Isolation and expansion of pure and functional γδ T cells.

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vδ2+ T cells, while Vδ1+ T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vδ1+ and Vδ2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-γ, TNF-α and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

Distinct dynamics of antigen-specific induction and differentiation of different CD11c+Tbet+ B-cell subsets.

BACKGROUND: CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. OBJECTIVES: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts. METHODS: Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures. RESULTS: In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts. CONCLUSION: Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.

Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates.

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

Termination of CD40L co-stimulation promotes human B cell differentiation into antibody-secreting cells.

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (TFH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and TFH cells are transient; after TFH contact, B cells cycle between GC light and dark zones where TFH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets.

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

Human B cells promote T-cell plasticity to optimize antibody response by inducing coexpression of T(H)1/T(FH) signatures.

BACKGROUND: B cells mediate humoral immunity against pathogens but also direct CD4(+) T-cell responses. Recent plasticity studies in mice have challenged the concept of strict fate commitment during CD4(+) T-cell differentiation into distinct subsets. OBJECTIVE: We sought to elucidate the contribution of human antigen-primed B cells in CD4(+) T-cell responses that support humoral immunity. METHODS: CD4(+) T-cell differentiation by primary human B cells was investigated in in vitro cocultures by using tetanus toxoid and Salmonella species as antigen models. T-cell differentiation was assessed by using intracellular cytokines and subset-specific transcription factors and markers. IgM and IgG formation was analyzed by means of ELISA. RESULTS: Human B cells, but not dendritic cells, induce prominent and stable coexpression of TH1 and follicular helper T (TFH) cell characteristics during priming and on antigen recall. TH1/TFH cells coexpress the TH1 and TFH effector cytokines IFN-γ and IL-21 and the TFH marker CXCR5, demonstrating that the coexpressed TH1 and TFH subset-specifying transcription factors T-box transcription factor (T-bet) and B cell lymphoma 6 are both functionally active. B cell-derived IL-6 and IL-12 controlled respective expression of IL-21 and IFN-γ, with IL-21 being key for humoral immunity. CONCLUSION: Human B cells exploit CD4(+) T-cell plasticity to create flexibility in the effector T-cell response. Induction of a T-cell subset coexpressing IL-21 and IFN-γ might combine IL-21-mediated T-cell aid for antibody production while maintaining TH1 cytokine expression to support other cellular immune defenses.

Selective infection of antigen-specific B lymphocytes by Salmonella mediates bacterial survival and systemic spreading of infection.

BACKGROUND: The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer's Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading. METHODOLOGY/PRINCIPAL FINDINGS: Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood. CONCLUSIONS/SIGNIFICANCE: This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection.

CD5 costimulation induces stable Th17 development by promoting IL-23R expression and sustained STAT3 activation.

IL-17-producing CD4(+) T helper (Th17) cells are important for immunity against extracellular pathogens and in autoimmune diseases. The factors that drive Th17 development in human remain a matter of debate. Here we show that, compared with classic CD28 costimulation, alternative costimulation via the CD5 or CD6 lymphocyte receptors forms a superior pathway for human Th17-priming. In the presence of the Th17-promoting cytokines IL-1ß, IL-6, IL-23, and transforming growth factor-ß (TGF-ß), CD5 costimulation induces more Th17 cells that produce higher amounts of IL-17, which is preceded by prolonged activation of signal transducer and activator of transcription 3 (STAT3), a key regulator in Th17 differentiation, and enhanced levels of the IL-17-associated transcription factor retinoid-related orphan receptor-γt (ROR-γt). Strikingly, these Th17-promoting signals critically depend on CD5-induced elevation of IL-23 receptor (IL-23R) expression. The present data favor the novel concept that alternative costimulation via CD5, rather than classic costimulation via CD28, primes naive T cells for stable Th17 development through promoting the expression of IL-23R.

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation.

BACKGROUND: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+) T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8(+) T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8(+) T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8(+) T cells is dependent on CD4(+) T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis. CONCLUSIONS/SIGNIFICANCE: B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.

Urinary granzyme A mRNA is a biomarker to diagnose subclinical and acute cellular rejection in kidney transplant recipients.

The distinction between T-cell-mediated rejection (TCMR) and other causes of kidney transplant dysfunction such as tubular necrosis requires biopsy. Subclinical rejection (SCR), an established risk factor for chronic allograft dysfunction, can only be diagnosed by protocol biopsy. A specific non-invasive biomarker to monitor immunological graft status would facilitate diagnosis and treatment of common transplantation-related complications. To identify possible markers, we measured urinary mRNA levels of several cytolytic proteins by quantitative PCR. Our cohort of 70 renal transplant recipients had biopsy proven type I and type II TCMR, acute tubular necrosis, SCR, calcineurin inhibitor-toxicity, cytomegalovirus infection, and stable graft function with normal histology. Granzyme A (GzmA) mRNA was significantly higher in subclinical and acute cellular rejection compared to patients with stable grafts or those with tubular necrosis with 80% sensitivity and up to 100% specificity. Granzyme B and perforin mRNA levels could significantly discriminate acute rejection from stable or tubular necrosis, but were not significantly elevated during SCR. Importantly, only GzmA mRNA remained below detection limits from grafts that were stable and most with tubular necrosis. Hence, the presented data indicate that urinary GzmA mRNA levels may entail a diagnostic non-invasive biomarker to distinguish patients with subclinical and acute cellular rejection from those with tubular necrosis or stable grafts.

B cell receptor-mediated internalization of salmonella: a novel pathway for autonomous B cell activation and antibody production.

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.