DN Thymocytes Research Articles

Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies

AbstractLMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arf tumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4+/CD8+ malignancies. Inactivation of the Arf tumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arf promoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arf promoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.

Chromatin conformations and contacts that support Tcra/d locus rearrangement (62.2)

Abstract The Tcra/d locus rearranges in DN thymocytes to assemble Tcrd genes and in DP thymocytes to assemble Tcra genes. We used 3D-FISH to show that the 3’ portion of the locus is contracted in both DN and DP thymocytes, whereas the 5’ portion is contracted in DN but decontracts in DP thymocytes. We proposed that the fully contracted conformation in DN thymocytes allows dispersed Vαs to be used in a single round of Vδ-Dδ-Jδ rearrangement, whereas the 3’-contracted, 5’-decontracted conformation in DP thymocytes allows for multiple rounds of Vα-to-Jα rearrangement initiating with 3' Vαs. For high resolution analysis, we used the 3C method to detect molecular interactions between different sites in the locus. The Tcra enhancer (Eα) activates the T early α promoter (TEA) to target 5’Jαs for initial rearrangement. Eα also activates proximal Vαs over 500 kb. We detected molecular interactions between Eα and TEA, between Eα and proximal Vαs, and between different proximal Vαs, in DP but not DN thymocytes. All pairwise interactions depended on Eα. With TEA deleted, Eα also interacted with downstream Jαs. We propose that Eα nucleates a chromatin hub that supports transcription and ordered Vα-to-Jα recombination. However, deletion of Eα does not impact Tcra/d locus 3’end contraction as measured by 3D-FISH. Thus, overall locus conformation is independent of Eα, but this conformation may facilitate Eα interactions with distant sites.

IL-18 and IL-12 promote development of early thymic progenitors and can substitute for IL-7 in short-term OP9-DL4 co-cultures (64.7)

Abstract Determining the most effective parameters for in vitro propagation of T cell precursors is critical for realizing the promise of many envisioned T cell-based immunotherapies. To this end, the consequences of culturing ETP and DN2 thymocytes with IL-18 and IL-1β in the context of the OP9-DL4 co-culture model were evaluated after inference of a hypothesized role for these molecules in thymopoiesis based on results obtained via a novel microarray/literature mining algorithm. Sort-purified mouse ETPs and DN2 thymocytes were cultured on OP9-DL4 stromal layers ±IL-18, IL-1β, IL-12 and/or IL-7 for one week. IL-18 and IL-12 alone promoted proliferation and differentiation of ETPs to the DN3a stage and these cytokines could compensate for absence of IL-7. When IL-18 was used in conjunction with IL-7, synergistic proliferation was observed. IL-1β proved to be deleterious to development and was not overcome by inclusion of IL-7. To evaluate the developmental potential of the derived pre-T cells, culture-generated DN3a-like cells were sort-purified then cultured for an additional 8 days. In these assays, the pre-T cells demonstrated differentiation to the DP stage. Overall, these results indicate that IL-18 and IL-12 can positively impact T cell development and should prove useful in strategies to augment in vitro T cell development in the OP9-DL4 system.

Therapeutic potential of zoledronic acid in enhancing immune function in aged mice (106.3)

Abstract Hematopoietic stem cells originate in the bone marrow and give rise to immune cells. Zoledronic acid (ZA) is a bisphosphonate shown to modulate skeletal and immune systems. Hip fracture patients treated with ZA had a significant increase in bone density and increased longevity over placebo. We hypothesize ZA treatment will promote bone density in aged mice which in turn will enhance immunity. Young/aged mice were treated with saline or ZA (IV 100ug/kg) and euthanized at 1, 3 and 6 months to determine the impact of ZA on immune homeostasis. Compared to control, ZA-treated young mice showed significant improvement in bone volume. ZA-treated aged mice showed a qualitative improvement in bone volume. Frequency of DN thymocytes and sjTREC were increased over control in ZA-treated aged mice, suggesting improved thymopoiesis. Potential of ZA for improving immunity was also explored. Mice were treated with ZA (3 mo), immunized with flu HA protein (IN, d0,7,14) and then challenged with PR8 influenza (d42). HA-specific Ab response and survival was assessed. No difference in response was observed with ZA treatment in young mice. ZA-treated aged mice however had a stronger HA-specific response over control, suggesting ZA enhanced host response to immunization. Survival of ZA-treated aged mice was also higher than control, suggesting a single infusion of ZA enhances immunity. Given these data, further investigation of ZA as a potential therapeutic for immune senescence is warranted.

