DP Cells Research Articles

Role of KDM2B and Non-Canonical PRC1.1 As a Tumor Suppressor in T-ALL

Introduction: Polycomb repressive complexes (PRCs) play an important role for the transcriptional repression of their target genes through histone modification. KDM2B is a component of non-canonical PRC1.1 and has a role for the recruitment of the complex to the target gene loci. It has a zinc finger-CxxC (ZF-CxxC) domain which specifically binds to unmethylated sequences in CpG islands (CGIs) and deletion of the CxxC domain induces complete loss of KDM2B occupancy and removal of other PRC1.1 components from CGIs. Recent studies revealed that loss of function mutations of several PRC component genes such as EZH2, EED, SUZ12 and BCOR were frequently detected in human T-cell acute lymphoblastic leukemia (T-ALL), which suggested PRCs have a tumor suppressive role in T cell development. Our group have reported conditional knock out of Bcor, encoding a component protein of non-canonical PRC1.1, induced T-ALL in mice. However, it is still unknown how KDM2B and non-canonical PRC1.1 regulate T cell leukemogenesis. Therefore, we performed detailed analysis of mice deficient for Kdm2b ZF-CxxC domain (Kdm2bΔCxxC/ΔCxxC) specifically in hematopoietic cells.Methods: We used the conditional Kdm2b allele (Kdm2bfl) mice, which contains LoxP sites flanking Kdm2b exon 13 encoding the ZF-CxxC domain. To generate hematopoietic cell specific Kdm2b KO mice, we transplanted Kdm2bfl/fl;Cre-ERT total BM cells into lethally irradiated CD45.1+ recipient mice and deleted Kdm2b 4 weeks after transplantation by intraperitoneally injecting of tamoxifen.Results: During the observation period of 300 days, almost all of the Kdm2b KO mice developed lethal T-ALL. They showed thymomegaly and splenomegaly and presented infiltration of donor-derived leukemic cells into the bone marrow, spleen, thymus and peripheral blood. Flow cytometric analysis revealed that T-ALL cells were mainly CD4 and CD8-double positive (DP). Notch1 active mutations in exons 26, 27, 28 or 34 were found in over a half of the T-ALL cases, indicating the Notch1 activation could be a driver for leukemic transformation in this mouse model. RNA sequence analysis of the DP cells revealed activation of Myc, which plays a key role in the development of T-ALL, and their downstream target genes in Kdm2b-deficient T-ALL. ChIP sequence analysis of DP thymocytes expressing 3xFLAG-KDM2B confirmed the binding peaks of KDM2B at the promotor of Myc. Peak calling analysis of the ChIP sequence data revealed that KDM2B was mainly located at transcript start sites (TSS), where KDM2B was co-localized with H2AK119ub1 and H3K27me3 histone marks. In addition, ChIP sequencing of H3K4me3 revealed that the KDM2B target genes include more bivalent genes than non-target genes. We next compared histone modification status around TSS in WT and Kdm2b KO DP cells and Kdm2b-deficient T-ALL cells. Global levels of H2AK119ub1 were significantly decreased in T-ALL cells and the reduction was mainly observed at the promoters of KDM2B target genes. Direct target genes of NOTCH1 including Myc also lost H2AK119ub1 at their promotors in T-ALL cells. Peak calling analysis of KDM2B, BCOR and NOTCH1 ChIP sequence data revealed that their target genes were closely overlapped at the promotor region. Moreover, EZH2 binding peaks were also overlapped with those of KDM2B and NOTCH1, suggesting that non-canonical PRC1.1 and PRC2 cooperatively antagonize NOTCH1-mediated gene activation. KEGG pathway analysis of the genes with overlapping binding peaks among KDM2B, EZH2 and NOTCH1 showed significant enrichment of T cell receptor signaling, Notch1 signaling and cell cycle pathway, all of which play an important role for the development of T-ALL. Of interest, H3K27me3 levels of the common target genes of EZH2, KDM2B, and NOTCH1 were much lower than the EZH2-specific target genes, indicating that H2AK119ub1 plays a key role in the repression of NOTCH1 targets.Conclusions: Our findings suggest that KDM2B recruits non-canonical PRC1.1 at the promotor regions of NOTCH1 targets to restraint thymocytes from transformation in concert with PRC2. DisclosuresNo relevant conflicts of interest to declare.

