The relative contribution of direct versus indirect presentation in priming alloreactive T cell responses has yet to be clearly defined. This study reveals cellular dynamics that underlie the indirect pathway of alloantigen presentation by host DCs after transplantation. We used an immunoimaging approach to demonstrate that donor-derived DCs are killed within dLNs as a result of direct NK–DC cytolytic interactions. We found that NK cells actively patrol the LNs with average three-dimensional velocities of 10 μm/min either in the presence or absence of allogeneic DCs and that their motility enables them to rapidly scan the LN microenvironment and eliminate allogeneic DC targets. These three-dimensional NK cell velocities are consistent with our previously reported two-dimensional average velocity of 7 μm/min (4
). Furthermore, as in our previous study with foreign B cells (4
), NK cells preferentially formed prolonged interactions with allogeneic targets that led to cell lysis. As a result of such interactions with NK cells, allogeneic DCs were eliminated within hours following adoptive transfer or skin transplantation. We examined the functional consequence of this rapid elimination and found that allogeneic DCs were unable to directly stimulate either primary or recall T cell responses. In determining the mechanism by which allorecognition occurs following organ transplantation, we showed that DC-depleted CD11c-DTR+
donor cardiac allografts were rejected with normal kinetics. In contrast, survival of unmanipulated donor hearts into DC-depleted CD11c-DTR+
recipients was prolonged. Taken together, these results validate the patrolling function of rapidly motile NK cells and indicate that direct presentation by donor DCs is dispensable for acute rejection.
Delineation of the mechanisms underlying alloantigen presentation will aid clinical development of tolerogenic interventions to minimize transplant rejection. Traditionally, direct presentation of alloantigens by donor-derived DCs has been a primary focus of transplant immunology and has been regarded as the driving mechanism underlying T cell-mediated allograft rejection. Several lines of evidence have sustained this view. For instance, data from in vitro experiments have demonstrated that the majority of alloreactive T cells respond directly to allogeneic APCs (12
). However, the high precursor frequency of T cells with direct alloreactivity does not necessarily indicate that alloreactive T cells are effectively activated through direct presentation in vivo. Further evidence for the primacy of the direct pathway in acute rejection originates from transplant studies demonstrating prolonged survival rates of donor DC-depleted allografts (16
). As suggested by Auchincloss et al. (15
), however, the defect in allorecognition after donor DC depletion may not just be a consequence of the absence of direct presentation but rather a reflection of the requirement for donor DCs to provide alloantigen to the indirect pathway. That is, donor DCs may act largely as vehicles for transporting sufficient concentrations of allopeptides to endogenous DCs to drive indirect presentation. Therefore, the delay in allograft rejection following donor DC depletion does not necessarily substantiate the premise that direct presentation is required for allograft rejection. Additionally, support for the importance of the direct pathway in allorecognition derives from data indicating normal allograft survival in MHC class II-deficient recipients lacking the indirect pathway of presentation. However, analysis of MHC class II-deficient mice has revealed a profound dysregulation in T cell responsiveness (37
). Therefore, it has been proposed that MHC class II-deficient mice may use a novel pathway of alloreactivity (39
Recently, a third pathway of alloantigen presentation termed “semidirect” has been proposed that may account for indirect allorecognition in MHC class II-deficient recipients (32
). In the semidirect pathway, recipient DCs acquire and present intact allogeneic MHC:peptide molecules to responding T cells. Thus, MHC class II-deficient cells could acquire Ag-presenting function through the transfer of MHC:peptide complexes from exosomes or directly from short-lived migratory DCs draining the allograft (40
). Importantly, the semidirect pathway may also help explain our finding that 4C T cells with direct allospecificity are capable of associating with endogenous DCs, although this issue clearly will require further study.
It is becoming increasingly apparent that prolonged stimulation is essential for T cell activation, acquisition of effector function, and establishment of effective memory (28
). Celli et al. (28
) reported that a contact time of 6 h was required to elicit a single round of T cell division, with maximal proliferation and effector function differentiation achieved only following prolonged DC-T cell interactions persisting >24 h. Therefore, the demonstration of allogeneic DC susceptibility to host-mediated immune attack (6
), together with our finding that this occurs within hours of arriving in dLNs, suggests an inability of donor-derived DCs to induce effective T cell stimulation through direct presentation. A straightforward hypothesis, therefore, would be that in the absence of effector cell killing, restoration of direct presentation combined with intact indirect presentation would result in subsequently enhanced T cell expansion. This is in fact what has been described recently in two studies that used anti–NK1.1-depleting Ab in CD8-deficient mice (7
). Interestingly, however, augmented direct alloreactivity did not accelerate allograft rejection. In addition, our suggestion for a predominant role for indirect presentation is further supported by recent evidence using an allospecific TCR transgenic system to examine T cell activation events. Brennan et al. (19
) found that following allograft transplantation, T cells were preferentially primed through indirect presentation. Importantly, allospecific T cells with indirect reactivity were enriched in the effector and memory T cell compartments. Therefore, the critical threshold of engagement required to induce robust T cell activation appears to occur preferentially through the indirect pathway of alloantigen presentation.
Our findings provide insight into the role of innate immunity in shaping alloreactivity and emphasize the importance of indirect presentation in generating alloimmune T cell responses. It should be emphasized, however, that although NK cells appear to be important early effectors, this does not exclude overlap with CTL later in the response (49
). A suggestion substantiated by our finding that 3 d following DC immunization, NK1.1+
cell depletion in WT animals resulted in comparable levels of allogeneic DC elimination as in isotype-treated controls (unpublished observation). Thus, given sufficient time to develop, CTL may have a complementary role in killing allogeneic DCs. Taken together, our results support the concept that host-mediated immune attack limits direct alloantigen presentation.
In summary, these data refine our understanding of the mechanisms underlying solid organ transplant rejection and may enable more effective tolerance induction protocols to be engineered. In this regard, results from our experiments are in accord with studies indicating that certain tolerizing regimens require indirect presentation (50
). Although further investigation is required to assess a potential role for direct presentation in an Ag-experienced recipient containing a complete repertoire of naive as well as memory T cells, our data demonstrate that rapid elimination of donor DCs down-modulates direct Ag presentation, favoring alloreactivity via the indirect pathway. The translation of our findings into a clinically relevant therapeutic intervention emphasizes the adaptation of current immunomodulatory approaches to target the indirect pathway of allopresentation.