ILT3.Fc inhibits the production of exosomes containing inflammatory microRNA in supernatants of alloactivated T cells.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Allospecific CD8 T suppressor cells induced by multiple MLC stimulation or priming in the presence of ILT3.Fc have similar gene expression profiles.

Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8(+) CD28(-) T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity.

Downregulation of inflammatory microRNAs by Ig-like transcript 3 is essential for the differentiation of human CD8(+) T suppressor cells.

We have investigated the mechanism underlying the immunoregulatory function of membrane Ig-like transcript 3 (ILT3) and soluble ILT3Fc. microRNA (miRNA) expression profile identified genes that were downregulated in ILT3-induced human CD8(+) T suppressor cells (Ts) while upregulated in T cells primed in the absence of ILT3. We found that miR-21, miR-30b, and miR-155 target the 3'-untranslated region of genes whose expression was strongly increased in ILT3Fc-induced Ts, such as dual specificity phosphatase 10, B cell CLL/lymphoma 6, and suppressor of cytokine signaling 1, respectively. Transfection of miRNA mimics or inhibitors and site-specific mutagenesis of their 3'-untranslated region binding sites indicated that B cell CLL/lymphoma 6, dual specificity phosphatase 10, and suppressor of cytokine signaling 1 are direct targets of miR-30b, miR-21, and miR-155. Primed CD8(+) T cells transfected with miR-21&30b, miR-21&155, or miR-21&30b&155 inhibitors displayed suppressor activity when added to autologous CD3-triggered CD4 T cells. Luciferase reporter assays of miR-21 and miR-155 indicated that their transcription is highly dependent on AP-1. Analysis of activated T cells showed that ILT3Fc inhibited the translocation to the nucleus of the AP-1 subunits, FOSB and c-FOS, and the phosphorylation of ZAP70 and phospholipase C-γ 1. In conclusion, ILT3Fc inhibits T cell activation and induces the generation of Ts targeting multiple inflammatory miRNA pathways.

Gene profile analysis of CD8(+) ILT3-Fc induced T suppressor cells.

Gene profile analysis of ILT3-Fc-induced Ts revealed a significant upregulation of Zink finger proteins, most of which act as transcriptional repressors. Included among these repressors is BCL6, which was shown to play a critical role in the differentiation of ILT3-Fc-induced T suppressor (Ts) cells. Genes implicated in cell cycle progression were downregulated. Genes encoding numerous inflammatory cytokines and chemokines were also downregulated. In contrast, antiapoptotic genes, as well as members of the WNT and transforming growth factor-ß pathways, were upregulated. This study elucidates certain important aspects of Ts differentiation and function.

BCL6 is required for differentiation of Ig-like transcript 3-Fc-induced CD8+ T suppressor cells.

Ig-like transcript 3 (ILT3) is an inhibitory receptor expressed by tolerogenic dendritic cells. When human CD8(+) T cells are allostimulated in the presence of recombinant ILT3-Fc protein, they differentiate into antigenic specific T suppressor (Ts) cells that inhibit CD4 and CD8 T cell effector function both in vitro and in vivo. ILT3-Fc-induced CD8(+) Ts cells express high amounts of BCL6 that are crucial to their function. Knockdown of BCL6 from unprimed human T cells prevents their differentiation into Ts cells, whereas ex vivo overexpression of BCL6 converts CD8(+) T cells into Ts cells. NOD/SCID mice transplanted with human pancreatic islets and humanized by injection of human PBMCs tolerate the graft and develop BCL6(high) CD8(+) Ts cells when treated with ILT3-Fc before or after the onset of rejection. This indicates that ILT3-Fc acts through BCL6 and is a potent immunosuppressive agent for reversing the onset of allo- or possibly autoimmune attacks against pancreatic islets.

Suppression of xenogeneic graft-versus-host disease by treatment with immunoglobulin-like transcript 3-Fc.

Allogeneic hematopoietic cell transplantation represents an important therapy for certain malignant and nonmalignant diseases. However, graft-versus-host disease (GVHD) is a major cause of mortality and morbidity. The search for agents that can efficiently suppress GVHD has been going on for more than half a century. GVHD is particularly strong in xenogeneic donor-recipient combinations, given the unlimited number of potentially immunogenic antigens donor lymphocytes encounter in the host. Using a hu-nonobese diabetic/severe combined immunodeficiency (hu-NOD/SCID) gamma-null model of xenogeneic GVHD, we have demonstrated that treatment with recombinant immunoglobulin-like transcript 3-Fc protein induces the differentiation of CD8(+) T suppressor cells and blocks the cellular and humoral arm of the GVH reaction.

