Donor Dendritic Cells Home to Lymphoid Organs in Allogeneic Transplant Recipients, Enhance Donor T-Cell Chimerism, and Polarize Donor T-Cell Activation.

The fate of donor dendritic cells (DC) contained in the allograft, and their role in regulating immunity following allogeneic transplant has not been fully explored. We have previously demonstrated that donor BM CD11b⁻ DC added to purified hematopoietic stem cells (HSC) and T-cells polarize donor T-cells to Th1 immunity, and improve leukemia survival among tumor-bearing transplant recipients compared with recipients of allogeneic CD11b⁺ donor antigen presenting cells (APC) or grafts with no donor APC (Li, et al Blood 2008, 112:1252). Objective: In this study, we studied the homing of donor DC in the primary and secondary lymphoid organs of transplant recipients in murine allogeneic HSC transplantation. Methods: In this study we transplanted between 5 × 10⁴ and 1 × 10⁶ FACS-sorted lineage⁻ CD11c⁺ CD11b⁻ DC (predominately plasmacytoid DC (pDC) and pDC precursors) and lineage⁻ CD11c⁺ CD11b⁺ APC and APC precursors (CD11b⁺ APC) purified from GFP⁺ or luciferase⁺ transgenic donors in combination with 3 × 10³ lineage⁻ c-kit⁺ Sca-1⁺ HSC and 1 × 10⁶ CFSE stained donor T-cells in B6□B10BR, B6□BALB/C, and FVB□B10BR allogeneic transplant models to explore homing, maturation, and persistence of donor DC in the primary and secondary lymphoid organs of transplant recipients. Homing and short-term maturation of donor DC was assessed 3 days and 10 days post-transplant by FACS analysis gating on GFP⁺ donor DC. In vivo maturation of DC was assessed by FACS gating on GFP⁺ donor APC progeny in transplant recipients and compared with DC subsets cultured with CD40L (1μg/mL) or LPS (10ng/mL). Proliferation and IFN-ψ production of CFSE-labeled donor T-cells isolated from recipients' peripheral blood, lymph nodes, and spleen was assessed by flow cytometric analysis of CFSE and intracellular cytokine staining. Persistence of donor DC was assessed by bioluminescence imaging 7, 14, 21, and 28 days post-transplant in FvB□B10BR allogeneic transplant model using 5 × 10⁴ pDC and CD11b⁺ APC from FvB luciferase⁺ transgenic donors transplanted with 3 × 10³ HSC and 3 × 10⁵ T-cells from FvB wild-type donors. Results: The frequencies of GFP⁺ pDC and CD11b⁺ APC recovered from transplant recipients at day 10 post-transplant were 2.2 ± 0.2% and 1.8 ± 0.3%, respectively in BM and 4.9 ± 1.5% and 1.9 ± 0.5%, respectively in recipient spleen. Absolute numbers of GFP⁺ donor DC were measured in the sections of spleen by confocal microscopy and were similar comparing recipients of pDC and CD11b⁺ APC (9.0/μL and 7.3/μL spleen, respectively, at day 10). The GFP⁺ CD11b⁺ APC had a scatter profile consistent with myelo-monocytic differentiation while GFP⁺ pDC retained mononuclear properties. The expression of MHC-II and CD80/CD86 on GFP⁺ CD11b⁺ APC recovered from transplant recipients was minimal, indicating predominant differentiation to myeloid-derived suppressor cells rather than to DC. FACS analysis of lineage expression patterns in donor GFP⁺ pDC and CD11b⁺ APC recovered from recipient BM were similar compared with sorted APC cultured with CD40L or LPS. PDC up-regulated MHC-II, CD80 and CD86 expression, while CD11b⁺ APC expressed high levels of PDL1 and PDL2 and up-regulated F4/80 and Gr-1-associated markers. In contrast, the same donor APC populations recovered from the spleen expressed lower levels of DC-associated markers. Bioluminescent imaging showed that luciferase⁺ donor DC persisted and expanded in the lymph nodes and spleen of transplant recipients for up to one month. The immunological effect of transplanting pDC led to increased donor T-cell proliferation, IFN-ψ production and CD25 expression compared with transplanting grafts containing HSC, T-cells and CD11b⁺ APC, or grafts containing only HSC and T-cells in both MHC-mismatched murine HSCT models. In conclusion, the results indicate that different subsets of donor APC and DC home to lymphoid tissues and up-regulate a pattern of co-stimulatory and co-inhibitory molecules consistent with their observed immunological immune activity in vivo. Maturation of pDC in vivo was slower than that in vitro, and pDC maturation was slower in the recipients' BM compared with recipient's spleen suggesting that the post-transplant microenvironment may impair DC differentiation. Less mature pDC in the stem cell graft that home to the BM may facilitate donor engraftment and immune reconstitution, while more mature pDC that home to the spleen may polarize donor T-cell Th1 responses.

Waller:NIH-National Heart, Lung, and Blood Institute, NHLBIP01H1086773: Research Funding.