Loss of p19Arf Increases the Self-Renewal Capacity of Immature Thymocytes That Over-Express LMO2

AbstractAbstract 4198LMO2 is a lim-only domain transcription factor that mediates protein-protein interactions in a multimeric complex containing LDB1, E47, Tal1 and either GATA1 or GATA2. It was discovered as a result of chromosomal translocations in T-cell leukemia; where it becomes abnormally regulated by T-cell receptor elements and is involved in approximately 9% of cases. LMO2 transgenic mice develop T-cell leukemia with a latency of between 10–12 months indicating that other co-operating genetic events are required. In recent X-SCID gene therapy trials 4 patients developed T-cell leukemia due to vector–induced activation of LMO2 gene expression. Two out of the 4 patients had loss of the CDKN2A locus with another having an activating insertion in BMI1, a known repressor of the CDKN2A locus. The CDKN2A locus encodes both p19Arf and p16INK4A, and p19Arf suppression is known to increase self-renewal in a variety of different stem cell systems. Because LMO2 has been shown to promote thymocyte self-renewal (Curtis et al, Science 2010); we investigated whether Arf loss could collaborate with LMO2 in regulating self-renewal of thymocyte progenitors. We utilized OP9-DL1 stromal cells that express the Notch ligand; Delta-ligand 1 which enables differentiation of immature bone marrow cells and thymocytes into mature T-cells. When cultured on OP9-DL1 stromal cells LMO2+Arf+/+ and LMO2+Arf-/- populations had a block in progression to mature T-cells at the DN2 stage of thymocyte differentiation. These DN2 thymocytes were sorted and replated onto fresh OP9-DL1 stromal cells and analysis 7 days later showed that both populations had 99% DN2 stage thymocytes, indicating that LMO2 was inducing a complete block in thymocyte differentiation. LMO2+ Arf-/- DN2 thymocytes had 3 fold more thymocytes than the Arf+/+ population 11 days after the thymocytes had been sorted. This indicates that Arf loss may be increasing the self-renewal of the DN2 population. We next transplanted either 2×105 LMO2+ Arf-/- or 2×105 LMO2 Arf+/+ DN2 thymocytes together with 2×105 WT bone marrow cells into 2 groups of 10 WT lethally irradiated syngenic recipients. After only three weeks LMO2+ Arf-/-(n=7) cells were detected in the thymi of recipient mice. Marked cells came to represent about 18% of total thymocytes between weeks 6 to 15 post-transplant, these LMO2+ Arf-/- thymocytes were able to differentiate into mature T cell lineages in the thymus. By week 18 the LMO2+ Arf-/- cells dramatically increased to 97% of total thymocytes indicating that they maybe a preleukemic pool. In contrast, no thymic repopulation with LMO2 Arf+/+ (N=7) cells was noted. Serial transplant experiments were performed by isolating mCherry+, LMO2+ thymocytes 12 weeks post transplant. Then 2×105 LMO2+ thymocytes were transplanted into secondary Rag2-/-γc-/- recipients. After 6 weeks the thymi of 2 secondary mice were analyzed and were 100% mCherry+, LMO2+ and contained mature T-cells. These LMO2+ Arf-/- thymocytes had the capacity to be serially transplanted demonstrating that Arf loss was contributing to the self-renewal potential of these LMO2+DN2 thymocytes. Comparing our data to the published result for LMO2+Arf+/+, a 100 fold less thymocytes were transplanted in our assay potentially explaining the lack of engraftment with our LMO2+Arf+/+ cells. Furthermore we observed activation of the Arf locus when these thymocytes were transplanted in vivo, indicating that somehow these LMO2+thymocytes were inducing Arf activation at later times after transplant. p19Arf expression in thymocytes has been reported to induce p53 dependent apoptosis (Miyazaki et al, Immunity 2008). This supports the concept that Arf loss contributes to the self-renewal potential of thymocytes in and above the context of LMO2 over-expression. The combination of an increase in self-renewal induced by LMO2 and loss of the gatekeeper function of self-renewal through p19Arf loss leads to acceleration in the pathogenesis of LMO2 induced T-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Critical Requirement of GABPα for Normal T Cell Development

GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Ralpha expression, GABP is critically required for TCR rearrangements and hence normal T cell development.

Basis for lineage-specific control of PTEN expression by Rpl22 (36.64)

Abstract Mutations in a number of ribosomal proteins impair erythropoiesis (e.g., Rps19 mutations in Diamond Blackfan Anemia); however, such mutations have not been reported to disrupt T cell development. We have shown that ablation of ribosomal protein L22 (Rpl22) results in a strikingly specific defect in T cell development, selectively arresting αβ lineage cells through translational de-repression of the tumor suppressor p53 while sparing cells of the γδ-lineage. We performed comparative analysis of Rpl22-/- αβ and γδ precursors to assess the basis for lineage restriction of the blockade. Initial studies revealed a marked decrease in expression of PTEN in mixed-lineage CD4-8- (DN) progenitors from Rpl22-/- mice. This result is paradoxical, as PTEN is a positive regulator of p53 expression, yet its expression is reduced in cells where p53 is elevated. We hypothesized that the reduction in PTEN and the increase in p53 were actually occurring in distinct populations within the DN pool. Indeed, we found that PTEN levels were not reduced in purified αβ-lineage DN3 thymocytes from Rpl22-/- mice, suggesting the loss of PTEN occurs specifically in γδ lineage cells. In agreement, real-time PCR analysis revealed that PTEN mRNA levels are reduced in Rpl22-/- γδ T cells. Taken together, these data indicate that one important factor contributing to the sparing of γδ lineage development in Rpl22-/- mice is the selective reduction in γδ lineage cells of PTEN expression, which destabilizes p53.

Auto-reconstitution of the T-cell compartment by radioresistant hematopoietic cells following lethal irradiation and bone marrow transplantation

In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells. Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell-derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell-dependent antibody response. Both thymus-derived and peripheral T-cell-derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell-dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7-dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny. We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post-bone marrow transplantation.

Thymic development beyond β-selection requires phosphatidylinositol 3-kinase activation by CXCR4

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection.

The HOX11/TLX1 Transcription Factor Oncogene Induces Chromosomal Aneuploidy in T-ALL.

