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.

Serum elevation of B lymphocyte stimulator does not increase regulatory B cells in glioblastoma patients undergoing immunotherapy.

Regulatory B cells that secrete IL-10 (IL-10(+) Bregs) represent a suppressive subset of the B cell compartment with prominent anti-inflammatory capacity, capable of suppressing cellular and humoral responses to cancer and vaccines. B lymphocyte stimulator (BLyS) is a key regulatory molecule in IL-10(+) Breg biology with tightly controlled serum levels. However, BLyS levels can be drastically altered upon chemotherapeutic intervention. We have previously shown that serum BLyS levels are elevated, and directly associated, with increased antigen-specific antibody titers in patients with glioblastoma (GBM) undergoing lymphodepletive temozolomide chemotherapy and vaccination. In this study, we examined corresponding IL-10(+) Breg responses within this patient population and demonstrate that the IL-10(+) Breg compartment remains constant before and after administration of the vaccine, despite elevated BLyS levels in circulation. IL-10(+) Breg frequencies were not associated with serum BLyS levels, and ex vivo stimulation with a physiologically relevant concentration of BLyS did not increase IL-10(+) Breg frequency. However, BLyS stimulation did increase the frequency of the overall B cell compartment and promoted B cell proliferation upon B cell receptor engagement. Therefore, using BLyS as an adjuvant with therapeutic peptide vaccination could promote humoral immunity with no increase in immunosuppressive IL-10(+) Bregs. These results have implications for modulating humoral responses in human peptide vaccine trials in patients with GBM.

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.

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.

Multiepitope supramolecular peptide nanofibers eliciting coordinated humoral and cellular antitumor immune responses.

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8+ T cell, and CD4+ T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

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.

A conjoined universal helper epitope can unveil antitumor effects of a neoantigen vaccine targeting an MHC class I-restricted neoepitope.

Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.

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.

Effective effectors: How T cells access and infiltrate the central nervous system.

Several Phase II and III clinical trials have demonstrated that immunotherapy can induce objective responses in otherwise refractory malignancies in tumors outside the central nervous system. In large part, effector T cells mediate much of the antitumor efficacy in these trials, and potent antitumor T cells can be generated through vaccination, immune checkpoint blockade, adoptive transfer, and genetic manipulation. However, activated T cells must still traffic to, infiltrate, and persist within tumor in order to mediate tumor lysis. These requirements for efficacy pose unique challenges for brain tumor immunotherapy, due to specific anatomical barriers and populations of specialized immune cells within the central nervous system that function to constrain immunity. Both autoimmune and infectious diseases of the central nervous system provide a wealth of information on how T cells can successfully migrate to the central nervous system and then engender sustained immune responses. In this review, we will examine the commonalities in the efferent arm of immunity to the brain for autoimmunity, infection, and tumor immunotherapy to identify key factors underlying potent immune responses.

Promising vaccines for treating glioblastoma.

INTRODUCTION: Conventional therapies for glioblastoma  (GBM) typically fail to provide lasting antitumor benefits, owing to their inability to specifically eliminate all malignant cells. Cancer vaccines are currently being evaluated as a means to direct the adaptive immune system to target residual GBM cells that remain following standard-of-care treatment. AREAS COVERED: In this review, we provide an overview of the more noteworthy cancer vaccines that are under investigation for the treatment of GBM, as well as potential future directions that may enhance GBM-vaccine effectiveness. EXPERT OPINION: To date, no cancer vaccines have been proven effective against GBM; however, only a few have reached phase III clinical testing. Clinical immunological monitoring data suggest that GBM vaccines are capable of stimulating immune responses reactive to GBM antigens, but whether these responses have an appreciable antitumor effect on GBM is still uncertain. Nevertheless, there have been several promising outcomes in early phase clinical trials, which lend encouragement to this area of study. Further studies with GBM vaccines are, therefore, warranted.

CD27 stimulation unveils the efficacy of linked class I/II peptide vaccines in poorly immunogenic tumors by orchestrating a coordinated CD4/CD8 T cell response.

