Visualizing the functional diversification of CD8+ T cell responses in lymph nodes.

CD8(+) T cell responses generate effector cells endowed with distinct functional potentials but the contribution of early events in this process is unclear. Here, we have imaged T cells expressing a fluorescent reporter for the activation of the interferon-γ (IFN-γ) locus during priming in lymph nodes. We have demonstrated marked differences in the efficiency of gene activation during stable T cell-dentritic cell (DC) contacts, influenced in part by signal strength. Imaging the first cell division, we have demonstrated that heterogeneity in T cell functional potential was largely apparent as T cells initiated clonal expansion. Moreover, by analyzing the fate of single activated T cells ex vivo, we have provided evidence that these early differences resulted in clonal progenies with distinct functional properties. Thus, the early set of T cell-DC interactions in lymph nodes largely contribute to the heterogeneity of T cell responses through the generation of functionally divergent clonal progenies.

Distinct influences of peptide-MHC quality and quantity on in vivo T-cell responses.

The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency.

Cutting Edge: A dual role for type I IFNs during polyinosinic-polycytidylic acid-induced NK cell activation.

NK cells are cytotoxic lymphocytes that are most efficient at fulfilling their functions after a phase of priming provided by cytokines and/or accessory cells. Although type I IFNs are known to be important in this process, it remains unclear whether they act directly on NK cells or indirectly on accessory cells. We used adoptive transfer experiments and mixed bone marrow chimeras to dissect the requirement for type I IFN signaling in response to the dsRNA analog polyinosinic-polycytidylic acid. We demonstrate that optimal NK cell priming requires type I IFNs to signal on both NK cells and accessory cells. In the absence of IL-15, the residual NK cell activation was strictly dependent on cell-intrinsic IFNAR signaling in NK cells. Our results suggest that type I IFNs produced following viral infection simultaneously target accessory cells for IL-15 transpresentation and NK cells themselves and that these two pathways cooperate for NK cell priming.

Decoding the dynamics of T cell-dendritic cell interactions in vivo.

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.

Dynamic behavior of NK cells during activation in lymph nodes.

During infection, Toll-like receptor agonists induce natural killer (NK)-cell activation by stimulating dendritic cells (DCs) to produce cytokines and transpresent IL-15 to NK cells. Yet the cellular dynamics underlying NK-cell activation by DCs in secondary lymphoid organs are largely unknown. Here, we have visualized NK-cell activation using mice in which NK cells and DCs express different fluorescent proteins. In response to poly I:C or lipopolysaccharide, NK cells maintained a vigorous migratory behavior, establishing multiple short contacts with maturing DCs. Furthermore, mature antigen-loaded DCs that made long-lived interactions with T cells formed short-lived contacts with NK cells. The different behaviors of T cells and NK cells during activation was correlated with distinct calcium responses upon interaction with DCs. That NK cells become activated while remaining motile may constitute an efficient strategy for sampling local concentrations of cytokines around DCs in secondary lymphoid tissues.

Termination of T cell priming relies on a phase of unresponsiveness promoting disengagement from APCs and T cell division.

T cells are primed in secondary lymphoid organs by establishing stable interactions with antigen-presenting cells (APCs). However, the cellular mechanisms underlying the termination of T cell priming and the initiation of clonal expansion remain largely unknown. Using intravital imaging, we observed that T cells typically divide without being associated to APCs. Supporting these findings, we demonstrate that recently activated T cells have an intrinsic defect in establishing stable contacts with APCs, a feature that was reflected by a blunted capacity to stop upon T cell receptor (TCR) engagement. T cell unresponsiveness was caused, in part, by a general block in extracellular calcium entry. Forcing TCR signals in activated T cells antagonized cell division, suggesting that T cell hyporesponsiveness acts as a safeguard mechanism against signals detrimental to mitosis. We propose that transient unresponsiveness represents an essential phase of T cell priming that promotes T cell disengagement from APCs and favors effective clonal expansion.

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.

Cutting edge: cognate CD4 help promotes recruitment of antigen-specific CD8 T cells around dendritic cells.

The cellular orchestration underlying help provided by CD4 T lymphocytes to CD8 T cell responses is not fully understood. We documented that the formation of three-cell clusters occurred as soon as day 1 and relied on long-lasting CD4 and CD8 T cell interactions with dendritic cells (DCs). The influence of CD4 help on CD8 T cell differentiation could be observed as early as the second round of cell division. Importantly, our results identify a new facet to the phenomenon of CD4 help in which DCs, upon cognate interactions with CD4 T cells, increase their ability to attract/retain Ag-specific CD8 T cells. Our results support a model in which CD4 help operates rapidly, in part by favoring CD8 T cells recruitment around those DCs that are the most competent for priming.