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Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity.

Background:The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional B cells (cTrB) have been described in patients with longstanding RA.Objectives:To examine the frequency and evolution of cTrB cells in the peripheral blood of early RA (ERA) patients.Methods:Freshly isolated PBMCs from 48 steroid and DMARD-naïve ERA patients with a disease duration below 24 weeks and 48 healthy controls (HC) were examined by flow cytometry. Cocultures of isolated memory B cells were established with autologous T cells, in the absence or presence of TrB cells.Results:As compared with HC, ERA patients demonstrated an increased frequency of cTrB cells. cTrBs of ERA and HC displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFNγ and IL-21 production, together with ACPA secretion in autologous B/T cell cocultures. Basal frequencies of cTrBs above the median value observed in HC were associated with a good EULAR response to MTX at 12 months (RR=2.91; 95% CI, 1.37-6.47). A significant reduction of cTrBs was observed 12 months after initiating MTX, when the cTrB cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone.Conclusion:An increased frequency of regulatory cTrB cells is apparent in untreated ERA, and the baseline cTrB cell frequency is associated with the clinical response to MTX at 12 months.References:[1]Matsushita T, et al. J Clin Invest. 2008;118:342. Flores-Borja F, et al. Sci Transl Med. 2013;5:173ra23.Disclosure of Interests:Paula Fortea-Gordo Grant/research support from: BMS, Alejandro Villalba: None declared, Laura Nuño: None declared, Maria-Jose Santos-Bornez Grant/research support from: BMS, Diana Peiteado: None declared, Irene Monjo: None declared, Amaya Puig-Kröger: None declared, Paloma Sanchez-Mateos: None declared, Emilio Martín-Mola Grant/research support from: BMS, Roche, Alejandro Balsa Grant/research support from: BMS, Roche, Consultant of: AbbVie, Gilead, Lilly, Pfizer, UCB, Sanofi, Sandoz, Speakers bureau: AbbVie, Lilly, Sanofi, Novartis, Pfizer, UCB, Roche, Nordic, Sandoz, Maria-Eugenia Miranda-Carus Grant/research support from: BMS, Roche

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the alpha 4 beta 1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are beta 1-integrin-binding partners that regulate beta 1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of alpha 4 beta 1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by alpha 4 beta 1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on alpha 4 beta 1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of alpha 4 beta 1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating alpha 4 beta 1 activity.

BackgroundA novel population of CD4 +T cells with B cell helping capacity has been described in the synovial tissues and peripheral blood of seropositive RA patients with an established disease, and termed ‘peripheral helper’ (Tph) cells. (Rao DA et al, Nature 2017) Tph cells are characterised by the lack of CXCR5 together with a bright expression of PD-1 (CD4 +CXCR5-PD-1hi T cells). As opposed to CD4 +CXCR5+PD-1hi follicular helper T cells (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Tph cell numbers have not been previously examined in early RA (eRA).ObjectivesTo study the frequency of circulating CD3 +CD4+CXCR5-PD-1hi Tph cells (cTph), in patients with eRA.MethodsPeripheral blood was drawn from DMARD-naïve early RA patients (eRA) (2010 ACR criteria) with a disease duration <24 weeks (n=42), and healthy controls (HC) matched for age and gender (n=42). For comparison, blood was also drawn from 66 patients with established RA (disease duration >2 years), 45 patients with Spondyloarthritis (SpA), and their age and gender-matched HC (one HC per patient). In addition, synovial fluid from 7 patients with established RA and 3 patients with SpA was examined. Established RA patients were receiving low-dose oral methotrexate and were naïve for biological agents. SpA patients were receiving NSAIDs, low-dose oral methotrexate and/or sulphasalazine and were naïve for biologicals. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, ICOS and PD-1, and examined by flow cytometry.ResultsThe frequency of circulating CXCR5- cells gated for CD4 +T cells was not different among the studied groups. In contrast, eRA patients demonstrated an increased frequency of circulating CD4 +CXCR5-PD-1hi Tph and CD4 +CXCR5-PD-1hiICOS+ T cells. When examining seropositive (RF +and/or ACPA+, n=25) and seronegative eRA patients (RF- and ACPA-, n=17) separately, it was evident that the above described alterations were only apparent in seropositive eRA. Likewise, increased cTph numbers were observed in seropositive (n=47) but not seronegative (n=19) established RA, and not in SpA patients (n=45), which is consistent with data reported by Rao et al. Interestingly, this increased cTph cell frequency was observed only in seropositive RA patients with an active disease (DAS28 >2.6, n=24), whereas the numbers of cTph cells in established RA patients who had achieved remission (DAS28 <2.6, n=23) were not different from HC. Furthermore, Tph cells were present in the synovial fluid of seropositive RA (n=4) but not of seronegative RA (n=3) or SpA (n=3).ConclusionsTph cells may play an important role in the pathogenesis of seropositive but not seronegative RA. An increased cTph cell frequency is a marker of active, seropositive RA.Reference[1] Rao DA, et al. Nature2017;542(7639):110–114.Disclosure of InterestNone declared

