Susanna Celli

Quantitative imaging of Plasmodium transmission from mosquito to mammal

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.

Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice.

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 Förster 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.

CD4 T cells integrate signals delivered during successive DC encounters in vivo

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-2Rα expression and IFN-γ production. Thus, repeated encounters with Ag-bearing DCs can occur in vivo and modulate CD4 T cell differentiation programs.

Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy

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.

The mechanism of anti-CD20–mediated B cell depletion revealed by intravital imaging

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.

Competition for antigen determines the stability of T cell–dendritic cell interactions during clonal expansion

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.

Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging

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.—Li, T., Bourgeois, J.‐P., Celli, S., Glacial, F., Le Sourd, A.‐M., Mecheri, S., Weksler, B., Romero, I., Couraud, P.‐O., Rougeon, F., and Lafaye, P. Cell‐penetrating anti‐GFAP VHH and corresponding fluorescent fusion protein VHH‐GFP spontaneously cross the blood‐brain barrier and specifically recognize astrocytes: application to brain imaging. FASEB J. 26, 3969–3979 (2012). www.fasebj.org

How many dendritic cells are required to initiate a T cell response

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.

Decoding the dynamics of T cell–dendritic cell interactions in vivo

Summary: 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.

Immunogenicity and tolerance following HIV-1/HBV plant-based oral vaccine administration

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.