Organ transplantation is a life-saving procedure for patients with end-stage organ failure. The introduction of novel immunosuppressive drugs has led to significant improvement in short-term survival rates of solid organ allografts. Nevertheless, these drugs result in increased rates of cancer and opportunistic infections. Furthermore, they do not prevent chronic allograft dysfunction, the leading cause of graft failure after 1 year posttransplantation. To improve transplantation outcomes, it is critical to continue the development of novel strategies to prevent acute and chronic rejection. To accomplish this goal, it is necessary to elucidate the exact basic mechanisms leading to acute and chronic rejection, and tolerance. It is clear that T cells play a central role in the process of allograft rejection. However, donor dendritic cells (dDC) are the most potent antigen-presenting cells and have proven to be major regulators in determining the fate of the alloimmune response. The ability of dDC to mount an efficient alloimmune response relies on their capacity to be recruited in the donor tissue and their ability to traffic to secondary lymphoid tissues in the recipients, both of which are tightly controlled by specific chemokine pathways.

Chemokines play important roles in selectively recruiting leukocytes to the site of inflammation as well as in the activation of leukocytes following recruitment (1–3). Chemokines have also been increasingly recognized for their role in regulating migration of circulating monocytes and their transformation to tissue dendritic cells (DCs). The CX3CR1 signaling has been demonstrated to be important in the generation of lymphoid tissue DC by regulating the trafficking of circulating monocytes into tissue (4–6) (Figure 1).

Our specific hypothesis is that the CX3CR1 signaling plays an important role in the seeding of dDC in heart tissue as well as their function posttransplantation, hence playing a key role in directing the alloimmune response toward rejection and tolerance. Based on advances in cellular imaging, it is now feasible to follow some of these key cellular and molecular events in vivo in the beating heart (7, 8). We propose to study the chemokine-mediated regulation of dDC function and trafficking to recipient lymphoid tissues and transplanted graft.

We previously showed that recipients of heart allografts from CX3CR1^−/− donors show longer survival (12). In addition, the same recipients with heart allografts from CX3CR1^−/− donors are resistant to the induction of tolerance by T cell costimulatory blockade, which indicated the importance of the CX3CR1 pathway in the generation of heart tissue DCs and the divergent role of tissue/DCs in rejection versus tolerance (12). CX3CR1⁺ cells were thought to be DC precursors that play an immunomodulatory role in the induction of tolerance. No data are yet available on the importance of chemokine pathways in regulating homeostasis of DC in heart transplants.

To better differentiate the chain of events underlying the body’s tolerance of transplanted tissue versus the events leading up to rejection, we employed the new powerful imaging tool to detect cell behaviors following transplant in the current study.

We transplanted CX3CR1^GFP/+ DC hearts into complete MHC-mismatched BALB/c recipients. We performed several imaging sessions at 3, 24, and 72 h later of transplant under anesthesia. There were few significant changes in both 3 and 24 h imaging sessions (Figure 2; Video S1 in Supplementary Material); however, images from 72 h at cardiac graft showed like a “passengers at the airport.” GFP cells were trying to prepare for exiting from the graft (Figure 3; Videos S2–S5 in Supplementary Material). In addition, GFP⁺ cell motility and shape change was detected (triangle) in transplanted heart graft (Figures 3A–C). In contrast to earlier images of GFP⁺ cells (Figure 2), the morphology of GFP⁺ cell in the allograft was spiky and come to be a typical DC shape, which suggested that the first 3 days of posttransplant (24–72 h) may be a transition phase of DC maturation by allorecognition. In addition, we took images from the secondary lymphoid tissues to investigate further understanding of unsolved transplant immunological questions. Mice survive the procedure in our model, thereby permitting imaging of the same recipients between the beating cardiac allograft and secondary lymphoid tissues via the systemic circulation over time. Unfortunately, we almost failed to see donor DC at secondary lymphoid tissues by our IVM system. Only a few GFP⁺ cells were detected in the vasculature and lymph nodes (Figures 3D,E). From three lymph nodes imaged – two mesentric, one inguinal – only one GFP⁺ cell was observed (Figure 3E). Compared with same kind of experiment done by MHC class II GFP⁺ donor model, this is very small number (data not shown).

Since the maximum imaging depth of fluorescence microscopy is limited to 100 μm, the penetration depth is sufficient to access most of the major coronary vessels and capillaries and the myocardium in our experimental transplant model. However, there may be other immunological events in deeper tissues and areas where we cannot access to observe them with our IVM system. For the further investigation, we have already performed adoptive transfer models using colored recipient CD4 or CD8⁺ T cells to see cellular interactions between dDC and those T cells (Ueno et al., American Transplant Congress 2012, 2013, 2014, 2015). There is also a possibility that the CX3CR1 is not an optimal marker for the entire DC population in the heart. Furthermore, the lymph node investigated in this study may not represent the draining lymph nodes.

Based on advances in intravital imaging, it is now feasible to follow some of these key cellular and molecular events longitudinally in mice, which will provide us with an improved understanding of the highly dynamic temporospatial events from DC mobilization to activation, rather than a static snapshot provided by traditional experimental approaches.

TU and PK participated in the performance of the research, performed the data collection, performed the statistical analysis, and contributed to the writing of the manuscript; MM and MY participated in the writing of the manuscript and performed review; TS and KJ participated in the statistical analysis; MS and AC helped in the design of the study; and RA and SY participated in the review.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.