Misregulation of the IgH Locus in Thymocytes.

Functional antigen receptor genes are assembled by somatic rearrangements that are largely lymphocyte lineage specific. The immunoglobulin heavy chain (IgH) gene locus is unique amongst the seven antigen receptor loci in undergoing partial gene rearrangements in the wrong lineage. Here we demonstrate that breakdown of lineage-specificity is associated with inappropriate activation of the Eμ enhancer during T cell development by a different constellation of transcription factors than those used in developing B cells. This is reflected in reduced enhancer-induced epigenetic changes, eRNAs, formation of the RAG1/2-rich recombination center, attenuated chromatin looping and markedly different utilization of DH gene segments in CD4+CD8+ (DP) thymocytes. Additionally, CTCF-dependent VH locus compaction is disrupted in DP cells despite comparable transcription factor binding in both lineages. These observations identify multiple mechanisms that contribute to lineage-specific antigen receptor gene assembly.

Abstract LB-053: Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system

Abstract Breast cancer stem cells (BCSCs) display plasticity allowing them to transition between quiescent mesenchymal- (M) and proliferative epithelial-like (E) states. This dynamic equilibrium of BCSCs in distinct M- and E-like states coordinately drives tumorigenesis, metastasis and treatment resistance. Despite the functional significance of CSC plasticity in tumor development and progression, tracing of BCSC state transitions under physiologic conditions is hampered by the lack of a functional dual fluorescent reporter to monitor state dynamics of BCSCs at single cell resolution. To this end, we developed a dual fluorescent reporter to assess EMT-MET state transitions of BCSCs in real-time. As E-BCSCs express high levels of ALDH1A3, we generated an ALDH1A3 fluorescent reporter system utilizing Crisper-Cas 9 mediated knockin of mCherry downstream of the ALDH1A3 promotor. To trace M-BCSCs that express high levels of CD44 and vimentin, we constructed a lentiviral reporter expressing GFP driven by a portion of the vimentin promotor that is specifically expressed in mesenchymal CD44+ cells. Dual reporter cells were generated by infection of ALDH1A3_mCherry expressing cells with the lentivirus expressing Vimentin_GFP. To assess stem cell characteristics of these reporter cells, we first examined their sphere-forming capacity. Both GFP-mCherry+ (mCherry+) and GFP+mCherry- (GFP+) cells have significantly higher capacity to form tumor spheres than GFP-mCherry- (DN) cells while the GFP+mCherry+ (DP) cells harbor the highest sphere-forming ability. To determine the relationship between cells expressing these reporters, we generated tumor spheres from single cells and examined the fluorescent pattern of resultant tumor spheres. While spheres generated from DP cells display mixed phenotypes of cells expressing mCherry and/or GFP, spheres derived from mCherry+ cells maintain mCherry+. Interestingly, spheres derived from GFP+ cells generated three distinct phenotypes with some spheres expressing strong GFP and weak mCherry fluorescence while others expressing strong mCherry but weak or no GFP signal. These distinct phenotypes of tumor spheres derived from GFP+ cells suggest that M-BCSCs possess capacity to transition to a dual (GFP+mCherry+) or epithelial-like (GFP-mCherry+) state in order to form tumor spheres at non-attachment culture conditions that are normally associated with increased oxidative stress. These results suggest that the DP cells are the most primitive, with the greatest tumor sphere forming capacity and multistate differentiation potential followed by the Vimentin_GFP+ cells, ALDH1A3_mcherry+ cells and DN cells. By time-lapse microscopy, we further monitored how metabolic/oxidative stress induced by glycolytic inhibitor 2DG or H2O2 affects BCSC state dynamics at single cell resolution. We found that oxidative stress promotes the transition of GFP+mCherry- M-BCSCs to GFP+mCherry+ (bipotent) and GFP-mCherry+ (E-like) BCSCs. In contrast, the antioxidant N-acetyl cysteine promoted the transition of bipotent and E-like BCSCs to a GFP+mCherry- M-like state. These studies demonstrate that the redox state of the tumor microenvironment regulates state transitions of BCSCs. This dual fluorescent reporter system should prove useful in monitoring CSC state transitions under more physiologically relevant in vivo conditions. Citation Format: Ming Luo, Michael Brooks, Joseph Burnett, Nathan Truchan, Duxin Sun, Max S. Wicha. Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-053.

