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Abstract Background The risk of influenza infection depends on biological characteristics, individual or collective behaviors and the environmental context. The Cohorts for Pandemic Influenza (CoPanFlu) France study was set up in 2009 after the identification of the novel swine-origin A/H1N1 pandemic influenza virus. This cohort of 601 households (1450 subjects) representative for the general population aims at using an integrative approach to study the risk and characteristics of influenza infection as a complex combination of data collected from questionnaires regarding sociodemographic, medical, behavioral characteristics of subjects and indoor environment, using biological samples or environmental databases. Methods/Design Households were included between December 2009 and July 2010. The design of this study relies on systematic follow-up visits between influenza seasons and additional visits during influenza seasons, when an influenza-like illness is detected in a household via an active surveillance system. During systematic visits, a nurse collects individual and environmental data on questionnaires and obtains blood samples from all members of the household. When an influenza-like-illness is detected, a nurse visits the household three times during the 12 following days, and collects data on questionnaires regarding exposure and symptoms, and biological samples (including nasal swabs) from all subjects in the household. The end of the follow-up period is expected in fall 2012. Discussion The large amount of data collected throughout the follow-up will permit a multidisciplinary study of influenza infections. Additional data is being collected and analyzed in this ongoing cohort. The longitudinal analysis of these households will permit integrative analyses of complex phenomena such as individual, collective and environmental risk factors of infection, routes of transmission, or determinants of the immune response to infection or vaccination.

The objective was to conduct a comprehensive review of the morbidity and mortality observed in published patients with gastrointestinal defects and immunodeficiency syndrome-1 (GIDID1) related to TTC7A abnormalities. This included phenotypic, genotypic, and therapeutic aspects. Twenty-seven articles were included, which represented a total of 83 patients. Mortality was of 65.8% of the cases with a mean death at 11.8 months. The mortality rate was 197.1 per 1,000 patients-years, which is significantly higher than other enteropathy types caused by defects in epithelial trafficking and polarity (such as MOY5B, STX3, EPCAM, SPINT2, TTC37 and SKIV2L ). Prematurity was also significant, with an average gestational age of 34.8 weeks. Antenatal signs were observed in 30 patients, including 14 cases of hydramnios. Three distinct phenotypic associations were identified: immune deficiency and multiple intestinal atresia without enteropathy (ID/MI), immune deficiency and enteropathy without atresia (ID/E), and immune deficiency with multiple intestinal atresia and enteropathy (ID/ MIA/E). The mortality rates for these groups were 91.6%, 47.3% and 55.5%, respectively ( p = 0.03), at earlier age of mortality for the ID/MIA phenotype and a later one for the ID/E phenotype. ELA syndrome (Enteropathy, Lymphopenia and Alopecia) was only observed in the ID/E group. Among the three genotypes (double variant Nonsense NS/NS, variant Missense/Nonsense MS/NS, double variant Missense MS/MS), NS/NS was significantly associated with the ID/MIA phenotype (77.8%), while MS/MS was associated with the ID/E phenotype (73.7%). Few therapies have been shown to be effective in treating enteropathy, particularly immunosuppressive therapies and hematopoietic stem cell transplants. The use of Leflunomide in one patient did not yield successful treatment outcomes. In conclusion, we confirm association between mortality and phenotype, which is itself linked to genotype., Competing Interests: None.The authors have no conflicts of interest to disclose., (2024, International Research and Cooperation Association for Bio & Socio - Sciences Advancement.)

