Christiane Ruedl

OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL Responses after virus infection.

OX40, a member of the TNF receptor superfamily, is expressed on activated T cells and implicated in stimulation of T cells and T-dependent humoral responses. We generated OX40-/- mice and found that the formation of extrafollicular plasma cells, germinal centers, and antibody responses was independent of OX40. After infection with LCMV and influenza virus, OX40-/- mice retain primary and memory cytotoxic T cell responses with normal expansion and decline of specific CTL. In contrast, CD4+ T cell proliferation and the number of IFN-gamma-producing CD4+ T cells were reduced in OX40-/- mice. Moreover, the number of CD4+ T cells infiltrating the lungs of influenza virus-infected OX40-/- mice was reduced. These results define a unique role of OX40 in the generation of optimal CD4+ T cell responses in vivo.

The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3

ABSTRACT Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

Nonmethylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects

DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags. CpGs are therefore among the most promising adjuvants known to date. Nevertheless, CpGs are characterized by two drawbacks. They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly. We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qβ is a simple and attractive method to reduce these two problems. CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability. In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity. In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8+ T cells (4–14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors. Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

Anatomical Origin of Dendritic Cells Determines Their Life Span in Peripheral Lymph Nodes

Dendritic cells (DCs) exhibit considerable heterogeneity in their anatomical location, surface phenotype, and functional properties. In this study, we demonstrate that peripheral lymph nodes contain at least four major, functionally separable, and independently derived, DC subsets, which can be clearly demarcated by their CD11c, CD40, and CD8 expression pattern. Surprisingly, all DCs derived directly from the bone marrow, the myeloid- and the lymphoid-related subsets, turned over fast with t1/2 of a couple of days. In contrast, DCs exported from the skin, both dermal and epidermal, accumulated 3- to 4-fold slower, turnover that is dramatically increased by cutaneous inflammation.

High-dimensional analysis of the murine myeloid cell system

Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning–aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage–colony stimulating factor GM-CSF (Csf2rb−/−), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system.

Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells

Macrophages are one of the most diverse cell populations in terms of their anatomical location and functional specialization during both homeostasis and disease. Although it has been shown in different fate mapping models that some macrophages present in adult tissues are already established during fetal development, their exact origins are still under debate. In the current study, we developed a fate mapping strain, based on the Kit locus, which allowed us to readdress the origins question. Different types of macrophages from various adult tissues were traced to their fetal or adult sources by inducing labeling in precursors at several time points either during fetal development or in adult mice. We show that all adult macrophages, resident or infiltrating, are progenies of classical hematopoietic stem cells (HSC) with the exception of microglia and, partially epidermal Langerhans cells, which are yolk sac (YS)-derived.

Critical Role for Activation of Antigen-Presenting Cells in Priming of Cytotoxic T Cell Responses After Vaccination with Virus-Like Particles

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo 51Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

The antigen dose determines T helper subset development by regulation of CD40 ligand

Although the amount of antigen and the strength of T cell stimulation have been suggested to regulate Th1 vs. Th2 polarization, it remains unclear how the antigen dose and the strength of signal is detected by the T cell and translated into differential cytokine production. Using co‐cultures of dendritic cells (DC) and ovalbumin (OVA)‐specific CD4+ T cells obtained from RAG‐2–u2009/u2009– DO11.10 mice, we show here that high‐dose antigen induced Th1 development by up‐regulation of CD40 ligand (CD40L), whereas low‐dose antigen stimulation failed to induce CD40L and promoted Th2 development. CD40‐CD40L interaction was essential for IL‐12 production by DC. In the absence, de novo IL‐4 production by T cells and autocrine Th2 development was induced. Furthermore, our results demonstrate that LFA‐1u2009/u2009ICAM interaction promotes Th1 differentiation by lowering the antigen dose required for CD40L up‐regulation. Thus, we propose that (1) peptide‐MHC density and (2) accessory molecules such as LFA‐1 determine T helper polarization by regulation of CD40L.

Cross-presentation of virus-like particles by skin-derived CD8– dendritic cells: a dispensable role for TAP

Virus‐like particles (VLP) induce efficient CTL responses although they do not carry any genetic information. Here, we analyzed MHC class I associated presentation of VLP‐derived CTL‐epitopes in vivo. After intradermal injection of VLP containing the immunodominant epitope (p33) of lymphocytic choriomeningitis virus (p33‐VLP), presentation of peptide p33 in draining lymph nodes was largely restricted to CD8– skin‐derived dendritic cells (DC). Surprisingly, and in contrast to findings with tumor cells, TAP1‐deficient DC and macrophages mediated efficient cross‐presentation of VLP‐derived p33 in vivo and in vitro. However, the ability of TAP1‐deficient DC to cross‐present p33‐VLP was reduced compared to wild‐type DC, indicating that in DC, both TAP‐dependent and TAP‐independent pathways were operative. In contrast, macrophages cross‐presented p33‐VLP normally in the absence of TAP. The TAP‐dependent pathway of cross‐presentation is therefore confined to DC while both macrophages and DC harbor the TAP‐independent pathway. In summary, the results show that VLP‐derived epitopes are cross‐presented by CD8– DC in vivo in a partial TAP‐independent fashion and highlight important differences in the processing machinery of DC versus macrophages.

Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines

BACKGROUND & AIMSnVirus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells.nnnMETHODSnWe used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients.nnnRESULTSnThe introduced TCRs were expressed on the cell surface, evidenced by Vβ and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells.nnnCONCLUSIONSnTCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.