Suppression of Experimental Autoimmune Encephalomyelitis by ILT3.Fc.

Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS that is characterized by demyelination, axonal loss, gliosis, and inflammation. The murine model of MS is the experimental autoimmune encephalopathy (EAE) induced by immunization of mice with myelin oligodendrocyte glycoprotein (MOG)35-55 Ig-like transcript 3 (ILT3) is an inhibitory cell surface receptor expressed by tolerogenic human dendritic cells. In this study, we show that the recombinant human ILT3.Fc protein binds to murine immune cells and inhibits the release of proinflammatory cytokines that cause the neuroinflammatory process that result in paralysis. Administration of ILT3.Fc prevents the rapid evolution of the disease in C57BL/6 mice and is associated with a profound reduction of proliferation of MOG35-55-specific Th1 and Th17 cells. Inhibition of IFN-γ and IL-17A in mice treated with ILT3.Fc is associated with delayed time of onset of the disease and its evolution to a peak clinical score. Neuropathological analysis shows a reduction in inflammatory infiltrates and demyelinated areas in the brains and spinal cords of treated mice. These results indicate that inhibition of Th1 and Th17 development provides effective suppression of EAE and suggests the feasibility of a clinical approach based on the use of ILT3.Fc for treatment of MS. Furthermore, our results open the way to further studies on the effect of the human ILT3.Fc protein in murine experimental models of autoimmunity and cancer.

Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection.

BACKGROUND: In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection. METHODS: We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses. RESULTS: In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P = 9.8×10-5). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor-recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P = 6.5×10-5). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P = 4.7×10-8). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses. CONCLUSIONS: We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3. (Funded by the Columbia University Transplant Center and others.).

The clinical and pathological features of plasma cell myeloma post solid organ transplantation.

Plasma cell neoplasms (PCNs), comprising plasma cell myelomas (PCMs) and plasmacytomas, which occur after solid organ transplantation, represent rare subtypes of monomorphic post-transplant lymphoproliferative disorders (M-PTLDs). Data regarding the clinical and pathological features of post-transplant (PT)-PCMs are limited. To gain a better understanding of disease biology, we performed comprehensive immunophenotypic analysis, reviewed cytogenetic analysis results and evaluated clinical outcomes of PT-PCMs diagnosed and treated at our institution. Fifteen PT-PCM (M: F - 4:1) and two PT-MGUS (two males) cases were identified. The median age of PT-PCM patients was 68 years (29-79 years) and PCMs presented at a median of 9.7 years (0.5-24.7 years) after transplantation. The PT-PCMs accounted for 11.6% of all M-PTLDs and the period prevalence was 9/3108 (0.29%), 3/1071 (0.28%), 2/1345 (0.15%) and 1/878 (0.11%) post kidney, heart, liver and lung transplantation. Lytic bone disease was observed in 1/11 (9%) patients. Marrow plasma cell infiltration ranged from 10%-70% (median 20%), with 10/15 (67%) and 5/15 (33%) cases manifesting immature and plasmablastic morphology. The immunophenotype of all cases and cytogenetic abnormalities, identified in 60% of cases, were similar to multiple myeloma (MM) of immunocompetent individuals. All PT-PCMs were EBER negative. Ten of 11 (91%) patients with active MM were treated, all with proteasome inhibitor-based therapy. Treatment response and 5-year overall survival (54.5%) was comparable to MM of immunocompetent individuals. However, the survival of patients with plasmablastic PCMs was inferior to those with immature PCMs. 0ur findings indicate PT-PCMs to be predominantly late onset PTLDs that have similar clinicopathologic characteristics as conventional MM.

ILT3.Fc-CD166 Interaction Induces Inactivation of p70 S6 Kinase and Inhibits Tumor Cell Growth.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28- T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166-CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9-based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.

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.

A novel HLA-B*58:141 allele identified by next-generation sequencing in a potential hematopoietic cell recipient.

We report a novel HLA-B*58 allele, now named B*58:141, identified by next-generation sequencing.

Optical genome mapping in acute myeloid leukemia: a multicenter evaluation.

Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for diagnostic subtyping, prognosis, and patient management. However, cytogenetic/cytogenomic techniques used to identify those aberrations, such as karyotyping, fluorescence in situ hybridization (FISH), or chromosomal microarray analysis (CMA), are limited by the need for skilled personnel as well as significant time, cost, and labor. Optical genome mapping (OGM) provides a single, cost-effective assay with a significantly higher resolution than karyotyping and with a comprehensive genome-wide analysis comparable with CMA and the added unique ability to detect balanced structural variants (SVs). Here, we report in a real-world setting the performance of OGM in a cohort of 100 AML cases that were previously characterized by karyotype alone or karyotype and FISH or CMA. OGM identified all clinically relevant SVs and copy number variants (CNVs) reported by these standard cytogenetic methods when representative clones were present in >5% allelic fraction. Importantly, OGM identified clinically relevant information in 13% of cases that had been missed by the routine methods. Three cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. In 4% of cases, OGM findings would have altered recommended clinical management, and in an additional 8% of cases, OGM would have rendered the cases potentially eligible for clinical trials. The results from this multi-institutional study indicate that OGM effectively recovers clinically relevant SVs and CNVs found by standard-of-care methods and reveals additional SVs that are not reported. Furthermore, OGM minimizes the need for labor-intensive multiple cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow.

Induction of antigen-specific human T suppressor cells by membrane and soluble ILT3.

Antigen-specific CD8 suppressor T cells (CD8(+) Ts) are adaptive regulatory T cells that are induced in vivo and in vitro by chronic antigenic stimulation of human T cells. CD8(+) Ts induce the upregulation of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells rendering these antigen presenting cells (APCs) tolerogenic. Tolerogenic APCs induce CD4(+) T helper anergy and elicit the differentiation of CD4(+) and CD8(+) T regulatory/suppressor cells. Overexpression of membrane ILT3 in APC results in inhibition of NF-κB activation, transcription of inflammatory cytokines and costimulatory molecules. Soluble ILT3-Fc which contains only the extracellular, Ig-like domain linked to mutated IgG1 Fc, is strongly immunosuppressive. ILT3-Fc, induces the differentiation of human CD8(+) Ts which inhibit CD4(+) Th and CD8(+) CTL effector function both in vitro and in vivo. The acquisition of Ts' function by primed CD8(+) T cells treated with ILT3-Fc was demonstrated to be the effect of the significant upregulation of BCL6, a transcriptional repressor of IL-2, IFN-gamma, IL-5 and granzyme B. The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8(+) Ts. These genes are known inhibitors of cytokine production and TCR signaling and are targeted by miRNAs which are suppressed by ILT3-Fc. ILT3-Fc induces tolerance to allogeneic human islets and reverses rejection after its onset in a humanized NOD/SCID mouse model. Based on these findings we postulate that ILT3-Fc may become an important new agent for treatment of autoimmunity and transplant rejection.

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.

Quantifying protein abundance on single cells using split-pool sequencing on DNA-barcoded antibodies for diagnostic applications.

Proteins play critical roles across all facets of biology, with their abundance frequently used as markers of cell identity and state. The most popular method for detecting proteins on single cells, flow cytometry, is limited by considerations of fluorescent spectral overlap. While mass cytometry (CyTOF) allows for the detection of upwards of 40 epitopes simultaneously, it requires local access to specialized instrumentation not commonly accessible to many laboratories. To overcome these limitations, we independently developed a method to quantify multiple protein targets on single cells without the need for specialty equipment other than access to widely available next generation sequencing (NGS) services. We demonstrate that this combinatorial indexing method compares favorably to traditional flow-cytometry, and allows over two dozen target proteins to be assayed at a time on single cells. To showcase the potential of the technique, we analyzed peripheral blood and bone marrow aspirates from human clinical samples, and identified pathogenic cellular subsets with high fidelity. The ease of use of this technique makes it a promising technology for high-throughput proteomics and for interrogating complex samples such as those from patients with leukemia.

Previous SARS-CoV-2 infection or a third dose of vaccine elicited cross-variant neutralising antibodies in vaccinated solid-organ transplant recipients.