Ig-like transcript 3 regulates expression of proinflammatory cytokines and migration of activated T cells.

Ig-like transcript 3 (ILT3), an inhibitory receptor expressed by APC is involved in functional shaping of T cell responses toward a tolerant state. We have previously demonstrated that membrane (m) and soluble (s) ILT3 induce allogeneic tolerance to human islet cells in humanized NOD/SCID mice. Recombinant sILT3 induces the differentiation of CD8(+) T suppressor cells both in vivo and in vitro. To better understand the molecular mechanisms by which ILT3 suppresses immune responses, we have generated ILT3 knockdown (ILT3KD) dendritic cells (DC) and analyzed the phenotypic and functional characteristics of these cells. In this study, we report that silencing of ILT3 expression in DC (ILT3KD DC) increases TLR responsiveness to their specific ligands as reflected in increased synthesis and secretion of proinflammatory cytokines such as IL-1alpha, IL-1beta, and IL-6 and type I IFN. ILT3KD-DC also secretes more CXCL10 and CXCL11 chemokines in response to TLR ligation, thus accelerating T cell migration in diffusion chamber experiments. ILT3KD-DC elicit increased T cell proliferation and synthesis of proinflammatory cytokines IFN-gamma and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibition of NF-kappaB and, to a lesser extent, MAPK p38 pathways in DC. Our results suggest that ILT3 plays a critical role in the control of inflammation.

Immunoglobulin-like transcript 3-Fc suppresses T-cell responses to allogeneic human islet transplants in hu-NOD/SCID mice.

OBJECTIVE: The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS: NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS: Although human T-cell engraftment was similar in all groups, ILT3-Fc-treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8(+) T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc-treated mice and control recipients rejecting the grafts. CONCLUSIONS: The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.

Soluble Ig-like transcript 3 inhibits tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients.

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

Immunosuppressive activity of recombinant ILT3.

Tolerogenic antigen presenting cells (APC) are characterized by high expression of the inhibitory receptors ILT3 and ILT4. We have engineered ILT3 and ILT4 cytoplasmic deletion mutants (ILT3delta and ILT4delta), which were transfected in the dendritic-like cell line KG1, to investigate ILT3 and ILT4's capacity to signal extracellularly. KG1.ILT3delta, similar to untruncated ILT3, inhibits T cell responses such as proliferation and cell-mediated cytotoxicity. In contrast, KG1.ILT4delta lost the suppressive activity of untruncated ILT4. This indicates that the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs. We further demonstrated that recombinant soluble ILT3 inhibits T helper and cytotoxic function while inducing the differentiation of CD8(+) Ts cells. Hence, Ts modulate APC function inducing inhibitory receptors, which in turn elicit the generation of Ts.

Recombinant Ig-like transcript 3-Fc modulates T cell responses via induction of Th anergy and differentiation of CD8+ T suppressor cells.

The Ig-like transcript (ILT)3 is crucial to the tolerogenic activity acquired by dendritic cells exposed to allospecific T suppressor (Ts) cells. We have explored the immunomodulatory property of the extracellular region of ILT3 using a cytoplasmic deletion mutant of ILT3 (ILT3delta), expressed as membrane-bound ILT3 on KG1 cells, and a rILT3-Fc fusion protein. We found that both membrane-bound and soluble ILT3 inhibited T cell proliferation in primary and secondary MLC inducing anergy in CD4+ Th cells and suppressing the differentiation of IFN-gamma-producing CD8+ CTL. Furthermore, membrane-bound and soluble ILT3 induced the differentiation of CD8+ FOXP3+ Ts cells in primary 7-day MLC. The suppressive activity of these CD8+ Ts cells is alloantigen specific and mediated by their capacity to induce the up-regulation of ILT3 and down-regulation of costimulatory molecules such as CD86 in APC from the stimulator used for priming, but not on control HLA-mismatched APC. Our finding that ILT3-Fc has potent immunosuppressive activity in vitro and that it acts on T cells only upon activation suggests the possibility that this agent may be of use for specific suppression of the immune response in autoimmunity or transplantation.

