Benchmarking computational methods for B-cell receptor reconstruction from single-cell RNA-seq data.

Multiple methods have recently been developed to reconstruct full-length B-cell receptors (BCRs) from single-cell RNA sequencing (scRNA-seq) data. This need emerged from the expansion of scRNA-seq techniques, the increasing interest in antibody-based drug development and the importance of BCR repertoire changes in cancer and autoimmune disease progression. However, a comprehensive assessment of performance-influencing factors such as the sequencing depth, read length or number of somatic hypermutations (SHMs) as well as guidance regarding the choice of methodology is still lacking. In this work, we evaluated the ability of six available methods to reconstruct full-length BCRs using one simulated and three experimental SMART-seq datasets. In addition, we validated that the BCRs assembled in silico recognize their intended targets when expressed as monoclonal antibodies. We observed that methods such as BALDR, BASIC and BRACER showed the best overall performance across the tested datasets and conditions, whereas only BASIC demonstrated acceptable results on very short read libraries. Furthermore, the de novo assembly-based methods BRACER and BALDR were the most accurate in reconstructing BCRs harboring different degrees of SHMs in the variable domain, while TRUST4, MiXCR and BASIC were the fastest. Finally, we propose guidelines to select the best method based on the given data characteristics.

Persistently activated, proliferative memory autoreactive B cells promote inflammation in rheumatoid arthritis.

Autoreactive B cells mediate autoimmune pathology, but exactly how remains unknown. A hallmark of rheumatoid arthritis (RA), a common autoimmune disease, is the presence of disease-specific anticitrullinated protein antibodies (ACPAs). Here, we showed that ACPA-positive B cells in patients with RA strongly expressed T cell-stimulating ligands, produced abundant proinflammatory cytokines, and were proliferative while escaping inhibitory signals. This activated state was found at different degrees in different stages of disease: highest in patients with recent-onset RA, moderate in patients with established RA, and far less pronounced in ACPA-positive individuals "at risk" for developing disease. The activated autoreactive B cell response persisted in patients who achieved clinical remission with conventional treatment. ACPA-positive B cells in blood and synovial fluid secreted increased amounts of the chemoattractant interleukin-8, which attracted neutrophils, the most abundant immune cell in arthritic joints. Tetanus toxoid-specific B cells from the same patients exhibited properties of memory B cells without the activation and proliferation phenotype, but these cells transiently acquired a similar proliferative phenotype upon booster vaccination. Together, these data indicated that continuous antigenic triggering of autoreactive B cells occurs in human autoimmune disease and support the emerging concept of immunological activity that persists under treatment even in clinical remission, which may revise our current concept of treatment targets for future therapeutic interventions. In addition, our data pointed to a pathogenic role of ACPA-positive B cells in the inflammatory disease process underlying RA and favor approaches that aim at their antigen-specific inactivation or depletion.

Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue.

Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4+ T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6+ CD4+ T cells from SVF display a more activated phenotype than the IL-6- T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4+ T cells cannot be assigned to a conventional Th subset. TCRß gene analysis revealed that IL-6+ and IL-6- CD4+ T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4+ T cells. Thus, IL-6+ CD4+ T cells are TCRαß T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint.

Activation of human basophils by combined toll-like receptor- and FcεRI-triggering can promote Th2 skewing of naive T helper cells.

Basophils are mostly known for their involvement in allergic reactions. Recent studies in mice indicate a role for basophils in the induction of adaptive immunity, especially T helper 2 (Th2) responses. Therefore, it would be highly important to understand how basophils respond to pathogen-associated molecules, such as ligands for toll-like receptors (TLRs), and if the basophils could promote Th2 responses via these stimuli. To this end, the activation of basophils via TLRs in combination with activation via IgE was studied, as well as its effect on T helper cell skewing. Using quantitative PCR, we demonstrated the presence of mRNA for TLRs 1-8 in human basophils. Basophils responded to TLR triggering with differential cytokine production, but not with degranulation. Simultaneous triggering of TLRs and IgE led to synergy in production of IL-4, IL-8, IL-13, and RANTES. Furthermore, the synergistic effects on basophils mediated by IgE and TLR-4 triggering allowed robust Th2 skewing upon activation of naïve human CD4⁺ T cells. Our data show that human basophils respond to TLR ligands in synergy with IgE-mediated activation and that the cytokines produced can promote Th2 differentiation. These results indicate a role for basophils in the regulation of T-cell responses in humans.

Genetic variation of the Fc gamma receptor 3B gene and association with rheumatoid arthritis.

BACKGROUND: Fc gamma receptors (FcγRs) play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs) in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA). METHODOLOGY/PRINCIPAL FINDINGS: CNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA) in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy) in 6.7% and high copy number (≥3 copies) in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively). Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel) that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08). CONCLUSIONS/SIGNIFICANCE: The current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the possible protective effect of the -256A>TG indel polymorphism must be addressed in larger studies.

Immunomodulatory dendritic cells inhibit Th1 responses and arthritis via different mechanisms.

Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-gamma-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity.

Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans.