Objectives: The SARS-CoV-2 pandemic poses a great threat to global health, particularly in solid organ transplant recipients (SOTRs). A 3-dose mRNA vaccination protocol has been implemented for the majority of SOTRs, yet their immune responses are less effective compared to healthy controls (HCs). Methods: We analyzed the humoral immune responses against the vaccine strain and variants of concern (VOC), including the highly mutated-omicron variant in 113 SOTRs, of whom 44 had recovered from COVID-19 (recovered-SOTRs) and 69 had not contracted the virus (COVID-naïve). In addition, 30 HCs, 8 of whom had recovered from COVID-19, were also studied. Results: Here, we report that three doses of the mRNA vaccine had only a modest effect in eliciting anti-viral antibodies against all viral strains in the fully vaccinated COVID-naive SOTRs (n = 47). Only 34.0% of this group of patients demonstrated both detectable anti-RBD IgG with neutralization activities against alpha, beta, and delta variants, and only 8.5% of them showed additional omicron neutralizing capacities. In contrast, 79.5% of the recovered-SOTRs who received two doses of vaccine demonstrated both higher anti-RBD IgG levels and neutralizing activities against all VOC, including omicron. Conclusion: These findings illustrate a significant impact of previous infection on the development of anti-SARS-CoV-2 immune responses in vaccinated SOTRs and highlight the need for alternative strategies to protect a subset of a lesser-vaccine responsive population.

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.

HLA antibodies in pediatric heart transplantation.

We have analyzed the impact of anti-HLA antibodies present in the patients' circulation prior and/or following heart transplantation in a population of 108 pediatric recipients. Anti-HLA class I and class II antibodies were monitored by traditional CDC using donor and panel T and B lymphocytes and by SPA for detection of DSA. There was a highly significant correlation between the development of AMR and presence of CDC- or SPA-detected DSA. However, the fraction of the transplant population which remained AMR-free was much higher among patients with SPA-detected compared to CDC-detected DSA. Furthermore, long-term graft survival was negatively affected only by cytotoxic, complement-fixing anti-HLA class I antibodies developing following transplantation. Anti-HLA class I or class II antibodies detected by SPA had no effect on long-term survival rates.

Immunophenotypic Spectrum and Genomic Landscape of Refractory Celiac Disease Type II.

Refractory celiac disease type II (RCD II), also referred to as "cryptic" enteropathy-associated T-cell lymphoma (EATL) or "intraepithelial T-cell lymphoma," is a rare clonal lymphoproliferative disorder that arises from innate intraepithelial lymphocytes. RCD II has a poor prognosis and frequently evolves to EATL. The pathogenesis of RCD II is not well understood and data regarding the immunophenotypic spectrum of this disease and underlying genetic alterations are limited. To gain further biological insights, we performed comprehensive immunophenotypic, targeted next-generation sequencing, and chromosome microarray analyses of 11 RCD II cases: CD4-/CD8- (n=6), CD8+ (n=4), and CD4+ (n=1), and 2 of 3 ensuing EATLs. Genetic alterations were identified in 9/11 (82%) of the RCD II cases. All 9 displayed mutations in members of the JAK-STAT signaling pathway, including frequent, recurrent STAT3 (7/9, 78%) and JAK1 (4/9, 44%) mutations, and 9/10 evaluable cases expressed phospho-STAT3. The mutated cases also harbored recurrent alterations in epigenetic regulators (TET2, n=5 and KMT2D, n=5), nuclear factor-κB (TNFAIP3, n=4), DNA damage repair (POT1, n=3), and immune evasion (CD58, n=2) pathway genes. The CD4-/CD8- and other immunophenotypic subtypes of RCD II exhibited similar molecular features. Longitudinal genetic analyses of 4 RCD II cases revealed stable mutation profiles, however, additional mutations were detected in the EATLs, which occurred at extraintestinal sites and were clonally related to antecedent RCD II. Chromosome microarray analysis demonstrated copy number changes in 3/6 RCD II cases, and 1 transformed EATL with sufficient neoplastic burden for informative analysis. Our findings provide novel information about the immunophenotypic and genomic characteristics of RCD II, elucidate early genetic events in EATL pathogenesis, and reveal potential therapeutic targets.

Sinus Lift and Implant Insertion on 3D-Printed Polymeric Maxillary Models: Ex Vivo Training for In Vivo Surgical Procedures.

