Frequency of dendritic cell subsets and ILT3, ILT4 gene expression in two different immunosuppressive protocols in kidney transplant recipients. A cohort report.

Dendritic cells (DCs) have a major role in the initiation of an immune response and Immunoglobulin-like transcript 3&4 (ILT3&ILT4) are inhibitory receptors that induce tolerance in DCs. Recent studies show that immunosuppressive agents affect frequency of DCs. Herein, we compared the effect of mycophenolate mofetil (MMF) and sirolimus (SRL) in tacrolimus (TAC)-based immunosuppression on DC subsets frequency and ILT3/ILT4 gene expression in kidney transplant recipients. We enrolled 24 adult transplant recipients who received MMF/TAC (n = 14) or SRL/TAC (n = 10). Peripheral blood samples were obtained from recipients, 24-48 h before transplantation and 4 months after transplantation. The frequency of DC subsets was analyzed by flow cytometry and gene expression of ILT3/ILT4 were estimated by real-time PCR. Our results showed that MMF vs. SRL treated recipient showed an increase in pDC % with increased in the expression of ILT3/ILT4 which is in favor of better allograft survival; However, for confirming the results of this preliminary study, a cohort study with larger sample size is necessary.

Human CD43+ B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally in healthy individuals.

CD20(+)CD27(+)CD43(+) B (CD43(+) B) cells have been newly defined among PBMCs and proposed to be human B1 cells. However, it is controversial as to whether they are orthologs of murine B1 cells and how they are related to other B-cell populations, particularly CD20(+)CD27(+)CD43(-) memory B cells and CD20(low)CD27(high)CD43(high) plasmablasts. Our objective is to identify phenotypically the position of CD43(+) B cells among peripheral B-lineage cell compartments in healthy donors, with reference to B-cell subsets from patients with systemic lupus erythematosus (SLE). We found that CD43(+) B cells among PBMCs from healthy subjects were indistinguishable phenotypically from memory B cells in terms of surface markers, and spontaneous in vitro Ig and IL-10 secretion capability, but quite different from plasmablasts. However, a moderate correlation was found in the frequency of CD43(+) B cells with that of plasmablasts in healthy donors but not in SLE patients. An in vitro differentiation experiment indicated that CD43(+) B cells give rise to plasmablasts more efficiently than do memory B cells, suggesting that they are more closely related to plasmablasts developmentally than are memory B cells, which is also supported by quantitative PCR analysis of mRNA expression of B-cell and plasma cell signature genes. Thus, we conclude that, in healthy individuals, CD43(+) B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally, although the developmental origin of CD43(+) B cells is not necessarily the same as that of plasmablasts.

Prognostic impact of LILRB4 expression on tumor-infiltrating cells in resected non-small cell lung cancer.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4/ILT3) is an up-and-coming molecule that promotes immune evasion. We have previously reported that LILRB4 facilitates myeloid-derived suppressor cells (MDSCs)-mediated tumor metastasis in mice. This study aimed to investigate the impact of the LILRB4 expression levels on tumor-infiltrating cells on the prognosis of non-small cell lung cancer (NSCLC) patients. METHODS: We immunohistochemically evaluated the LILRB4 expression levels of completely resected 239 NSCLC specimens. Whether the blocking of LILRB4 on human PBMC-derived CD33+ MDSCs inhibited the migration ability of lung cancer cells was also examined using transwell migration assay. RESULTS: The LILRB4 high group, in which patients with a high LILRB4 expression level on tumor-infiltrating cells, showed a shorter overall survival (OS) (p = 0.013) and relapse-free survival (RFS) (p = 0.0017) compared to the LILRB4 low group. Multivariate analyses revealed that a high LILRB4 expression was an independent factor for postoperative recurrence, poor OS and RFS. Even in the cohort background aligned by propensity score matching, OS (p = 0.023) and RFS (p = 0.0046) in the LILRB4 high group were shorter than in the LILRB4 low group. Some of the LILRB4 positive cells were positive for MDSC markers, CD33 and CD14. Transwell migration assay demonstrated that blocking LILRB4 significantly inhibited the migration of human lung cancer cells cocultured with CD33+ MDSCs. CONCLUSION: Together, signals through LILRB4 on tumor-infiltrating cells, including MDSCs, play an essential role in promoting tumor evasion and cancer progression, impacting the recurrence and poor prognosis of patients with resected NSCLC.