Id Proteins Suppress E2A-Driven Invariant Natural Killer T Cell Development prior to TCR Selection.

A family of transcription factors known as E proteins, and their antagonists, Id proteins, regulate T cell differentiation at critical developmental checkpoints. Id proteins promote the differentiation of conventional αβ T cells and suppress the expansion of innate-like αβ T cells known as invariant natural killer T (iNKT) cells. However, it remains to be determined whether Id proteins differentially regulate these distinct lineage choices in early stages of T cell development. In this manuscript, we report that in Id-deficient mice, uninhibited activity of the E protein family member E2A mediates activation of genes that support iNKT cell development and function. There is also biased rearrangement in Id-deficient DP cells that promotes selection into the iNKT lineage in these mice. The observed expansion of iNKT cells is not abrogated by blocking pre-TCR signaling, which is required for conventional αβ T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, our study reveals a previously unappreciated role of E2A in coordinating the development of the iNKT lineage at an early stage, prior to their TCR-mediated selection alongside conventional αβ T cells.

Titrated extract of Centella asiatica increases hair inductive property through inhibition of STAT signaling pathway in three-dimensional spheroid cultured human dermal papilla cells.

Dermal papilla (DP) is a pivotal part of hair follicle, and the smaller size of the DP is related with the hair loss. In this study, we investigated the effect of titrated extract of Centella asiatica (TECA) on hair growth inductive property on 3D spheroid cultured human DP cells (HDP cells). Significantly increased effect of TECA on cell viability was only shown in 3D sphered HPD cells, not in 2D cultured HDP cells. Also, TECA treatment increased the sphere size of HDP cells. The luciferase activity of STAT reporter genes and the expression of STAT-targeted genes, SOCS1 and SOCS3, were significantly decreased. Also, TECA treatment increased the expression of the hair growth-related signature genes in 3D sphered HDP cells. Furthermore, TECA led to downregulation of the level of phosphorylated STAT proteins in 3D sphered HDP cells. Overall, TECA activates the potential of hair inductive capacity in HDP cells.

Gsdma3 regulates hair follicle differentiation via Wnt5a-mediated non-canonical Wnt signaling pathway.

Hair follicle is a mini-organ that consists of complex but well-organized structures, which are differentiated from hair follicle progenitor or stem cells. How non-canonical Wnt signaling pathway is involved in regulating hair follicle differentiation remains elusive. Here we showed that Wnt5a regulates hair follicle differentiation through an epithelial-mesenchymal interaction mechanism in mice. We first observed that Wnt5a is expressed in the epithelial and dermal papilla cells during hair follicle development and growth. For the upstream of Wnt5a, RT-PCR and immunohistochemistry staining showed that Wnt5a expression is significantly decreased in the Gsdma3-mutant mice in vivo. Overexpression of Gsdma3 results in a significantly increased expression of Wnt5a in the cultured epidermal cells in vitro. We also checked the downstream factors of Wnt5a by adenovirus-mediated overexpression of Wnt5a to the dermal papilla cells isolated from the mouse whisker. We found that overexpression of Wnt5a suppresses canonical Wnt signaling pathway effectors such as β-catenin and Lef1. In addition, genes involved in maintaining cell quiescent state are also significantly decreased in their expression to the DP cells which were treated by Wnt5a. Our study indicates that Wnt5a mediates epithelia-expressed Gsdma3 to influence DP cell behaviors, which in turn regulate hair follicle epithelia differentiation in mice.

