CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells.

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.

The ICOS-ICOSL pathway tunes thymic selection.

Negative selection of developing T cells plays a significant role in T-cell tolerance to self-antigen. This process relies on thymic antigen-presenting cells which express both self-antigens and cosignaling molecules. Inducible T-cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL). The ICOS signaling pathway plays important roles in shaping the immune response to infections, but its role in central tolerance is less well understood. Here we show that ICOSL is expressed by subsets of thymic dendritic cells and medullary thymic epithelial cells as well as thymic B cells. ICOS expression is upregulated as T cells mature in the thymus and correlates with T-cell receptor signal strength during thymic selection. We also provide evidence of a role for ICOS signaling in mediating negative selection. Our findings suggest that ICOS may fine-tune T-cell receptor signals during thymic selection contributing to the generation of a tolerant T-cell population.

Differential interferon-gamma production potential among naïve CD4+ T cells exists prior to antigen encounter.

Individual CD4+ T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4+ T-cell lineage. We show that ex vivo-activated CD5lo CD4+ T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5hi counterparts. In addition, a greater percentage of CD5lo effector CD4+ T cells produce IFNγ in both polyclonal and monoclonal CD4+ T-cell populations after antigen challenge in vivo. These results suggest that differential IFNγ production potential exists among CD4+ T cells prior to activation and independent of TCR affinity for foreign antigen.

Alterations in the Thymic Selection Threshold Skew the Self-Reactivity of the TCR Repertoire in Neonates.

Neonatal and adult T cells differ in their effector functions. Although it is known that cell-intrinsic differences in mature T cells contribute to this phenomenon, the factors involved remain unclear. Given emerging evidence that the binding strength of a TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine whether altered thymic selection influences the self-reactivity of the TCR repertoire during ontogeny. We found that conventional and regulatory T cell subsets in the thymus of neonates and young mice expressed higher levels of cell surface CD5, a surrogate marker for TCR avidity for self-pMHC, as compared with their adult counterparts, and this difference in self-reactivity was independent of the germline bias of the neonatal TCR repertoire. The increased binding strength of the TCR repertoire for self-pMHC in neonates was not solely due to reported defects in clonal deletion. Rather, our data suggest that thymic selection is altered in young mice such that thymocytes bearing TCRs with low affinity for self-peptide are not efficiently selected into the neonatal repertoire, and stronger TCR signals accompany both conventional and regulatory T cell selection. Importantly, the distinct levels of T cell self-reactivity reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in thymic selection in young versus adult mice skew the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates.

Hoxa cluster genes determine the proliferative activity of adult mouse hematopoietic stem and progenitor cells.

Determination of defined roles for endogenous homeobox (Hox) genes in adult hematopoietic stem and progenitor cell (HSPC) activity has been hampered by a combination of embryonic defects and functional redundancy. Here we show that conditional homozygous deletion of the Hoxa cluster (Hoxa(-/-)) results in a marked reduction of adult HSPC activity, both in vitro and in vivo. Specifically, proliferation of Hoxa(-/-) HSPCs is reduced compared with wild-type (WT) cells in vitro and they are less competitive in vivo. Notably, the loss of Hoxa genes had little impact on HSPC differentiation. Comparative RNA sequencing analyses of Hoxa(-/-) and WT hematopoietic stem cells (CD150(+)/CD48(-)/Lineage(-)/c-kit(+)/Sca-1(+)) identified a large number of differentially expressed genes, three of which (Nr4a3, Col1a1, and Hnf4a) showed >10-fold reduced levels. Engineered overexpression of Hoxa9 in Hoxa(-/-) HSPCs resulted in partial phenotypic rescue in vivo with associated recovery in expression of a large proportion of deregulated genes. Together, these results provide definitive evidence linking Hoxa gene expression to proliferation of adult HSPCs.

Hoxa9 collaborates with E2A-PBX1 in mouse B cell leukemia in association with Flt3 activation and decrease of B cell gene expression.