Abstract 142T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of transcription factor oncogenes. TLX1/HOX11 was originally isolated from the recurrent t(10;14)(q24;q11) in T-ALL and is aberrantly expressed in 5% to 10% of pediatric and up to 30% of adult T-ALL cases. Tlx1 plays an important role during embryonic development and acts as a master transcriptional regulator necessary for the genesis of the spleen. TLX1 positive T-ALLs have a distinct gene expression profile resembling that of thymocytes blocked at the early double positive stage of development. This observation supports the hypothesis that aberrant expression of TLX1 contributes to the pathogenesis of T-ALL by interfering with critical transcriptional regulatory networks involved in cell proliferation, differentiation and survival during T-cell development. However, the identity of such oncogenic pathways and the mechanisms though which they operate are still largely unknown. In order to gain further insight into the mechanisms of transformation induced by TLX1, we generated a transgenic model of TLX1 induced T-ALL. In this model, a human TLX1 cDNA was expressed in developing thymocytes under the control of the proximal LCK promoter. TLX1 transgenic mice displayed a specific defect in T-cell development characterized by reduced thymic size and cellularity with increased apoptosis and a differentiation arrest at the CD4/CD8 double negative, CD25/CD44 double positive stage of differentiation (DN2 thymocytes). Long term follow up revealed that TLX1 transgenic mice develop tumors with a median latency of 29 weeks. TLX1 induced leukemias are characterized by increased thymic size and diffuse infiltration of bone marrow, spleen and peripheral organs by lymphoblasts expressing T-cell markers. Analysis of TCRβ expression and transplantation into isogenic recipients demonstrated that TLX1 tumors are clonal and transplantable. Microarray gene expression profiling of mouse and human T-ALLs showed that tumors from TLX1 transgenic mice have a gene expression signature that is highly related to that of human T-ALLs with aberrant expression of TLX1. Moreover, ChIP-on-chip analysis of promoters bound by TLX1 showed this genetic program is dominated by the downregulation of TLX1 direct target genes. Mutation profiling of T-cell oncogenes and array CGH analysis in mouse TLX1 tumors demonstrated the presence of cooperative mutations including Pten deletions, activating mutations in Notch1 and loss of Bcl11b. Strikingly, SKY analysis and array CGH revealed that 80% of TLX1 induced tumors had numerical chromosomal abnormalities including a high frequency of trisomy 15. Analysis of gene expression of ChIP-on-chip TLX1 direct target genes in double negative thymocytes from preleukemic mice showed downregulation of genes primarily involved in the control of chromosomal segregation during mitosis such as Bub1, Brca2, Chek1, Kntc1, Kif23, CenpE and Plk1. These data suggest that aberrant TLX1 expression directly promotes aneuploidy and contributes to T-cell transformation by interfering with the expression of genes responsible for chromosomal segregation during mitosis. Importantly, T-cell lymphoblasts from TLX1 transgenic mice failed to undergo a mitotic cell cycle arrest after treatment with taxol, a cell cycle inhibitor that interferes with microtubule remodeling during mitosis. Strikingly, karyotype analysis of a series of 59 pediatric T-ALLs demonstrated a high frequency of chromosomal gains and losses in TLX1 and TLX3 positive human T-cell tumors compared with other genetic subgroups of T-ALL (P <0.001). Thus, aberrant expression of TLX1 in T-cell precursors seems to impair the function of the mitotic checkpoint and facilitate the acquisition of chromosomal gains and losses during T-cell transformation. These results establish for the first time a mechanistic link between the activity of a leukemogenic transcription factor oncogene and the development of chromosomal aneuploidy in the pathogenesis of human leukemia. Disclosures:Ferrando:Merck, Pfizer: Research Funding.

Molecular Mechanisms of the Pre-TCR Alpha Chain (pTa) Enhancer Transcriptional Regulation by Notch1.

