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

Ulrich H. von Andrian, M.D., Ph.D.

CBR Institute for Biomedical Research, Inc.

Funded in June, 2003: $200000 for 2 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

ABSTRACT

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

Peripheral lymph nodes (PLN) are the key sites where naive T cells are exposed to antigens presented by dendritic cells (DC). The kinetics, geometry, and microenvironment in which interactions between T cells and DC occur are thought to be critical factors that shape the type and strength of immune responses. However, these parameters have not been analyzed in vivo.

During the past 18 months, we have developed a new technique that employs multi-photon intravital microscopy (MP-IVM) to visualize and quantitate lymphocyte-DC interactions in lymph nodes of anesthetized mice. This technique allows us, for the first time, to examine the dynamics and spatial relationships of T cell interactions with DC. In Aim 1, we will compare the dynamics of naive versus central memory CD8 T cell-interactions with DC. Aim 2 will characterize the dynamics of T cell-DC interactions leading to tolerance and compare these to T cell-DC interactions leading to immunity. Aim 3 will visualize the effect of CD4 help during the priming of CD8 T cells.

This project will provide fundamental information on cellular immune responses in vivo with significantly higher spatial and temporal resolution than has been achieved previously. Results from this study may ultimately contribute to improved vaccination strategies and a better understanding of autoimmune diseases and infections.

HYPOTHESIS

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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

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Scientific Results:
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.

SELECTED PUBLICATIONS

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Mrass P., Takano H., Ng L.G., Daxini S., Lasaro M.O., Iparraguirre A., Cavanagh L.L., von Andrian U.H., Ertl H.C., Haydon P.G., and Weninger W. Random migration precedes stable target cell interactions of tumour infiltrating T cells. J Exp Med. 2006 Nov 27;203(12):2749-61.

Mempel T.R., Pittet M.J., Khazaie K., Weninger W., Weissleder R., von Boehmer H., and von Andrian U.H. Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity. 2006 Jul;25(1):129-41.

Cariappa A., Mazo I.B., Chase C., Shi H.N., Liu H., Li Q., Rose H., Leung H., Cherayil B.J., Russell P., von Andrian U.H., and Pillai S. Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.  Immunity. 2005 Oct;23(4):397-407.

Halin C., Scimone M.L., Bonasio R., Gauguet J.-M., Mempel T.R., Quackenbush E.J., Proia R., Mandala S. and von Andrian U.H. The S1P-analog FTY720 differentially modulates T cell homing via HEV: T cell-expressed S1P1 amplifies integrin activation in peripheral lymph nodes but not in Peyer’s patches.  Blood. 2005 Aug 15;106(4):1314-22.

Cavanagh L.L., Bonasio R., Mazo I.B., Halin C., van der Velden A.W.M., Cariappa A., Chase C., Russell P., Starnbach M.N., Leung H., Pillai S., Weninger W., and von Andrian U.H. Activation of bone marrow-resident memory T cells by circulating dendritic cells.  Nat Immunol. 2005 Oct;6(10):1029-37.

Mazo I.B., Honczarenko M., Leung H., Cavanagh L.L., Bonasio R., Weninger W., Engelke K., Xia L., McEver R., Koni P.A., Silberstein L.E. and von Andrian U.H. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.  Immunity. 2005 Feb;22(2):259-70.

Mempel T.R., Henrickson S.E., and von Andrian U.H.  T cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004 Jan 8;427(6970):154-9.