Expression of the transcription factor Foxp3 (forkhead box P3) has been implicated as a key element for CD25(+) T regulatory cell function in mice. However, literature over similar involvement of FOXP3 expression in human T regulatory cells is limited. We found that, unlike murine cells, FOXP3 mRNA expression could be induced in human CD25(-) and CD8(+) peripheral blood mononuclear cells, which were both negative for FOXP3 mRNA expression after isolation. Expression of FOXP3 mRNA began as soon as 24-40 hours after stimulation, demonstrating a correlation between activation and FOXP3 mRNA expression in human cells. In order to determine whether FOXP3 expression is confined to CD4(+)CD25(+) T cells with a regulatory phenotype, we analyzed several well-defined T-cell clones and lines with various specificities. Surprisingly, expression of FOXP3 mRNA was detected in all clones and limited to the CD25(hi) populations. Nonetheless, the CD25(hi) fraction did not display regulatory properties because both the CD25(hi) and CD25(low) populations exhibited a similar proliferative- and interferon-gamma-secreting potential after antigenic stimulation. These results indicate that FOXP3 expression in humans, unlike mice, may not be specific for cells with a regulatory phenotype and may be only a consequence of activation status.

Functional regulatory immune responses against human cartilage glycoprotein-39 in health vs. proinflammatory responses in rheumatoid arthritis.

The class of immune response against autoantigens could profoundly influence the onset and/or outcome of autoimmune diseases. Until now, there is only limited information on the antigen-specific balance between proinflammatory and regulatory responses in humans. Here we analyzed the natural immune response against a candidate autoantigen in rheumatoid arthritis, human cartilage glycoprotein-39 (HC gp-39). Peripheral blood mononuclear cells from healthy individuals reacted against HC gp-39 with the production of IL-10 but not IFN-gamma. Ex vivo assays indicated that the naturally occurring HC gp-39-specific immune response in bulk is powerful enough to suppress antigen-specific recall responses, demonstrating that rather than being unresponsive, the HC gp-39-directed immune response in healthy individuals shows a strong bias toward a regulatory phenotype. Moreover, CD4(+) T cell lines directed against HC gp-39 expressed CD25, glucocorticoid-induced tumor necrosis factor receptor, and Foxp3 molecules and were capable of suppressing antigen-specific T cell responses. Cell-cell contact was required for this suppression. As opposed to healthy individuals, the HC gp-39-directed immune response in 50% of patients with rheumatoid arthritis exhibits polarization toward a proinflammatory T helper 1 phenotype and is significantly less powerful in suppressing antigen-specific recall responses. Together these findings indicate that the presence of HC gp-39-specific immune responses in healthy individuals may have an inhibitory effect on inflammatory responses in areas where HC gp-39 is present. Furthermore, these data indicate that the class of HC gp-39-directed immune response in rheumatoid arthritis patients has shifted from an antiinflammatory toward a proinflammatory phenotype.

Allele-specific expression of the IL-1 alpha gene in human CD4+ T cell clones.

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1alpha gene expression.

Analysis of allelic expression patterns of IL-2, IL-3, IL-4, and IL-13 in human CD4+ T cell clones.

The occurrence of monoallelic expression of cytokine genes in single cells has been convincingly demonstrated, but there have been few reports of this phenomenon in T cell clones. Here we describe studies on the expression of alleles of the human genes encoding IL-2, IL-3, IL-4, and IL-13 in human CD4(+) T cell clones. In contrast to the results reported in mouse T cell clones and single human T cells, we found no evidence for the monoallelic expression of the IL-2, IL-3, and IL-13 genes. The gene for IL-4 showed an imbalance in expression from each allele, indicating differential expression of IL-4 alleles within or between IL-4-expressing cells.

Role of tumor necrosis factor-alpha and interleukin-10 promoter gene polymorphisms in leprosy.

Single-nucleotide polymorphisms within the genes coding for tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 have been associated with several infectious diseases. To determine whether such polymorphisms are associated with leprosy, genotyping was performed at the -308 and -238 positions of the promoter of the TNF-alpha gene in 210 and 191 patients with multibacillary (MB) leprosy, respectively; 90 and 79 patients with paucibacillary (PB) leprosy; and 92 control subjects. For the -592 and -819 positions within the promoter of the IL-10 gene, 143 patients with MB leprosy, 79 patients with PB leprosy, and 62 control subjects were included in the analysis. TNF2 allele frequency was significantly higher among control subjects than among all patients with leprosy or in the MB group (P<.05 and P<.01). For the IL-10 gene, the frequency of the homozygous -819TT genotype was significantly higher among patients than among control subjects. These data indicate that a relationship exists between TNF-alpha and IL-10 promoter polymorphisms and the development of PB leprosy.

Role of tumor necrosis factor - alpha and interleukin-10 promoter gene polymorphisms in leprosy

Single-nucleotide polymorphisms within the genes coding for tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 have been associated with several infectious diseases. To determine whether such polymorphisms are associated with leprosy, genotyping was performed at the -308 and -238 positions of the promoter of the TNF-alpha gene in 210 and 191 patients with multibacillary (MB) leprosy, respectively; 90 and 79 patients with paucibacillary (PB) leprosy; and 92 control subjects. For the -592 and -819 positions within the promoter of the IL-10 gene, 143 patients with MB leprosy, 79 patients with PB leprosy, and 62 control subjects were included in the analysis. TNF2 allele frequency was significantly higher among control subjects than among all patients with leprosy or in the MB group (P smaller.05 and P smaller.01). For the IL-10 gene, the frequency of the homozygous -819TT genotype was significantly higher among patients than among control subjects. These data indicate that a relationship exists between TNF-alpha and IL-10 promoter polymorphisms and the development of PB leprosy.