Abstract 3649Poster Board III-585The Notch1 pathway and pre-TCR signalling are critical regulators of thymocyte development that have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Although the pre-TCR is required for Notch-dependent T-ALL, the role of pre-TCR alpha (pTa) as a Notch1 target remains controversial. Previous work in our laboratory has shown that the pTa gene is a target gene of the E2A-HEB transcription factors and that the SCL and LMO1 or LMO2 oncogenic transcription factors inhibits E2A activity and pTa expression, thereby causing thymocyte differentiation arrest during the pre-leukemic stage. SCL and LMO1/2 are implicated in 25% of T-ALL cases and induce aggressive T-ALL in transgenic (tg) mice when inappropriately expressed in the thymus. To define the mechanism regulating pTa expression by Notch1 and SCL, we used pTa regulatory sequences as a tool. To this end, we have performed transcriptional assays by co-delivering a luciferase reporter gene construct driven by the 200 bp pTa enhancer sequence with or without SCL or Notch1 intracellular domain (N1ICD), the functional fragment of the Notch1 receptor. Sequence analysis of the pTa enhancer revealed the presence of four E boxes that are potential binding sites for E2A-HEB and a CSL consensus sequence that can recruit the CSL/RBP-Jk transcription factor. Transcriptional assays in double negative thymomas indicated that the integrity of E box 2 and 3 (that recruit E2A-HEB) are essential for pTa enhancer activity in DN thymocytes, confirming the importance of E2A-HEB for pTa expression in these cells. In contrast, the CSL consensus sequence is important but not essential for pTa enhancer activity in these cells, consistent with previous results based on a conditional Notch1 knock out. In the presence of N1ICD, pTa enhancer sequences are strongly activated, either in fibroblasts or in double negative T cell lines in a dose dependent manner. This activation requires the integrity of the CSL consensus sequence but not of the E boxes. Moreover, we found that N1ICD strongly overrides the inhibitory activity of SCL on the pTa enhancer. Inhibition by SCL requires the integrity of E2 and E3 but not the CSL, confirming that SCL inhibits E2A-HEB activity, without affecting N1ICD. Together, our observations indicate that the regulation of pTa enhancer activity by Notch1 and E2A/HEB occur though independent mechanisms. Disclosures:No relevant conflicts of interest to declare.

Accelerated thymic atrophy as a result of elevated homeostatic expression of the genes encoded by the TNF/lymphotoxin cytokine locus

TNF, lymphotoxin (LT)-alpha, LT-beta and LIGHT are members of a larger superfamily of TNF-related cytokines that can cross-utilize several receptors. Although LIGHT has been implicated in thymic development and function, the role of TNF and LT remains incompletely defined. To address this, we created a model of modest homeostatic overexpression of TNF/LT cytokines using the genomic human TNF/LT locus as a low copy number Tg. Strikingly, expression of Tg TNF/LT gene products led to profound early thymic atrophy characterized by decreased numbers of thymocytes and cortical thymic epithelial cells, partial block of thymocyte proliferation at double negative (DN) 1 stage, increased apoptosis of DN2 thymocytes and severe decline of T-cell numbers in the periphery. Results of backcrossing to TNFR1-, LTbetaR- or TNF/LT-deficient backgrounds and of reciprocal bone marrow transfers implicated both LT-alpha/LT-beta to LTbetaR and TNF/LT-alpha to TNFR1 signaling in accelerated thymus degeneration. We hypothesize that chronic infections can promote thymic atrophy by upregulating LT and TNF production.

Altered CD4/CD8 cell fate decision in the absence of MAZR due to redirected differentiation of MHC class I-restricted thymocytes (85.9)

Abstract Cell fate specifications during thymocyte differentiation are linked with the tightly controlled expression of CD4 and CD8. We have previously identified that the transcriptional regulator MAZR is an important regulator of the activation of the Cd8ab gene complex during the DN to DP transition. To investigate the role of MAZR in more detail, MAZR-/- mice were generated. MAZR-/- mice were smaller in size and were born at reduced mendelian frequency. MAZR-/- DN thymocytes did not show premature expression of CD8, indicating that the deletion of MAZR is not sufficient to activate CD8 expression in DN cells. However, variegated CD8 expression in DP thymocytes of Cd8 enhancer E8I,E8II doubly deficient mice was dramatically reduced in the absence of MAZR. In addition, we observed elevated CD4/CD8 ratios in thymocytes and in peripheral T cells of MAZR-/- mice. Using MHC class I-restricted TCR transgenic mice, we demonstrated that the altered CD4/8 ratio in MAZR-/- mice was, in part, due to redirected differentiation of a fraction of MHC-class I restricted thymocytes into the CD4 lineage. Thus, our data provide genetic evidence that MAZR controls CD8 expression during thymocyte development. In addition, our results may indicate that MAZR is part of the transcription factor network that controls CD4/CD8 cell fate decisions of DP thymocytes. Supported by the Austrian Science Fund (P19930, SFB-F2305 and START Program Y-163).

