Leukocyte Adhesion Deficiency Type Research Articles

Impairment of cytokine production following immunological synapse formation in patients with Wiskott-Aldrich syndrome and leukocyte adhesion deficiency type 1

T cells following immunological synapse (IS) formation with antigen-presenting cells produce multiple cytokines through T cell receptor, integrin, and costimulatory signaling. Here, we investigated the cytokine profiles following IS formation in response to staphylococcal superantigen exposure in three adolescent patients with classical Wiskott-Aldrich syndrome (WAS) and in one patient with leukocyte adhesion deficiency (LAD) type 1. All WAS patients showed lower Th1 and Th2-skewed cytokine production; similar results were observed in the flow cytometric analysis of IFNγ- and IL-4-producing T cells. The patient with LAD type 1 with somatic mosaicism in 2% of CD8+ T cells showed lower Th1 and Th2 cytokine production than healthy controls. The patients with WAS were susceptible to infections and atopic manifestations, and the patients with LAD type 1 showed cold abscess on their skin, our findings using patient samples provide clinical insights into the mechanisms underlying immunodeficiency related to the symptoms of each disease.

Preclinical safety and efficacy of lentiviral-mediated gene therapy for leukocyte adhesion deficiency type I

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene, which encodes for the CD18 subunit of β2-integrins. Deficient expression of β2-integrins results in impaired neutrophil migration in response to bacterial and fungal infections. Using a lentiviral vector (LV) that mediates a preferential myeloid expression of human CD18 (Chim.hCD18-LV), we first demonstrated that gene therapy efficiently corrected the phenotype of mice with severe LAD-I. Next, we investigated if the ectopic hCD18 expression modified the phenotypic characteristics of human healthy donor hematopoietic stem cells and their progeny. Significantly, transduction of healthy CD34+ cells with the Chim.hCD18-LV did not modify the membrane expression of CD18 nor the adhesion of physiological ligands to transduced cells. Additionally, we observed that the repopulating properties of healthy CD34+ cells were preserved following transduction with the Chim.hCD18-LV, and that a safe polyclonal repopulation pattern was observed in transplanted immunodeficient NOD scid gamma (NSG) mice. In a final set of experiments, we demonstrated that transduction of CD34+ cells from a severe LAD-I patient with the Chim.hCD18-LV restores the expression of β2-integrins in these cells. These results offer additional preclinical safety and efficacy evidence supporting the gene therapy of patients with severe LAD-I.

Healing With Complication: An Unusual Case of Nasal Tip Ulceration in Leukocyte Adhesion Deficiency Type 1

A 2-month-old girl presented with a necrotizing lesion over the tip of the nose that started as redness. The lesion subsequently increased in size to form a vesicle and ruptured to form a chronic, progressive, nonhealing ulcer without pus discharge (Figure 1, A). The girl was born at term, birth weight 2.5 kg. There was a history of delayed separation of the umbilical cord at day 22 of life. She developed an episode of omphalitis and another episode of sepsis with pneumonia on days 6 and 20 of life, respectively.

Identification of the causative mutation in the ITGB2 gene in a LAD1 patient by whole exome sequencing

Leukocyte adhesion deficiency type 1 (LAD1) is a rare congenital immunodeficiency disease. The cause of disease is determined to be the mutations in the ITGB2 gene that codes for CD18, the beta chain of beta-2 integrins, leads to decreased expression or functioning of CD18. This deficiency leads to severe impairment of leukocyte adhesion to the vascular wall and leukocyte migration to sites of infection and inflammation. LAD1 has also been associated with inhibition of interleukin-23 and interleukin-17 resulting in a hyperinflammatory and chronic inflammation. Patients with LAD1 typically present in early infancy with recurrent, life threatening infections that are frequently fatal before 2 years of age without hematopoietic stem cell transplant (HSCT). However, LAD1 is difficult to diagnose and many LAD1 patients die at a young age despite intensive antibiotic therapy. Accurate diagnosis requires detailed clinical information (delayed umbilical cord loss, severe periodontitis, delayed wound healing and sores, skin abscesses, and recurrent infection), and confirmation the absence of integrins by flow cytometric analysis. A better understanding of the molecular characteristics of this disease is needed to raise awareness and definitive diagnosis infants with LAD1. To definitive diagnosis, whole exome sequencing and Sanger sequencing were performed in an eighteen-month-old boy with severe leukocytosis, recurrent infections, delayed wound healing, and hepatosplenomegaly associated with an acquired cytomegalovirus infection. Two variants: One previously reported mutation (c.533C>T, p.Pro178Leu) and one novel variant (c.59-1G>A), in the ITGB2 gene were detected. These results can be used for definitive genetic diagnosis, genetic counseling, as well as a prenatal diagnosis in LAD1 patients.

Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis.

β2-integrins are heterodimeric surface receptors that are expressed specifically by leukocytes and consist of a variable α (CD11a-d) and a common β-subunit (CD18). Functional impairment of CD18, which causes leukocyte adhesion deficiency type-1 results in an immunocompromised state characterized by severe infections, such as invasive pulmonary aspergillosis (IPA). The underlying immune defects have largely been attributed to an impaired migratory and phagocytic activity of polymorphonuclear granulocytes (PMN). However, the exact contribution of β2-integrins for PMN functions in-vivo has not been elucidated yet, since the mouse models available so far display a constitutive CD18 knockout (CD18-/- or CD18hypo). To determine the PMN-specific role of β2-integrins for innate effector functions and pathogen control, we generated a mouse line with a Ly6G-specific knockdown of the common β-subunit (CD18Ly6G cKO). We characterized CD18Ly6G cKO mice in-vitro to confirm the PMN-specific knockdown of β2-integrins. Next, we investigated the clinical course of IPA in A. fumigatus infected CD18Ly6G cKO mice with regard to the fungal burden, pulmonary inflammation and PMN response towards A. fumigatus. Our results revealed that the β2-integrin knockdown was restricted to PMN and that CD18Ly6G cKO mice showed an aggravated course of IPA. In accordance, we observed a higher fungal burden and lower levels of proinflammatory innate cytokines, such as TNF-α, in lungs of IPA-infected CD18Ly6G cKO mice. Bronchoalveolar lavage revealed higher levels of CXCL1, a stronger PMN-infiltration, but concomitantly elevated apoptosis of PMN in lungs of CD18Ly6G cKO mice. Ex-vivo analysis further unveiled a strong impairment of PMN effector function, as reflected by an attenuated phagocytic activity, and a diminished generation of reactive oxygen species (ROS) and neutrophil-extracellular traps (NET) in CD18-deficient PMN. Overall, our study demonstrates that β2-integrins are required specifically for PMN effector functions and contribute to the clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lungs.

Molecular dissection of two Pakistani families segregating Leukocyte Adhesion Deficiency Type-I

BACKGROUND & OBJECTIVE: Leukocyte adhesion deficiency (LAD1) is an autosomal recessive type of inherited disorder caused by total or partial deficiency of CD18 expression. LAD1 is characterized by recurrent bacterial and fungal infections, in some cases delayed umbilical cord separation, delayed wound healing due to blockade in leukocyte migration to site of inflammation and infection. The present study involves genetic analysis of two unrelated families suffering from LAD1.
 METHODOLOGY: In the present study, two separated and unrelated Pakistani families are included suffering from Leukocyte Adhesion Deficiency type -1 (PAD1). After detailed clinical evaluation, whole blood samples were collected from patients, parents and available healthy siblings. Genomic DNA was extracted from all the blood samples, and using a specific primer all the coding ITGB2 gene exons were PCR amplified.
 RESULTS: The amplified products were sanger sequenced. DNA sequencing analysis revealed a nonsense mutation c.186C > A, p.(Cys62*) in exon four and a missense mutation c.382G>T, p.(Asp128Tyr) in exon five of the gene. The mutation is segregating in autosomal recessive pattern in the family.
 CONCLUSION: Recurrent mutations on a specific locus (gene) changes the allele frequency from a healthy allele to a disease allele and hence play role in new genotype. This research study demonstrates the allelic heterogeneity of the ITGB2 gene in Pakistani patients diagnosed with LAD1. The research findings in the present study suggest that every population should develop national registry of patients suffering from primary immune deficiencies and a mutation database for rare genetic disorders. This will facilitate early diagnosis and genetic counseling to the patient family.