Rat CD8+ FOXP3+ T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR-B in APC and rendering the graft invulnerable to rejection.

Human CD8+ FOXP3+ T suppressor cells (TS) were previously shown to induce the expression of the inhibitory receptors, Immunoglobulin-like transcript (ILT) 3 and ILT4 on dendritic and endothelial cells, rendering them tolerogenic to allogeneic T cells. We have demonstrated the importance of CD8+ TS in a rat model of heart allo-transplantation. Tolerance was induced in ACI recipients by multiple transfusions of UVB-irradiated blood from Lewis heart donors. CD8+ T cells from tolerant ACI rats expressed FOXP3, transferred tolerance to naive secondary hosts and induced the upregulation of the inhibitory receptor, paired immunoglobulin-like receptor (PIR)-B, an ILT4 orthologue, in Lewis dendritic cells (DC) and heart endothelial cells (EC). When long-term surviving Lewis heart allografts with PIR-B+ EC were retransplanted from a primary to a secondary ACI recipient they did not elicit rejection. This study focuses attention on the need to develop agents that act directly on graft EC in order to achieve tolerance.

Identification of tissue-restricted transcripts in human islets.

The purpose of our study was to identify transcripts specific for tissue-restricted, membrane-associated proteins in human islets that, in turn, might serve as markers of healthy or diseased islet cell masses. Using oligonucleotide chips, we obtained gene expression profiles of human islets for comparison with the profiles of exocrine pancreas, liver, and kidney tissue. As periislet presence of type 1 interferon is associated with the development of type 1 diabetes, the expression profile of human islets treated ex vivo with interferon-alpha2beta (IFNalpha2beta) was also determined. A set of genes encoding transmembrane- or membrane-associated proteins with novel islet-restricted expression was resolved by determining the intersection of the islet set with the complement of datasets obtained from other tissues. Under the influence of IFNalpha2beta, the expression levels of transcripts for several of the identified gene products were up- or down-regulated. One of the islet-restricted gene products identified in this study, vesicular monoamine transporter type 2, was shown to bind [3H]dihydrotetrabenazine, a ligand with derivatives suitable for positron emission tomography imaging. We report here the first comparison of gene expression profiles of human islets with other tissues and the identification of a target molecule with possible use in determining islet cell masses.

Gene expression profiling and functional activity of human dendritic cells induced with IFN-alpha-2b: implications for cancer immunotherapy.

PURPOSE: In this study, we have compared patterns of gene expression and functional activity of human dendritic cells (DCs) cultured under defined conditions in IFN-alpha-2b and recombinant human granulocyte macrophage colony-stimulating factor (DCA) with cells grown in granulocyte macrophage colony-stimulating factor and IL-4 (DC4) as an initial step in evaluating the clinical utility of DCA in cancer immunotherapy. EXPERIMENTAL DESIGN AND RESULTS: Comparison of mRNA transcript profiles between DCA and DC4 revealed different expression patterns for cytokines, chemokines, chemokine receptors, costimulatory molecules, and adhesion proteins. Many genes involved in antigen (Ag) processing were equally expressed in both populations; however, expression of transcripts involved in Ag presentation was increased in DCA. DCA also showed up-regulation of Toll-like receptor 2 and 3, as well as several tumor necrosis factor family ligands. Consistent with expression profiling, functional assays demonstrated that DCAs were more potent stimulators of naive T-cell responses than DC4 in an interleukin 15 and interleukin 1beta-dependent manner. DCA-mediated tumor cell-directed cytotoxicity induced apoptosis in different human tumor cell lines and internalized apoptotic bodies to a greater extent than DC4. Lastly, in vitro priming experiments, using apoptotic cells or peptide as sources of Ag, showed that DCA drove the expansion of tumor peptide Ag-specific autologous CD8+ T cells to a greater extent than DC4. CONCLUSIONS: The unique phenotype conferred by culturing DCs in IFN-alpha-2b may be useful in adoptive transfer regimens where the destruction of tumor cells in situ, initiation of T-cell responses toward tumor tissue with unknown Ags, and/or enhancement of pre-existing Ag-specific memory responses are desired outcomes.