BackgroundCD19+CD24highCD38high B cells have been described to have a regulatory capacity and their frequency is altered in the peripheral blood of patients with various autoimmune diseases. The pathogenesis of AS is not well understood, and evidence suggesting the implication of either autoinflammatory or autoimmune mechanisms has been reported. In addition, increased frequencies of circulating B cells bearing a regulatory phenotype has recently been described in spondyloartrhitis (1).ObjectivesTo study the frequency of circulating CD19+CD24high CD38high B cells (Breg) in patients with Ankylosing Spondylitis (AS), and test the regulatory capacity of this B cell subset.MethodsPeripheral blood was drawn from AS patients naïve for TNF blockers (AS/nb) (n=37) and healthy controls (HC) (n=37), that were matched with patients for age and gender. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by flow cytometry. For functional studies, total CD19+ B cells were isolated from PBMCs of 3 HC by magnetical sorting. Breg-depleted CD19+ B cells were obtained after total CD19+ B cells were depleted of CD19+CD24highCD38high B cells by cytometry in a FacsVantage sorter (Beckton Dickinson). Total CD19+ B cells or Breg-depleted CD19+ B cells were established in culture and stimulated through their BCR. Secretion of IFNγ was determined by ELISA in culture supernatants.ResultsWhen compared with healthy controls, AS/nb patients demonstrated a significantly increased frequency of CD19+CD24highCD38high B cells (Breg). The frequency of circulating Breg was increased not only in AS/nb patients with high or very high disease activity (ASDAS-CRP) >2.1 but also in AS patients with low activity or no activity (ASDAS-CRP<2.1). The frequency of circulating Breg cells did not correlate significantly with ASDAS-CRP, ASDAS-ESR, BASDAI, CRP or ESR values. Functional in vitro studies showed that the secretion of IFNγ was significantly higher in Breg-depleted CD19+ as compared with total CD19+ B cells, indicating that Breg have the capacity to downmodulate B cell pro-inflammatory cytokine secretion.ConclusionsAn increased frequency of circulating CD19+CD24highCD38high B cells is observed in AS/nb patients, that is not related with disease activity. Functional in vitro studies confirmed that CD19+CD24highCD38high B cells are able to downmodulate B cell pro-inflammatory cytokine secretion.ReferencesCantaert T, Doorenspleet ME, Francosalinas G, Paramarta JE, Klarenbeek PL, Tiersma Y, van der Loos CM, De Vries N, Tak PP, Baeten DL. Increased numbers of CD5+ B lymphocytes with a regulatory phenotype in spondylarthritis. Arthritis Rheum. 2012;64:1859–68.Disclosure of InterestNone declared

