A CD153+CD4+ T follicular cell population with cell-senescence features plays a crucial role in lupus pathogenesis via osteopontin production.

Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.

Automated analysis of flow cytometry data with minimal training files: Research evaluation of an elastic image registration algorithm for TBNK, stem cell enumeration, and lymphoid screening tube assays.

Automated analysis of flow cytometry data can improve objectivity and reduce analysis time but has generally required work by software and algorithm experts. Here, we investigated the performance of BD ElastiGate™ Software (hereafter ElastiGate), which allows users to automate gating by selecting gated training files, then uses elastic image registration to gate new files. Three assays of increasing complexity were examined: TBNK, stem cell enumeration (SCE), and lymphoid screening tube (LST). For TBNK analysis, 60 peripheral blood (PB) samples from normal, HIV+, and controls were tested with ground truth analysis by an existing automated method. For SCE, 128 samples including bone marrow (BM), cord blood (CB), and apheresis were tested with analysis by multiple manual analysts. For LST, 80 PB and 28 BM samples were tested with manual analysis. For ElastiGate, a minimal number of training files was selected. Results were compared by Bland-Altman or F1 score analysis. For TBNK, ElastiGate using three training files (1 control, 1 normal, 1 HIV+) showed mean %bias across all reported populations between -1.48% and 7.13% (average 2.08%). For SCE, ElastiGate using three BM and two CB training files showed median F1 scores >0.93 in comparison to >0.94 and >0.92 for two other manual analysts. For LST, ElastiGate using four training files for each of PB and BM showed median F1 scores >0.945 for 13 of 14 PB populations and 10 of 14 BM populations, with generally similar or better performance for normal samples compared to abnormal; populations with lower scores were often associated with lower agreement between manual analysts. Based on analysis of three assays with four sample types of increasing complexity, ElastiGate with minimal training files may perform as an automated gating assistant. The results reported here are for research use only, not for use in diagnostic or therapeutic procedures.

Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.

Molecular dissection of antibody responses against pneumococcal surface protein A: evidence for diverse DH-less heavy chain gene usage and avidity maturation.

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA(3-286)) from strain R36A. Abs to PspA(3-286) were encoded by diverse V(H) and V(kappa) families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 +/- 0.5) and 8-9 (8.7 +/- 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D(H) gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D(H) gene-derived amino acids did not prevent anti-PspA(3-286) mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA(3-286) polyclonal Ab response in PspA(3-286)- and R36A-immunized mice. Compared with PspA(3-286)-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D(H) genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D(H)-less Abs.

Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment.

Vascular-deposited IgG immune complexes promote neutrophil recruitment, but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1, in its bent state, interacts with the IgG receptor FcγRIIA in cis to reduce the affinity of FcγRIIA for IgG and inhibit FcγRIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of FcγRIIA, as does a Mac-1 ligand and a mutation in Mac-1's ligand binding αI-domain. Sialylated complex glycans on FcγRIIA interact with the αI-domain via divalent cations, and this interaction is required for FcγRIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented FcγRIIA-dependent recruitment to IgG-coated endothelium. In mice, CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary, cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases.

Modification of Gene Expression, Proliferation, and Function of OP9 Stroma Cells by Bcr-Abl-Expressing Leukemia Cells.

Expression of the Bcr-Abl fusion gene in hematopoietic progenitor cells (HPCs) results in the development of chronic myelogenous leukemia (CML), for which hematopoietic microenvironment plays an important role. We investigated the specific effects of an HPC line transduced with Bcr-Abl, KOBA, on BM-derived OP9 stroma cells. DNA microarray analysis revealed that OP9 cells co-cultured with KOBA cells (OP9/L) show diverse changes in the gene expression. OP9/L cells showed significant down-regulation of Cdkn genes and up-regulation of Icam1, leading to the increased proliferation capacity of OP9 cells and enhanced transmigration of leukemia cells through them. The effects were attributed to direct Notch activation of OP9 cells by KOBA cells. OP9/L cells also showed a markedly altered cytokine gene expression pattern, including a robust increase in a variety of proinflammatory genes and a decrease in hematopoietic cytokines such as Cxcl12, Scf, and Angpt1. Consequently, OP9/L cells promoted the proliferation of KOBA cells more efficiently than parental OP9 cells, whereas the activity supporting normal myelopoiesis was attenuated. In mice bearing KOBA leukemia, the characteristic genetic changes observed in OP9/L cells were reflected differentially in the endothelial cells (ECs) and mesenchymal stroma cells (MCs) of the BM. The ECs were markedly increased with Notch-target gene activation and decreased Cdkn expression, whereas the MCs showed a marked increase in proinflammatory gene expression and a profound decrease in hematopoietic genes. Human CML cell lines also induced essentially similar genetic changes in OP9 cells. Our results suggest that CML cells remodel the hematopoietic microenvironment by changing the gene expression patterns differentially in ECs and MCs of BM.