Here we sought to decipher the rules governing the transition from a phase dominated by serial T cell–DC contacts (phase one) to a phase of stable T cell–DC interactions (phase two). We found that the duration of phase one correlated inversely with the overall antigen dose by comparing the effect of DCs pulsed with native LCMV-derived C-peptide and the altered peptide ligand M-peptide. M-peptide has a prolonged half-life in H-2Db
and is thus retained at much higher density during the voyage of DCs to lymph nodes. A dose-response analysis of the effect of peptide-pulsing concentrations on the ability of pulsed DCs to elicit phase two–like activity identified a low threshold concentration of M-peptide that supported efficient T cell activation and effector differentiation while inducing prolonged phase one–like activity. Higher doses of M-peptide yielded a rapid transition to phase two. Furthermore, activation of naive T cells could be controlled by the amount of antigen carried by each DC and by the density of antigen-presenting DCs. However, these two variables do not influence T cell activation equally (H.Z., B. Jin, S.E.H., A. Perelson, U.H.v.A. and A.K.C., unpublished data). The dependence on DC density is linear, whereas the dependence on the number of cognate pMHC complexes per DC is nonlinear with a sharp threshold between nonactivating and activating pMHC density per DC32,37
(H.Z., B. Jin, S.E.H., A. Perelson, U.H.v.A. and A.K.C., unpublished data). We propose that phase one constitutes a period of signal integration allowing T cells to ‘decide’ how to respond to the overall antigen dose encountered.
What is the function of the antigen environment in T cell priming? Coordinated changes in T cell interaction dynamics with antigen-presenting DCs, consistent with our three-phase model, have been noted by other groups studying CD4+
. Such studies have provided clues that multiphase interactive programs may be modified by antigen context. For example, when antigen is presented by tolerogenic DCs20
or in the presence of the coinhibitory molecule CTLA-4 (ref. 38
), T cells do not transition to phase two. Another study exploring anergy induction has shown that T cell activity depends on the potency of the peptide; only high-potency pMHC complexes induce calcium-dependent T cell deceleration. Thus, in certain tolerance-inducing settings, T cells maintain transient contacts without transitioning to phase two. Multiphoton microscopy has also been used to study the effect of antigen dose and APC density on P14 T cell interactions with DCs in explanted lymph nodes15
. That study showed that DCs pulsed with high-dose antigen (1 μM M-peptide) form large clusters with T cells (indicative of phase two) and induce full proliferation of P14 cells, in contrast to results obtained with low-dose antigen (30 pM M-peptide). Our results have confirmed and refined those observations and provide quantitative information on how antigen dose influences the character of T cell activation.
T cell activation seems to require very few cognate DCs. Although the low frequency of DCs in our DC dilution experiments made it impractical to obtain statistically meaningful MP-IVM data, we can extrapolate from our observations with higher DC densities to propose a mechanism in which at low DC abundance, a T cell must scan a larger volume to find cognate APCs. However, as T cells migrate, they may encounter draining sinusoids through which they can egress from the lymph node and, if not activated by 20 h, 90% of T cells will have left a given lymph node7
. Thus, although DCs pulsed with large amounts of cognate antigen probably promote tight adhesion of T cells after only a few contacts, T cells presumably need more contacts to form long-lasting conjugates with DCs bearing small numbers of cognate pMHC complexes and may leave the lymph node before this happens.
It remains unclear how many pMHC complexes are needed to induce T cell activation in vivo40
. In vitro
work with effector T cells has shown that single TCR-pMHC contacts yield calcium flux, about 10 pMHC complexes are required for immune synapse formation41,42
, and cytotoxic T lymphocytes can lyse target cells with three cognate pMHC complexes43
. To estimate the number of pMHC complexes required for T cell activation, we estimated here that naive CD8+
T cells interacted with DCs presenting fewer than 4 × 103
cognate pMHC complexes. The true numbers are probably lower because our calculations assumed that all available H-2Db
molecules on a given peptide-pulsed DC had their endogenous self-peptide replaced. A saturating amount (such as 10 μM) of M-peptide might approach this extreme, but this seems unlikely for the threshold activating dose (200 pM). We can extrapolate from the similar effects on T cell activity exerted by DCs pulsed with 200 pM M-peptide and those pulsed with 10 μM C-peptide, and the fact that the TCR affinity for M-peptide and C-peptide is similar28
, to conclude that at 24 h after DC injection (around the time of phase-two initiation), there were approximately the same number of cognate pMHC complexes remaining on DCs initially pulsed with 200 pM M-peptide or 10 μM C-peptide (H.Z., B. Jin, S.E.H., A. Perelson, U.H.v.A. and A.K.C., unpublished data). We can therefore calculate that DCs pulsed with 200 pM M-peptide probably start with about 480 pMHC complexes at the time of DC pulsing and retain about 60 pMHC complexes 18 h later (H.Z., B. Jin, S.E.H., A. Perelson, U.H.v.A. and A.K.C., unpublished data). Given that this is probably an overestimation of pMHC complexes, T cells can be activated by very few pMHC complexes.