Background and Objectives: Real-life studies noted that the risk of disease activity in multiple sclerosis (MS) after switching to rituximab (RTX) or ocrelizumab (OCR) may be unequal depending on prior disease-modifying therapy (DMT), with a higher risk associated with fingolimod (FING)., Methods: We performed a retrospective analysis of a structured prospective data collection including all consecutive patients with relapsing MS who were prescribed RTX/OCR in the MS center of Marseille. Cox proportional hazards models were applied to clinical and MRI outcomes., Results: We included 321 patients with a median (interquartile range [IQR]) follow-up of 3.5 years (1.5-5) after RTX/OCR initiation. At the first RTX/OCR infusion, the mean (SD) age of patients was 37 (10) years, and the median (IQR) disease duration was 8 years (3-15): 68 patients did not receive treatment before RTX/OCR and 108 switched from FING, 47 from low efficacy therapy, and 98 from natalizumab. For statistical analysis, the group "FING" was divided into "short-FING" and "long-FING" groups according to the median value of the group's washout period (27 days). On Cox proportional hazards analysis, for only the "long-FING" group, the risk of relapse within the first 6 months of RTX/OCR was increased as compared with patients without previous DMT (hazard ratio [HR]: 8.78; 95% CI 1.72-44.86; p < 0.01). Previous DMT and washout period duration of FING had no effect on B-cell levels at 6 months. Beyond the first 6 months of RTX/OCR, age <40 years was associated with increased risk of relapse (HR: 3.93; 95% CI 1.30-11.89; p = 0.01), male sex with increased risk of new T2 lesions (HR: 2.26; 95% CI 1.08-4.74; p = 0.03), and EDSS ≥2 with increased risk of disability accumulation (HR: 3.01; 95% CI 1.34-6.74; p < 0.01). Previous DMT had no effect on the effectiveness of RTX/OCR beyond 6 months after initiation., Discussion: For patients switching from FING to RTX/OCR, the risk of disease reactivation within the first 6 months of treatment was increased as compared with patients with other DMT or no previous DMT only when the washout period exceeded 26 days. Neither FING nor other previous DMT reduced the effectiveness of RTX/OCR beyond the first 6 months of treatment.

