Kendra L. Congdon

Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance

A fundamental question in hematopoietic stem cell (HSC) biology is how self-renewal is controlled. Here we show that the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation, can be uncoupled, and we identify Notch signaling as a key factor in inhibiting differentiation. Using transgenic Notch reporter mice, we found that Notch signaling was active in HSCs in vivo and downregulated as HSCs differentiated. Inhibition of Notch signaling led to accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo. Finally, intact Notch signaling was required for Wnt-mediated maintenance of undifferentiated HSCs but not for survival or entry into the cell cycle in vitro. These data suggest that Notch signaling has a dominant function in inhibiting differentiation and provide a model for how HSCs may integrate multiple signals to maintain the stem cell state.

Regulation of myeloid leukaemia by the cell-fate determinant Musashi

Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi–Numb signalling axis. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

A pilot study of IL-2Rα blockade during lymphopenia depletes regulatory T-cells and correlates with enhanced immunity in patients with glioblastoma.

Background Preclinical studies in mice have demonstrated that the prophylactic depletion of immunosuppressive regulatory T-cells (TRegs) through targeting the high affinity interleukin-2 (IL-2) receptor (IL-2Rα/CD25) can enhance anti-tumor immunotherapy. However, therapeutic approaches are complicated by the inadvertent inhibition of IL-2Rα expressing anti-tumor effector T-cells. Objective To determine if changes in the cytokine milieu during lymphopenia may engender differential signaling requirements that would enable unarmed anti-IL-2Rα monoclonal antibody (MAbs) to selectively deplete TRegs while permitting vaccine-stimulated immune responses. Methodology A randomized placebo-controlled pilot study was undertaken to examine the ability of the anti-IL-2Rα MAb daclizumab, given at the time of epidermal growth factor receptor variant III (EGFRvIII) targeted peptide vaccination, to safely and selectively deplete TRegs in patients with glioblastoma (GBM) treated with lymphodepleting temozolomide (TMZ). Results and Conclusions Daclizumab treatment (n = 3) was well-tolerated with no symptoms of autoimmune toxicity and resulted in a significant reduction in the frequency of circulating CD4+Foxp3+ TRegs in comparison to saline controls (n = 3)( p = 0.0464). A significant (p<0.0001) inverse correlation between the frequency of TRegs and the level of EGFRvIII specific humoral responses suggests the depletion of TRegs may be linked to increased vaccine-stimulated humoral immunity. These data suggest this approach deserves further study. Trial Registration ClinicalTrials.gov NCT00626015

Monoclonal antibody blockade of IL-2 receptor α during lymphopenia selectively depletes regulatory T cells in mice and humans.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

Wnt Signaling in Normal and Malignant Hematopoiesis

One of the most remarkable characteristics of stem cells is their ability to perpetuate themselves through self-renewal while concomitantly generating differentiated cells. In the hematopoietic system, stem cells balance these mechanisms to maintain steady-state hematopoiesis for the lifetime of the organism, and to effectively regenerate the system following injury. Defects in the proper control of self-renewal and differentiation can be potentially devastating and contribute to the development of malignancies. In this review, we trace the emerging role of Wnt signaling as a critical regulator of distinct aspects of self-renewal and differentiation, its contribution to the maintenance of homeostasis and regeneration, and how the pathway can be hijacked to promote leukemia development. A better understanding of these processes could pave the way to enhancing recovery after injury and to developing better therapeutic approaches for hematologic malignancies.

Activation of Wnt Signaling in Hematopoietic Regeneration

Hematopoietic stem cells (HSCs) respond to injury by rapidly proliferating and regenerating the hematopoietic system. Little is known about the intracellular programs that are activated within HSCs during this regenerative process and how this response may be influenced by alterations in signals from the injured microenvironment. Here we have examined the regenerating microenvironment and find that following injury it has an enhanced ability to support HSCs. During this regenerative phase, both hematopoietic and stromal cell elements within the bone marrow microenvironment show increased expression of Wnt10b, which can function to enhance growth of hematopoietic precursors. In addition, regenerating HSCs show increased activation of Wnt signaling, suggesting that microenvironmental changes in Wnt expression after injury may be integrated with the responses of the hematopoietic progenitors. Cumulatively, our data reveal that growth signals in the hematopoietic system are re‐activated during injury, and provide novel insight into the influence of the microenvironment during regeneration.

Divide and Conquer: How asymmetric division shapes cell fate in the hematopoietic system

A fundamental mechanism by which cells can give rise to daughters with different fates is via asymmetric division. During asymmetric division, a mother cell generates daughter cells that go on to adopt different fates because of differential segregation of cell fate determinants. Although originally characterized in invertebrates, asymmetric division has recently been shown to regulate cell fate decisions in the mammalian hematopoietic system, playing crucial roles in stem cell renewal, lymphocyte activation, and leukemogenesis. These discoveries have opened new doors to understanding how regulation of division pattern contributes to the normal development and function of the immune system as well as how its dysregulation can lead to cancer.

Humoral autoimmunity to basement membrane antigens is regulated in C57BL/6 and MRL/MpJ mice transgenic for anti-laminin Ig receptors.

Basement membrane proteins are targeted in organ-limited and systemic autoimmune nephritis, yet little is known about the origin or regulation of immunity to these complex extracellular matrices. We used mice transgenic for a nephrotropic systemic lupus erythematosus (SLE) Ig H chain to test the hypothesis that humoral immunity to basement membrane is actively regulated. The LamH-Cμ Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin α1. To determine the fate of transgene-bearing B cells in vivo, transgenic mice were outcrossed onto nonautoimmune B6 and SLE-prone MRL backgrounds and exposed to potent mitogen or Ag in adjuvant. In this work we demonstrate that transgenic autoantibodies are absent in serum from M6 and M29 lineage transgenic mice and transgenic B cells hypoproliferate and fail to increase Ig production upon exposure to endotoxin or when subjected to B cell receptor cross-linking. Administration of LPS or immunization with autologous or heterologous laminin, maneuvers that induce nonoverlapping endogenous anti-laminin IgG responses, fails to induce a transgenic anti-laminin response. The marked reduction in splenic B cell number suggests that selected LamH-Cμ H chain and endogenous L chain combinations generate autospecificities that lead to B cell deletion. It thus appears that SLE-like anti-laminin B cells have access to and engage a tolerizing self-Ag in vivo. Failure to induce autoimmunity by global perturbations in immune regulation introduced by the MRL autoimmune background and exposure to potent environmental challenge suggests that humoral immunity to nephritogenic basement membrane epitopes targeted in systemic autoimmunity is tightly regulated.

Epidermal growth factor receptor and variant III targeted immunotherapy

Immunotherapeutic approaches to cancer have shown remarkable promise. A critical barrier to successfully executing such immune-mediated interventions is the selection of safe yet immunogenic targets. As patient deaths have occurred when tumor-associated antigens shared by normal tissue have been targeted by strong cellular immunotherapeutic platforms, route of delivery, target selection and the immune-mediated approach undertaken must work together to maximize efficacy with safety. Selected tumor-specific targets can spare potential toxicity to normal tissue; however, they are far less common than tumor-associated antigens and may not be present on all patients. In the context of immunotherapy for high-grade glioma, 2 of the most prominently studied antigens are the tumor-associated epidermal growth factor receptor and its tumor-specific genetic deletion variant III. In this review, we will summarize the immune-mediated strategies employed against these targets as well as the caveats particular to these approaches.