BACKGROUND AND OBJECTIVES: The aim of this study is to demonstrate the increased efficiency achieved by dental practitioners when carrying out an ex vivo training process on 3D-printed maxillaries before performing in vivo surgery. MATERIALS AND METHODS: This developed ex vivo procedure comprises the following phases: (i) scanning the area of interest for surgery; (ii) obtaining a 3D virtual model of this area using Cone Beam Computed Tomography (CBCT); (iii) obtaining a 3D-printed model (based on the virtual one), on which (iv) the dental practitioner simulates/rehearses ex vivo (most of) the surgery protocol; (v) assess with a new CBCT the 3D model after simulation. The technical steps of sinus augmentation and implant insertion could be performed on the corresponding 3D-printed hemi-maxillaries prior to the real in vivo surgery. Two study groups were considered, with forty patients divided as follows: Group 1 comprises twenty patients on which the developed simulation and rehearsal procedure was applied; Group 2 is a control one which comprises twenty patients on which similar surgery was performed without this procedure (considered in order to compare operative times without and with rehearsals). RESULTS: Following the ex vivo training/rehearsal, an optimal surgery protocol was developed for each considered case. The results of the surgery on patients were compared with the results obtained after rehearsals on 3D-printed models. The performed quantitative assessment proved that, using the proposed training procedure, the results of the in vivo surgery are not significantly different (p = 0.089) with regard to the ex vivo simulation for both the mezio-distal position of the implant and the distance from the ridge margin to sinus window. On the contrary, the operative time of Group 1 was reduced significantly (p = 0.001), with an average of 20% with regard to in vivo procedures performed without rehearsals (on the control Group 2). CONCLUSIONS: The study demonstrated that the use of 3D-printed models can be beneficial to dental surgeon practitioners, as well as to students who must be trained before performing clinical treatments.

Detection of Nonhematologic Neoplasms by Routine Flow Cytometry Analysis.

OBJECTIVES: We investigated the ability of routine flow cytometry (FC) to detect nonhematologic neoplasms (non-HN) using antibody panels routinely used for the diagnosis of hematologic neoplasms. METHODS: FC analyses of 4,000 various diagnostic samples were retrospectively reviewed to identify cases in which an aberrant, viable CD45-negative, nonhematologic neoplastic population was detected by FC panels designed to evaluate hematologic neoplasms. RESULTS: A total of 57 (1.4%) diverse non-HNs were identified, representing neuroendocrine tumors (33/57) and carcinomas (9/57), as well as other malignancies (15/57) such as sarcoma and melanoma. The majority of neoplasms were positive for at least one antibody, typically CD56 (43/51, 84.3%), followed by CD117 (15/34, 44.1%) and CD138 (6/33, 18.2%). CONCLUSIONS: Our findings highlight the importance of carefully inspecting CD45-negative events to identify non-HNs by routine FC analysis. This can help expedite further downstream immunophenotypic analysis of specimens and triage samples for appropriate genetic and molecular studies.

CD8+ T suppressor cells and the ILT3 master switch.

Similar to helper and cytotoxic T cells, CD8(+) T suppressor cells (Ts) acquire antigen specificity via direct interaction with antigen-presenting cells (APC). They induce the upregulation of the inhibitory receptor immunoglobulin-like transcript (ILT)3 on professional and nonprofessional APC, rendering these cells tolerogenic and able to induce the differentiation of further waves of regulatory and suppressor T cells. This review sums up evidence that ILT3 is the centerpiece of CD8(+) Ts-driven suppression and acts as a master switch in the regulation of CD8(+) and CD4(+) T-cell responses to antigens in transplantation, autoimmunity, allergy, and cancer.

CD8+ suppressor and cytotoxic T cells recognize the same human leukocyte antigen-A2 restricted cytomegalovirus peptide.

We explored the possibility that antigen-specific human CD8(+) T cells, which display cytotoxic or suppressor function, can recognize the same peptide epitope. Using the human leukocyte antigen-A0201 restricted immunodominant cytomegalovirus epitope pp65-NLVPMVATV for pulsing either mature/immunogenic or ILT3(high)ILT4(high) tolerogenic dendritic cells (DC), we generated cytotoxic and suppressor CD8(+) T-cell lines, respectively. Our data indicate that modulating the functional state of DC is crucial to the development of tolerogenic or immunogeneic peptide-specific vaccines.