Interplay between pre-TCR, CXCR4 and laminin10/11 during thymic β selection (85.8)

Abstract The βselection checkpoint during thymocyte development is a key step when immature thymocytes at double negative 3 (DN3) stage are selected to progress to the DN4 stage and beyond. While the role for the pre-T cell receptor (pre-TCR) during βselection has been established, how other receptors contribute to βselection is not well understood. Here, we identify a previously unrecognized role for CXCR4 during βselection using mice with a conditional loss of CXCR4 expression in DN thymocytes. CXCR4 physically and functionally associates with the pre-TCR and influences βselection via at least three mechanisms: regulating steady-state localization of immature thymocytes within thymic subregions, providing survival signals, and influencing efficient thymocyte proliferation post-β selection. We also show co-localization of SDF-1α, laminin 10/11 and DN3 thymocytes at the subcortical thymic regions, where the βselection events are thought to take place. We characterize the adapter protein ShcA and ERK kinase as functionally relevant signaling molecules downstream of CXCR4/pre-TCR/integrins. These data identify CXCR4 as a novel costimulator during βselection that functions together with pre-TCR and integrin ligands to orchestrate thymic development. Taken together, these data identify a novel interplay between the pre-TCR, CXCR4 and integrin receptors in regulating the βselection checkpoint.

Leptin protects against endotoxin-induced thymic remodeling (86.9)

Abstract Thymic atrophy is highly inducible by stress. While prolonged thymus atrophy can contribute to T cell deficiency, no treatment is available to enhance thymopoiesis during stress events. Leptin deficiency in mice results in chronic thymic atrophy and suppressed immunity, suggesting a role for leptin in regulating immune homeostasis. Specific roles however for intrathymic leptin signaling are currently undefined. We report here the novel finding that leptin receptor expression in the thymus is not present on developing thymocytes, but restricted to medullary thymic epithelial cells. We found leptin protected against endotoxin-induced acute thymic atrophy by preventing the loss of medullary thymic epithelial cells, protecting DP thymocytes from endotoxin-induced apoptosis, and increasing proliferation in DN thymocytes. We demonstrated that these thymoprotective effects are mediated through a leptin receptor isoform b-specific mechanism, and that leptin treatment increased thymic steady-state mRNA levels for IL-7. Taken together, our data suggested a novel intrathymic role for leptin in indirectly modulating thymocytes during thymic injury via a direct effect on medullary thymic epithelial cells. Overall, these findings support the use of leptin as a potential therapeutic to enhance T cell reconstitution in settings of stress-induced thymic atrophy. Supported by AG025150.

Real time multi-photon imaging reveals coordination between thymocyte development, location and migration through intrathymic microenvironments (85.6)

Abstract As they develop, CD4-CD8- (DN) thymocytes are found progressively from the corticomedullary junction towards the capsule. Following beta selection, CD4 and CD8 are expressed, generating double positive (DP) thymocytes that are found throughout the cortex. If a DP thymoycte expresses T cell receptor (TCR) and passes positive-selection, it becomes a single positive (SP) thymocyte, which is located in the medulla. Static images have revealed this intrathymic localization, suggesting a model in which DN thymocytes migrate directionally towards the capsule, while DP thymocytes move towards the medulla, which they enter as SP thymocytes. Using multi-photon imaging of thymocyte subsets in thick thymic slices, we directly test this model. Using thymuses expressing EGFP, we identify the precise microenvironments in which thymocyte subsets are localized. For the first time, we now image within the thymic medulla. We find that DN and DP thymocytes migrate without directional bias throughout the cortex, but DP velocities are influenced by cortical micorenvironmental cues. Interestingly, only SP thymocytes enter the medulla, where they migrate very rapidly. SPs also migrate rapidly in the cortex, where their movement is biased towards the medulla. Importantly, medullary entry is strictly dependent on G-protein-coupled receptor activity, though CCR7 signaling cannot entirely account for this requirement. L.I.R.E is a Leukemia and Lymphoma Society Special Fellow

