation Impairs Donor Hematopoietic Stem Cell Engraftment

Antithymocyte globulin (ATG) is often included in the conditioning regimen to prevent graft-vs.-host disease in allogeneic hematopoietic stem cell (HSC) transplantation. However, because ATG contains antibodies targeting a wide range of antigens on human cells, its potential off-target effects remain a concern. Here, we explored this question in humanized mice that were made by transplantation of human fetal thymic grafts (under the renal capsule) and CD34+ human fetal liver cells (i.v.). We showed that ATG binds to almost all lineages of human hematopoietic cells including HSCs, and accordingly, it is capable of depleting almost all human hematopoietic cells. Interestingly, the efficacy of ATG was highly variable depending on the tissue of residence, with human cells in bone marrow significantly less susceptible than those in the blood and spleen. Recovery of multilineage human lymphohematopoietic reconstitution in humanized mice that received ATG 3 weeks after HSC transplantation indicates that ATG had a minimal effect on human HSCs that have settled in bone marrow niches. However, efficient human HSC depletion and engraftment failure were seen in mice receiving ATG immediately following transplantation. Our data indicate that the efficacy of ATG is tissue-dependent, and suggest a risk of impairing donor hematopoietic engraftment when ATG is used in preparative conditioning regimens in HSC transplantation. DisclosuresNo relevant conflicts of interest to declare.

Fetal thymus graft enables recovery from age-related hearing loss and expansion of CD4-Positive T cells expressing IL-1 receptor type 2 and regulatory T Cells

BackgroundAccumulating evidence has indicated the relationship between the systemic immune system and the central nervous system including the inner ear.ResultsWe have shown that age-related developments of T-cell dysfunction, hearing loss, and degeneration of cochlear spiral ganglion (SG) neurons observed in 6-month-old mice were recovered in 12 months old mice which previously given fetal thymus transplants twice. We have also demonstrated that CD4+ T cells expressing interleukin 1 receptor type 2 (IL-1R2) and naturally occurring regulatory T cells (nTregs), which expanded in aged 12-month-old mice, were reduced in the thymus-grafted mice of the same age.ConclusionIt is conceivable that the rejuvenation of systemic immune function by fetal thymus grafts contributes not only to the activation of cellular immunity but also to the decrease of IL-1R2+ CD4+ T cells or nTregs, which cells accelerate both age-related hearing loss (AHL) and neurodegeneration of the cochlear neurons. Further studies on the interactions among IL-1R2 expression on CD4+ T cells, Tregs, and neuronal cells and also on the relationships between fetal thymus grafting and the rejuvenation of systemic immunity should be designed in order to advance towards therapeutic effects on neurosenescence, including AHL.

Isolation and transplantation of different aged murine thymic grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Suspension of thymic emigration promotes the maintenance of antigen-specific memory T cells and the recall responses

Thymic involution is evolutionarily conserved and occurs early in life. However, the physiological significance remains elusive of this seemingly detrimental process. The present study investigated the potential impact of altered thymic output on T cell memory using ovalbumin (OVA) expressed by Listeria monocytogenes as a model antigen. Suspension of thymic emigration by thymectomy was shown to lead to a marked increase in the frequency and total number of OVA-specific memory T cells. In contrast, oversupply of thymic emigrants through thymic grafting caused a significant decrease of such cells. When rechallenged with L. monocytogenes expressing OVA, the thymectomized mice mounted a more potent recall response as evidenced by the enlarged population of OVA-specific tetramer+ cells and the accelerated clearance of the bacteria. Notably, the memory-enhancing effect of thymectomy was abrogated following weekly adoptive transfer of naive T cells. Together, data from the present study indicate that reduced thymic output favors the maintenance of the memory T cell pool.

Revisiting thymus function.

OPINION article Front. Immunol., 28 August 2014Sec. T Cell Biology Volume 5 - 2014 | https://doi.org/10.3389/fimmu.2014.00411

363 Human Hematopoietic Stem Cells With a Defined Immunodeficiency and Enteropathy Transfer Clinical Phenotype to a Novel Humanized Mouse Strain