A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

Anti-immunoglobulin-like transcript 3 induced acute myocarditis-A case report.

To the best of our knowledge, this is the first published report of anti-immunoglobulin-like transcript 3 (ILT3)-induced myocarditis. A 48-year old female patient with refractory acute myeloid leukemia who was given a single dose of anti-ILT3 monotherapy presented with fever, hypotension, chest pain, and elevated cardiac biomarkers. Systolic bi-ventricular function was in normal limits. The patient was promptly treated with pulse dose steroids with a rapid hemodynamic and clinical improvement and declining levels of cardiac biomarkers. The diagnosis of acute myocarditis was confirmed using cardiac magnetic resonance imaging applying the revised Lake Lewis criteria. While larger-scale data are needed in order to assess the incidence, management and prognosis of anti-ILT-3 induced myocarditis, we believe a high level of suspicion for adverse non-target cardiac effects is required in patients receiving this novel class of drugs.

Distorted frequency of dendritic cells and their associated stimulatory and inhibitory markers augment the pathogenesis of pemphigus vulgaris.

The objective of this study was to investigate the frequency and functionality of DCs and its associated stimulatory and inhibitory markers in the pathogenesis of PV Active PV patients (n = 30) having both skin and oral lesions, and 30 healthy controls were recruited in the study. The frequency of DCs was determined by flow cytometry followed by the primary culture by using recombinant IL-4 (250 IU/ml) and GM-CSF (600 IU/ml). The culture supernatant was used for ELISA. RNA was isolated from sorted DCs and used for the mRNA expression of DC-associated stimulatory (CD40 and CD80) and inhibitory (PSGL1 and ILT3) markers. Tissue localization of Langerhans cells was done by immunohistochemistry. In this study, altered frequency of myeloid DC (mDC) and plasmacytoid DC (pDC) was seen in the circulation of PV patients. The primary culture of patient-derived DCs showed anomalous cytokine profiling. In the culture supernatant of DCs, elevated levels of TNF-ɑ and IL-12 were detected in PV patients. Meanwhile, reverse trend was found in the case of IFN-ɑ and IL-10 cytokine levels. Similarly, a discrepancy in the expression of DC-associated stimulatory (CD40 and CD80) and inhibitory (PSGL1 and ILT3) markers suggested their possible involvement in the immunopathogenesis of PV. An elevated number of tissue localizing Langerhans cells was also observed in the perilesional skin. This study indicates the distorted frequency and functionality of DCs in the immunopathogenesis of PV. Targeting these functional markers in the future may generate novel therapeutic options for better management of PV.

Synergistic Effects of Interferon-γ and Vitamin D3 Signaling in Induction of ILT-3highPDL-1high Tolerogenic Dendritic Cells.

In the past, interferon (IFN)-γ and vitamin D3 (vit D3) have both been associated with induction of tolerogenic characteristics in human dendritic cells (DCs). Although there are only a few reports on interdependency of their actions, the interplay between IFN-γ and vit D3 has been clearly demonstrated in certain aspects of immune reactivity. Since both agents have been associated with regulation of immune responses, we set out to examine their functional and mechanistic interactions in context of principal regulators of immunity, the DCs. Combined treatment with vit D3 and IFN-γ caused an extensive expression of immunoglobulin-like transcript (ILT)-3 and programmed death ligand (PDL)-1 on γ/D3DCs, significantly greater than that caused by vit D3 alone. Such γ/D3DCs retained all general DC characteristics. After CD40 ligand-induced activation, they produced increased amounts of IL-10 with almost absent production of IL-12p70. On the other hand, the co-stimulatory potential of γ/D3DCs was weak, with cells possessing the capacity to inhibit CD4+ T cell, CD8+ T cell, as well as memory T cell responses. Naive CD4+ T cells stimulated with γ/D3DCs produced increased amounts of IL-10 with concomitantly low IFN-γ production, upon T cell receptor activation. Additionally, γ/D3DCs completely inhibited granzyme B expression by CD8+ T cells. The percentage of FoxP3-positive cells in co-cultures with naive CD4+ T cells was significantly higher where γ/D3DCs were used as stimulators compared to DCs treated with vit D3 alone and it could be partially reversed by PDL-1 blockade. Interestingly, γ/D3DCs were inefficient at suppressing mDC-induced CD4+ T cell proliferation, but were twice as effective as D3DCs at suppressing mDC-induced CD8+ T cell proliferation. Blockade of indoleamine-2,3-dioxygenase did not reduce the tolerogenic phenotype induced by IFN-γ and vit D3 treatment. Examination of signaling pathways activation revealed a tendency toward increased ERK and Akt phosphorylation in γ/D3DCs. Inhibition of MEK/ERK and PI3K/mTOR pathways significantly reduced the expression of ILT-3 and PDL-1 on γ/D3DCs. In summary, we present the first evidence for existing synergy between IFN-γ and vit D3 in shaping a unique tolerogenic DC activation state.