BACKGROUND: The fusion protein E2A-PBX1 induces pediatric B cell leukemia in human. Previously, we reported oncogenic interactions between homeobox (Hox) genes and E2A-PBX1 in murine T cell leukemia. A proviral insertional mutagenesis screen with our E2A-PBX1 B cell leukemia mouse model identified Hoxa genes as potential collaborators to E2A-PBX1. Here we studied whether Hoxa9 could enhance E2A-PBX1 leukemogenesis. RESULTS: We show that Hoxa9 confers a proliferative advantage to E2A-PBX1 B cells. Transplantation experiments with E2A-PBX1 transgenic B cells overexpressing Hoxa9 isolated from bone marrow chimeras showed that Hoxa9 accelerates the generation of E2A-PBX1 B cell leukemia, but Hoxa9 is unable to transform B cells alone. Quantitative-reverse transcriptase polymerase chain reaction analysis demonstrated a strong repression of B cell specific genes in these E2A-PBX1/Hoxa9 leukemias in addition to Flt3 activation, indicating inhibition of B cell differentiation in combination with enhanced proliferation. Overexpression of Hoxa9 in established E2A-PBX1 mouse leukemic B cells resulted in a growth advantage in vitro, which was also characterized by an enhanced expression of Flt3. CONCLUSIONS: we show for the first time that Hoxa9 collaborates with E2A-PBX1 in the oncogenic transformation of B cells in a mouse model that involves Flt3 signaling, which is potentially relevant to human disease.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Developing the right tools for the job: Lin28 regulation of early life T-cell development and function.

T cells comprise a functionally heterogeneous cell population that has important roles in the immune system. While T cells are broadly considered to be a component of the antigen-specific adaptive immune response, certain T-cell subsets display innate-like effector characteristics whereas others perform immunosuppressive functions. These functionally diverse T-cell populations preferentially arise at different stages of ontogeny and are tailored to the immunological priorities of the organism over time. Many differences in early life versus adult T-cell phenotypes can be attributed to the cell-intrinsic properties of the distinct progenitors that seed the thymus throughout development. It is becoming clear that Lin28, an evolutionarily conserved, heterochronic RNA-binding protein that is differentially expressed among early life and adult hematopoietic progenitor cells, plays a substantial role in influencing early T-cell development and function. Here, we discuss the mechanisms by which Lin28 shapes the T-cell landscape to protect the developing fetus and newborn. Manipulation of the Lin28 gene regulatory network is being considered as one means of improving hematopoietic stem cell transplant outcomes; as such, understanding the impact of Lin28 on T-cell function is of clinical relevance.

Investigating T Cell Receptor Signals In Situ by Two-Photon Microscopy of Thymocytes Expressing Genetic Reporters in Low-Density Chimeras.

T cell development is a dynamic process accompanied by extensive thymocyte migration, cellular interactions, and T cell receptor (TCR) signaling. In particular, thymic selection processes that ensure a functional, self-tolerant repertoire require TCR interactions with self-peptide presented by major histocompatibility complex molecules expressed by specialized thymic antigen-presenting cells. The quantity and quality of these TCR signals influence T cell fate. Two-photon microscopy, which enables live imaging of cells in intact tissue, has emerged as a powerful tool to gain insights into thymocyte migration and TCR signaling during T cell development in situ. Here we describe the generation of non-irradiated, low-density chimeric mice by neonatal injection of adult bone marrow engineered to express fluorescent TCR signaling reporters for imaging by two-photon microscopy. We also describe how the thymic lobes from chimeric mice are prepared for live imaging of thymocyte behavior and TCR signaling events. While we focus on imaging TCR signals associated with T cell development in the thymus, these techniques can also be adapted to study TCR signaling in mature T cells in peripheral lymphoid organs.

HOXA4 provides stronger engraftment potential to short-term repopulating cells than HOXB4.

