Holger Kanzler

Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists

The identification of the antigen recognition receptors for innate immunity, most notably the Toll-like receptors, has sparked great interest in therapeutic manipulation of the innate immune system. Toll-like receptor agonists are being developed for the treatment of cancer, allergies and viral infections, and as adjuvants for potent new vaccines to prevent or treat cancer and infectious diseases. As recognition grows of the role of inappropriate Toll-like receptor stimulation in inflammation and autoimmunity, significant efforts have begun to develop antagonists to Toll-like receptors as well.

Somatic hypermutation in normal and transformed human B cells.

Summary: In the human, most IgM+IgD+ as well as CD5* peripheral blood B cells express unmutated V genes and thus can be assigned to a pre‐germinal centre (GC) stage of development. The memory B‐cell compartment generated in die GC reaction and characterized by cells bearing somatically mutated V‐region genes consists not only of class‐switched cells, but also of lgM‐only B cells and perhaps a subset of IgM+IgD+ B cells expressing the CD27 antigen. Comparison of the rearranged V‐region genes of human B‐cell lymphomas with those of the normal B‐cell subsets allows the identification of the progenitor cells of these tumours in terms of their stage of maturation. On this basis, most B‐cell on‐Hodgkin lymphomas, and in addition Hodgkin and Reed‐Stern berg (HRS) cells in Hodgkins disease (HD). are derived from B cells ac a GC or post‐GC stage of development. The mutation pattern indicates that the precursors of the tumour clones have been stringently selected for expression of a functional antigen receptor with one notable exception: HRS cells in classical (but: not lymphocyte‐predominant) HD appear to be derived from “crippled” GC B cells. Sequence analysis of rearranged V genes amplified from single tonsillar GC B cells revealed that the somatic hypermutation process introduces deletions and/or insertions into V‐region genes more frequently that indicated by previous investigations. Presumably, this feature of the hypermutation mechanism is often responsible for the generation of heavy chain disease, and also several types of chromosomal translocations of oncogenes into immunoglobulin loci in human B‐cell lymphomas.

Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors

Type 1 interferon-producing cells (IPCs), also known as plasmacytoid dendritic cell (DC) precursors, represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection, autoimmune SLE, and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3), IL-7, stem cell factor (SCF), macrophage-colony-stimulating factor (M-CSF), and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture, they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L, inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system, combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors, permits the generation of more than 10(9) IPCs from a single blood donor.

CpG Oligodeoxynucleotides Alter Lymphocyte and Dendritic Cell Trafficking in Humans

Purpose: CpG oligodeoxynucleotides (CpG-ODN) are being investigated as cancer vaccine adjuvants because they mature plasmacytoid dendritic cells (PDC) into potent antigen-presenting cells. CpG-ODN also induce PDC to secrete chemokines that alter lymphocyte migration. Whether CpG-ODN TLR signals enhance antigen-specific immunity and/or trafficking in humans is unknown. Experimental Design: We conducted a phase I study of CpG-ODN (1018 ISS) given as a vaccine adjuvant with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce T-cell immunity to a peptide vaccine from the tumor-associated antigen hTERT. Results: The adjuvant effect was limited; only 1 of 16 patients showed a high-frequency hTERT-specific tetramer CD8+ T-cell response. However, CpG-ODN induced marked, transient peripheral blood lymphopenia. Biopsies showed dense lymphocytic infiltration at the vaccine site clustered around activated PDC. In vitro, CpG-ODN-treated PDC induced T-cell migration, showing that CpG-ODN stimulation of human PDC was sufficient to chemoattract T cells. Conclusions: Our results show that (a) CpG-ODN with GM-CSF may not be an effective adjuvant strategy for hTERT peptide vaccines but (b) GM-CSF/CpG-ODN causes a PDC-mediated chemokine response that recruits T-cell migration to the peripheral tissues. These findings suggest a novel therapeutic role for targeted injections of CpG-ODN to direct lymphocyte migration to specific sites such as the tumor bed.

CpG-containing immunostimulatory DNA sequences elicit TNF-α–dependent toxicity in rodents but not in humans

CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

Virus overrides the propensity of human CD40L‐activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN‐γ and Th1 chemokine production

Depending on the activation status, plasmacytoid dendritic cells (PDC) and myeloid DC have the ability to induce CD4 T cell development toward T helper cell type 1 (Th1) or Th2 pathways. Thus, we tested whether different activation signals could also have an impact on the profile of chemokines produced by human PDC. Signals that induce human PDC to promote a type 1 response (i.e., viruses) and a type 2 response [i.e., CD40 ligand (CD40L)] also induced PDC isolated from tonsils to secrete chemokines preferentially attracting Th1 cells [such as interferon‐γ (IFN‐γ)‐inducible protein (IP)‐10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein‐1β/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation‐regulated chemokine/CCL17 and monocyte‐derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus‐activated PDC, and supernatants of CD40L‐activated PDC attracted memory CD4+ T cells, particularly the CD4+CD45RO+CD25+ T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN‐γ by PDC, which induces another Th1‐attracting chemokine monokine‐induced by IFN‐γ/CXCL9 and cooperates with endogenous type I IFN for IP‐10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L‐induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN‐γ.

Detection of identical Hodgkin-Reed Sternberg cell specific immunoglobulin gene rearrangements in a patient with Hodgkin's disease of mixed cellularity subtype at primary diagnosis and in relapse two and a half years later

BACKGROUND The malignant nature of Hodgkin-Reed Sternberg (H-RS) cells has been questioned due to their scarcity in lymphoma tissues. Recently, using micromanipulation of H-RS cells and single cell PCR evidence was obtained that H-RS cells represent a clonal B-cell population. In these studies H-RS cells were isolated from each one lymph node for a given case. In classical Hodgkins disease (HD) it thus could not be ruled out that H-RS cell clonality reflected a locally restricted clonal proliferation. We analysed biopsy specimens from a patient suffering from HD for the presence of clonally related H-RS cells at primary diagnosis and during relapse of the disease. MATERIALS AND METHODS In 1994 the H-RS cell line L1236 was generated from the peripheral blood of a patient suffering from a disseminating relapse of HD of mixed cellularity subtype. The patient had relapsed despite intensive treatment including high dose chemotherapy and autologous bone marrow transplantation. The clonal identity of this cell line with H-RS cells in situ was proven by amplifying identical Ig gene rearrangements of the cell line as well as of single H-RS cells picked from the patients bone marrow. Primers covering the CDR3 region were chosen from the H-RS cell specific VH1 gene rearrangement to detect H-RS cells of the identical clone by amplifying the rearranged VH1 genes in tissue samples obtained during disseminating relapsing disease and at primary diagnosis of HD in 1991. RESULTS The H-RS cell specific DNA sequence was detected in all affected tissues analysed including the cervical lymph node which has been exstirpated at primary diagnosis. CONCLUSION This finding indicates the existence of a clonal H-RS cell population during the first manifestation of HD and persistence and dissemination of this clone despite aggressive treatment. Thus, in the described case the malignant nature of H-RS cells defined by dissemination and recurrence of the identical H-RS cell clone in relapsing disease is proven.

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.