AGAPE, a new T cell specific Grb2 binding protein that plays a critical role in T cell development (85.7)

Abstract We report here the identification of a T-cell lineage protein designated AGAPE (ä-gä-pa; Another Grb2-Associated Protein Enigma). AGAPE contains a proline rich sequence that associates with the adaptor protein Grb2. It lacks any consensus catalytic domains but contains a nuclear localization sequence required for its translocation to the nucleus. AGAPE deficient mice were generated by homologous recombination in ES cells. AGAPE-/- mice have normal numbers of DN and DP thymocytes but markedly reduced numbers of CD4 SP and CD8 SP thymocytes and pheripheral T cells. Positive selection is dramatically reduced in both class II- and class I-restricted TCR transgenic AGAPE-/- mice. The proportion of CD4+CD8lo thymocytes was reduced in AGAPE-/- MHCII-/- mice suggesting that AGAPE plays and important role at the DP to SP transition stage. Interestingly, TCR induced activation responses are unaffected in AGAPE-/- thymocytes indicating that the developmental block is not due to a defect in signaling downstream of the TCR? Although DP thymocytes from AGAPE-/- mice show enhanced susceptibility to antibody-induced cell death, the introduction of the Bcl-2 transgene did not rescue T cell development. Expression of the lineage specific transcription factors ThPok and Gata-3 (but not Tox or Runx1/3) was reduced in CD69+ thymocytes purified from AGAPE-/- mice. However, enforced expression of ThPok or Gata-3 did not rescue CD4 SP development in AGAPE-/- mice. Thus, AGAPE is a new T cell specific nuclear protein that has a critical function during late thymocyte selection prior to CD4/CD8 lineage specification.

A streamlined method for rapid and sensitive chromatin immunoprecipitation

We report a streamlined procedure to efficiently carry samples from chromatin to qPCR-compatible DNA in as little as 4 h. We use this streamlined ChIP to quantify histone H3 modifications at active (cad) and repressed (T early alpha) promoters in a Rag1-deficient pro-T cell line after 1–2 h IP. We further show that the protocol readily quantified histone modifications in chromatin from 104 Rag-deficient DN thymocytes. Taken together, these data outline a simple, cost-effective procedure for efficient ChIP analysis.

Tyrosine Kinase Inhibition with Sunitinib Facilitates Thymic Engraftment by Modulating Thymic Niche Accessibility