Increased TCR signal strength in DN thymocytes promotes development of gut TCR\u03b1\u03b2(+)CD8\u03b1\u03b1(+) intraepithelial lymphocytes

CD4(+)CD8(+) “double positive” (DP) thymocytes differentiate into diverse αβ T cell sub-types using mechanistically distinct programs. For example, conventional αβ T cells develop from DP cells after partial-agonist T cell receptor (TCR) interactions with self-peptide/MHC, whereas unconventional αβ T cells, such as TCRαβ(+)CD8αα(+) intraepithelial lymphocytes (IELs), require full-agonist TCR interactions. Despite this, DP cells appear homogeneous, and it remains unclear how distinct TCR signalling instructs distinct developmental outcomes. Moreover, whether TCR signals at earlier stages of development, for example in CD4(−)CD8(−) double negative (DN) cells, impact on later fate decisions is presently unknown. Here, we assess four strains of mice that display altered TCR signal strength in DN cells, which correlates with altered generation of unconventional TCRαβ(+)CD8αα(+) IELs. FVB/n mice (compared to C57BL/6 animals) and mice with altered preTCRα (pTα) expression, both displayed weaker TCR signalling in DN cells, an inefficient DN-to-DP transition, and reduced contribution of TCRαβ(+)CD8αα(+) IELs to gut epithelium. Conversely, TCRαβ(+)CD8αα(+) IEL development was favoured in mice with increased TCR signal strength in DN cells. Collectively, these data suggest TCR signal strength in DN cells directly impacts on subsequent DP cell differentiation, fundamentally altering the potential of thymocyte progenitors to adopt conventional versus unconventional T cell fates.

CD117+CD44+ Stem T Cells Develop in the Thymus and Potently Suppress T-cell Proliferation by Modulating the CTLA-4 Pathway

BackgroundCD117 is expressed on double-negative (DN; CD4–CD8–) cells (Nat Rev Immunol 14:529–545; 2014), but whether it is expressed in other stages and its subsequent functions are unclear. We used an improved method of flow cytometry to analyze different populations of thymocytes (Sci Rep 4:5781; 2014). The expression of CD117 and CTLA-4 were directly assayed in the early stage of thymocytes.MethodsFlow cytometry was used to analyze different populations of thymocytes, and T-cell proliferation assays, RT-PCR, and real-time RT-PCR were used to characterize the stem cells and examine the function of CD44+CD117+ cells.ResultsIn DN cells, CD117 expression was greatest on CD44+CD25+ cells (DN2), followed by CD44+CD25– (DN1), CD44–CD25+ (DN3), and CD44–CD25– (DN4) cells. In thymocytes, CD117 expression was highest in DN cells, followed by single-positive (SP; CD4 or CD8) and double-positive (DP; CD4+CD8+) cells. Especially, CD117 expression was positively associated with CD44 and CTLA-4 expression. CTLA-4 expression was highest in DN cells, followed by SP and DP cells. CTLA-4 expression was positively associated with CD25, CD44, and Foxp3 expression. CD44+CD117+ T cells expressed more CTLA-4, which suppressed T-cell proliferation and blocked CTLA-4 to cause antibody-induced T-cell proliferation.ConclusionThese results suggest that CD44+CD117+ T cells are stem cells and a specific T-cell phenotype that initially develops in the thymus, but they do not progress through DN3 and DN4 stages, lack a DP stage, and potently suppress T-cell proliferation and modulate the CTLA-4 pathway.