BackgroundIL-17-expressing FoxP3 regulatory T cells have been described, and their suppressive capacity has been questioned. An inflammatory environment seems to favor IL-17 secretion by regulatory CD4+CD25+FoxP3+ T cells.ObjectivesTo assess the suppressive function and IL-17 producing capacity of CD4+CD25+CD127-FoxP3+ T cells in the synovial fluid of RA patients (RASFd).MethodsSynovial fluid was drawn from 35 patients with established RA who were receiving methotrexate and low-dose oral prednisone. The frequency of CD4+CD25+CD127-FoxP3+ T cells was assessed by flow cytometry. Total CD4+ T cells, CD4+CD25+CD127- T reg cells and CD4+CD25- Tresp cells were isolated by Ficoll-Hypaque gradient, followed by sorting. After isolation, cells were stimulated for 5 hours with PMA+ionomycin or cultured for 5 days in flat-bottom 96-well plates coated with an anti-CD3 monoclonal antibody. Treg cell function was assessed using two different approaches: A.The regulatory function of natural proportions of Tregs was inferred by comparing the proliferative and cytokine responses of total CD4+ T cells (TCD4T) versus CD25+ depleted CD4+ T cells (CD4+CD25-T cells or Tresp cells); B. The per cell suppressor potency of Tregs was assessed in cocultures of isolated Tregs with Tresp, established at different Treg/Tresp ratios. Proliferation was determined by 3Hthymidine incorporation and CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants.ResultsA high proportion of CD4+CD25+CD127- T cells was present in RASFd (mean ± SD, 21.1%±6.2), which is significantly higher than reported frequencies of this cell population in the peripheral blood of both RA and healthy subjects. These RASFd CD4+CD25+CD127- T cells expressed FoxP3 but did not express CD69. The proliferation rate, TNFα and IFNγ secretion were significantly higher for isolated Tresp as compared with TCD4T cells, indicating that natural proportions of Treg cells present in the synovial fluid of RA are funcionally suppressive. Surprisingly, TCD4T cells secreted higher amounts of IL-17 as compared with Tresp cells, although the difference did not reach statistical significance. On a per cell basis, RAPB Tregs were potent suppressors of Tresp proliferation, TNFα and IFNγ but not of IL-17 secretion; in fact IL-17 secretion did not decrease but was enhanced in the presence of increasing proportions of Treg cells. Isolated Treg cells did not proliferate or produce cytokines when cultured alone in anti-CD3 coated plates. However, in the presence of plate-bound anti-CD3 plus anti-CD28 and recombinant human IL-2, isolated Treg cells secreted significant amounts of IL-17 whereas no TNFα or IFNγ could be detected in supernatants.ConclusionsCD4+CD27+CD127- FoxP3+ Treg cells present in the synovial fluid of RA patients are potent suppressors of Tresp proliferation, TNFα and IFNγ secretion, and at the same time produce significant amounts of IL-17.References Voo KS, et al. Proc Natl Acad Sci U S A. 2009;106:4793.Beriou G et al. Blood. 2009;113:4240.Wang T, et al. Ann Rheum Dis. 2015;74:1293.Komatsu N, et al. Nat Med. 2014;20:62.Yang BH, et al. Mucosal Immunol. 2016;9:444. Disclosure of InterestNone declared

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the [alpha]4[beta]1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are [beta]1-integrin-binding partners that regulate [beta]1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of [alpha]4[beta]1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by [alpha]4[beta]1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on [alpha]4[beta]1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of [alpha]4[beta]1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating [alpha]4[beta]1 activity.

Necrotizing enterocolitis (NEC) is an inflammatory bowel disease and a leading cause of morbidity and mortality in preterm infants. In this study, a randomized double-blind parallel-group (1:1) trial was carried out in two neonatal intensive care units of two tertiary hospitals. Two hundred and twenty-five preterm newborns with an expected functional gastrointestinal tract were recruited and received an enteral dose of 75 mg of docosahexaenoic acid (DHA)/kg body weight or high-oleic sunflower oil daily for 14 days from the first enteral feed after birth. Confirmed NEC was evaluated with Bell’s scale from stage ≥ IIa. Two hundred and fourteen randomized infants were analyzed in terms of the intent-to-treat (DHA-group: n = 105; control-group: n = 109); data for two hundred infants were analysed per protocol. Confirmed NEC was lower in infants from the DHA-group compared with the control-group (0/100 vs. 7/100; p = 0.007), with RR = 0.93 (95% CI 0.881 to 0.981), risk difference = −7%, (95% CI −12.00 to −1.99), and number needed-to-treat = 15 (95% CI 8.3 to 50). Intent-to-treat analysis showed a lower level of treatment failure in the DHA-group compared with the control-group (6/105 (6%) vs. 16/109 (15%); p = 0.03, RR = 0.905, (95% CI 0.826 to 0.991)). The results after multivariate-regression analysis remained significant. Adverse events (apart from the incidence of NEC) were not different between groups. A daily dose of DHA for 14 days starting with the first enteral feed may prevent NEC in preterm infants.