Stable mixed chimerism is likely to be sufficient for clinical benefit when HSCT is undertaken for benign disease. However, potential short and long term morbidities of current ablative or non-myeloablative conditioning regimens present an unfavorable risk:benefit ratio when HSCT is undertaken in this setting. We sought to develop an entirely non-cytotoxic approach to achieve stable mixed chimerism by modulating accessibility of thymic niches. Previous work identified occupancy of the double negative (DN)3 niche (CD3-4-8-44-25+) as a primary barrier to thymic engraftment based upon evidence that IL-7Rα−/− and γc−/− mice (who have an accessible DN3 niche) are receptive to thymic engraftment, whereas RAG−/− mice (who have a full DN3 niche) are resistant to thymic engraftment. Our results are consistent with this paradigm, since transfer of 5 × 106 TCD BM cells from WT into IL-7Rα−/− mice vs RAG−/− mice showed higher rates of thymic chimerism (mean donor % 83±8 vs 3±3 respectively, p=0.001). In an attempt to “open” the DN3 niche, we pre-treated RAG−/− recipients of WT BM with PC61, an anti-CD25 MoAb that diminished DN3 cell numbers (pre-44±11 × 105, post-14±1 × 105). Anti-CD25 therapy modestly enhanced donor chimerism (20±9%, n=12 vs 12±6%, n=10, p=NS) and significantly increased thymic engraftment (90±20 vs 41±11 x106 thymocytes, p=0.05) in anti-CD25 treated RAG−/− mice vs controls respectively. Importantly however, modulation of the DN3 niche via anti-CD25 treatment in RAG−/− mice did not increase thymic chimerism to the levels achieved in IL-7Rα−/− mice which have defects in the DN1/DN2 niches, implicating a role for DN1/DN2 niche accessibility as well. Indeed, RAG−/−γc−/− mice were receptive to thymic engraftment, with mean donor chimerism of 81.84±14% and a 13-fold increase in thymic cellularity after transplantation of WT BM cells without any prior conditioning. We postulated therefore that the limited efficacy of anti-CD25 alone when RAG−/− recipients were used was due to the fact that donor thymic engraftment is limited by accessibility to the entire range of DN1, DN2 and DN3 niches, while anti-CD25 selectively modulates the DN3 niche. Current models hold that growth factors critical for DN1 and DN2 thymocytes include SCF, FLT3L, and IL-7, among others. To test whether targeted pharmacotherapy could diminish the competitive advantage of resident DN1 and DN2 thymocytes and render the DN1/DN2 niche accessible, we treated RAG−/− recipients with Sunitinib, a receptor tyrosine-kinase (TK) inhibitor that inhibits c-kit and Flt-3, which signal SCF and FLT3L respectively. Sunitinib treatment of donors (60 mg/kg/d on Days -4, -3, -2, -1) followed by transfer of 5 × 106 TCD WT BM cells on Day 0 and 1 enhanced donor chimerism and thymic engraftment (10±4%, n=9 vs 2±0.7%, n=8 donor chimerism, p=0.01; 43±5 vs 13±1 × 106 thymocytes, p<0.0001) in Sunitinib vs. vehicle treated recipients, respectively. Combined anti-CD25 and Sunitinib therapy was superior to Sunitinib alone prior to transplantation of TCD WT BM (15±4%, n=5 vs 7±2%, n=5 donor chimerism, p=NS; 74±14 vs 25±6 × 106 thymocytes, p=0.02) We postulate that Sunitinib plus anti-CD25 renders DN1-DN3 thymic niches accessible to transferred thymic progenitors. This therapy is virtually non-toxic, and could provide new opportunities for HSCT for benign disease. Ongoing studies are underway to clarify effects of this regimen on marrow stem cell engraftment, durability of engraftment, and activity in a minor-mismatched model.

Thymic Involution Correlates with Severe Ulcerative Colitis Induced by Oral Administration of Dextran Sulphate Sodium in C57BL/6 Mice but not in BALB/c Mice

There is accumulating evidence to support the interactions between psychological stress and inflammatory bowel disease (IBD). In order to elucidate the relationship between psycoimmunological stress and IBD, we examined the alteration of immune system during the disease course of experimental Ulcerative colitis(UC)-model induced by dextran sulfate sodium (DSS). When C57BL/6 mice were treated with 4.5% DSS, they developed progressive weight loss. In contrast, the same treatment applied to BALB/c mice led to a small weight loss from which they soon recovered. Surprisingly, we found significant involution of the thymus and a reduction in the number of double positive thymocytes in DSS-treated C57BL/6 mice but not in DSS-treated BALB/c mice. Double negative thymocytes, especially DN1 (CD25-CD44+) and DN2 (CD25+CD44+) thymocytes, were relatively upregulated. The weights of spleens were slightly increased in both C57BL/6 and BALB/c mice following oral administration of DSS. In C57BL/6 spleens, both CD4 and CD8 single positive T cells gradually decreased (day 3), then recovered (day 14) after treatment. Because oral administration causes starvation, we examined the effects of starvation on the thymus and spleen. Although involution of thymus was observed both in starvation and DSS-treatment, the weight of spleen was reduced only in starvation. Also, the population changes in thymocytes in starvation was different from DSS-treatment. The administration of the steroid inhibitor RU486 partially reversed the thymic involution in C57BL/6 mice, thus DSS-treated UC might induce psycoimmunological changes partly through hypothalamic-pituitary-adrenal axis.