With only dozens to a few thousand cognate pMHC complexes per DC, how many cognate pMHC complexes would a T cell encounter in a given interaction? The surface area of an immature DC can be estimated on the basis of its volume44
to be about 500 μm2
, and the surface area of the T cell–DC contact area is about 50 μm2
). Immature DCs pulsed with 10 μM C-peptide presented about 127 cognate pMHC complexes at 18 h, whereas DCs pulsed with 10 μM M-peptide presented about 3,133 cognate pMHC complexes. Thus, the average number of antigenic TCR ligands that may be experienced immediately after T cell transfer per contact region is about 12.7 or about 313 for DCs pulsed with 10 μM C-peptide or M-peptide, respectively. These numbers should decrease further as the peptides dissociate from H-2Db
, and although very small, they are in agreement with in vitro
. Given that pMHC complexes tend to cluster in immune synapses and such synapses may include noncognate pMHC complexes, we may be underestimating the number of pMHC complexes in contact zones. On the basis of in vitro
experiments with cognate pMHC molecules on lipid bilayers, it has been suggested that TCR microclusters initiate signaling46,47
. Our imaging studies cannot resolve the issue of whether there are any in vivo
microclusters of TCRs on the T cell or pMHC complexes on the DC. However, given that transition to phase two (and subsequent T cell proliferation) is stimulated by very small numbers of cognate pMHC complexes, if microclusters do form, they most likely involve endogenous ligands.
The physiological importance of pMHC half-life for T cell immunity is unclear. Most MHC class I peptides are synthesized by APCs and are continually replenished. However, DCs can also cross-present MHC class I antigens from exogenous sources48
. If a peripheral tissue DC engulfs pathogen-derived material and carries it to a draining lymph node, the antigenic cargo is limited49
. Moreover, when DCs receive maturation signals, they substantially reduce endocytosis and focus on moving MHC molecules to the surface12
. Thus, migrant DCs that enter lymph nodes may initially present a broad repertoire of peptides, which ‘skews’ toward the longest-lived peptides over time. Prolonged phase-one interactions provide T cells with a strategy for finding optimal APCs when cognate pMHC complexes are limited or have short half-lives.
An important unresolved issue is whether and how cognate T cell–DC contacts are counted and ‘remembered’. Activation could require a single, full signal from one APC if there is no cellular ‘memory’ of previous interactions. In this case, the phase one–type interactions could reflect contacts in which T cells fail to hit the ‘sweet spot’ on DCs. We consider this unlikely for many reasons. First, even if there were only a few sites on DCs that supported tight T cell binding, the high frequency of contacts in our experiments should have allowed some T cells to rapidly transition to phase two. The delayed and coordinated timing of phase two, particularly at low antigen densities, is inconsistent with this idea. Second, we have shown that the time necessary for antigen-specific T cells to transition to phase two while interacting with DCs pulsed with a threshold dose of antigen shrunk considerably when a second population of DCs pulsed with a high dose of antigen was also present. This supports the idea of signal integration by T cells: as T cells undergo serial interactions with DCs pulsed with both high- and low-dose peptide, they accumulate incremental activation signals until they reach a threshold that triggers stable interactions. The antigen density on the DC with which phase-two interactions are subsequently initiated seems to be irrelevant, as long as the overall signal is sufficient. Third, imaging experiments have shown that even transient encounters trigger persistent antigen-dependent calcium fluxes, regardless of whether T cells are in contact with DCs, which indicates that TCR signaling does not necessarily result in instant arrest50
. In addition, with the loss of pMHC complexes over time there should be a reduction in the frequency of suitable sites for T cell binding; however, there is an increase in tight contacts at late time points. Finally, in silico
modeling has shown that memory of previous cognate encounters substantially improves the efficiency of T cell responses to small amounts of antigen (H.Z., B. Jin, S.E.H., A. Perelson, U.H.v.A. and A.K.C., unpublished data).
Which has the most influence on the ‘decision’ to engage in tight interactions, T cells or DCs? Our data support the idea that T cells have the main function. First, the length of phase one was modulated by antigen dose even with a fixed time for DC maturation. So, if DCs determined the onset of phase two, they would have had to determine the number and/or intensity of cognate interactions with T cells and distinguish cognate encounters. That seems unlikely, because the duration of phase one would be inversely correlated with the concentration of cognate T cells in the lymph node (although there is evidence of this type of effect on the transition from phase two to phase three35
). Although in published studies the number of adoptively transferred TCR-transgenic T cells have been well above physiological numbers, as cognate T cells are rare in polyclonal naive T cell populations, the rate of cognate contacts experienced by individual DCs would be low and, consequently, the hypothetical threshold signal allowing DCs to switch to phase two might not be reached before pMHC complexes dissociate or DCs perish. In addition, our finding that individual DCs simultaneously supported both phase-one and phase-two interactions with two populations of T cells of different antigen specificity also undermines the idea that DCs ‘drive’ the length of phase one. However, these data do not preclude the possibility that DCs contribute to the quality and strength of cognate interactions (perhaps by reorganizing pMHC complexes into microdomains51
What are the consequences if the ‘decision’ to enter phase two is made by T cells? Any new antigen will be recognized by a small subset of T cells with varied TCR affinity. Given the inverse relationship between TCR affinity and the dose of antigen needed to activate a T cell10,11
, although high-affinity T cells may need only a few encounters with antigen-presenting DCs to commit to activation, T cells with intermediate or low affinities must somehow ‘decide’ whether to be recruited into the ongoing immune response or ignore the weak signal. Phase one provides a mechanism that allows migrating T cells to ‘measure’ the overall abundance of antigens and make an informed ‘decision’ about how to respond.