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International audience; BACKGROUND:Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap.OBJECTIVE:We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved.METHODS:hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion.RESULTS:The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade.CONCLUSION:Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction.

Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

International audience; Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b(+) mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8(+) T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8(+) T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8(+) T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.

International audience; Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a curative treatment for hematologic malignancies relies on donor cytotoxic T lymphocyte (CTL)-mediated graft-versus-leukemia (GVL) effect. Major complications of HSCT are graft-versus-host disease (GVHD) that targets specific tissues and tumor relapses. However, the mechanisms dictating the anatomical features of GVHD and GVL remain unclear. Here, we show that after HSCT, CTLs exhibited different killing activity in distinct tissues, being highest in the liver and lowest in lymph nodes. Differences were imposed by the microenvironment, partly through differential PD-1 ligands expression, which was strongly elevated in lymph nodes. Two-photon imaging revealed that PD-1 blockade restored CTL sensitivity to antigen and killing in lymph nodes. Weak CTL activity in lymph nodes promoted local tumor escape but could be reversed by anti-PD-1 treatment. Our results uncover a mechanism generating an anatomical segregation of CTL activity that may dictate sites of GVHD and create niches for tumor escape.

International audience; T cells interact with dendritic cells (DCs) for periods lasting from minutes to hours. However, a causal link between the duration of this interaction and the efficiency of T cell activation has not been established in vivo. Employing intravital two-photon imaging, we manipulated T cell-DC interactions in real time and found that the first T cell-DC encounter often resulted in a long-lived interaction. Moreover, the cessation of T cell receptor-major histocompatibility complex signals promoted cellular dissociation, suggesting that antigen availability on DCs regulates contact duration. Finally, at least 6 hr of in vivo T cell-DC interaction were required for naive CD4 + T cells to undergo clonal expansion. These results establish the importance of prolonged T cell-DC interactions for efficient CD4 + T cell activation in vivo.

In the absence of thymopoiesis, T lymphocytes are nevertheless present, mainly in the gut epithelium. Ontogeny of the extrathymic pathway and the extent of its involvement in euthymic mice are controversial. These questions have been addressed by assessing the expression of recombinase activating gene (RAG) through the use of green fluorescent protein RAG2 transgenic mouse models. In athymic mice, T lymphopoiesis occurs mainly in the mesenteric lymph node and less in the Peyer's patches. Ontogenic steps of this lymphopoiesis resemble those of thymopoiesis, but with an apparent bias toward gamma delta T cell production and with a paucity of oligoclonal alpha beta T cells possibly resulting from a deficit in positive selection. Whether in athymic or euthymic mice, neither T intraepithelial lymphocytes (IEL) nor cryptopatch cells (reported to contain precursors of IEL) displayed fluorescence indicating recent RAG protein synthesis. Newly made T cells migrate from the mesenteric node into the thoracic duct lymph to reach the gut mucosa. In euthymic mice, this extrathymic pathway is totally repressed, except in conditions of severe lymphocytic depletion. Thus, in normal animals, all gut T IEL, including CD8 alpha alpha(+) cells, are of thymic origin, CD8 alpha alpha(+) TCR alpha beta(+) IEL being the likely progeny of double negative NK1-1(-) thymocytes, which show polyclonal V alpha and V beta repertoires.

International audience; Anti-CD20 Ab therapy has proven successful for treating B cell malignancies and a number of autoimmune diseases. However, how anti-CD20 Abs operate in vivo to mediate B cell depletion is not fully understood. In particular, the anatomical location, the type of effector cells, and the mechanism underlying anti-CD20 therapy remain uncertain. Here, we found that the liver is a major site for B cell depletion and that recirculation accounts for the decrease in B cell numbers observed in secondary lymphoid organs. Using intravital imaging, we established that, upon anti-CD20 treatment, Kupffer cells (KCs) mediate the abrupt arrest and subsequent engulfment of B cells circulating in the liver sinusoids. KCs were also effective in depleting malignant B cells in a model of spontaneous lymphoma. Our results identify Ab-dependent cellular phagocytosis by KCs as a primary mechanism of anti-CD20 therapy and provide an experimental framework for optimizing the efficacy of therapeutic Abs.

