Immunosuppressive activity of recombinant ILT3

doi:10.1016/j.intimp.2006.07.017

International Immunopharmacology

Volume 6, Issues 13–14, 20 December 2006, Pages 1889-1894

https://doi.org/10.1016/j.intimp.2006.07.017 Get rights and content

Abstract

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.

Introduction

The interaction between antigen-specific CD8⁺CD28⁻ FOXP3⁺ T suppressor cells (Ts) with allogeneic dendritic cells (DC) or activated endothelial cells (EC) results in tolerization of DC or EC inducing the upregulation of the immunoglobulin-like transcripts ILT3 and ILT4 [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. We have demonstrated that tolerogenic DC or EC anergize alloreactive CD4⁺CD45RO⁺CD25⁺ T cells converting them into FOXP3⁺ regulatory T cells (Treg), which continue the cascade of suppression [5], [14]. Interleukin-10 (IL-10) and interferon (IFN)-alpha also induce ILT3 and ILT4 upregulation in DC and EC [5], [11], [14]. This implies a common mechanism of EC and DC-mediated suppression of T cell alloreactivity. This finding and the observation that in organ allograft recipients quiescence is associated with the presence in the circulation of donor-specific Ts and Tr emphasize the importance of the cross-talk between tolerogenic DC and EC with T cells in suppression of allorecognition [11], [13]. This concept was further substantiated in a rat model of heart allotransplantation. Tolerance was induced in ACI recipients by multiple transfusions of UVB-irradiated blood from Lewis heart donors [12]. CD8⁺ T cells from tolerant ACI rats expressed FOXP3, transferred tolerance to naïve secondary hosts and induced the upregulation of PIR-B, an ILT4 orthologue in Lewis DC and heart EC. When long-term surviving Lewis heart allografts with PIR-B⁺ EC were retransplanted from a primary to a secondary ACI recipient, they were indefinitely tolerated, demonstrating that the bi-directional interaction between Ts and APC is crucial to the induction and maintenance of tolerance [12]. Agents which elicit this interaction, such as human recombinant ILT3, may be important for achieving long-term tolerance. We have explored this possibility by testing the immunosuppressive activity of membrane and soluble ILT3 and ILT4 molecules [15].

Section snippets

Construction and expression of membrane and soluble ILT molecules

The cytoplasmic domain of ILT3 and ILT4 which contains the ITIM motifs was deleted by PCR amplification and cloning in the expression vector pc DNA4/TO/myc-His in frame with a c-myc epitope and polyhistidine (6 × His) region. The resulting mutants called ILT3delta (M1-E544) and ILT4delta (M1-E529) encodes proteins which contain the putative leader peptide, the extracellular, transmembrane and a stretch of 48 and 47 amino acids of the cytoplasmic domain of ILT3 and ILT4, respectively, fused with

Results

CD3⁺ T cells from healthy blood donors were cultured for 6 days with irradiated KG1, KG1.ILT3 (transfected with full length ILT3) or KG1.ILT3delta (the cytoplasmic deletion mutant of ILT3). KG1 cells induced strong T cell proliferation, while KG1.ILT3 and KG1.ILT3delta elicited only weak reactivity. T cell responsiveness to KG1.ILT3 and KG1.ILT3delta was enhanced when rIL2 was added to the assay indicating that T cell interaction with the extracellular domain of ILT3 results in anergy. In

Discussion

Over the last 10 years, the literature has been dominated by studies emphasizing the importance of naturally occurring CD4⁺CD25⁺ regulatory T cells. The prevailing dogma has been that this is a unique T cell subset, which derives from CD4⁺CD25⁺ thymocytes and is endowed with the omnipotent capacity of inhibiting auto and allo-reactivity, against self- and non-self-antigens. These natural Tregs were shown to act directly on other activated CD4⁺ and CD8⁺ T cells. High expression of CD25 was

Acknowledgements

This work was supported by grants from the NIH (AI25210-19, AI55234-03) and the Interuniversity Organ Transplantation Consortium, Rome, Italy.

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Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4

