CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

A universal TLR7-nanoparticle adjuvant promotes broad immune responses against heterologous strains of Influenza and SARS-CoV-2

Fully effective vaccines for viruses such as Influenza and SARS-CoV-2 must elicit a diverse repertoire of antibodies against multiple drifted virus strains. However, how to achieve a diverse response has no general solution except to combine multiple strains, which risks diluting the response for all strains included. Here, we describe the synthesis of a universal, toll-like receptor 7 agonist (TLR7)-nanoparticle adjuvant, TLR7-NP, constructed from TLR7 agonist-initiated ring-opening polymerization of lactide and self-assembly with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid). When mixed with Alum-adsorbed antigens, this TLR7-NP adjuvant elicited cross-reactive antibodies for both dominant and subdominant epitopes, as well as antigen-specific CD8+ T cell responses. TLR7-NPs adjuvanted influenza subunit vaccine successfully protected mice from heterologous viral challenge. TLR7-NPs also enhanced the antibody response to a SARS-CoV-2 subunit vaccine against multiple variants and revealed the mobilization of a virus-like response. We further demonstrate enhanced antigen-specific responses in human tonsil organoids with this novel adjuvant.

AIM2 deficiency in B cells ameliorates systemic lupus erythematosus by regulating Blimp-1–Bcl-6 axis-mediated B-cell differentiation

Absent in melanoma 2 (AIM2) has been reported to be a component of inflammasomes in innate immune cells. Surprisingly, AIM2 is expressed by B cells, and higher AIM2 expression is observed in the B cells from lupus patients. To date, the inflammasome-independent function of AIM2 in B cells remains unclear. Here, we report increased expression of AIM2 in human tonsil memory and germinal center (GC) B cells and in memory B cells and plasma cells from the circulation and skin lesions of lupus patients. Conditional knockout of AIM2 in B cells reduces the CD19+ B-cell frequency in lymph nodes and spleens, and dampens KLH-induced IgG1-antibody production. In a pristane-induced mouse model of lupus, AIM2 deficiency in B cells attenuates lupus symptoms and reduces the frequency of GC B cells, T follicular helper (Tfh) cells, plasmablast cells, and plasma cells. Furthermore, the loss of AIM2 in human B cells leads to the increased expression of Blimp-1 and reduces the expression of Bcl-6. However, the silencing of Blimp-1 and Bcl-6 has no significant effect on AIM2 expression, indicating that AIM2 might be the upstream regulator for Blimp-1 and Bcl-6. In addition, IL-10 is found to upregulate AIM2 expression via DNA demethylation. Together, our findings reveal that AIM2 is highly expressed in the B cells of lupus patients and promotes B-cell differentiation by modulating the Bcl-6–Blimp-1 axis, providing a novel target for SLE treatment.

Transcriptomic analysis of pathways associated with alpha(v) integrin-related non-canonical autophagy in human B cells

Autophagy proteins have been linked with development of immune-mediated diseases including lupus, but the mechanisms for this are unclear. We have previously shown that non-canonical autophagy induced by alpha(v)-integrins regulates B cell activation by viral and self- antigens in mice. Here we investigated the involvement of this pathway in B cells from human tissue. Our data revealed that autophagy is specifically induced in germinal-center and memory B cell sub-populations from human tonsil and spleen. Transcriptomic analysis showed that induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of non-canonical autophagy in B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells we found that alpha(v)-integrin-related non-canonical autophagy limits activation of specific pathways while promoting others. These data provide new mechanistic links for autophagy and immune dysregulation in diseases such as lupus.

Anti-Aging Effects of Nanovesicles Derived from Human Tonsil-Derived Mesenchymal Stem Cells

Growing evidence has demonstrated that biomimetic nanovesicles produced from specific cells show bioactive properties such as anti-tumor or anti-inflammatory activities. However, the properties of these nanovesicles are very diverse, depending on their cell sources. In this study, human tonsil-derived mesenchymal stem cells (TMSCs) were used in the production of functional biomimetic nanovesicles with anti-senescence. TMSCs were isolated from human tonsil tissue obtained by tonsillectomy. TMSC-derived nanovesicles (TMSC-NVs) were produced by serial extrusion using a mini-extruder. Western blotting and particle analysis were performed for characterization of TMSC-NVs. They were applied to both replicative and ultraviolet B-induced senescent human dermal fibroblasts in vitro. Following six days of treatment, analysis of the proliferation and senescence level of fibroblasts was performed using cell counting and senescence-associated β-galactosidase assay, respectively. Treatment with TMSC-NVs enhanced the cell proliferation and reduced the activity of senescence-associated β-galactosidase in both replicative and ultraviolet B-induced senescent cells. Treatment with TMSC-NVs resulted in increased expression of extracellular matrix and anti-oxidant genes. Treatment with TMSC-NVs resulted in reduced expression of vinculin in focal adhesion. These results show that TMSC-NVs have an effect on recovering from cellular senescence by oxidative stress and can be applied as useful materials for the development of skin rejuvenation.

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20μm pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20μm pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.