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium–T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri , via its type III secretion system, impairs the migration pattern of CD4 + T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4 + T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4 + T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.

T-cell activation in lymph nodes relies on encounters with antigen (Ag)–bearing dendritic cells (DCs) but the number of DCs required to initiate an immune response is unknown. Here we have used a combination of flow cytometry, 2-photon imaging, and computational modeling to quantify the probability of T cell–DC encounters. We calculated that the chance for a T cell residing 24 hours in a murine popliteal lymph nodes to interact with a DC was 8%, 58%, and 99% in the presence of 10, 100, and 1000 Ag-bearing DCs, respectively. Our results reveal the existence of a threshold in DC numbers below which T-cell responses fail to be elicited for probabilistic reasons. In mice and probably humans, we estimate that a minimum of 85 DCs are required to initiate a T-cell response when starting from precursor frequency of 10−6. Our results have implications for the rational design of DC-based vaccines.

International audience; Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

This study analyzes the mechanisms involved in xenotransplantation rejection between closely related species. Hamster hearts were transplanted heterotopically into both normal rats and rats previously sensitized by the transfusion of donor blood. Sequential ultrastructural and immunohistochemical analyses were performed on the grafts, spleens, and sera. The data obtained support the view that induced antibodies directed against the xenograft endothelium play a very important role in producing graft damage. Moreover, the demonstration of antibodies against myocyte determinants suggests that it is possible, in this particular model, that the antiendothelial antibodies are not the only ones involved in the injury process.

International audience; Transgenic tobacco plants expressing a HIV-1 polyepitope associated with hepatitis B (HBV) virus-like particles (VLPs) were previously described. It is demonstrated here that oral administration of these transgenic plants to humanized HSB mice to boost DNA-priming can elicit anti-HIV-1 specific CD8+ T cell activation detectable in mesenteric lymph nodes. Nevertheless, a significant regulatory T cell activation was induced in vivo by the vaccination protocols. The balance between tolerance and immunogenicity remains the main concern in the proof of concept of plant-based vaccine.

Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of natural killer (NK) cells and dendritic cells (DC) in the lymph nodes of live, anesthetized mice. Intravital two-photon imaging is a powerful tool to study the migration and cell interactions of immune cells such as NK and DC in physiological settings.

International audience; T lymphocytes receive activation signals during their encounters with antigen-bearing dendritic cells (DCs) in secondary lymphoid organs. With the recent application of two-photon imaging to visualize immune responses as they happen, the dynamics of T cell-DC interactions have been dissected in several mouse models. As we are integrating the results of these new studies, we are learning that the dynamics of T cell-DC interactions are regulated by multiple immunological parameters and, most importantly, that the spatiotemporal characteristics of these cell-cell contacts encode part of the T-cell fate.

International audience; Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of natural killer (NK) cells and dendritic cells (DC) in the lymph nodes of live, anesthetized mice. Intravital two-photon imaging is a powerful tool to study the migration and cell interactions of immune cells such as NK and DC in physiological settings.

CTLs have the potential to attack tumors, and adoptive transfer of CTLs can lead to tumor regression in mouse models and human clinical settings. However, the dynamics of tumor cell elimination during efficient T cell therapy is unknown, and it is unclear whether CTLs act directly by destroying tumor cells or indirectly by initiating the recruitment of innate immune cells that mediate tumor damage. To address these questions, we report real-time imaging of tumor cell apoptosis in vivo using intravital 2-photon microscopy and a Forster resonance energy transfer-based (FRET-based) reporter of caspase 3 activity. In a mouse model of solid tumor, we found that tumor regression after transfer of in vitro-activated CTLs occurred primarily through the direct action of CTLs on each individual tumor cell, with a minimal bystander effect. Surprisingly, the killing of 1 target cell by an individual CTL took an extended period of time, 6 hours on average, which suggested that the slow rate of killing intrinsically limits the efficiency of antitumor T cell responses. The ability to visualize when, where, and how tumor cells are killed in vivo offers new perspectives for understanding how immune effectors survey cancer cells and how local tumor microenvironments may subvert immune responses.

Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of lymph node T cells during either priming or tolerance, in live, anesthetized mice. Intravital imaging of T lymphocytes is a powerful tool to study the cellular orchestration of adaptive immune responses in physiological settings. This method should provide new insights into the regulation of lymphocyte migration and cell-cell interactions in various immunological contexts.

The regulation of T cell–dendritic cell (DC) contacts during clonal expansion is poorly defined. Although optimal CD4 T cell responses require prolonged exposure to antigen (Ag), it is believed that stable T cell–DC interactions occur only during the first day of the activation process. Here we show that recently activated CD4 T cells are in fact fully competent for establishing contact with Ag-bearing DC. Using two-photon imaging, we found that whereas prolonged interactions between activated T cells and Ag-bearing DCs were infrequent at high T cell precursor frequency, they were readily observed for a period of at least 2 days when lower numbers of T cells were used. We provide evidence that, when present in high numbers, Ag-specific T cells still gained access to the DC surface but were competing for the limited number of sites on DCs with sufficient peptide–MHC complexes for the establishment of a long-lived interaction. Consistent with these findings, we showed that restoration of peptide–MHC level on DCs at late time points was sufficient to recover interactions between activated T cells and DCs. Thus, the period during which CD4 T cells continue to establish stable interactions with DCs is longer than previously thought, and its duration is dictated by both Ag levels and T cell numbers, providing a feedback mechanism for the termination of CD4 T cell responses.

International audience; Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of lymph node T cells during either priming or tolerance, in live, anesthetized mice. Intravital imaging of T lymphocytes is a powerful tool to study the cellular orchestration of adaptive immune responses in physiological settings. This method should provide new insights into the regulation of lymphocyte migration and cell-cell interactions in various immunological contexts.

Comment in :- Malaria parasites up close. [Nat Med. 2006]; International audience; Plasmodium, the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.

International audience; The cellular mode of T cell priming in vivo remains to be characterized fully. We investigated the fate of T cell-dendritic cell (DC) interactions in the late phase of T cell activation in the lymph node. In general, CD4 T cells detach from DCs before undergoing cell division. Using a new approach to track the history of antigen (Ag)-recognition events, we demonstrated that activated/divided T cells reengage different DCs in an Ag-specific manner. Two-photon imaging of intact lymph nodes suggested that T cells could establish prolonged interactions with DCs at multiple stages during the activation process. Importantly, signals that are delivered during subsequent DC contacts are integrated by the T cell and promote sustained IL-2R ␣ expression and IFN-␥ production. Thus, repeated encounters with Ag-bearing DCs can occur in vivo and modulate CD4 T cell differentiation programs.

To prevent bystander destruction of healthy host tissues, cytotoxic CD8(+) T lymphocytes are fitted with specific receptors that direct their destructive forces specifically against chosen targets. We show here, however, that anti-H-Y monospecific, H-2(b-restricted MataHari CD8(+) T cells reject H-2(k) male skin grafts, with which they cannot directly interact. Such rejection is interferon-gamma-dependent and only occurs if the recipient endothelium expresses H-2(b). The findings suggest an alternate indirect effector pathway that requires processing and presentation of the donor H-Y antigen by recipient endothelium and have implications for both transplantation and autoimmune disease.

Biliary atresia is the most common indication for liver transplantation in infants and children, despite the advent of the Kasai operation. Coexisting anomalies, which have been noted in up to 27% of patients with biliary atresia, may form an association known as the "polysplenia syndrome," which includes (1) polysplenia, (2) midgut malrotation, (3) preduodenal portal vein, (4) absent prerenal inferior vena cava with azygos continuation, (5) situs inversus, (6) symmetric liver, (7) hepatic arterial anomalies, and (8) bilobed right lung with hyparterial bronchus. Two of 31 patients undergoing orthotopic liver transplantation for biliary atresia following failed portoenterostomy over the past 11 years manifested the polysplenia syndrome with absent prerenal inferior vena cava. The clinical course of these patients, constellation of anomalies, and technical adjustments required to perform liver transplantation are described. We do not believe that these complex congenital anomalies preclude liver transplantation.