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Background DBA/2J kidney allografts, but not heart allografts, are spontaneously accepted indefinitely in C57BL/6 (B6) mice, through regulatory tolerance mechanism dependent on Foxp3 cells. In contrast, B6 kidneys are rejected within a week in DBA/2J recipients. We hypothesized that the tolerogenic difference of the kidneys might be due to differences in number or function of plasmacytoid dendritic cells (pDCs), because these cells are potent inducers of Foxp3 cells. Methods pDCs from murine bone marrow, native kidneys, and spontaneously accepted kidney allografts were analyzed using flow cytometry and immunohistochemical staining. Naive T cells were cocultured with pDCs in specific strain combinations and analyzed for FoxP3 induction and functionality. MEK/ERK and NFκB inhibitors were used to assess the regulatory T-cell induction pathways. pDCs and T-cell cultures were adoptively transferred before heterotopic heart transplantation to assess allograft survival. Results DBA/2J pDCs were more potent in inducing Foxp3 in B6 T cells than the reverse combination, correlating with survival of the kidney allografts. Foxp3 induction by pDCs in vitro was dependent on pDC viability, immaturity, and class II MHC mismatch and blocked by MEK/ERK and NFκB inhibition. pDC-induced Foxp3 T cells suppressed proliferation of B6 T cells in vitro, and adoptive transfer into B6 recipients 2 weeks before heterotopic DBA/2J heart transplantation resulted in prolonged allograft survival. Conclusions These data suggest that pDC-induced regulatory T cells are dependent on downstream signaling effects and on strain-dependent, MHC class II disparity with naive T cells, which may explain organ- and strain-specific differences in spontaneous tolerance.

Intragraft events thought to be relevant to the development of tolerance are here subjected to a comprehensive mechanistic study during long-term spontaneous tolerance that occurs in C57BL/6 mice that receive life sustaining DBA/2 kidneys. These allografts rapidly develop periarterial Treg-rich organized lymphoid structures (TOLS) that form in response to class II but not to class I MHC disparity and form independently of lymphotoxin α and lymphotoxin β receptor pathways. TOLS form in situ in the absence of lymph nodes, spleen, and thymus. Distinctive transcript patterns are maintained over time in TOLS including transcripts associated with Treg differentiation, T cell checkpoint signaling, and Th2 differentiation. Pathway transcripts related to inflammation are expressed in early stages of accepted grafts but diminish with time, while B cell transcripts increase. Intragraft transcript patterns at one week posttransplant distinguish those from kidneys destined to be rejected, that is, C57BL/6 allografts into DBA/2 recipients, from those that will be accepted. In contrast to inflammatory tertiary lymphoid organs (iTLOs) that form in response to chronic viral infection and transgenic Lta expression, TOLS lack high endothelial venules and germinal centers. TOLS represent a novel, pathogenetically important type of TLO that are in situ markers of regulatory tolerance.

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8+ T-cells, regulatory T-cells, and most notably, polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). While both combination therapies led to further increases in CD8+ T-cells, only the lenvatinib and anti-PD-1 combination decreased PMN-MDSCs. PMN-MDSC expansion was also seen in the blood of mice and one patient receiving lenvatinib therapy for ATC. RNA-Seq of the ATC cell line used in our mouse model demonstrated that lenvatinib has multifaceted effects on angiogenesis, response to hypoxia, the epithelial-to-mesenchymal transition, and on multiple pathways implicated in inflammation and host immunity. Combination of lenvatinib with anti-Gr-1 antibody ameliorated lenvatinib's expansion of MDSCs and significantly improved lenvatinib's anti-tumor effect. These data suggest that MDSCs play a negative role in ATC's response to lenvatinib and support future study of their role as a potential biomarker and treatment target.

Background Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC. Methods We again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth. Results Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2–3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation. Conclusions Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

Supplemental digital content is available in the text., Background Belatacept-based therapy in kidney transplant recipient has been shown to increase long-term renal allograft and patient survival compared with calcineurin inhibitor–based therapy, however, with an increased risk of acute T cell-mediated rejection (aTCMR). An improved understanding of costimulation blockade-resistant rejections could lead to a more personalized approach to belatacept therapy. Here, immunomic profiles of aTCMR biopsies of patients treated with either tacrolimus or belatacept were compared. Methods Formalin-fixed paraffin-embedded renal transplant biopsies were used for immunohistochemistry and gene expression analysis using the innovative NanoString technique. To validate NanoString, transcriptomic profiles of patients with and without biopsy-proven aTCMR were compared. Biopsies from 31 patients were studied: 14 tacrolimus-treated patients with aTCMR, 11 belatacept-treated patients with aTCMR, and 6 controls without rejection. Results A distinct pattern was seen in biopsies with aTCMR compared to negative controls: 78 genes had a higher expression in the aTCMR group (false discovery rate P value

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8

The enteric nervous system (ENS) is composed of neural crest-derived neurons (also known as ganglion cells) the cell bodies of which are located in the submucosal and myenteric plexuses of the intestinal wall. Intramucosal ganglion cells are known to exist but are rare and often considered ectopic. Also derived from the neural crest are enteric glial cells that populate the ganglia and the associated nerves, as well as the lamina propria of the intestinal mucosa. In Hirschsprung disease (HSCR), ganglion cells are absent from the distal gut because of a failure of neural crest-derived progenitor cells to complete their rostrocaudal migration during embryogenesis. The fate of intramucosal glial cells in human HSCR is essentially unknown. We demonstrate a network of intramucosal cells that exhibit dendritic morphology typical of neurons and glial cells. These dendritic cells are present throughout the human gut and express Tuj1, S100, glial fibrillary acidic protein, CD56, synaptophysin, and calretinin, consistent with mixed or overlapping neuroglial differentiation. The cells are present in aganglionic colon from patients with HSCR, but with an altered immunophenotype. Coexpression of Tuj1 and HNK1 in this cell population supports a neural crest origin. These findings extend and challenge the current understanding of ENS microanatomy and suggest the existence of an intramucosal population of neural crest-derived cells, present in HSCR, with overlapping immunophenotype of neurons and glia. Intramucosal neuroglial cells have not been previously recognized, and their presence in HSCR poses new questions about ENS development and the pathobiology of HSCR that merit further investigation.

The relative contribution of central and peripheral mechanisms to the generation and maintenance of allograft tolerance is of considerable interest. Here, we present new evidence that regulatory T cells (Foxp3(+) ) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor- and recipient-derived Foxp3(+) cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells. Foxp3(+) cell depletion did not result in the rejection of skin grafts of only MHC-disparate donors (B6.C-H2(d) /bByJ), indicating that MHC antigens were not the target in the graft. We conclude that two different mechanisms of tolerance are present in mixed chimeras. Hematopoietic chimerism, resistant to Foxp3(+) depletion, is probably due to deletional tolerance to MHC antigens, as supported by previous studies. In contrast, regulatory tolerance mechanisms involving Foxp3(+) cells are required to control reactivity against non-MHC antigens not present on hematopoietic lineages.

It is well established that the secretion of thrombospondin-1 (TSP-1) by activated stromal cells and its accumulation in the tumor microenvironment during dysplasia inhibits primary tumor growth through inhibition of angiogenesis. This inhibitory function of TSP-1 is actuated either by inhibiting MMP9 activation and the release of VEGF from extracellular matrix or by an interaction with CD36 on the surface of endothelial cells resulting in an increase in apoptosis. In contrast, several published articles have also shown that as tumor cells become more invasive and enter the early stage of carcinoma, they up-regulate TSP-1 expression, which may promote invasion and migration. In our in vivo studies using the polyoma middle T antigen (PyT) transgenic mouse model of breast cancer, we observed that the absence of TSP-1 significantly increased the growth of primary tumors, but delayed metastasis to the lungs. In this study, we propose a mechanism for the promigratory function of TSP-1 in mouse mammary tumor cells in vitro. We demonstrate the correlations between expression of TSP-1 and its receptor integrin α3β1, which is considered a promigratory protein in cancer cells. In addition we propose that binding of TSP-1 to integrin α3β1 is important for mediating actin filament polymerization and therefore, cell motility. These findings can help explain the dual functionality of TSP-1 in cancer progression.