Genes of the HOX4 paralog group have been shown to expand hematopoietic stem cells (HSCs). Endogenous expression of HOXA4 is 10-fold higher than HOXB4 in embryonic primitive hematopoietic cells undergoing self-renewal suggesting a more potent capacity of HOXA4 to expand HSC. In this study, we provide evidence by direct competitive bone marrow cultures that HOXA4 and HOXB4 induce self-renewal of primitive hematopoietic cells with identical kinetics. Transplantation assays show that short-term repopulation by HOXA4-overexpressing multilineage progenitors was significantly greater than HOXB4-overexpressing progenitors in vivo, indicating differences in the sensitivity of the cells to external signals. Small array gene expression analysis showed an increase in multiple Notch and Wnt signaling -associated genes, including receptors and ligands, as well as pluripotency genes, for both HOXA4- and HOXB4-overexpressing cells, which was more pronounced for HOXA4, suggesting that both HOX proteins may assert their affects through intrinsic and extrinsic pathways to induce self-renewal of primitive hematopoietic cells. Thus, HOXA4 increases short-term repopulation to higher levels than HOXB4, which may involve Notch signaling.

Inability of HOXB4 to enhance self-renewal of malignant B cells: favorable profile for the expansion of autologous hematopoietic stem cells.

Leukemic stem cells share self-renewal properties and slow proliferation with hematopoietic stem cells. Based on expression signatures, it has been suggested that these cells use the same molecular pathways for these processes. However, it is not clear whether leukemic stem cells also respond to factors known to enhance the self-renewal activity of hematopoietic stem cells. The transcription factor homeobox B4 (HOXB4) is known to induce expansion of mouse hematopoietic stem cells. The recombinant TAT-HOXB4 protein also expands human CD34+ cells. In this study we investigated whether overexpression of HOXB4 could increase leukemic initiating cell numbers, an issue that is crucial to its clinical usage. A transgenic mouse model for E2A-PBX1 induced pre-B acute lymphoblastic leukemia was used in combination with HOXB4 transgenic mice to test oncogenic interactions between HOXB4 and E2A-PBX1. The frequency of leukemic initiating cells retrovirally overexpressing HOXB4 was measured by transplantation at limiting dilution and evaluation of leukemia development in recipient mice. Moreover, human B cell lines were evaluated for their colony forming cell potential upon exposure to TAT-HOXB4 protein. Our data with the mouse models show that HOXB4 neither accelerates the generation of E2A-PBX1 B cell leukemia nor expands the number of leukemia initiating cells. Additionally, the growth or colony forming cell proportions of human B cell lines was not changed by HOXB4, suggesting that human B leukemic initiating cells are not affected by HOXB4.

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation.

HOXA4 induces expansion of hematopoietic stem cells in vitro and confers enhancement of pro-B-cells in vivo.

Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.

HoxA cluster is haploinsufficient for activity of hematopoietic stem and progenitor cells.

OBJECTIVE: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knockouts. Because HoxB genes are dispensable for hematopoiesis, and most HoxA genes are expressed an order of magnitude higher than other cluster genes in hematopoietic stem cell (HSC)-enriched populations, we hypothesize that maintenance of HoxA cluster expression is important for adult hematopoiesis and that global decrease of HoxA gene expression levels affects steady-state hematopoiesis. MATERIALS AND METHODS: Expression levels of HoxA cluster genes have been determined in primitive hematopoietic populations derived from adult mice using quantitative reverse transcriptase polymerase chain reaction. Furthermore, the functional effect of single allelic deletion of the entire HoxA cluster on hematopoietic cells was analyzed by competitive repopulation assays using HoxA(+/-) mice. RESULTS: We show that the HoxA cluster is predominantly expressed in long-term HSCs and that expression declines with progression to short-term HSCs and early progenitors in a quantifiable manner. Monoallelic deletion of the HoxA cluster caused a general increase in primitive hematopoietic cell populations, but a decrease in side populations. In addition exhaustion of B-cell progenitors with age was observed, resulting in less mature B cells. Moreover, bone marrow of HoxA(+/-) mice had a significant larger population of Mac1/Gr1 neutrophils, which might be caused by accelerated maturation of myeloid progenitors. Transplantation assays demonstrated that HoxA(+/-) HSCs were less competitive in long-term repopulation of myeloablated recipients, which appeared intrinsic to HSCs. CONCLUSION: These results show for the first time that maintenance of adult HSCs and progenitors is particularly sensitive to HoxA gene levels, suggesting a specific role for the HoxA cluster in primary regulation of definitive hematopoiesis.