Understanding the Role of LFA-1 in Leukocyte Adhesion Deficiency Type I (LAD I): Moving towards Inflammation?

LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the ITGB2 (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.

Premature Loss of Deciduous Teeth as a Symptom of Systemic Disease: A Narrative Literature Review.

Background: Premature loss of primary teeth can occur as a consequence of dental trauma, neonatal tooth extraction, early childhood caries, or periodontal problems, or it can be a manifestation of systemic disease. This review aims to present systemic disorders that can lead to premature loss of deciduous teeth in children and to provide a comprehensive resource for clinical practice for both physicians and dentists. Methods: This study is a narrative review of original studies and case reports published in English and Polish between 1957 and 2021 that was conducted by searching electronic scientific resources: PubMed, Google Scholar, Web of Science, and Science Direct. The schema of the qualification process is represented by a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). In total, 196 articles were identified; after provisional assessment of the titles and abstracts by two reviewers, 46 were found to be relevant to the topic, including 1 review, 16 original papers, and 27 case reports regarding systemic disease resulting in premature tooth loss. Results: In this study, 16 systemic diseases were linked to premature primary tooth loss in children: Papillon–Lefèvre syndrome, mucocutaneous dyskeratosis, Coffin–Lowry syndrome, congenital adrenal hyperplasia, Langerhans cell histiocytosis, cherubism, hypophosphatasia, acatalasia, Chediak–Higashi syndrome, cyclic neutropenia, erythromelalgia, Down syndrome, Hajdu–Cheney syndrome, short bowel syndrome, leukocyte adhesion deficiency type 1 (LAD-1), and Wiedemann–Steiner syndrome (WSS).

Examining the Effect of Kindlin-3 Binding Site Mutation on LFA-1-ICAM-1 Bonds by Force Measuring Optical Tweezers.

Integrins in effector T cells are crucial for cell adhesion and play a central role in cell-mediated immunity. Leukocyte adhesion deficiency (LAD) type III, a genetic condition that can cause death in early childhood, highlights the importance of integrin/kindlin interactions for immune system function. A TTT/AAA mutation in the cytoplasmic domain of the β2 integrin significantly reduces kindlin-3 binding to the β2 tail, abolishes leukocyte adhesion to intercellular adhesion molecule 1 (ICAM-1), and decreases T cell trafficking in vivo. However, how kindlin-3 affects integrin function in T cells remains incompletely understood. We present an examination of LFA-1/ICAM-1 bonds in both wild-type effector T cells and those with a kindlin-3 binding site mutation. Adhesion assays show that effector T cells carrying the kindlin-3 binding site mutation display significantly reduced adhesion to the integrin ligand ICAM-1. Using optical trapping, combined with back focal plane interferometry, we measured a bond rupture force of 17.85 ±0.63 pN at a force loading rate of 30.21 ± 4.35 pN/s, for single integrins expressed on wild-type cells. Interestingly, a significant drop in rupture force of bonds was found for TTT/AAA-mutant cells, with a measured rupture force of 10.08 ± 0.88pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects catch bond formation and bond strength of integrin–ligand bonds. As a consequence of this reduced binding, CD8+ T cell activation in vitro is also significantly reduced.

Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. DisclosuresNo relevant conflicts of interest to declare.