Nab2 maintains thymus cellularity with aging and stress.

Thymic cellularity is influenced by a variety of biological and environmental factors, such as age and stress; however, little is known about the molecular genetic mechanisms that regulate this process. Immediate early genes of the Early growth response (Egr) family have critical roles in immune function and response to environmental stress. The transcription factors, Egr1, Egr2 and Egr3, play roles in the thymus and in peripheral T-cell activation. Nab2, which binds Egrs 1, 2, and 3 as a co-regulator of transcription, also regulates peripheral T-cell activation. However, a role for Nab2 in the thymus has not been reported. Using Nab2-deficient (KO) mice we found that male Nab2KO mice have reduced thymus size and decreased numbers of thymocytes, compared with age-matched wildtype (WT) mice. Furthermore, the number of thymocytes in Nab2KO males decreases more rapidly with age. This effect is sex-dependent as female Nab2KO mice show neither reduced thymocyte numbers nor accelerated thymocyte loss with age, compared to female WT littermates. Since stress induces expression of Nab2 and the Egrs, we examined whether loss of Nab2 alters stress-induced decrease in thymic cellularity. Restraint stress induced a significant decrease in thymic cellularity in Nab2KO and WT mice, with significant changes in the thymocyte subset populations only in the Nab2KO mice. Stress reduced the percentage of DP cells by half and increased the percentage of CD4SP and CD8SP cells by roughly three-fold in Nab2KO mice. These findings indicate a requirement for Nab2 in maintaining thymocyte number in male mice with age and in response to stress.

The Checkpoint for Agonist Selection Precedes Conventional Selection in Human Thymus

The thymus plays a central role in self-tolerance by preventing strongly self-reactive thymocytes from accumulating as naïve T cell receptor (TCR) αβ+ T cells in the periphery. The elimination of auto-reactive T cells from the naïve pool is in part mediated by deletion during conventional negative selection. Alternatively, self-reactive thymocytes can also be positively selected in response to strong TCR signals during agonist selection and functionally differentiate to innate TCRαβ+ T cells such as the CD8αα+ double negative (DN) T cells. How thymocytes discriminate between these opposite outcomes remains unclear.We identified a novel agonist-selected PD-1+ CD8αα+ subset of mature CD8+ T cells in human thymus. Using the same markers a similar population was also identified in cord blood at about the same frequency as TCRγδ+ cells. This population expresses high levels of Helios, indicative of strong TCR engagement, and displays an effector phenotype associated with agonist selection. Indeed, PD-1+CD8αα+ T cells exhibit innate production of IFN-γ and an elevated T-bet to Eomes ratio typical of effector CD8 T cells. These cells are CD62L-, CXCR3+ and Hobit high suggesting that these cells leave the thymus and home to the tissues. Interestingly, in vitro CD3/TCR stimulation of sorted early post-β-selection thymocyte blasts uniquely gives rise to this innate subset, whereas small CD4+CD8+ double positive precursors fail to survive strong TCR signals. The generation of the innate subset seems to arise also in vivo from early post-β-selection thymocyte blasts as these two populations have an identical TCRα repertoire: ex vivo isolated PD-1+CD8αα+ thymocytes are skewed for early 3’ TRAV and 5’ TRAJ rearrangements compared to conventional CD8 T cells. A similar skewing was found in early post-β-selection thymocyte blasts. As TCRα rearrangements are terminated by TCR engagement of agonist selection, this is strong evidence for a precursor progeny relationship.Together, we conclude that human CD8αα+ T cells are preferentially selected by strong TCR engagement on a subset of progenitors that express a full TCRαβ early on, leading to the generation of a post-selection T cell population with innate functional capacity and a markedly distinct TCR repertoire. These findings uncover the heterogeneity among DP precursors in their potential to survive strong selection signals and suggests that the decision making in the thymus to divert immature thymocytes to the agonist selection pathway occurs early before conventional selection of DP cells. We propose that progression through the immature thymic developmental program influences the outcome of TCR engagement with early post-β-selection thymocytes triggered by strong TCR signals preferentially giving rise to innate CD8αα+ T cells in humans. DisclosuresNo relevant conflicts of interest to declare.

Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis.

The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. Here, we found that the Dpp target gene sal is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. We further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, we demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology.

FBXL12 regulates T-cell differentiation in a cell-autonomous manner.

Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells including embryonic, adult tissue and cancer stem cells. The SCF(FBXL) (12) complex is an authentic ubiquitin ligase that targets ALDH3 for degradation. FBXL12 is essential for the differentiation of trophoblast stem cells into specific cell types in the placenta during mouse embryogenesis, but its physiological functions in adult tissues have remained unknown. We have now investigated the role of the FBXL12-ALDH3 axis in the thymus, in which FBXL12 was most abundant among adult mouse tissues examined. During T-cell differentiation, FBXL12 is most abundant in CD4(+) CD8(+) (DP) cells, with its expression declining as these cells differentiate into CD4(+) CD8(-) or CD4(-) CD8(+) (SP) cells. T cells of FBXL12-null mice manifested a differentiation block at the DP-SP transition that was associated with ALDH3 accumulation in DP cells. This differentiation block was also apparent in wild-type mouse recipients of FBXL12-null bone marrow transplants as well as in FBXL12-null fetal thymic organ culture, suggesting that it is a cell-autonomous phenomenon in the thymus rather than an indirect effect of altered systemic conditions. Our results thus indicate that, in addition to its role in placental development, the FBXL12-ALDH3 axis is required for maturation of undifferentiated thymocytes.

Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression.

Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL.

Chimeric Antigen Receptor Transgenic, T Cell Receptor/CD3 Negative Monospecific T Cells Generated from Cord Blood CD34 Positive Cells

Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T cells can be generated in vitro that do not express an endogenous T cell receptor to avoid alloreactivity causing graft versus host reactions.We compared carcino-embryonic antigen (CEA)- specific CARs of the first generation (intracellular CD3ζ signaling chain), of the 2nd generation (intracellular CD3ζ and CD28 signaling chain) and of the 3rd generation (intracellular CD3ζ, CD28 and OX40 signaling chain). CD34+ progenitor cells were isolated from human cord blood or postnatal thymus and subsequently transduced with one of the three green fluorescent protein (GFP)-encoding CAR constructs. Transduced cells were subsequently co-cultured on OP9DL1 in the presence of stem cell factor, Flt3-ligand and interleukin-7. Unlike T cell receptor transduced precursors (1), expansion was not enhanced by transduction of the chimeric receptor. Expansion was highest with first generation CARs whereas second and third generation CARs displayed only restricted expansion. Similar to T cell receptor transduced progenitors, CAR transduced cells show an accelerated differentiation during co-culture compared to the non-transduced cells: first committed CD5+ CD7+ T precursors appear, then CD4+ CD8+ double positive cells (DP) and finally CD1- CD27- single positive or double negative (DN) mature T cells. In cultures transduced with 2nd and 3rd generation CARs, few transduced cells passed through the proliferative DP pathway but rather differentiated to mature CD1- CD27- non-proliferative DN cells without passing through the DP stage. This phenomenon is responsible for the limited expansion seen with precursors transgenic for 2nd or 3rd generation CARs. However, in all cultures CAR+ DP cells were generated and, as shown for TCR transgenic cells (1), we were able to induce CEA specific maturation after co-culturing these DP cells with a cell line expressing CEA or by antibody-induced cross-linking of the CAR, giving rise to CD1- CD27+ matured cells. The observations described above are compatible with data obtained in mice showing that strong T cell receptor (TCR) activation during thymocyte differentiation inhibits the generation of DP cells and induces maturation to DN cells. Both the spontaneously and induced mature CAR+ cells were TCR and CD3 negative, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. Moreover, these cells lack NK marker expression (CD56, NKG2D) and show expression of T cell markers (CD5, CD7, CD2), confirming their T cell nature. In conclusion, the CAR+ CD3/TCR negative cells are T cells as these are derived from T cell precursors (CD5+, DP cells) and express various membrane and nuclear T cell markers.Mature CD1- CD27- CAR+ cells can be expanded to large cell numbers using T cell expansion protocols. They displayed cytokine production specific for CEA+ tumor lines as well as specific cytotoxicity. Moreover, the 2nd and 3rd generation CAR expressing cells showed increased specific cytokine production when compared to the first generation CAR expressing cells. These results show that the cord blood-derived CAR+ cells have potent functional activity similar to peripheral blood derived CAR+ T cells.We believe that these in vitro generated CAR+ cells developed from HLA-matched cord blood progenitors may be ideal as an adjunct to cord blood transplantation.(1) Snauwaert et al, Leukemia, 2014 DisclosuresNo relevant conflicts of interest to declare.