Introduction: Anti-MAG neuropathies are associated with an IgM monoclonal gammopathy of undetermined significance (MGUS) or with a malignant haemopathy. Our objective was to determine whether the presence of a haemopathy or somatic mutations of MYD88 and CXCR4 genes influences disease presentation and response to rituximab (RTX)., Methods: We included 79 patients (mean age 74 years, disease duration 9.68 years) who had a bone marrow aspiration with morphologic and immunophenotypic analysis. MYD88 L265P and CXCR4 mutations were analysed in peripheral B cells. Information collected included: inflammatory neuropathy cause and treatment sensory sum score (ISS), MRC testing, overall neuropathy limitation scale (ONLS), Rash-built Overall Disability Score (RODS), ataxia score, anti-MAG titres, peak IgM dosage, neurofilament light chain levels, motor and sensory amplitudes, motor unit index (MUNIX) and motor unit size index (MUSIX) sum scores. Efficacy of RTX was evaluated at 12 months in 26 patients., Results: Malignant haematological disorders were discovered in 17 patients (22%): 13 Waldenstrom macroglobulinemia, 3 marginal zone lymphoma and one mantle cell lymphoma. MYD88 L265P mutation was detected in 29/60 (48%) patients and CXCR4 in 1 single patient. Disease severity, biological and electrophysiological data and response to RTX were comparable in patients with MGUS/lymphoma and patients with/without MYD88 L265P mutation. ISS was lower and MUSIX higher in patients improved by RTX., Conclusions: MYD88 L265P mutation and underlying haemopathies are not predictive of a more severe disease. However, in cases of resistant and progressive neuropathy, they provide an opportunity to prescribe newly available drugs such as Bruton tyrosine kinase inhibitors., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Background and Objectives: Patients with multiple sclerosis (PwMS) receiving extended dosing of rituximab (RTX) have exhibited no return of disease activity, which suggests that maintenance of deep depletion of circulating B cells is not necessary to maintain the efficacy of RTX in MS., Methods: This was a prospective monocentric observational study including all consecutive PwMS who started or continued RTX after 2019, when the medical staff decided to extend the dosing interval up to 24 months for all patients. Circulating B-cell subsets were monitored regularly and systematically in case of relapse. The first extended interval was analyzed., Results: We included 236 PwMS (81% with relapsing-remitting MS; mean [SD] age 43 [12] years; median [range] EDSS score 4 [0-8]; mean relapse rate during the year before RTX start 1.09 [0.99]; 41.5% with MRI activity). The median number of RTX infusions before extension was 4 (1-13). At the time of the analysis, the median delay in dosing was 17 months (8-39); the median proportion of circulating CD19 + B cells was 7% (0-25) of total lymphocytes and that of CD27 + memory B cells was 4% (0-16) of total B cells. The mean annual relapse rate did not differ before and after the extension: 0.03 (0.5) and 0.04 (0.15) ( p = 0.51). Similarly, annual relapse rates did not differ before and after extension in patients with EDSS score ≤3 (n = 79) or disease duration ≤5 years (n = 71) at RTX onset. During the "extended dosing" period, MRI demonstrated no lesion accrual in 228 of the 236 patients (97%). Five patients experienced clinical relapse, which was confirmed by MRI. In these patients, the level of B-cell subset reconstitution at the time of the relapse did not differ from that for patients with the same extension window., Discussion: The efficacy of RTX outlasted substantial reconstitution of circulating B cells in PwMS, which suggests that renewal of the immune system underlies the prolonged effect of RTX in MS. These findings suggest that extended interval dosing of RTX that leads to a significant reconstitution of circulating B cells is safe in PwMS, could reduce the risk of infection, and could improve vaccine efficacy., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Objectives: Severe COVID-19 is associated with exaggerated complement activation. We assessed the efficacy and safety of avdoralimab (an anti-C5aR1 mAb) in severe COVID-19., Design: FOR COVID Elimination (FORCE) was a double-blind, placebo-controlled study., Setting: Twelve clinical sites in France (ICU and general hospitals)., Patients: Patients receiving greater than or equal to 5 L oxygen/min to maintain Sp o2 greater than 93% (World Health Organization scale ≥ 5). Patients received conventional oxygen therapy or high-flow oxygen (HFO)/noninvasive ventilation (NIV) in cohort 1; HFO, NIV, or invasive mechanical ventilation (IMV) in cohort 2; and IMV in cohort 3., Interventions: Patients were randomly assigned, in a 1:1 ratio, to receive avdoralimab or placebo. The primary outcome was clinical status on the World Health Organization ordinal scale at days 14 and 28 for cohorts 1 and 3, and the number of ventilator-free days at day 28 (VFD28) for cohort 2., Measurements and Main Results: We randomized 207 patients: 99 in cohort 1, 49 in cohort 2, and 59 in cohort 3. During hospitalization, 95% of patients received glucocorticoids. Avdoralimab did not improve World Health Organization clinical scale score on days 14 and 28 (between-group difference on day 28 of -0.26 (95% CI, -1.2 to 0.7; p = 0.7) in cohort 1 and -0.28 (95% CI, -1.8 to 1.2; p = 0.6) in cohort 3). Avdoralimab did not improve VFD28 in cohort 2 (between-group difference of -6.3 (95% CI, -13.2 to 0.7; p = 0.96) or secondary outcomes in any cohort. No subgroup of interest was identified., Conclusions: In this randomized trial in hospitalized patients with severe COVID-19 pneumonia, avdoralimab did not significantly improve clinical status at days 14 and 28 (funded by Innate Pharma, ClinicalTrials.gov number, NCT04371367)., Competing Interests: Dr. Carvelli received support for article research from Innate Pharma. Drs. Carvelli, Allardet-Servent, Barbar, Desgrouas, Camou, Piperoglou, Viotti, Boyer-Chammard, Lacombe, Le Dault, Schleinitz, and Vivier disclosed the off-label product use of avdoralimab. Dr. Guervilly received funding from Xenios FMC. Dr. Demaria’s institution received funding from BPI. Drs. Demaria, Karakunnel, Fares, Batista, Boyer-Chammard, and Vivier received funding from innate pharma. Drs. Demaria, Karakunnel, Fares, Batista, Rotolo, Viotti, Boyer-Chammard, and Vivier disclosed that they are employees of Innate Pharma. Dr. Karakunnel received funding from Primevax Precision Biologics. Dr. Rotolo received funding from Sanofi. Dr. Viotti disclosed work for hire. Dr. Lacombe received funding from MSD, Gilead, Janssen, and ViiV Healthcare. Dr. Vivier disclosed that he is a cofounder, shareholder, and employee of Innate Pharma and that his spouse is a shareholder of Innate Pharma. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.)

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis., (© 2020 by The American Society of Hematology.)

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146-deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor-induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF-induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR-2/Ve-cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve-cadherin network in melanoma extravasation across the vessel barrier that identifies CD146-targeted therapy as a potential strategy for the treatment of melanoma metastasis., (© 2014 UICC.)

The CoPanFlu-France cohort of households was set up in 2009 to study the risk factors for infection by the pandemic influenza virus (H1N1pdm) in the French general population. The authors developed an integrative data-driven approach to identify individual, collective and environmental factors associated with the post-seasonal serological H1N1pdm geometric mean titer, and derived a nested case-control analysis to identify risk factors for infection during the first season. This analysis included 1377 subjects (601 households). The GMT for the general population was 47.1 (95% confidence interval (CI): 45.1, 49.2). According to a multivariable analysis, pandemic vaccination, seasonal vaccination in 2009, recent history of influenza-like illness, asthma, chronic obstructive pulmonary disease, social contacts at school and use of public transports by the local population were associated with a higher GMT, whereas history of smoking was associated with a lower GMT. Additionally, young age at inclusion and risk perception of exposure to the virus at work were identified as possible risk factors, whereas presence of an air humidifier in the living room was a possible protective factor. These findings will be interpreted in light of the longitudinal analyses of this ongoing cohort.

Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.

Severe sepsis and septic shock are still deadly conditions urging to develop novel therapies. A better understanding of the complex modifications of the immune system of septic patients is needed for the development of innovative immunointerventions. Natural killer (NK) cells are characterized as CD3(-)NKp46(+)CD56(+) cells that can be cytotoxic and/or produce high amounts of cytokines such as IFN-γ. NK cells are also engaged in crosstalks with other immune cells, such as dendritic cells, macrophages, and neutrophils. During the early stage of septic shock, NK cells may play a key role in the promotion of the systemic inflammation, as suggested in mice models. Alternatively, at a later stage, NK cells-acquired dysfunction could favor nosocomial infections and mortality. Standardized biological tools defining patients' NK cell status during the different stages of sepsis are mandatory to guide potential immuno-interventions. Herein, we review the potential role of NK cells during severe sepsis and septic shock.

Rationale: CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined., Objective: The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential., Methods and Results: Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell-derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow., Conclusions: Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases.

Objectives: During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration. Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown., Methods and Results: TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes. Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes., Conclusions: Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation.

The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the tyrosine phosphatase SHP-1. We now identified Src and SHP-2 as sst2-associated molecules and demonstrated their role in sst2 signaling. Surface plasmon resonance and mutation analyses revealed that SHP-2 directly associated with phosphorylated tyrosine 228 and 312, which are located in sst2 ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This interaction was required for somatostatin-induced SHP-1 recruitment and activation and consequent inhibition of cell proliferation. Src interacted with sst2 and somatostatin promoted a transient Gbetagamma-dependent Src activation concomitant with sst2 tyrosine hyperphosphorylation and SHP-2 activation. These steps were abrogated with catalytically inactive Src. Both catalytically inactive Src and SHP-2 mutants abolished somatostatin-induced SHP-1 activation and cell growth inhibition. Sst2-Src-SHP-2 complex formation was dynamic. Somatostatin further induced sst2 tyrosine dephosphorylation and complex dissociation accompanied by Src and SHP-2 inhibition. These steps were defective in cells expressing a catalytically inactive Src mutant. All these data suggest that Src acts upstream of SHP-2 in sst2 signaling and provide evidence for a functional role for Src and SHP-2 downstream of an inhibitory G protein-coupled receptor.

A small fraction of T cells expresses killer-cell Ig-like receptors (KIR), a family of MHC class I-specific receptors that can modulate TCR-dependent activation of effector functions. Although KIR(+) cells are enriched within Ag-experienced T cell subsets, the precise relationships between KIR(+) and KIR(-) T cells and the stage of KIR induction on these lymphocytes remain unclear. In this study, we compared KIR(-) and KIR(+) alphabeta T cell clones, sorted by means of the CD158b (KIR2DL2/KIR2DL3/KIR2DS2) specific mAb GL183. We isolated several pairs of CD158b(+) and CD158b(-) alphabeta T cell clones sharing identical productive and nonproductive TCR transcripts. We showed that expression of functional KIR on T cells is regulated after termination of TCR rearrangements. Transcriptional regulation of KIR genes was documented in multiple T cell clones generated from the same donor, and the presence of KIR transcripts was also detected in KIR(-) T cells. These results document a complex regulation of KIR expression in T cells at both pre and posttranscriptional levels, under the control of yet undefined signals provided in vivo.

FcgammaRIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of FcgammaRIIB with immunoreceptor tyrosine-based activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic FcgammaRIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the FcgammaRIIB ITIM and the N-terminal ITIM of a killer cell Ig-like receptor that does not bind SHIPs. Loss of function and gain of function substitutions identified the Y+2 leucine, in the FcgammaRIIB ITIM, as determining the binding of both SHIP1 and SHIP2, but not the binding of SHP-1 or SHP-2. Conversely, the Y-2 isoleucine that determines the in vitro binding of SHP-1 and SHP-2 affected neither the binding nor the recruitment of SHIP1 or SHIP2. One hydrophobic residue, in the ITIM of FcgammaRIIB therefore determines the affinity for SHIPs. This residue is symmetrical to the hydrophobic residue that determines the affinity of all ITIMs for SHPs. It defines a SHIP-binding site, distinct from a SHP-binding site, that enables FcgammaRIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.