Gene Delivery Using Baculovirus in Human Hematopoietic Stem and Progenitor Cells Requires Inhibition of Cellular Innate Immune Pathways

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) has the potential to treat inherited blood disorders not amendable to CRISPR-Cas9 gene inactivation or single base editing. For many diseases, one of the major hurdles is viral delivery of large DNA templates needed for gene correction. Due to limited adeno-associated virus (AAV) packaging capacity other delivery approaches are needed.Baculovirus (BV), specifically Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a large double-stranded DNA (dsDNA) virus widely used for protein expression and AAV production. In addition, BV has been proposed as a potential therapeutic vector (Ono, Viruses 2018). BV does not replicate in mammalian cells, can deliver large quantities of DNA with virtually unlimited packaging capacity, and can express genes under the control of mammalian promoters. While capable of transducing human hepatic cells and some cell lines (Chen, Biotechnol Adv. 2011), to our knowledge BV transduction efficiency has not been tested in human CD34+ HSPCs or shown in any hematopoietic cell line.Here we show for the first time that BV can be used as a gene delivery vector for primary human CD34+ cells mobilized from healthy donors. We constructed VSV-G pseudotyped BV with a copGFP reporter flanked by 4kb homology arms (HAs) to ITGB2, a locus mutated in leukocyte adhesion deficiency type I (LAD-1) (Fig. A, top). As measured by qPCR, viral DNA was detected in CD34+ cells after transduction at a multiplicity of infection (MOI) of 50, suggesting vector binding and entry in these cells. However, although toxicity was not observed, GFP expression as assessed by flow cytometry was mostly undetectable (less than 0.1%). In contrast, robust (>70%) GFP expression was measured in 293A cells using the same BV vector, suggesting that an inhibitory cellular process was uniquely triggered in primary CD34+ cells following transduction with BV.Recent work has shown that BV can activate cellular innate immune pathways including toll-like receptors (TLRs) (Abe, J Virol 2009) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (Amalfi S, JVI 2020) resulting in viral clearance and attenuation of gene expression (Ono, JVI 2014). We hypothesized that inhibition of activated cellular innate immune pathways may allow for more efficient BV gene expression in human CD34+ HSPCs. To examine this possibility, we tested over a dozen small molecule inhibitors at multiple doses targeting the major dsDNA sensing innate immune pathways including cGAS-STING (Fig. A, bottom). We found that a 45-minute pre-treatment with the STING inhibitor, H-151, while slightly toxic, enhanced GFP expression several fold, from less than 0.1% to an average of 1.5% in multiple independent donors (Fig. B-C). To improve viability, we also targeted cell death pathways. We tested the pan-caspase inhibitor, zVAD-FMK, which can inhibit both innate activation of gasdermin D (GSDMD), a major dsDNA sensing pathway, as well as apoptotic cell death. We additionally tested the necroptosis inhibitor Nec-1, as necroptosis can be activated in settings of apoptotic inhibition and inflammation. Notably, the combination of both inhibitors with H-151 improved not only cell viability, but also substantially enhanced GFP expression (8%), suggesting a synergistic benefit by inhibiting both innate immune activation and cell death pathways (Fig. D-E). To assess whether BV can efficiently transduce HSPCs with long-term repopulating activity, we pre-stimulated CD34+ cells for 48 hours in culture followed by transduction with BV at an MOI of 25 with our optimized drug cocktail. We examined GFP positivity in both CD34+CD38+ progenitors and CD34+CD38- HSC enriched populations by flow cytometry. After 24 hours, we found an average of 28% GFP+ CD34+CD38- cells and 8% GFP+ CD34+CD38+ progenitors (Fig. F-G). These data suggest that using our optimized approach, BV can target more primitive HSPCs.Collectively, our results lay the groundwork for future studies characterizing innate immune responses to dsDNA viruses in CD34+ cells, and highlight the potential use of BV as a delivery system for homology-directed gene editing in HSPCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

M179 UNSUCCESSFUL ALLOGENEIC HEMATOPOIETIC STEM-CELL TRANSPLANT IN PATIENT WITH LEUKOCYTE ADHESION DEFICIENCY TYPE 1 FROM ECUADOR

Patients with severe Leukocyte adhesion deficiency type 1 (LAD-1) rarely live longer than two years of age without hematopoietic stem cell transplant (HSCT). Those who undergo a HSCT transplant have a 75% of survival. LAD-1 is a disease caused by a deficiency of expression, or function of CD18. CD18 is a common subunit of the beta 2-integrin coded by ITGF2 gene. Integrins mediate leukocyte adhesion, extravasation, and migration into tissues thus making the patients prone to severe cutaneous and mucosal infections.

Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency.

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of β2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.

Challenges in the gene therapy of bone marrow failure syndromes

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Juan Bueren 200x200.png" data-image="1"></div><div class="author-det"> <b>Juan Bueren</b> is the Head of the Division of Hematopoietic Innovative Therapies of CIEMAT and CIBER of Rare Diseases. Dr Bueren is also Coordinator of the Mixed Unit of Advanced Therapies CIEMAT/IIS-Jiménez Díaz Foundation and consultant of Rocket Pharmaceuticals Inc, and currently serves as Vice-President of the European Society for Cell and Gene Therapy. Dr Bueren is Scientific Director of gene therapy trials for patients with Fanconi anemia and also for patients with the primary immunodeficiency, leukocyte adhesion deficiency type I, and has participated in the development of therapeutic lentiviral vectors designed as Orphan Drugs by the European Medicines Agency and by the FDA for these two diseases. Both ODs have been licensed to Rocket Pharmaceuticals which is currently developing different global gene therapy programs in Europe and in the USA for Fanconi anemia and LAD-I. </div></div></div>

Hematopoietic Stem Cell Transplantation in 65 Patients with Leukocyte Adhesion Deficiency Type I and Type III — A Retrospective Multi-Center Study

Leukocyte adhesion deficiency (LAD) is an autosomal recessive, primary immunodeficiency disorder, characterized by lethal deep tissue infections, leukocytosis with impaired pus-formation, delayed wound healing and hyper-inflammation. Life expectancy of affected patients is severely shortened. LAD-I is caused by defective expression of beta-2 integrin on immune cells, resulting in impaired leukocyte adhesion and migration. LAD-II is a metabolic disease with syndromal features. LAD-III is caused by mutations in the FERMT3 gene resulting in activation defect of all beta-integrins. LAD-III is characterized by leukocyte adhesion/migration deficiency and an additional Glanzmann's thrombasthenia-like syndrome.In this retrospective multi-center study, data were collected from the European Group for Blood and Marrow Transplantation (EBMT) registry. This study included 65 children from 14 European and Asian centers with either LAD-I (n=60) or LAD-III (n=5), all receiving an allogeneic hematopoietic stem-cell transplantation (alloHSCT) from 2008 to 2015.Median age at diagnosis was 0.3 years (yrs); interval from diagnosis to alloHSCT was 8.1 months (median; range 0.6 to 134.5 months). An HLA-identical sibling or matched non-sibling family donor was available for 35 (54%) patients; 28 (43%) received the allograft from a matched unrelated donor and 2 (3%) patients were transplanted from an HLA-haplo-identical family donor. Stem cell source was bone marrow in 37 (58%) patients, peripheral blood in 17 (26%) and cord blood in 10 (16%) of the cohort. Reduced intensity conditioning (RIC) was used in 19 (29%) and myeloablative conditioning (MAC) in 46 (71%) patients. Half of the cohort (n=33) received a busulfan-based conditioning regimen; treosulfan was used in 18 (28%) patients. Serotherapy was applied to 71% of the cohort and consisted primarily of ATG (85%).The overall survival of the cohort was 80% (95%CI: 70-91%) at 24 months, and 77% (95%CI: 65-89%) at 60 months post alloHSCT. In total 12 patients died. The cause of death was GVHD (n=6) and infections (n=6).Primary or secondary graft failure occurred in 9 patients. The incidence of graft failure at 24 months was 11% (95% CI: 7-24%) in RIC and 14% (95% CI: 3-24%) in MAC group. A second alloHSCT was performed in 8 patients; it was successful in 7 cases.At 100 days, 10% (95% CI: 2-17%) and 16% (95% CI: 7-25%) of patients developed grade I-II and grade III+IV aGVHD, respectively . Extensive chronic GVHD was reported in 2 cases.AlloHSCT restored leukocyte function in LAD-I and both leukocyte and thrombocyte function in LAD-III leading to complete remission of symptoms with hematological and immunological reconstitution. Transplantation should be performed early in the course of the disease and prior to the occurrence of life-threatening infectious complications. DisclosuresBader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