Investigation into the pathogenesis of IBD has been limited by the absence of humanized murine IBD models. The development of immunodeficient mouse strains has facilitated the study of human immune reconstitution in murine hosts. However, adaptive immune responses and antibody class switching are generally weak in models that lack autologous fetal thymic grafts, possibly due to human CD4+ T cell selection in the mouse thymus occurring on murine major histocompatibility complex II (MHCII). To address this, we modified the widely utilized NOD.Prkdc.Il2rγ (NSG) immunocompromised strain to be deficient for murine MHCII (NSGAbo) and instead expressed human leukocyte antigen-DR1 under the control of the murine MHCII promoter (NSGAboDR1). We tested whether CD4+ T cell development and adaptive immune responses are improved following reconstitution using human HLA-matched CD34+ hematopoietic stem cells (HSCs) and whether immune reconstitution using HSCs from a patient with IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked), a well-defined immunodeficiency, that is caused by a mutation in the transcription factor FOXP3, would transfer the clinical phenotype to NSGAboDR1 mice. Results: At 20 weeks following HSC injection into radiation-conditioned neonates, we found that the development of human T cells and dendritic cells were increased in NSGAboDR1 mice and that these mice displayed delayed-type hypersensitivity response to a secondary recall antigen challenge. There was an increase in T cell clonotype diversity as determined by CDR3 sequencing of the TCRβ gene in CD4+ T cells in NSGAboDR1 mice compared to NSG mice. In addition, NSGAboDR1 mice had an increase in mature B cells and antibody class switching compared to NSG and NSGAbo mice. We then reconstituted NSGAboDR1 mice using HSCs from a patient with IPEX that is associated with functional defects in Treg and assessed if the clinical phenotype would be transferred to NSGAboDR1 mice. All mice reconstituted with IPEX CD34+ HSCs were deceased by 18 weeks while all control mice receiving healthy bone marrow CD34+ cells remained viable. IPEX mice developed lymphocytic infiltrate in lung, portal tract and bile ducts of the liver, and slight accumulation of lymphocytes in the small intestine similar to scurfy mice, which also harbor a mutation in Foxp3. CD4+ T cell in IPEX mice had an expanded effector memory T cell population and concomitant decrease in Naive CD4+ T cells. Conclusions: We demonstrate that human HSCs from a patient with a defined genetic immunodeficiency and enteropathy can transfer the disease phenoytpe in a novel humanized mouse strain. This strain does not require grafts of human fetal tissue nor is it restricted to autologous fetal HSCs allowing for reconstitution using human HSCs with defined genetics to study human immune cells and IBD pathogenesis in vivo.

Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Thymus Xenografts

Transplantation of xenogeneic thymus tissue allows xenograft tolerance induction in the highly disparate pig-to-mouse model. Fetal swine thymus (SW THY) can support the generation of a diverse human T cell repertoire that is tolerant of the pig in vitro. We demonstrate that SW THY generates all human T cell subsets, including regulatory T cells (Tregs), in similar numbers as fetal human thymus (HU THY) grafts in immunodeficient mice receiving the same human CD34(+) cells. Peripheral T cells are specifically tolerant to the mouse and to the human and porcine donors, with robust responses to nondonor human and pig Ags. Specific tolerance is observed to pig skin grafts sharing the THY donor MHC. SW THY-generated peripheral Tregs show similar function, but include lower percentages of naive-type Tregs compared with HU THY-generated Tregs. Tregs contribute to donor-pig specific tolerance. Peripheral human T cells generated in SW THY exhibit reduced proportions of CD8(+) T cells and reduced lymphopenia-driven proliferation and memory-type conversion, accelerated decay of memory-type cells, and reduced responses to protein Ags. Thus, SW thymus transplantation is a powerful xenotolerance approach for human T cells. However, immune function may be further enhanced by strategies to permit positive selection by autologous HLA molecules.

Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)(+) T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable β chain (TCR-Vβ) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3(+) and total CD4(+) T cells showed restricted TCR-Vβ expression. The development of naive T cells and diverse CD4(+) TCR-Vβ repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vβ family expression was also observed in FoxP3(+) CD4(+) T cells. Interestingly, the percentages of each of the TCR-Vβ families expressed on FoxP3(+) and total CD4(+) T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vβ family became significantly similar between FoxP3(+) and total CD4(+) T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3(+) and FoxP3(-) T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3(+) and FoxP3(-) cells, and pretransplantation FoxP3(+) and FoxP3(-) clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3(+) T cells in cDGA, corresponding with immunological and clinical recovery.

Recovery of Thymic Epithelial Cells and T Cell Immune Reconstitution After Cord Blood Transplantation in Adults Depends On Angiogenesis and Neovascularization