Expression of ILT3 predicts poor prognosis and is inversely associated with infiltration of CD45RO+ T cells in patients with colorectal cancer.

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.

Blood tolerogenic monocytes and low proportions of dendritic cell subpopulations are hallmarks of human tuberculosis.

BACKGROUND: The immune mechanisms underlying the pathogenesis of tuberculosis (TB) need better understanding to improve TB management, as the disease still causes more than 1.5 million deaths annually. This study tested the hypothesis that a modulation of the proportions or activation status of APC during Mycobacterium tuberculosis infection may impact on the course of the disease. PROCEDURE: Proportions of circulating APC subsets and the expression of stimulatory (CD86), inhibitory (ILT-3, ILT-4, ILT-7), or apoptosis-inducing (PDL-1, PDL-2) molecules were analyzed in 2 independent cohorts, on blood monocytes and dendritic cell (DC) subsets from patients with active or latent TB infection (aTB /LTBI) and from uninfected subjects. RESULTS: Higher proportions of classical CD14+ CD16- and intermediate CD14+ CD16+ monocytes, and lower proportions of plasmacytoid DC (pDC) and type 2 myeloid DC were observed in the blood from untreated patients with aTB compared with those with LTBI and with healthy subjects, with an early normalization of the proportions of pDC during treatment. In addition, monocytes from M. tuberculosis-infected subjects expressed higher levels of ILT-3, ILT-4, and PDL-1 compared with healthy controls, these differences being more important for patients with aTB than for those with LTBI. CONCLUSIONS: These results confirm the hypothesis of a modulation of the proportions and activation status of APC during M. tuberculosis infection and suggest that these cells could play a role in driving the course of M. tuberculosis infection.

TLR3 Signaling Promotes the Induction of Unique Human BDCA-3 Dendritic Cell Populations.

Conventional and plasmacytoid dendritic cells (cDCs and pDCs) are the two populations of DCs that can be readily identified in human blood. Conventional DCs have been subdivided into CD1c(+), or blood dendritic cells antigen (BDCA) 1 and CD141(+), or BDCA-3, DCs, each having both unique gene expression profiles and functions. BDCA-3 DCs express high levels of toll-like receptor 3 and upon stimulation with Poly I:C secrete IFN-ß, CXCL10, and IL-12p70. In this article, we show that activation of human BDCA-3 DCs with Poly I:C induces the expression of activation markers (CD40, CD80, and CD86) and immunoglobulin-like transcript (ILT) 3 and 4. This Poly I:C stimulation results in four populations identifiable by flow cytometry based on their expression of ILT3 and ILT4. We focused our efforts on profiling the ILT4(-) and ILT4(+) DCs. These ILT-expressing BDCA-3 populations exhibit similar levels of activation as measured by CD40, CD80, and CD86; however, they exhibit differential cytokine secretion profiles, unique gene signatures, and vary in their ability to prime allogenic naïve T cells. Taken together, these data illustrate that within a pool of BDCA-3 DCs, there are cells poised to respond differently to a given input stimulus with unique output of immune functions.

Molecular and Cellular Characterization of Human CD8 T Suppressor Cells.

