Two Photon Intravital Microscopy Reveals CD4⁺ T Cells Making Cognate Interactions With Tissue Dendritic Cells In Skin Graft-Versus-Host-Disease

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs (SLO), we have consistently observed MHCII⁺ donor-derived tissue APCs (t-APCs), including tissue dendritic cells (t-DCs) in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells. We hypothesize that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease locally. We could not use knockout or APC depletion approaches to study T cell: t-APCs interactions as they impact APCs systemically and might therefore affect T cell stimulation in SLO. We therefore utilized two-photon intravital microscopy to analyze interactions between fluorescent donor CD4⁺ T cells and t-DCs in skin. 129 (H-2^b) hosts were irradiated and reconstituted with B6 (H-2^b) CD11c-YFP transgenic (Tg) Bone Marrow (BM) with B6 RFP Tg CD4 cells and nonfluorescent B6 CD8 cells. We imaged ear skin in GVHD mice 4 weeks later. In general CD4 cells co-localized with DCs. We observed CD4⁺ T cells that were highly motile and only in transient contact with DCs and others that made stable contact with DCs. To determine how much TCR: MHCII interactions drive sustained CD4⁺ T cell: DC interactions and arrest CD4⁺ T cell motility, mice were imaged and then injected with an MHCII blocking antibody (Ab; Y3P) with continued imaging of the same regions. After injection, T cell mean speed significantly increased and the proportion of T cells in stable contact with DCs decreased, indicating that transient disruption of TCR: MHCII is sufficient to restore motility to some T cells. In a second approach to assess the specificity of CD4⁺ T cell: t-DC interactions we transplanted 129 mice with B6 RFP⁺ CD4 cells, nonfluorescent CD8 cells and a mix of CD11c-YFP MHCII^-/- and RFP wt BM or a mix of CD11c-YFP wt and RFP Tg MHCII^-/- BM. We are currently comparing the motility of CD4 cells that make contact with MHCII⁺ as compared to MHCII^- DCs, with the prediction that contact times will be shorter with the latter. Our data suggest that CD4⁺ T cells make cognate interactions with t-DCs in skin and we hypothesize that these interactions promote GVHD locally. Because the graft-versus-leukemia effect occurs primarily in bone marrow and secondary lymphoid tissues, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL.