Kai Yu

Receptor Engagement on Cells Expressing a Ligand for the Tolerance-Inducing Molecule OX2 Induces an Immunoregulatory Population That Inhibits Alloreactivity In Vitro and In Vivo

Increased survival of C57BL/6 renal allografts following portal vein donor-specific pretransplant immunization of C3H mice is associated with increased expression of the molecule OX2 seen on host dendritic cells, along with a marked polarization in cytokine production from lymphocytes harvested from the transplanted animals, with preferential production of IL-4, IL-10, and TGF-β on donor-specific restimulation in vitro, and decreased production of IL-2, IFN-γ, and TNF-α compared with non-portal vein-immunized control transplanted mice. The increased renal allograft survival and the altered cytokine production are abolished by infusion of anti-mouse OX2 mAb (3B6). Infusion of a soluble OX2:Fc immunoadhesin can itself produce significant prolongation of xeno- and allografts in mice. We have used FITC-conjugated OX2:Fc to characterize cells expressing a ligand (OX2L) for OX2, and provide evidence that subpopulations of LPS-stimulated splenic macrophages, Con A-activated splenic T cells, and the majority (>80%) of γδTCR+ T cells express this ligand. We show below that F4/80+, OX2L+ splenic macrophages, admixed with OX2:Fc, represent a potent immunosuppressive population capable of causing more profound inhibition of alloreactivity in vitro or in vivo than that seen using either OX2:Fc or OX2+ (or OX2L+) cells alone. Immunoregulation by this OX2L+ population occurs in an MHC-restricted fashion.

Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice

CD200 has been characterized as an important immunoregulatory molecule, increased expression of which can lead to decreased transplant rejection, autoimmunity, and allergic disease. Elevated CD200 expression has been reported to be associated with poor prognosis in a number of human malignancies. We have found that cells of the transplantable EMT6 mouse breast cancer line growing in vitro express low levels of CD200, but levels increase markedly during growth in immunocompetent mice. Similar increased in vivo expression does not occur in NOD-SCID.IL-2γr−/− mice or mice with generalized over-expression of a CD200 transgene. In both mice, tumor growth occurs faster. Altered CD200 expression in control versus transgenic mice is accompanied by reproducible changes in tumor-infiltrating host cells, and altered cell composition in lymph nodes draining the tumor (DLN). Neutralization of expressed CD200 by anti-CD200mAbs leads to decreased tumor growth in immunocompetent mice, with improved detection of cytotoxic anti-tumor immune cells in DLN. Finally, we report that tumor growth in vivo can be monitored by levels of soluble CD200 (sCD200) in serum of tumor-bearing animals.

Graft-infiltrating cells expressing a CD200 transgene prolong allogeneic skin graft survival in association with local increases in Foxp3+Treg and mast cells

Expression of the molecule CD200 has been reported to increase allograft survival by suppression of inflammation and acquired immunity. In previous studies we have shown that increased skin and cardiac allograft survival in transgenic mice over-expressing CD200 (CD200(tg)) occurs in association with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation. We investigated changes in graft-infiltrating cells, Treg and mast cells in skin grafts post transplantation into control or CD200(tg) mice, using focused gene array and real-time PCR to assess altered gene expression, and FACS, immunohistology and MLC to determine numbers/function of those cells. Graft-infiltrating cells isolated from CD200(tg) recipients suppressed induction of CTL from control lymph node cells in vitro, and contained increased numbers of infiltrating, non-degranulating, mast cells and Foxp3(+)Treg. Mast cells were also evident in graft tissue of control animals, but there these cells showed evidence for degranulation, and fewer Foxp3(+)Treg were present than was the case of CD200(tg) mice. The infusion of a competitive inhibitor of CD200:CD200R interactions, CD200(tr), at high concentrations (50μg/mouse iv) caused rapid rejection of grafts in CD200(tg) mice, mast cell degranulation within graft tissue, and a decrease in Treg infiltrates. These effects were attenuated by simultaneous infusion of the mast cell stabilizer, sodium cromoglycate. We conclude that CD200 expression contributes to graft prolongation through local suppression of mast cell degranulation, attraction/expansion of Treg, and attenuation of T cell effector activation.

Further evidence for a role of tumor CD200 expression in breast cancer metastasis: decreased metastasis in CD200R1KO mice or using CD200-silenced EMT6

Previous studies reported that CD200 expression on cells of the transplantable EMT6 mouse breast cancer line was increased during growth in immunocompetent mice. Low levels of expression persisted in NOD-SCID.IL-2γr−/− mice or mice with generalized over-expression of a CD200 transgene (CD200tg mice), despite the faster tumor growth in both of these latter strains. We also showed that CD200 expression (by the host and/or tumor cells) led to increased seeding of tumor cells to DLN in immunocompromised (CD200tg or NOD-SCID.IL-2γr−/−) vs immunocompetent mice, using limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN). Evidence for an important role for CD200 expression in this increased metastasis came from the observation that neutralization of CD200 by anti-CD200mAbs decreased tumor metastasis and increased levels of cytotoxic anti-tumor immune cells in DLN. In the current studies, we have extended these observations by exploring tumor growth/metastasis in CD200R1 KO mice in which we have previously shown, in a transplant model, that expression of CD200 fails to deliver an immunosuppressive signal. In addition, we have studied local and metastatic growth in healthy control mice of EMT6 tumor cells stably transduced with shRNA able to silence CD200 expression. In both scenarios, decreased metastasis was observed, with increased immunity to EMT6 detected by cytotoxicity assays. In addition, adoptive transfer of DLN to control mice attenuated EMT6 metastases implying a potential therapeutic benefit from neutralizing CD200 expression in breast cancer.