Neutrophil granulocytes: participation in homeostatic and reparative processes. Part II

A supportive homeostatic function of neutrophilic granulocytes is accomplished in the physiology of diverse tissues and body systems. Neutrophils are found along the entire female reproductive tract (FRT), gradually declining in numbers from the upper parts towards the vagina. At the same time, both quantity and activity of FRT mucosal neutrophils are controlled by hormonal changes at different phases of menstrual cycle. Tissue neutrophils serve as an important source of broad-spectrum proteolytic enzymes such as matrix metalloproteinases and elastase necessary for extracellular matrix remodeling as well as vascular endothelial growth factor (VEGF) required for physiological FRT angiogenesis. During pregnancy, decidual neutrophils play a prominent role in vascular remodeling in pregnant uterus as well as development of maternal-fetal immune tolerance. The influx of neutrophils into the intestinal mucosa due to its trauma or infection not only ensures defense against pathogens, but also leads to increased proliferation of intestinal epithelial cells. Neutrophilic granulocytes elicit signals and events protective for the epithelium by marking them with a “hypoxic signature” to trigger transcription of the gene set responsible for production of mucins, mucin-modifying peptides, antimicrobial proteins, в-defensins, ultimately contributing to lesion healing and recovery of epithelial barrier function. “Inflammatory hypoxia” initiated by neutrophils and subsequent stabilization of the transcription factor hypoxia-induced factor (HIF) in intestinal epithelial cells trigger mechanisms of self-limited and resolved inflammation, which prevent excessive accumulation of neutrophils in the intestinal lumen and development of chronic inflammatory process. Neutrophilic granulocytes dominate in the oral cavity mucosa and comprise more than 95% of total leukocyte population recruited into the gingival sulcus and gingival fluid. Neutrophils maintain physiological amount and stability of symbiotic microflora composition in dental and gingival biofilms, counteracting pathogenic bacteria via phagocytosis, degranulation and extracellular trap formation, thereby ensuring healthy state in periodontal structures. Finally, similar to some other congenital disorders affecting neutrophil quantity and functions it was shown that in case of leukocyte adhesion deficiency type 1 (LAD-1) pathogenesis of periodontitis may not only be associated with a defect in their protective effector activity, but also with altered immunoregulatory function of tissue neutrophils.

Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47–SIRPα Checkpoint Disruption

The CD47-signal regulatory protein-alpha (SIRPα) immune checkpoint constitutes a therapeutic target in cancer, and initial clinical studies using inhibitors of CD47-SIRPα interactions in combination with tumor-targeting antibodies show promising results. Blockade of CD47-SIRPα interaction can promote neutrophil antibody-dependent cellular cytotoxicity (ADCC) toward antibody-opsonized targets. Neutrophils induce killing of antibody-opsonized tumor cells by a process identified as trogoptosis, a necrotic/lytic type of cancer cell death that involves trogocytosis, the antibody-mediated endocytic acquisition of cancer membrane fragments by neutrophils. Both trogocytosis and killing strictly depend on CD11b/CD18-(Mac-1)-mediated neutrophil-cancer cell conjugate formation, but the mechanism by which CD47-SIRPα checkpoint disruption promotes cytotoxicity has remained elusive. Here, by using neutrophils from patients with leukocyte adhesion deficiency type III carrying FERMT3 gene mutations, hence lacking the integrin-associated protein kindlin3, we demonstrated that CD47-SIRPα signaling controlled the inside-out activation of the neutrophil CD11b/CD18-integrin and cytotoxic synapse formation in a kindlin3-dependent fashion. Our findings also revealed a role for kindlin3 in trogocytosis and an absolute requirement in the killing process, which involved direct interactions between kindlin3 and CD18 integrin. Collectively, these results identified a dual role for kindlin3 in neutrophil ADCC and provide mechanistic insights into the way neutrophil cytotoxicity is governed by CD47-SIRPα interactions.

