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Leukocyte adhesion requires beta2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e., not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation.

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand, foot, and mouth disease (HFMD). In our previous study, intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity, while immunization with the inactivated CA16 vaccine did not. In this report, we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different, but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-kappaB pathway signaling molecules, which were identified as being capable of activating DCs, ILCs, and T cells, were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly, ILC colocalization decreased over time, while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore, neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100{\%} protected against wild-type EV71 or CA16 viral challenge. Together, our results provide new insights into the development of vaccines for HFMD.

PURPOSE Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (Kd {\textless} 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS A combinatorial costimulatory design allows the use of very low-affinity binding domains (Kd {\textless} 1 mumol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.

OBJECTIVES Anti-citrullinated protein antibodies (ACPAs) are a group of autoantibodies targeted against citrullinated proteins/peptides and are informative rheumatoid arthritis (RA) biomarkers. ACPAs also play a crucial role in RA pathogenesis, and their underlying mechanism merits investigation. METHODS Immunohistochemical (IHC) assays were carried out to determine IL-1beta levels in ACPA+ and ACPA- RA patients. PBMC-derived monocytes were differentiated into macrophages before stimulation with ACPAs purified from RA patients. The localization and interaction of molecules were analyzed by confocal microscopy, co-IP, and surface plasmon resonance. RESULTS In our study, we found that IL-1beta levels were elevated in ACPA+ RA patients and that ACPAs promoted IL-1beta production by PBMC-derived macrophages. ACPAs interacted with CD147 to enhance the interaction between CD147 and integrin beta1 and, in turn, activate the Akt/NF-kappaB signaling pathway. The nuclear localization of p65 promoted the expression of NLRP3 and pro-IL-1beta, resulting in priming. Moreover, ACPA stimulation activated pannexin channels, leading to ATP release. The accumulated ATP bound to the P2X7 receptor, leading to NLRP3 inflammasome activation. CONCLUSIONS Our study suggests a new hypothesis regarding IL-1beta production in RA involving ACPAs, which may be a potential therapeutic target in RA treatment.

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells, as the most immature na{\{i}}ve B cell subset upstream of pathogenic B cells display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated mostly Caucasian SLE patients we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition early transitional B cells from the bone marrow displayed the highest interferon score reflecting a B cell interferon burden of central origin. Hence the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass ultimately leading in periphery to the expansion of autoantibodies-secreting cells."

The upper respiratory tract is continuously exposed to a vast array of potentially pathogenic viruses and bacteria. Influenza A virus (IAV) has particular synergism with the commensal bacterium Streptococcus pneumoniae in this niche, and co-infection exacerbates pathogenicity and causes significant mortality. However, it is not known whether this synergism is associated with a direct interaction between the two pathogens. We have previously reported that co-administration of a whole-inactivated IAV vaccine (gamma-Flu) with a whole-inactivated pneumococcal vaccine (gamma-PN) enhances pneumococcal-specific responses. In this study, we show that mucosal co-administration of gamma-Flu and gamma-PN similarly augments IAV-specific immunity, particularly tissue-resident memory cell responses in the lung. In addition, our in vitro analysis revealed that S. pneumoniae directly interacts with both gamma-Flu and with live IAV, facilitating increased uptake by macrophages as well as increased infection of epithelial cells by IAV. These observations provide an additional explanation for the synergistic pathogenicity of IAV and S. pneumoniae, as well as heralding the prospect of exploiting the phenomenon to develop better vaccine strategies for both pathogens.

We investigated the contribution of host immune cells to bacterial killing in a whole-blood bactericidal activity (WBA) assay, an ex vivo model used to test efficacy of drugs against mycobacterium tuberculosis (Mtb). We performed WBA assays with immuno-magnetic depletion of specific cell types, in the presence or absence of rifampicin. Innate immune cells decreased Mtb growth in absence of drug, but appeared to diminish the cidal activity of rifampicin, possibly attributable to intracellular bacterial sequestration. Adaptive immune cells had no effect with or without drug. The WBA assay may have potential for testing adjunctive host-directed therapies acting on phagocytic cells.

Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/beta-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation acting as a negative regulator of the Wnt/beta-catenin pathway."

Associations between chronic antigen stimulation, T cell dysfunction, and the expression of various inhibitory receptors are well characterized in several mouse and human systems. During chronic hepatitis B virus (HBV) infection (CHB), T cell responses are blunted with low frequencies of virus-specific T cells observed, making these parameters difficult to study. Here, using mass cytometry and a highly multiplexed combinatorial peptide-major histocompatibility complex (pMHC) tetramer strategy that allows for the detection of rare antigen-specific T cells, we simultaneously probed 484 unique HLA-A*1101-restricted epitopes spanning the entire HBV genome on T cells from patients at various stages of CHB. Numerous HBV-specific T cell populations were detected, validated, and profiled. T cells specific for two epitopes (HBVpol387 and HBVcore169) displayed differing and complex heterogeneities that were associated with the disease progression, and the expression of inhibitory receptors on these cells was not linearly related with their extent of T cell dysfunction. For HBVcore169-specific CD8+ T cells, we found cellular markers associated with long-term memory, polyfunctionality, and the presence of several previously unidentified public TCR clones that correlated with viral control. Using high-dimensional trajectory analysis of these cellular phenotypes, a pseudo-time metric was constructed that fit with the status of viral infection in corresponding patients. This was validated in a longitudinal cohort of patients undergoing antiviral therapy. Our study uncovers complex relationships of inhibitory receptors between the profiles of antigen-specific T cells and the status of CHB with implications for new strategies of therapeutic intervention.

Multiple myeloma (MM) is a tumor of plasma cells (PCs). Due to the intense immunoglobulin secretion, PCs are prone to endoplasmic reticulum stress and activate several stress-managing pathways, including autophagy. Indeed, autophagy deregulation is maladaptive for MM cells, resulting in cell death. CK1alpha, a pro-survival kinase in MM, has recently been involved as a regulator of the autophagic flux and of the transcriptional competence of the autophagy-related transcription factor FOXO3a in several cancers. In this study, we investigated the role of CK1alpha in autophagy in MM. To study the autophagic flux we generated clones of MM cell lines expressing the mCherry-eGFP-LC3B fusion protein. We observed that CK1 inhibition with the chemical ATP-competitive CK1 alpha/delta inhibitor D4476 resulted in an impaired autophagic flux, likely due to an alteration of lysosomes acidification. However, D4476 caused the accumulation of the transcription factor FOXO3a in the nucleus, and this was paralleled by the upregulation of mRNA coding for autophagic genes. Surprisingly, silencing of CK1alpha by RNA interference triggered the autophagic flux. However, FOXO3a did not shuttle into the nucleus and the transcription of autophagy-related FOXO3a-dependent genes was not observed. Thus, while the chemical inhibition with the dual CK1alpha/delta inhibitor D4476 induced cell death as a consequence of an accumulation of ineffective autophagic vesicles, on the opposite, CK1alpha silencing, although it also determined apoptosis, triggered a full activation of the early autophagic flux, which was then not supported by the upregulation of autophagic genes. Taken together, our results indicate that the family of CK1 kinases may profoundly influence MM cells survival also through the modulation of the autophagic pathway.

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including interleukin-6 (IL-6), and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs), Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells, including clonal hematopoiesis.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). In this report, we show that TNFalpha promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFalpha antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFalpha and PAF sets the stage for reparative events mediated by PAFR signaling.