Bidirectional interactions between dendritic cells and Ag-experienced T cells initiate either a tolerogenic or immunogenic pathway. The outcome of these interactions is of crucial importance in malignancy, transplantation, and autoimmune diseases. Blockade of costimulation results in the induction of T helper cell anergy and subsequent differentiation of antigen-specific CD8+ T suppressor/regulatory cells (Ts). Ts, primed in the presence of inhibitory signals, exert their inhibitory function in an antigen-specific manner, a feature with tremendous clinical potential. In transplantation or autoimmunity, antigen-specific Ts can enforce tolerance to auto- or allo-antigens, while otherwise leaving the immune response to pathogens uninhibited. Alternatively, blockade of inhibitory receptors results in the generation of cytolytic CD8+ T cells, which is vital toward defense against tumors and viral diseases. Because CD8+ T cells are MHC Class I restricted, they are able to recognize HLA-bound antigenic peptides presented not only by APC but also on parenchymal cells, thus eliciting or suppressing auto- or allo-immune reactions.

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (ß7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

Immunoglobulin-like transcript 3 is expressed by myeloid-derived suppressor cells and correlates with survival in patients with non-small cell lung cancer.

Myeloid-derived suppressor cells (MDSCs) play an important role in immune suppression and accumulate under pathologic conditions such as cancer and chronic inflammation. They comprise a heterogeneous population of immature myeloid cells that exert their immunosuppressive function via a variety of mechanisms. Immunoglobulin-like transcript 3 (ILT3) is a receptor containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be expressed on antigen-presenting cells and is an important regulator of dendritic cell tolerance. ILT3 exists in a membrane-bound and a soluble form and can interact with a yet unidentified ligand on T cells and thereby induce T-cell anergy, regulatory T cells, or T suppressor cells. In this study, we analyzed freshly isolated peripheral blood mononuclear cells (PBMCs) of 105 patients with non-small cell lung cancer and 20 healthy controls and demonstrated for the first time that ILT3 is expressed on MDSCs. We show that increased levels of circulating MDSCs correlate with reduced survival. On the basis of ILT3 cell surface expression, an ILT3low and ILT3high population of polymorphonuclear (PMN)-MDSCs could be distinguished. Interestingly, in line with the immunosuppressive function of ILT3 on dendritic cells, patients with an increased proportion of PMN-MDSCs and an increased fraction of the ILT3high subset had a shorter median survival than patients with elevated PMN-MDSC and a smaller ILT3high fraction. No correlation between the ILT3high subset and other immune variables was found. ILT3 expressed on MDSCs might reflect a previously unknown mechanism by which this cell population induces immune suppression and could therefore be an attractive target for immune intervention.

Cervical (pre)neoplastic microenvironment promotes the emergence of tolerogenic dendritic cells via RANKL secretion.

The progression of genital human papillomavirus (HPV) infections into preneoplastic lesions suggests that infected/malignant cells are not adequately recognized by the immune system. In this study, we demonstrated that cervical/vulvar cancer cells secrete factor(s) that affect both the maturation and function of dendritic cells (DC) leading to a tolerogenic profile. Indeed, DC cocultured with cancer cell lines display both a partially mature phenotype after lipopolysaccharide (LPS) maturation and an altered secretory profile (IL-10high and IL-12p70low). In addition, tumor-converted DC acquire the ability to alter T-cell proliferation and to induce FoxP3+ suppressive T cells from naive CD4+ T cells. Among the immunosuppressive factors implicated in DC alterations in genital (pre)neoplastic microenvironment, we identified receptor activator of nuclear factor kappa-B ligand (RANKL), a TNF family member, as a potential candidate. For the first time, we showed that RANKL expression strongly increases during cervical progression. We also confirmed that RANKL is directly secreted by cancer cells and this expression is not related to HPV viral oncoprotein induction. Interestingly, the addition of osteoprotegerin (OPG) in coculture experiments reduces significantly the inhibition of DC maturation, the release of a tolerogenic cytokine profile (IL-12low IL-10high) and the induction of regulatory T (Treg) cells. Our findings suggest that the use of inhibitory molecules directed against RANKL in cervical/vulvar (pre)neoplastic lesions might prevent alterations of DC functionality and represent an attractive strategy to overcome immune tolerance in such cancers.