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

Bim expression in early DP thymocytes limits development of TCRαβ+DN thymocytes and CD8αα intestinal intraepithelial lymphocytes (HEM2P.243)

Abstract Thymocyte negative selection is thought to cull T cells with strong self-reactivity and maintain T cells with non-self-specificity. While initial work implicated Bim as a critical mediator of thymocyte negative selection, its role in the process has been controversial. Thus, we re-examined thymocyte development in mice with global and T cell specific loss of Bim. We found that global Bim-/- mice had a striking accumulation of DN4 thymocytes expressing high levels of surface TCR, which failed to generate DP cells in OP9-DL1 culture. Interestingly, CD4Cre-driven deletion of Bim promoted DN4 cells, but later, dLckCre-driven deletion of Bim did not. Combined, these data show that the aberrant DN4 cells in Bim-/- mice were once DP cells, but had lost co-receptor expression. Investigating the fate of these cells, we found that splenic DN T and gut CD8αα T cells were increased in Bim-/- mice. While IL-15 is critical for both DN/CD8αα T cells, the additional loss of Bim partially restored DN/CD8αα T cells in IL-15 deficient mice, suggesting that IL-15 signals antagonize Bim to promote DN T and CD8αα T cells. Finally, TCR or IL-15 signals were sufficient to promote expression of both CD8αα and the gut homing receptor CCR9 on splenic DN T cells. In conclusion, these data further delineate the role of Bim on thymocyte fate and DN/CD8αα T cell development; and show that dLckCre-Bimf/f mice can isolate the effects of Bim on peripheral T cell survival without affecting thymic selection.

PD-1+CD28H+ selectively identifies for superior tumor-reactive T lymphocytes in pancreatic cancer (TUM7P.1013)

Abstract Tumor-infiltrating lymphocytes (TILs) are enriched in tumor-specific T cells and have been used as a source for adaptive cell therapy (ACT) of cancer. TILs are a mixture of different T cell subsets with both anti-tumor and pro-tumor capacities. It is valuable to isolate potent tumor-reactive T cells while eliminating suppressor T cells from TILs to maximize anti-tumor immune response. CD28H is a newly discovered co-receptor of the B7 family that interacts with its ligand B7-H5 to costimulate human T cells. CD28H is constitutively expressed on native T cells. However, repetitive antigenic exposure induces the loss of CD28H expression in T cells. T cells that have lost CD28H have the phenotypic characteristics of terminally differentiated and replicative senescent cells. Here we characterized PBMCs and TILs from patients with pancreatic cancer based on the expression of CD28H and PD-1. We found that a subset of T cells, PD-1+CD28H+ (DP) cells, are exclusively enriched in TILs. Based on phenotypic and functional analysis, our preliminary results suggest that PD-1+CD28H+ cells represent a subset of superior tumor-reactive cells in TILs. By targeting these DP cells by CD28H agonistic mAb and/or with a PD-1 blocking antibody, we are expecting to reprogram TIL functions in human pancreatic cancer.

