Random migration and signal integration promote rapid and robust T cell recruitment

FWN – Publicaties zonder aanstelling Universiteit Leide

Biological second and third harmonic generation microscopy.

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Towards estimating the true duration of dendritic cell interactions with T cells

To initiate an adaptive immune response, T cells need to interact with dendritic cells (DCs), and the duration of these interactions plays an important role. In vitro and in vivo experiments have generally tried to estimate the required period of opportunity for T cell stimulation rather than the duration of individual T cell–DC interactions. Since the application of multi-photon microscopy (MPM) to living lymphoid tissues, the interactions between immune cells, as well as the duration thereof, can directly be observed in vivo. Indeed, long-lasting interactions between T cells and DCs were shown to be important for the onset of immune responses. However, because MPM imaging is typically restricted to experiments lasting 1 h, and because T cell–DC conjugates frequently move into and out of the imaged volume, it is difficult to estimate the true duration of interactions from MPM contact data. Here, we present a method to properly make such an estimate of (the average of) the distribution of contact durations. We validate the method by applying it to spatially explicit computer simulations where the true distribution of contact duration is known. Finally, we apply our analysis to a large experimental data set of T–DC contacts, and predict an average contact time of about three hours. However, we identify a mismatch between the experimental data and the model predictions, and investigate possible causes of the mismatch, including minor tissue drift during imaging experiments. We discuss in detail how future experiments can be optimized such that MPM contact data will be minimally affected by these factors

Conduits Mediate Transport of Low-Molecular-Weight Antigen to Lymph Node Follicles

SummaryTo track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4 + T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4 + T follicular helper (T FH) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid \u3b1-galactosylceramide (\u3b1GalCer). We previously observed that immunization of mice lacking CD4 + T cells (MHC II -/-) elicits specific IgG responses only when protein Ags are mixed with \u3b1GalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II -/- mice upon immunization with protein Ags mixed with \u3b1GalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II -/- mice display a typical CXCR5 hiprogrammed death-1 hiICOS hiBcl-6 hi T FH phenotype and induce germinal centers. The specific IgG response induced in MHC II -/- mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT FH cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT FH and CD4 + T FH cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses

Infection-stimulated infraosseus inflammation and bone destruction is increased in P-/E-selectin knockout mice

Infections of the dental pulp commonly result in infraosseus inflammation and bone destruction. However, the role of phagocytic leucocytes in the pathogenesis of pulpal infections has been uncertain. In this work we used P/E−/− selectin-deficient mice, which lack rolling adhesion of leucocytes to endothelium and mimic the human syndrome, leucocyte adhesion deficiency II (LAD-II), to test the hypothesis that phagocytic leucocytes protect against pulpal infection and subsequent periapical infraosseus bone resorption. P/E−/− mice and P/E+/+ wild-type controls were subjected to surgical pulp exposure, and both groups were infected with a mixture of pulpal pathogens including Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros and Streptococcus intermedius. Animals were killed after 20 days, and the extent of infraosseus bone destruction was quantified by histomorphometry. In two separate experiments, P/E−/− mice had significantly greater bone resorption than P/E+/+ controls. The increased bone destruction correlated with a twofold decrease in polymorphonuclear (PMN) infiltration into periapical inflammatory tissues of P/E−/− mice. P/E−/− mice had higher tissue levels of the bone resorptive cytokine, interleukin (IL)-1α. Tissue levels of IL-2, IL-4, IL-10, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were all higher in P/E−/− mice, but the increases were not statistically significant. Only IL-12 was higher in P/E+/+ mice, possibly reflecting a greater number of infiltrating monocytes in wild-type mice. These findings demonstrate that phagocytic leucocytes are protective in this model, and suggest that elevated expression of inflammatory cytokines is responsible for the observed bone destruction

Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes

The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation