Recruitment of Donor T Cells to Secondary Lymphoid Tissue Is Enhanced by Conditioning Therapy and Donor T Cells/Marrow

Abstract

Background: Previous investigators have demonstrated that secondary lymphoid tissue (SLT) including the spleen is critical for the initial activation of donor T cells after allogeneic stem cell transplantation (SCT) leading to GVHD. This work showed significant redundancy in the role of all SLT including the spleen and suggested that approaches to prevent GVHD would need to inhibit the migration of donor T cells to all SLT. The mechanisms mediating the migration of donor T-cells to SLT have not been well characterized and the specific role of conditioning therapy in this process has not been investigated. Thus, here we sought to evaluate the expression of trafficking proteins by host antigen presenting cells isolated from the MLN and spleen.Methods: B6D2 recipient mice that underwent conditioning were lethally irradiated the night prior to transplantation. The following day recipient mice were transplanted i.v. with 3 × 106 T cell depleted bone marrow cells with 5 × 106 column purified T-cells. T-cells were from eGFP C57Bl/6 transgenic mice. Six hours after transplantation, recipient mice were sacrificed and the MLN and spleen removed. Dendritic cells (DCs) were isolated by collagenase digestion and mechanical disruption and sorted on a high speed MoFlow sorter using anti-CD11c monoclonal antibodies. Gates were drawn around CD11c high cells that did not express eGFP. 3–5 × 104 DCs from the MLN and 1–2 × 105 DCs from the spleen were pooled from five different mice per group, lysed in an RNA amplification buffer and analyzed either by real-time PCR or Agilent Whole Mouse Genome Microarray Chips.Results: Four different groups of mice were analyzed including one group that received no treatment, one that received irradiation alone, one that received irradiation plus allogeneic bone marrow and T cells, and one that received allogeneic bone marrow and T cells without irradiation. By comparing recipients that received allogeneic bone marrow and T cells, with those that received conditioning therapy, allogeneic bone marrow and T cells, we found that conditioning therapy significantly induced the expression by host DCs of the chemokines, CXCL10, CCL17, CCL20 and CCL22 from DCs isolated from both the MLN and the spleen. CCL3 expression by host cells was only upregulated from DCs isolated from the MLN and not the spleen. Conditioning therapy downregulated the expression of CCL1, CCL2 and CCL4 from DCs isolated from both the MLN and spleen (Table 1). Conditioning therapy markedly enhanced the expression by splenic and MLN DCs of STAT4 and p40 IL-12.Table 1MLNSpleenCXCL1014.820CCL310.9−1.0CCL177.05.1CCL222.47.8CCL202.21.6CCL61.31.2CCL5−1.33.0CCL19−1.4−2.3CXCL9−1.5−1.3CCL1−2.22.2CCL2−3.7−3.0CCL4−6.0−1.4Next we analyzed the effect of allogeneic bone marrow and T cells on the expression of chemokine ligands and signaling proteins by splenic and MLN DCs. Interestingly, the receipt of allogeneic bone marrow plus T cells and conditioning therapy led to a marked increase in the expression of chemokine ligands by DCs isolated from the spleen with little effect on the expression of chemokine ligands by DCs isolated from the MLN compared to mice that only received irradiation but did not undergo allogeneic transplantationTable 2.MLNSpleenCXCL91.157CCL222.236CCL5−1.134CXCL102.130CCL174.38.1CCL4−1.17.2CCL61.46.7CCL36.01.0CCL202.41.0CCL12.5−1.5CCL19−4.2−4.0CCL2−6.7−7.1The receipt of irradiation, donor bone marrow and T cells markedly enhanced the expression by host DCs from the spleen of STAT4 and p40 IL-12 while downregulating the expression of STAT6.We confirmed that the expression of CXCL9, CXCL10, CCL4 and CCL5 were markedly increased by host APCs using real time PCR. Finally, we demonstrated in vitro that donor T cell production of IFN-g was critical to the generation of CXCL9 and CXCL10 by host APCs.Conclusion: Conditioning therapy markedly enhances the expression of chemokine ligands and proteins important in Th1 T cell activation by host APCs. However, the expression was enhanced the most in splenic DCs isolated from recipient mice that had received irradiation with allogeneic bone marrow and allogeneic T cells. Our work suggests that the processes mediating the migration of donor T cells to the spleen may differ from those in the MLN and that the CXCR3 binding chemokines, CXCL9 and CXCL10 along with the CCR5 binding chemokine, CCL3, may be quite critical for donor T cell homing to the spleen.