Impaired Th17 polarization of phenotypically naive CD4(+) T-cells during chronic HIV-1 infection and potential restoration with early ART.

BackgroundDepletion of mucosal Th17 cells during HIV/SIV infections is a major cause for microbial translocation, chronic immune activation, and disease progression. Mechanisms contributing to Th17 deficit are not fully elucidated. Here we investigated alterations in the Th17 polarization potential of naive-like CD4+ T-cells, depletion of Th17-commited subsets during HIV pathogenesis, and Th17 restoration in response to antiretroviral therapy (ART).ResultsPeripheral blood CD4+ T-cells expressing a naive-like phenotype (CD45RA+CCR7+) from chronically HIV-infected subjects receiving ART (CI on ART; median CD4 counts 592 cells/μl; viral load: <50 HIV-RNA copies/ml; time since infection: 156 months) compared to uninfected controls (HIV-) were impaired in their survival and Th17 polarization potential in vitro. In HIV- controls, IL-17A-producing cells mainly originated from naive-like T-cells with a regulatory phenotype (nTregs: CD25highCD127−FoxP3+) and from CD25+CD127+FoxP3− cells (DP, double positive). Th17-polarized conventional naive CD4+ T-cells (nT: CD25−CD127+FoxP3−) also produced IL17A, but at lower frequency compared to nTregs and DP. In CI on ART subjects, the frequency/counts of nTreg and DP were significantly diminished compared to HIV- controls, and this paucity was further associated with decreased proportions of memory T-cells producing IL-17A and expressing Th17 markers (CCR6+CD26+CD161+, mTh17). nTregs and DP compared to nT cells harbored superior levels of integrated/non-integrated HIV-DNA in CI on ART subjects, suggesting that permissiveness to integrative/abortive infection contributes to impaired survival and Th17 polarization of lineage-committed cells. A cross-sectional study in CI on ART subjects revealed that nTregs, DP and mTh17 counts were negatively correlated with the time post-infection ART was initiated and positively correlated with nadir CD4 counts. Finally, a longitudinal analysis in a HIV primary infection cohort demonstrated a tendency for increased nTreg, DP, and mTh17 counts with ART initiation during the first year of infection.ConclusionsThese results support a model in which the paucity of phenotypically naive nTregs and DP cells, caused by integrative/abortive HIV infection and/or other mechanisms, contributes to Th17 deficiency in HIV-infected subjects. Early ART initiation, treatment intensification with integrase inhibitors, and/or other alternative interventions aimed at preserving/restoring the pool of cells prone to acquire Th17 functions may significantly improve mucosal immunity in HIV-infected subjects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0164-6) contains supplementary material, which is available to authorized users.

Earlier-born secondary hair follicles exhibit phenotypic plasticity.

The mouse pelage is composed of four distinct hair types. The fact that the follicles that generate these hair types form in successive waves during late embryonic development suggested the model that distinct epigenetic states of the inductive mesenchyme fixed when the follicles are formed specify the distinctive hair morphologies. This model is inconsistent with the observation that many follicles produce different hair types in successive hair cycles. In this study, the characteristics of the hair follicles that switch between the production of different hair types were examined. These follicles were born earlier than those that do not switch between hair types and made longer hairs. They also expressed a higher level of Sox2 in the dermal papilla and had more DP cells per follicle. These observations are consistent with the hypothesis that different birthdates specify the potential of different follicles. However, rather than directly specifying hair type, birthdate correlates with three types: guard hairs, a plastic population that can make awl, auchene or zigzag hairs, and a population that normally makes only zigzag hairs. Although Sox2 expression levels in the DP identify this subset during the morphogenetic cycle, Sox2 expression is not a fixed epigenetic state specified when the follicle is first formed.