Abstract 4175Umbilical cord blood transplantation (UCBT) is increasingly used as alternative source for allogeneic hematopoietic stem cell transplantation. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. A significant component of immune reconstitution after UCBT involves the thymic regeneration, which can be measured by T-cell receptor rearrangement excision circles (TRECs). TREC levels are a strong predictive factor for overall survival in adult recipients of UCBT. HSCT compromises thymopoiesis due to injury of the thymic microenvironment, particularly thymic epithelial cells (TEC). Human UCB is enriched in endothelial precursors that can sustain thymopoiesis in immunodeficient mice transplanted with human thymic grafts, where they engraft and promote neovascularization and wound healing. We hypothesized that thymic neovascularization induced by the UCB-derived endothelial progenitors might be a critical factor involved in the recovery of TEC, which subsequently support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether markers of angiogenesis and neovascularization ANG1 and VEGF were associated with TEC function as determined by assessment of IL-7 and SCF levels and with thymic regeneration as determined by TREC values and recovery of T cell subsets. 27 evaluable patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to transplantation and at 1, 2, 3, 6 and 12 months after UCBT. At various time points after UCBT, serum levels of IL-7 and SCF displayed a strong correlation (p<0.005) with the levels of ANG1 and VEGF indicating that functional recovery of TEC is associated and coincides with angiogenesis and neovascularization. In contrast, serum levels of IL-7 and SCF displayed a strong inverse correlation (p<0.001) with TREC, CD4+, CD8+ and Treg numbers suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on immature T cell progenitors resulted in differentiation and expansion of these immature T cells. Similarly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. Conversely, VEGF and ANG1 levels positively correlated (p<0.001) with CD4+, CD4+CD25+ and CD4+CD45RA+ numbers at various time points after UCBT. Our results are consistent with a model of TEC recovery supported by VEGF and ANG1, leading to production of SCF and IL-7 by TEC, and uptake of these cytokines by T cell progenitors resulting in their differentiation and expansion. Thus, T cell immune reconstitution after UCBT depends on TEC and is associated with indicators of angiogenesis. These sequential steps in the process of thymic regeneration might represent novel therapeutic targets for improvement of immune reconstitution after UCBT. Disclosures:No relevant conflicts of interest to declare.

Self-renewal of thymocytes in the absence of competitive precursor replenishment

Soon after transplantation of wild-type thymi into immunodeficient mice lacking functional T cell receptors, productive T cell development in the donor thymus ceases. This observation underlies one of the central dogmas of T cell biology: because thymocytes are seemingly short-lived, intrathymic T cell development depends on continuous import of lymphoid progenitors from the bone marrow. New work reinterprets the outcome of this classical experiment as being the result of competition for intrathymic niches specifically supporting the DN3 stage of early T cell development. Surprisingly, when this niche space is uncontested by immigrating host progenitors, development of T cells in the thymus grafts continues. These new findings suggest that early thymocytes do indeed have substantial self-renewing potential.

Thymus-autonomous T cell development in the absence of progenitor import

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

A Model of Isolated, Vascular Whole Thymus Transplantation in Nude Rats

BackgroundWe used a model of vascularized thymus lobes as a whole isolated organ transplantation in rats. Materials and MethodsMale Fisher rats (F344, RT11v1; n = 10) and male homozygous Rowett nude rats (rnu/rnu; n = 10) were used as donors and recipients, respectively. Both vascular lobes of the thymus as a whole isolated organ were heterotopically transplanted to the neck of recipients. The right common carotid artery of the donor thymus was anastomosed end-to-end to the homonymous artery of the recipient. The anterior vena cava and the left brachiocephalic vein of the donor thymus were anastomosed end-to-side to the right and left external jugular veins of the recipient, respectively. Histological examination was used to monitor graft viability; graft function was assessed using flow cytometry (FCM) and immunologic effects by skin grafts in vivo. ResultsAll recipients survived. Preparation of the donors and recipients took 35.6 ± 5.5 minutes and 60.3 ± 15.1 minutes, respectively. The blood supply to the thymus graft was patent. Histology of the thymus on postoperative days 14, 56, and 112 revealed viable grafts with preserved microarchitecture. FCM analysis showed 37.18 ± 11.1% CD3+ T cells at day 21 after transplantation. Skin grafts from F344 and Rowett rats survived 8–10 and more than 30 days, respectively, whereas all third-party Sprague Dawley grafts were rejected within 5 days. ConclusionWe developed a novel model of isolated, direct vascularized whole thymus transplantation in nude rats, in which both lobes of the fully vascularized thymus were harvested en bloc for successful transplantation.

Fetal thymus graft prevents age-related hearing loss and up regulation of the IL-1 receptor type II gene in CD4+ T cells

We found that rejuvenation of the recipient immunity by inoculation of young CD4+ T cells or a fetal thymus graft led to down regulation of the interleukin 1 receptor type II (IL-1R2) gene in CD4+ T cells and reduced age-related hearing loss and degeneration of the spiral ganglion in SAMP1 mice, a murine model of human senescence. Our studies on the relationship between age-related systemic immune dysfunctions and neurodegeneration mechanisms open up new avenues of treatment of neurosenescence, including presbycusis, for which there is no effective therapy.

Vitiligo post-greffe thymique chez un enfant porteur d’un déficit en Foxn1

Foxn1 transcription factor deficit leads to immune deficiency, with hair and nail abnormalities. We report the case of a patient also presenting localized leucoderma. A 3-year-old boy underwent thymus transplantation at the age of 9 months for Foxn1 deficiency. He had developed urticaria and autoimmune hypothyroidism after thymus grafting. On examination, he had universal non-scarring alopecia, nail changes (atrophy, partial onycholysis and longitudinal grooves) and leucoderma on both big toes. This is the first description of leucoderma occurring in a patient with Foxn1 deficiency, as well as the first report of this pigment abnormality following thymus transplantation. The pathogenic hypotheses discussed were post-graft vitiligo and leucoderma induced by Foxn1 deficiency.

