Bryon D. Johnson

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A.

Summary.  Background: Previous studies from our laboratory have demonstrated that lineage‐targeted synthesis of factor VIII (FVIII) under the direction of the platelet‐specific integrin αIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. Objective: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII‐deficient (FVIIInull) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)‐based gene transfer cassette encoding 2bF8. Results: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV‐transduced BM survived tail clipping and we did not detected inhibitory or non‐inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIIInull secondary recipients demonstrated sustained platelet‐FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long‐term repopulating haematopoietic stem cells. Conclusions: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus‐mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.

Immunosuppressive Effects of Multiple Myeloma Are Overcome by PD-L1 Blockade

Multiple myeloma is an incurable plasma cell malignancy. Patients who fail conventional therapy are frequently treated with hematopoietic stem cell transplantation (HSCT), which results in reduced tumor burden, but the patients subsequently relapse from sites of chemotherapy-resistant disease. Using the 5T33 murine model of myeloma and a previously successful immunotherapy regimen consisting of autologous (syngeneic) HSCT and cell-based vaccine administration, we were unable to improve survival of myeloma-bearing mice. The 5T33 tumor line, similar to malignant plasma cells from myeloma patients, expresses high levels of programmed death receptor ligand-1 (PD-L1), which binds to the inhibitory receptor, PD-1. We observed that T cells from myeloma-bearing mice express high levels of PD-1, which has also been observed in patients with multiple myeloma. These PD-1(+) T cells were exhausted and produced IL-10. Based on these observations, we combined HSCT with whole-cell vaccination and PD-L1 blockade. Inhibition of the PD-1/PD-L1 pathway with HSCT and whole-cell vaccination increased the survival of myeloma-bearing mice from 0% to 40%. These data demonstrate a role for PD-L1 in suppressing immune responses to myeloma and suggest that blockade of this pathway may enhance immunotherapy for this disease.

Immune response modulation by curcumin in a latex allergy model

BackgroundThere has been a worldwide increase in allergy and asthma over the last few decades, particularly in industrially developed nations. This resulted in a renewed interest to understand the pathogenesis of allergy in recent years. The progress made in the pathogenesis of allergic disease has led to the exploration of novel alternative therapies, which include herbal medicines as well. Curcumin, present in turmeric, a frequently used spice in Asia has been shown to have anti-allergic and inflammatory potential.MethodsWe used a murine model of latex allergy to investigate the role of curcumin as an immunomodulator. BALB/c mice were exposed to latex allergens and developed latex allergy with a Th2 type of immune response. These animals were treated with curcumin and the immunological and inflammatory responses were evaluated.ResultsAnimals exposed to latex showed enhanced serum IgE, latex specific IgG1, IL-4, IL-5, IL-13, eosinophils and inflammation in the lungs. Intragastric treatment of latex-sensitized mice with curcumin demonstrated a diminished Th2 response with a concurrent reduction in lung inflammation. Eosinophilia in curcumin-treated mice was markedly reduced, co-stimulatory molecule expression (CD80, CD86, and OX40L) on antigen-presenting cells was decreased, and expression of MMP-9, OAT, and TSLP genes was also attenuated.ConclusionThese results suggest that curcumin has potential therapeutic value for controlling allergic responses resulting from exposure to allergens.

Programmed Death Receptor-1/Programmed Death Receptor Ligand-1 Blockade after Transient Lymphodepletion To Treat Myeloma

Early phase clinical trials targeting the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway to overcome tumor-mediated immunosuppression have reported promising results for a variety of cancers. This pathway appears to play an important role in the failure of immune reactivity to malignant plasma cells in multiple myeloma patients, as the tumor cells express relatively high levels of PD-L1, and T cells show increased PD-1 expression. In the current study, we demonstrate that PD-1/PD-L1 blockade with a PD-L1–specific Ab elicits rejection of a murine myeloma when combined with lymphodepleting irradiation. This particular combined approach by itself has not previously been shown to be efficacious in other tumor models. The antitumor effect of lymphodepletion/anti–PD-L1 therapy was most robust when tumor Ag–experienced T cells were present either through cell transfer or survival after nonmyeloablative irradiation. In vivo depletion of CD4 or CD8 T cells completely eliminated antitumor efficacy of the lymphodepletion/anti–PD-L1 therapy, indicating that both T cell subsets are necessary for tumor rejection. Elimination of myeloma by T cells occurs relatively quickly as tumor cells in the bone marrow were nearly nondetectable by 5 d after the first anti–PD-L1 treatment, suggesting that antimyeloma reactivity is primarily mediated by preactivated T cells, rather than newly generated myeloma-reactive T cells. Anti–PD-L1 plus lymphodepletion failed to improve survival in two solid tumor models, but demonstrated significant efficacy in two hematologic malignancy models. In summary, our results support the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel approach for improving the survival of patients with multiple myeloma.

CD25+ regulatory T cell inhibition enhances vaccine-induced immunity to neuroblastoma.

Evidence that CD4+CD25+ regulatory T (Treg) cells play a role in the progression of cancer continues to mount. There is a great deal of interest as to whether transient elimination or functional inhibition of these cells can improve the efficacy of immunotherapy for cancer. Our goals in this study were to test whether treatment of mice with anti-CD25 monoclonal antibody (mAb) (PC61) could induce rejection of a murine neuroblastoma, whether anti-CD25 treatment could increase tumor immunity when administered just before cell-based vaccination, and to learn how anti-CD25 treatment influences the vaccine-induced antitumor response. Treatment of mice with anti-CD25 mAb induced rejection of the mouse neuroblastoma, Neuro-2a, as 90% of anti-CD25-treated mice survived challenge with a lethal dose of tumor cells. In vivo anti-CD25 mAb treatment before the first of 2 weekly vaccines significantly improved the survival of tumor-vaccinated/challenged mice (75% vs. 33% survival), whereas antibody treatment before each of the 2 vaccines did not, suggesting that excessive treatment with anti-CD25 mAb interferes with activated antitumor effector cells. A detailed phenotypic analysis of tissues from anti-CD25-treated mice indicated that the antibody partially depletes CD4+Foxp3+ Treg cells (25% to 40%) in A/J mice, and that the antibody may inhibit the remaining cells by inducing loss of CD25 expression and blocking CD25 molecules, partially confirming recent data from other investigators. Importantly, we found that in vivo anti-CD25 mAb treatment significantly decreased the contribution of asialo GM1+ cells in the antitumor response. As we did not see a direct effect of anti-CD25 mAb on in vitro assays of immune cell function in spleen cells from treated animals, this indicates that inhibition of Treg cells amplifies the immune response in vivo in a manner that bypasses the requirement for innate immune activation, potentially mediated by natural killer cells, and allows for protective CD4+ and CD8+ cells to expand directly in response to cell-based vaccines.

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII

Summary.  von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within α‐granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet α‐granules. Isolex® selected CD34+ cells from human G‐CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B‐domain deleted form of human FVIII (BDD‐FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII‐transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor‐activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold‐conjugated antibodies colocalized FVIII and VWF within the α‐granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2–6 weeks post‐transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.

Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma

BackgroundMultiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.MethodsTo test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.ResultsTemporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.ConclusionsThese data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.

Rapamycin enriches for CD4+ CD25+ CD27+ Foxp3+ regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis

BACKGROUND CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells. METHODS CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity. RESULTS In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin. DISCUSSION The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.

Cloning and expression of Aspergillus fumigatus allergen Asp f 16 mediating both humoral and cell-mediated immunity in allergic bronchopulmonary aspergillosis (ABPA)

Background Aspergillus fumigatus, a ubiquitous fungus, is responsible for a number of lung disorders in atopic and non‐atopic individuals. Standardized, pure, and relevant allergens are desirable for reliable immunodiagnosis of the disease and to understand the structural and functional properties of these allergens and the role they play in causing ABPA.