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Immunoglobulin like transcript 3 (ILT3) was previously identified as an inhibitory receptor to induce T cell anergy in tranplantation, autoimmunity and allergy. Here we aimed to investigate the expression of ILT3 in colorectal cancer, analyze the association between ILT3 expression and clinicopathological variables and prognosis, and evaluate the correlation between the expression of ILT3 and CD45RO+ T cells density. Expression of ILT3 was identified on the cell membrane and/or in the cytoplasm. High expression ILT3 was identified in 55 of 85 (64.7%) tumor specimens, which was significantly higher than that in the adjacent normal tissues(5/30) (P < 0.001). High ILT3 expression was significantly associated with positive lymph node metastasis (N1-2; P = 0.03), advanced disease (stage III–IV; P = 0.03), and reduced OS in patients. The ILT3 expression level was an independent prognostic factor (P = 0.004) and inversely correlated with the number of CD45RO+ T cells (P = 0.019). In the present study, high ILT3 expression was observed in colorectal cancer and inversely associated with CD45RO+ T cells density and prognosis, suggesting that ILT3 played an important role in tumor progression by possible influence on CD45RO+ T cells in the tumor microenvironment.
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Soluble ILT3 protein bound to alloactivated CD4+ Th cell and induced anergy in them, in addition to inducing differentiation of antigen-specific CD8+ Ts cells and suppressing the differentiation of IFNγ-producing CD8+ CTL [52–54]. In contrast, the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs, as demonstrated by the lost of suppressive activity of a cytoplasmic deletion mutants of ILT4 [52,55]. Therefore, ILT4 acts only on the cell surface and exerts its inhibitory effects solely by signaling intracellularly and blocking the differentiation of mature immunogenic APC.
The immunolglobulin-like transcript (ILT) family of proteins are surface receptors expressed by antigen-presenting cells capable of modulating host cell functions through intracellular signaling. Here we discuss the recent studies regarding the role of dendritic cell (DC)–expressed ILT receptors in suppressor T (Ts) cells induced DC tolerization. DC-expressed ILT3 and ILT4 are stimulated by their cognate ligands such as major histocompatibility complex class I (MHC-I), HLA-G, and CD1d, and this stimulation is a prerequisite for DC tolerization. The molecular interaction between ILT and ligands seems to create a tri-molecule complex at the interface between Ts cell and DC, which consists of DC-expressed ILT receptor and MHC-I, as well as T-cell–expressed T cell receptor (TCR). Furthermore, ILT4 recognition of MHC-I and CD1d is shown to be antigen dependent, suggesting that T-cell antigen specificity possibly affects the outcome of DC tolerization. Therefore the ILT-MHC interface could be a potential novel target for improving vaccine efficiency and allograft survival, or for devising new treatments for autoimmune diseases.
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This effect could be mimicked by transiently overexpressing ILT3 in HUVEC or by adding soluble recombinant ILT3 to cocultures, suggesting that ILT3/ILT4 on human vascular EC might have an anergizing effect of EC on CD4 T cells by directly sending suppressive signals to T cells. This finding is in line with data demonstrating that for the inhibitory effect of ILT3 the cytoplasmatic domain is dispensable, whereas ILT4 needs its ITIM to display suppressive effects [24,27]. CD4 T cells coincubated with ILT3+ILT4+ HUVEC did not express foxp3; also expression of Th1/Th2 transcription factors T-bet and GATA3 were not changed.
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Engagement of the inhibitory receptors ILT2 and ILT4 alters the cytokine and chemokine secretion profile of monocytes and can inhibit Fc receptor signaling [15,22]. The role and function of ILT3 on DCs have been precisely described [8,12,18,23–28]. Although the ligand for ILT3 is unknown, ILT4 is known to bind to the third domain of HLA class I molecules (HLA-A, HLA-B, HLA-C, and HLA-G), competing with CD8 for MHC class I binding [29].
The expression of inhibitory immunoglobulin-like transcripts (ILTs) on dendritic cells (DCs) is a biomarker of tolerogenic DCs. In this article we discuss current knowledge on the function of ILTs and explore the molecular mechanisms involved in modulation of DCs via the inhibitory receptor and its natural ligand, rendering these cells tolerogenic. We propose a method to enhance targeting of inhibitory receptors on DCs using microparticles containing a preferential ligand, HLA-G, and monoclonal antibody against the pan-DCs marker CD11c. The double-coated microparticles increase the binding of ILT4 receptor and improve modulation of DCs in vitro and in vivo. This targeting concept can be used for regulation of specific immune responses to antigens in transplantation, autoimmunity, allergy, and cancer.
Changes in the Expression of the Immunoglobulin-like Transcript 3 (ILT3) and ILT4 Receptors in Renal Allograft Recipients: Effect of Donor and Recipient Aging
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It is possible that the poorer findings with ILT3 may be due to the small number of patients, but they also may relate to different mechanisms of action. ILT3 has been demonstrated to act in both membrane-bound and soluble fractions; whereas, ILT4 acts only on the cell surface.6 On the other hand, a functional and phenotypic overlap between tolerogenic DCs and endothelial cells in the induction of CD8+CD28− T cells has been reported that may interfere with our data.7
The aim of the present study was to investigate the number and phenotype of pre- and posttransplant peripheral blood dendritic cells (DCs) in kidney graft recipients to correlate with CD4⁺CD25^high Treg and CD8⁺CD28⁻ cells. Data were analyzed according to the age of the donor–recipient pairs.
A cohort of 49 cadaveric kidney transplant recipients was prospectively studied pretransplant and 6 months posttransplant by three-color flow cytometry with specific monoclonal antibodies. Patients were subgrouped according to age (elderly were considered above 60 years old and young below 55 years old) in the following donor–recipient pairs: aged/aged, young/aged, aged/young, young/young.
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Immunosuppressive activity of recombinant ILT3

Abstract

Introduction

Section snippets

Construction and expression of membrane and soluble ILT molecules

Results

Discussion

Acknowledgements

Transpl Immunol

Transpl Immunol

Hum Immunol

Hum Immunol

Hum Immunol

Transpl Immunol

Hum Immunol

Immunity

Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28− T cells

Int Immunol

CD8+CD28− T suppressor cells and the induction of antigen-specific, antigen-presenting cell-mediated suppression of Th reactivity

Immunol Rev

Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4

Nat Immunol

Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8⁺CD28⁻ T cells

CD8⁺CD28⁻ T suppressor cells and the induction of antigen-specific, antigen-presenting cell-mediated suppression of Th reactivity