Catherine Barbey

Background/Purpose: Plasmacytoid dendritic cells (pDC) produce large amounts of type I IFN (IFN-I), cytokines convincingly linked to systemic lupus erythematosus (SLE) pathogenesis. BIIB059 is a humanized mAb that binds BDCA2, a pDC-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. A first-in-human study was conducted to assess safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of single BIIB059 doses in healthy volunteers (HV) and patients with SLE with active cutaneous disease as well as proof of biological activity and preliminary clinical response in the SLE cohort. Methods: A randomized, double-blind, placebo-controlled, clinical trial was conducted in HV (n=54) and patients with SLE (n=12). All subjects were monitored for adverse events. Serum BIIB059 concentrations, BDCA2 levels on pDCs, and IFN-responsive biomarkers in whole blood and skin biopsies were measured. Skin disease activity was determined using the Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity (CLASI-A).Results: Single doses of BIIB059 were associated with a favorable safety and PK profile. BIIB059 administration led to BDCA2 internalization on pDCs, which correlated with circulating BIIB059 levels. BIIB059 administration in patients with SLE decreased expression of IFN response genes in blood, normalized MxA expression and reduced immune infiltrates in skin lesions, and decreased CLASI-A score. Conclusion: Single doses of BIIB059 were associated with favorable safety and PK/PD profiles, and robust target engagement and biological activity, supporting further development of BIIB059 in SLE. The data suggest that targeting pDCs may be beneficial for patients with SLE, especially those with cutaneous manifestations.

Background: Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. Objectives: To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. Methods: After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells in the induction of tolerance was analysed. Results: Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T_h1 and T_h2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4⁺CD25⁻ T cells, which strongly expressed membrane-bound transforming growth factor β (mTGFβ), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4⁺CD25⁺(Foxp3⁺) T cells during the process of tolerization was indispensable to CD4⁺CD25⁻ T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4⁺CD25⁻mTGFβ⁺ T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Conclusion: Nasal application of OVA in established asthma led to the induction of CD4⁺CD25⁻mTGFβ⁺ T cells with regulatory properties, able to confer protection upon allergen re-exposure.

Classical B lymphocyte activation is dependent on BCR cross-linking in combination with physical interaction with Th cells. Other B cell molecules that contribute to the activation are complement, cytokine, and TLRs recognizing specific pathogen-associated molecular patterns. Moraxella (Branhamella) catarrhalis is a common Gram-negative respiratory pathogen that induces proliferation in human IgD-expressing B cells independently of T cell help. The activation is initiated by the B cell superantigen Moraxella IgD-binding protein (MID) through a nonimmune cross-linking of IgD. However, IgD cross-linking alone is not sufficient to induce proliferation. In this study, we characterized the significance of TLRs in superantigen-dependent B cell activation using whole bacteria or rMID in the presence or absence of TLR ligands. IgD cross-linking by MID sensitized B cells obtained from children with tonsillar hyperplasia for mainly TLR9, whereas TLRs 1, 2, 6, and 7 were less important. The Moraxella-induced activation was inhibited when a dominant-negative TLR9 ligand was added. Interestingly, BCR-mediated endocytosis of whole Moraxella and degradation of live bacteria in naive B cells were observed with fluorescence, confocal, and transmission electron microscopy. This unique observation proved the strong intracellular TLR9 response as well as highlighted the Ag-presenting function of B cells. In conclusion, our findings suggest an important role of TLRs in the adaptive immune response and reveal novel insights into the T cell-independent B cell activation induced by bacteria.

Human cancer vaccines are often prepared with altered “analog” or “heteroclitic” antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity.

To monitor antigen-specific CD4⁺T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4⁺T cells, was performed. Grossly, twice as many TT-specific CD4⁺T-cell clones, ex vivo derived from the CCR7^+/−CD69⁺interleukin-2-positive (IL-2⁺) CD4⁺subsets, belonged to the central memory (T_CM; CD62L⁺CD27⁺CCR7⁺) compared to the effector memory population (T_EM; CD62L⁻CD27⁻CCR7⁻). After the boost, a predominant expansion of the T_CMpopulation was observed with more limited variations of the T_EMpopulation. TCR beta-chain-variable region (BV) spectratyping and sequencing confirmed a large concordance between most frequently expressed BV TCR-CDR3 from ex vivo-sorted CCR7^+/−CD69⁺IL-2⁺CD4⁺subsets and BV usage of in vitro-derived TT-specific CD4⁺T-cell clones, further demonstrating the highly polyclonal but stable character of the specific recall response to TT. Taken together, ex vivo flow cytometry analysis focused on the CCR7^+/−CD69⁺IL-2⁺CD4⁺subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28⁻) T cells strongly expressing granzyme/perforin, and two EM28⁺ subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28⁻-derived clones lysed target cells with high efficacy. In contrast, EM28⁺-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28⁺ conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8⁺ T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

The aim of T cell vaccines is the expansion of antigen‐specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine‐induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross‐reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross‐reactive potential of vaccine‐induced cells, we analyzed the reactivity of CD8⁺ T cells following vaccination of HLA‐A2⁺ melanoma patients with Melan‐A peptide, incomplete Freund's adjuvant and CpG‐oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan‐A‐reactive CD8⁺ T cells in vivo. A collection of predicted Melan‐A cross‐reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan‐A‐reactive CD8⁺ T cell clones. While Melan‐A‐reactive CD8⁺ T cells prior to vaccination are usually constituted of widely cross‐reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor‐reactive.

Summary: CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor‐α (TNF‐α) gene transcription in an epitope‐specific manner. To dissect the early signalling events leading to TNF‐α gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP‐70, CD3‐ζ, LAT and Vav. This was accompanied by down‐regulation of membrane‐associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP‐70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca²⁺ rise and phosphoinositide turnover. We conclude that down‐regulation of membrane‐associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope‐specific fashion, to unopposed tyrosine kinase activity and to the activation of the T‐cell receptor/CD3 complex signalling cascade, resulting in TNF‐α gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T‐cell functions.