Partial Tolerance Induced by Transplantation of Spatially Separated Thymuses: A Cue for T Cell Retolerization in Thymus Grafts

Thymus grafts made up of mixed syngeneic and xenogeneic thymus tissues could induce donor-specific tolerance to xenografts with no development of autoimmune syndrome (AIS). But the requirements for the simultaneous presentation of tissue antigens from both species in the process of T cell development in thymus grafts have not hitherto been defined. To do this, we setup a model in which xenothymus grafts from F344 rats were heterotopically implanted into BALB/c nude mice carrying syngeneic thymus grafts, and the grafts were either mixed together or spatially separately; next, we examined the induction of donor-specific tolerance, any pathological changes and the distribution of T lymphocytes. In contrast to the mixed thymus grafts, spatially separated thymus transplants could neither induce a long-term tolerance to skin grafts nor prevent AIS completely. (51) Cr-labelled cell-tracing experiments showed that mature peripheral T cells could re-enter into both kinds of thymus grafts, while the T cells isolated from the syngeneic thymus tended to concentrate in the xenothymus grafts. Hence, our data suggest that the immune tolerance induced by mixed thymus transplants could be partially reversed if the thymus tissues from donors and recipients were segregated by spatial telorism. The uneven recirculation of mature T cells might imply that T cells get retolerized within the thymus grafts.

Immune Reconstitution After Cord Blood Transplantation in Adults Depends on Activity of Thymic Epithelial Cells and Vascular Endothelial Elements,

Abstract 4075Umbilical cord blood (UCB) grafts are increasingly used as sources of hematopoietic stem cells. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. We recently determined that thymic regeneration as identified by T-cell receptor rearrangement excision circles (TREC) is a strong predictive factor for clearance of CMV viremia and for overall survival in adult UCBT recipients. We were able to detect TRECs by 100 days that was faster than in other adult UCBT studies reported previously, in which TRECs remained undetectable during the first year. A significant difference between our study and previous reports was the use of 2 UCB units in all our patients whereas only a single unit was used in the previous trials. Human UCB is enriched in endothelial precursors that sustain thymopoiesis in SCID mice transplanted with human thymic grafts where they engraft and promote neovascularization and wound healing. The loss of thymopoietic capacity after allogeneic stem cell transplantation results from injury of the thymic microenvironment, particularly of thymic epithelial cells (TEC). We hypothesized that thymic neovascularization induced by UCB-derived endothelial progenitors might be a critical factor promoting early recovery of TEC, which support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether thymic regeneration and immune reconstitution in adult recipients of double UCBT was associated with IL-7 and SCF levels and with activity of endothelial progenitors. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB units. GvHD prophylaxis was with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to and at 1, 2, 3, 6 and 12 months after UCBT. Serum levels of IL-7 displayed a 3-fold increase at 1 month after UCBT and gradually declined to the pre-transplant levels at 12 months. SCF peaked at 2 months after UCBT and gradually decreased to the pre-transplant levels by 12 months. Strikingly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with TREC, CD4, CD8 and Treg cell numbers at various time intervals after UCBT suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on the surface of immature T cell progenitors resulted in differentiation and expansion of these T cell populations. Moreover, there was a strong inverse correlation (p<0.001) of IL-7 and SCF levels with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. To determine whether TEC activity correlated with endothelial lineage engraftment and neovascularization, we assessed levels of serum thrombomodulin (TM), which is expressed by metabolically active vascular endothelial cells. Serum TM at 1, 2, 3 and 6 months strongly correlated (p<0.005) with the levels of IL-7 and SCF at various time points after UCBT. Thus, quantitative and qualitative T cell immune reconstitution after UCBT depends on IL-7 and SCF and is associated with activity of vascular elements. These results support the intriguing hypothesis that infusion of 2 UCB units might have a cell dosage effect on thymic regeneration and immune reconstitution similar to that seen with improved rates of hematologic engraftment in double versus single UCB recipients. Disclosures:No relevant conflicts of interest to declare.

Human Natural Regulatory T Cell Development, Suppressive Function, and Postthymic Maturation in a Humanized Mouse Model

CD4(+) regulatory T cells (Tregs) control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34(+) cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells and formation of secondary lymphoid organs, in NOD/SCID mice. Strong Ag-specific immune responses and homeostatic expansion of human T cells that are dependent on peripheral human APCs occur. We now demonstrate that FOXP3(+)Helios(+) "natural" Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen, and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, postthymic "naive" to "activated" phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.