Kindlin-3 recruitment to the plasma membrane precedes high-affinity β2-integrin and neutrophil arrest from rolling

AbstractIntegrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the β2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates β2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and β2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3–fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ β2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3–reconstituted, kindlin-3–knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity β2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ β2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity β2-integrin.

Clinical and Genetic Spectrum of a Large Cohort of Patients With Leukocyte Adhesion Deficiency Type 1 and 3: A Multicentric Study From India.

Leukocyte adhesion deficiency (LAD) syndrome is a group of inborn errors of immunity characterized by a defect in the cascade of the activation and adhesion leading to the failure of leukocyte to migrate to the site of tissue injury. Three different types of LAD have been described. The most common subtype is LAD type 1 (LAD1) caused due to defects in the ITGβ2 gene. LAD type 2 (LAD2) is caused by mutations in the SLC35C1 gene leading to a generalized loss of expression of fucosylated glycans on the cell surface and LAD type 3 (LAD3) is caused by mutations in the FERMT3 gene resulting in platelet function defects along with immunodeficiency. There is a paucity of data available from India on LAD syndromes. The present study is a retrospective analysis of patients with LAD collated from 28 different centers across India. For LAD1, the diagnosis was based on clinical features and flow cytometric expression of CD18 on peripheral blood leukocytes and molecular confirmation by Sanger sequencing. For patients with LAD3 diagnosis was largely based on clinical manifestations and identification of the pathogenic mutation in the FERMT3 gene by next-generation Sequencing. Of the total 132 cases diagnosed with LAD, 127 were LAD1 and 5 were LAD3. The majority of our patients (83%) had CD18 expression less than 2% on neutrophils (LAD1°) and presented within the first three months of life with omphalitis, skin and soft tissue infections, delayed umbilical cord detachment, otitis media, and sepsis. The patients with CD18 expression of more than 30% (LAD1+) presented later in life with skin ulcers being the commonest manifestation. Bleeding manifestations were common in patients with LAD3. Persistent neutrophilic leukocytosis was the characteristic finding in all patients. 35 novel mutations were detected in the ITGβ2 gene, and 4 novel mutations were detected in the FERMT3 gene. The study thus presents one of the largest cohorts of patients from India with LAD, focusing on clinical features, immunological characteristics, and molecular spectrum.

AA Amyloidosis Secondary to Primary Immune Deficiency: About 40 Cases Including 2 New French Cases and a Systematic Literature Review

Primary immune deficiencies (PIDs) are a heterogeneous group of disorders resulting from defects in immune system. They lead to increased susceptibility to infections and immune dysregulation. The resulting chronic inflammation can induce long-term complications, including AA amyloidosis (AAA). To present the French cases of PID-related AAA and perform a systematic literature review to determine its main features and predisposing factors. A systematic literature review was performed by searching MEDLINE up until 2019. New French cases were identified with the help of the Reference Center for Auto-Inflammatory Diseases and AA Amyloidosis and the Reference Center for Hereditary Immune Deficiencies. Forty patients were identified including 2 new French cases. PIDs were varied: immunoglobulin deficits (n= 30), chronic granulomatous disease (n= 3), hyper-IgM syndrome (n= 3), hereditary complete C4 deficiency (n= 1), leucocyte adhesion deficiency type 1 (n= 1), hyper-IgE syndrome (n= 1), and Chediak-Higashi syndrome (n= 1). The mean age at PID diagnosis was 22.2 ± 16.02 years. Renal involvement was the most common manifestation of AAA (80%). Infections were extremely heterogeneous; bacterial infection with pulmonary involvement was the most frequent. Bronchiectasis was particularly common (52.5%). The delay between the first symptoms of PID and AAA diagnosis was 16.18 ± 7 years. Thirteen concomitant diagnoses were made. Twenty patients died during follow-up. AAA is a rare life-threatening complication of PID, especially in cases of long diagnostic and therapeutic delays. Bronchiectasis should be considered as a warning sign of chronic inflammation and increased risk of AAA.