Despite their promise, tumor-specific peptide vaccines have limited efficacy. CD27 is a costimulatory molecule expressed on CD4+ and CD8+ T cells that is important in immune activation. Here we determine if a novel CD27 agonist antibody (αhCD27) can enhance the antitumor T cell response and efficacy of peptide vaccines. We evaluated the effects of αhCD27 on the immunogenicity and antitumor efficacy of whole protein, class I-restricted, and class II-restricted peptide vaccines using a transgenic mouse expressing human CD27. We found that αhCD27 preferentially enhances the CD8+ T cell response in the setting of vaccines comprised of linked class I and II ovalbumin epitopes (SIINFEKL and TEWTSSNVMEERKIKV, respectively) compared to a peptide vaccine comprised solely of SIINFEKL, resulting in the antitumor efficacy of adjuvant αhCD27 against intracranial B16.OVA tumors when combined with vaccines containing linked class I/II ovalbumin epitopes. Indeed, we demonstrate that this efficacy is both CD8- and CD4-dependent and αhCD27 activity on ovalbumin-specific CD4+ T cells is necessary for its adjuvant effect. Importantly for clinical translation, a linked universal CD4+ helper epitope (tetanus P30) was sufficient to instill the efficacy of SIINFEKL peptide combined with αhCD27, eliminating the need for a tumor-specific class II-restricted peptide. This approach unveiled the efficacy of a class I-restricted peptide vaccine derived from the tumor-associated Trp2 antigen in mice bearing intracranial B16 tumors. CD27 agonist antibodies combined with peptide vaccines containing linked tumor-specific CD8+ epitopes and tumor-specific or universal CD4+ epitopes enhance the efficacy of active cancer immunotherapy.

Hyaluronic acid based low viscosity hydrogel as a novel carrier for Convection Enhanced Delivery of CAR T cells.

Convection Enhanced Delivery (CED) infuses therapeutic agents directly into the intracranial area continuously under pressure. The convection improves the distribution of therapeutics such as those aimed at brain tumors. Although CED successfully delivers small therapeutic agents, this technique fails to effectively deliver cells largely due to cell sedimentation during delivery. To overcome this limitation, we have developed a low viscosity hydrogel (LVHydrogel), which is capable of retaining cells in suspension. In this study, we evaluated whether LVHydrogel can effectively act as a carrier for the CED of tumor-specific chimeric antigen receptor (CAR) T cells. CAR T cells were resuspended in saline or LVHydrogel carriers, loaded into syringes, and passed through the CED system for 5 h. CAR T cells submitted to CED were counted and the efficiency of delivery was determined. In addition to delivery, the ability of CAR T cells to migrate and induce cytotoxicity was evaluated. Our studies demonstrate that LVHydrogel is a superior carrier for CED in comparison to saline. The efficiency of cell delivery in saline carrier was only ∼3-5% of the total cells whereas delivery by the LVHydrogel carrier was much higher, reaching ∼45-75%. Migration and Cytotoxicity was similar in both carriers in non-infused samples but we found superior cytotoxicity in LVHydrogel group post-infusion. We demonstrate that LVHydrogel, a biodegradable biomaterial which does not cause acute toxicity on preclinical animal models, prevents cellular sedimentation during CED and presents itself as a superior carrier to the current carrier, saline, for the CED of CAR T cells.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.

A Supramolecular Vaccine Platform Based on α-Helical Peptide Nanofibers.

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of alpha-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multi-epitope nanofibers in a modular fashion.

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.

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 (T(Regs)) 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 T(Regs) 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 T(Regs) 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.

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.

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-Cmu Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin alpha1. 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-Cmu 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.

Kappa editing rescues autoreactive B cells destined for deletion in mice transgenic for a dual specific anti-laminin Ig.

We explored mechanisms involved in B cell self-tolerance in a double- and triple-transgenic mouse model bearing the LamH-C mu Ig H chain conventional transgene and a gene-targeted replacement for a functional V kappa 8J kappa 5 L chain gene. Whereas the H chain is known to generate anti-laminin Ig in combination with multiple L chains, the H + L Ig binds ssDNA in addition to laminin. Immune phenotyping indicates that H + L transgenic B cells are regulated by clonal deletion, receptor editing via secondary rearrangements at the nontargeted kappa allele, and anergy. Collectively, the data suggest that multiple receptor-tolerogen interactions regulate autoreactive cells in the H + L double-transgenic mice. Generation of H + LL triple-transgenic mice homozygous for the targeted L chain to exclude secondary kappa rearrangements resulted in profound B cell depletion with absence of mature B cells in the bone marrow. We propose that the primary tolerogen of dual reactive B cells in this model is not ssDNA, but a strongly cross-linking tolerogen, presumably basement membrane laminin, that triggers recombination-activating gene activity, L chain editing, and deletion.