Multi-Photon Visualization of T Cell-Dendritic Cell Interactions in Living Lymph Nodes

Hypothesis

Hypothesis

Goals:
Aim 1: To compare the dynamics of naive versus central memory CD8 T cell-interactions with dendritic cells (DC).

Aim 2: To characterize the dynamics of T cell-DC interactions leading to tolerance and compare these to T cell-DC interactions leading to immunity.

Aim 3: To visualize the effect of CD4 help during the priming of CD8 T cells.

Findings:
Our efforts for Aim 1 of our goals led to the unexpected observation that naive TCR transgenic P14 CD8 T cell priming by DC occurs in three distinct phases: transient serial encounters during a first activation phase that lasts several hours are followed by a second phase of stable contacts culminating in cytokine production, which transitions into a third phase of high motility and rapid proliferation (Mempel et al. 2004).

In light of this discovery, which could not have been predicted based on prior in vitro experiments of T cell activation, we focused further work on understanding the rules that govern T cell transition from one phase to the next. In particular, we set out to explore how and when T cells decide to switch from the brief serial encounre mode in phase one to long-lasting conjugation in phase two. Using DCs pulsed with different concentrations of altered peptide ligands for the P14 TCR, we generated evidence that the duration of phase one is inversely proportional to the antigen concentration and TCR affinity for the cognate peptide. This work was presented as an oral presentation at the 2006 AAI meeting in Boston (Henrickson et al. 2006).

We also explored how central memory cells respond to antigen-pulsed DC. Initially, we focused our studies on the bone marrow, because we discovered that both central memory CD T cells and circulating DC home avidly to that organ (Mazo et al. 2005; Cavanagh et al. 2005). We showed that DC form immediate tight contacts with bone marrow-resident central memory cells, and this mode of interaction was only seen in the presence of cognate antigen (Cavanagh et al. 2005).

In work for Aim 2, we developed a new intravital visualization technique to observe the kinetics and mechanisms of cytotoxic T cell (CTL)-mediated killing of antigen-presenting target cells (Mempel et al. 2006). Moreover, using this model, we demonstrated that the simultaneous activation of regulatory T cells (Treg) causes these cells to suppress CTL function. Interestingly, in contrast to published in vitro effects of Treg, suppression in vivo did not depend on inhibition of effector cell proliferation. Rather, the Treg selectively prohibited CTL from degranulating. Thus, even though the "regulated" CTL were fully differentiated, the presence of activated Treg resulted in a near-complete (and rapidly reversible) inability of CTL to kill their targets. This effect depended on the action of TGF-beta on CTL, because TCR transgenic CTL that expressed a dominant engative receptor for TGF-beta wer not suppressible in this model.

While gearing up to conduct experiments for Aim 3, we became aware of multi-photon intravital microscopy work from Dr. Ron Germain’s group (Castellino et al. Nature 440:890-895, 2006) that provided compelling observations on the mechanisms of CD4 help for CD8 T cells in lymph nodes. In light of this unexpected competition, we decided to focus our investigations on Aims 1 and 2. We may revisit some of the proposed studies in this aim at a later time.