Timothy J. Wilson

FCRL1 is a plasma membrane coreceptor on B cells that has been shown to potentiate B cell receptor (BCR)–driven calcium flux and negatively regulate ERK phosphorylation in a GRB2 and tyrosine-dependent manner. Of the proteins that associate with FCRL1, the recruitment of the inositol phosphatase SHIP-1 is GRB2 dependent, implicating SHIP-1 in FCRL1-mediated ERK regulation. Using immunoprecipitation and Western blotting, it was found that a proline-rich region in the C-terminus of SHIP-1, rather than its N-terminal SH2 domain, mediates recruitment of SHIP-1 to Y281 in FCRL1 in a manner dependent on GRB2. Interestingly, Y281 and GRB2 were also required for FCRL1 colocalization to the BCR. Translational fusions between tyrosine-mutated FCRL1 and the SH2 domain of SHIP-1 were sufficient to drive both colocalization of FCRL1 with the BCR as well as the ERK-inhibitory activity of FCRL1. Our data are consistent with a function for SHIP-1 as an adapter between FCRL1 and the BCR signalosome, and that this adapter function is responsible for BCR/FCRL1 colocalization after BCR stimulation and modulation of ERK signaling.

Treatment of antibiotic-resistant Acinetobacter baumannii infections has become exceedingly challenging, leading to higher morbidity and mortality. Therefore, the development of new therapeutics to treat these infections is critically needed, and immunotherapy is one potential treatment option. Immune responses against this pathogen, particularly lymphocyte- mediated responses, are not well understood. In this study, we investigated the role of B cells in innate resistance to A. baumannii pulmonary infection using a B cell-deficient (µMT) mouse model. B cell-deficient mice were impaired in clearing A. baumannii from the lung, liver, and spleen and failed to prevent extrapulmonary dissemination of A. baumannii after infection. Transcriptomic analyses indicated that the lack of B cells was associated with reduced expression of several genes encoding antimicrobial proteins. In addition, B cell deficiency was associated with pulmonary eosinophilia and increased pulmonary recruitment of Ly6C ⁺ NK cells following A. baumannii infection. This study demonstrates the significant role of B cells in providing early protection against A. baumannii infection and implicates B cell-dependent mechanisms beyond antibody-mediated bacterial resistance.

Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pan-drug resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell deficient (Rag2^-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. Our results showed that intranasally infected Rag2^-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours post infection compared to wild type mice. Animal pretreatment with normal mouse serum or purified antibodies from naïve mice rescued Rag2^-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. Overall, our study shows that natural antibodies mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

A direct, real-time reverse transcriptase PCR test on pooled saliva was validated in 2,786 participants against oropharyngeal swabs. Among asymptomatic/pre-symptomatic participants, the test was found to be in 99.21% agreement and 45% more sensitive than contemporaneous oropharyngeal swabs. The test was then used for surveillance testing on 44,242 saliva samples from asymptomatic participants. Those whose saliva showed evidence of SARS-CoV-2 within 50 cycles of amplification were referred for confirmatory testing, with 87% of those tested by nasal swab within 72 hours receiving a positive diagnostic result on Abbott ID NOW or real-time PCR platforms. Median Ct values on the saliva PCR for those with a positive and negative confirmatory tests was 30.67 and 35.92 respectively, however, binary logistic regression analysis of the saliva Ct values indicates that Ct thresholds as high as 47 may be useful in a surveillance setting. Overall, data indicate that direct RT-PCR testing of pooled saliva samples is an effective method of SARS-CoV-2 surveillance.

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1−/− mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.

Mononuclear phagocytes comprise an array of tissue‐resident and monocyte‐derived cells with important roles in tissue homeostasis and resistance to infection. Their diverse phenotypes make functional characterization within tissues challenging, because multiple surface markers are typically required for subset identification and isolation by cell sorting methods. Analysis of SLAMF9 expression within renal mononuclear phagocyte populations by multi‐parametric flow cytometry indicates that SLAMF9 is a specific marker for identification of kidney‐resident CD45⁺ CD11c⁺ MHC‐II⁺ cells corresponding to prominent tissue‐resident MPC populations derived from dendritic cell progenitors in adult mice. High SLAMF9 expression was sufficient to identify and sort these cells from disaggregated tissue using a user‐operated cell sorter. The population can be further subdivided according to expression of CD11b and CD14 to identify IRF8^high cDC1 cells and cleanly separate the CD11b^high F4/80^low and CD11b^int F4/80^high CD11c⁺ MPC subsets. Therefore, SLAMF9 expression allows for the identification and sorting of kidney‐resident CD11b⁺ CD11c⁺ CD64⁺ F4/80⁺ CX₃CR1⁺ MHC‐II⁺ MPCs without the need for complex antibody panels or reporter mice, simplifying isolation of these cells for study ex vivo.

Signaling lymphocyte activation molecule family member 9 (SLAMF9) is a cell surface protein of the CD2/SLAM family of leukocyte surface receptors. It is conserved throughout mammals and has roles in the initiation of inflammatory responses and regulation of plasmacytoid dendritic cell function. Through comparison of reference sequences encoding SLAMF9 in human, mouse, and primate sequences, we have determined that the SLAMF9 gene underwent successive mutation events, resulting in the loss of the protein and subsequent recovery of a less stable version. The mutations included a single base pair deletion in the second exon and a change in the splice acceptor site of that same exon. These changes would have had the effect of creating and later repairing a frameshift in the coding sequence. These events took place since the divergence of the human lineage from the chimpanzee-human last common ancestor and represent the first known case of the functional loss and recovery of a gene within the human lineage.

Summary: Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen‐presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b⁺, Ly6C⁻, CD11c^low, F4/80^low, MHC‐II⁺, CX₃CR1⁺ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80^low), but not large (F4/80^high), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9^−/− mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up‐regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA‐differentiated, SLAMF9 knockdown THP‐1 cells showed an essential role of SLAMF9 in production of granulocyte–macrophage colony‐stimulating factor, tumour necrosis factor‐α, and interleukin‐1β. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.

Summary: Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome‐wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val₆₀₂ variant of the non‐synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two‐fold lower affinity compared with the SLE‐associated Met₆₀₂ variant for the small adaptor protein SAP. Comparison of the two variants in T‐cell lines revealed the Val₆₀₂ variant to be significantly more highly expressed than CD229 Met₆₀₂. Activation was diminished in cells expressing CD229 Val₆₀₂ compared with CD229 Met₆₀₂ as measured by up‐regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine‐tuning in the immune system with significant functional consequences.

The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.