Cure of metastatic growth of EMT6 tumor cells in mice following manipulation of CD200:CD200R signaling

AbstractnIn previous studies, we observed that regulation of expression of CD200, both on cells of a transplantable breast cancer, EMT6, and of the host, as well as of the receptor, CD200R in host mice, regulated local tumor growth and metastasis in immunocompetent animals. This in turn led to an improved ability to document immunity to EMT6 in CD200R1KO mice. In the current study, we have explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1xa0cm3 in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells along with CpG as adjuvant. While control animals treated in this fashion developed significant pulmonary and liver metastases within 30xa0days of surgery, significant protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice on sacrifice at day 300. Following surgical resection and immunization, draining lymph nodes from control mice contained tumor cells cloned at limiting dilution in vitro even before pulmonary and hepatic metastasis was seen. In contrast, within the limits of detection of the assay used (sensitivity ~1 in 107 cells), no tumor cells were detected at limiting dilution in similarly treated CD200R1KO mice, and significant reductions were seen in CD200KO mice. Infusion of anti-CD4, but less so anti-CD8, mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically treated control mice also attenuated metastatic growth of tumor, which was abolished by pretreatment of transferred cells with anti-CD4 mAb. Our data suggest that CD200:CD200R attenuates a potentially tumor-protective CD4 host response to breast cancer.

Effect of CD200 and CD200R1 expression within tissue grafts on increased graft survival in allogeneic recipients

In transgenic mice over-expressing CD200 (CD200(tg)) graft survival is associated with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation (Foxp3; TGFβ; IL-10). Grafts are rejected in recipients lacking the inhibitory receptor for CD200, CD200R1. We compared grafts of C57BL/6 skin taken from control, CD200KO, CD200(tg), CD200R1KO or CD200(tg).CD200R1KO C57BL/6 donor mice transplanted to control or CD200(tg) BALB/c recipients. Animals received either low-dose rapamycin (0.5mg/kg), which only enhanced survival in CD200(tg) mice, or high dose rapamycin (1.5mg/kg) which increased graft survival in all recipients. Recipient draining lymph nodes (DLNs) were analyzed at 14days post grafting in mixed leukocyte cultures (MLCs) with irradiated BL/6 or C3H/HeJ stimulator cells, assaying antigen-specific CTL at day 5. MLC responses were correlated with changes in mRNA gene expression in skin tissue harvested from the same recipients, focusing on genes altered in graft-accepting CD200(tg) recipients. Tissue histology was used to assess graft infiltrating Foxp3(+) Tregs, mast cells (MCs) and their degranulation. CD200(tg) grafts were accepted in control but not CD200KO/CD200R1KO recipients, along with decreased degranulation in graft MCs, diminished DLN MLC responses, and augmented intragraft Foxp3, TGFβ, IL-10 and mast cell gene expression. Skin grafts from either CD200KO or CD200R1KO donors to control mice were rejected, with no change in DLN MLC responses, no altered graft gene expression from that seen using control skin grafts, and pronounced graft MC degranulation. Our data highlight a role for both graft and host CD200/CD200R expression in increased allograft survival.

CCR4 dependent migration of Foxp3+ Treg cells to skin grafts and draining lymph nodes is implicated in enhanced graft survival in CD200tg recipients.

We have previously reported that transgenic overexpression of CD200 in either mouse skin graft donors or recipients significantly enhances skin allograft survival. By focused microarray analysis we showed this enhanced graft survival is associated with increased expression of Foxp3, GITR, CTLA-4 and CCR4 mRNA, all genes related to T(reg) cell induction/function, and of Gata3, IL-4, IL-5, IL-13, and somewhat surprisingly, of T-bet, INF-γ and granzyme b. Gene-specific real-time PCR and immunohistochemistry analysis confirmed an increase in Foxp3(+) T(reg) cells in both the skin grafts and draining lymph nodes (DLNs) of CD200(tg) recipient mice at both 7/14 days post engraftment, as well as providing evidence for increased expression of the ligands for CCR4, CCL17 and CCL22 in both locations. Following lentivirus-mediated shRNA treatment of Dox-treated CD200(tg) mice to attenuate expression of CCR4 mRNA, the increased localization of T(reg) cells in skin/DLN of CD200(tg) recipients was abolished, and the enhanced graft survival similarly reversed. We conclude that enhanced CCR4 dependent migration of Foxp3(+) T(reg) to grafted tissue and DLNs is an essential step in the graft prolongation afforded by overexpression of CD200.

Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

Background and aim CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of knockoutof CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS)—induced colitis, a mouse model of inflammatory bowel disease (IBD). Methods Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA. Results CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3+ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3+ regulatory T (Treg) cells was seen in the colon of CD200tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection. Conclusions The CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice.