Sophie Stephenson

BLIMP-1 is a target of cellular stress and downstream of the unfolded protein response.

B lymphocyte‐induced maturation protein‐1 (BLIMP‐1) acts during differentiation of B cells and monocytes, but was originally identified as a repressor of the IFN‐β promoter induced during viral infection. A central regulator of the intracellular response to viral infection is the interferon‐inducible double‐stranded RNA activated protein kinase (PKR). PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2‐alpha (eIF2α) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum resident PKR‐homologue, is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B‐cell differentiation. This suggested that BLIMP‐1 might be a target of stress responses involving PERK. We demonstrate that BLIMP‐1 is rapidly up‐regulated during the UPR in human myeloid and B‐cell lines. This response is conserved in murine B‐cells and murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp‐1. During the UPR, BLIMP‐1 mRNA is induced at the level of transcription. This response is dependent on an intact PERK signaling pathway, independent of new protein synthesis and blocked by an inhibitor of NF‐κB. Our data provide evidence for a novel pathway linking cellular stress to BLIMP‐1, a regulator of differentiation in macrophages and B cells.

In Vitro Generation of Long-lived Human Plasma Cells

Plasma cells (PCs), the terminal effectors of humoral immunity, are short-lived unless supported by niche environments in which they may persist for years. No model system has linked B cell activation with niche function to allow the in vitro generation of long-lived PCs. Thus, the full trajectory of B cell terminal differentiation has yet to be investigated in vitro. In this article, we describe a robust model for the generation of polyclonal long-lived human PCs from peripheral blood B cells. After a proliferative plasmablast phase, PCs persist in the absence of cell division, with viability limited only by elective culture termination. Conservative predictions for PC life expectancy are 300 d, but with the potential for significantly longer life spans for some cells. These long-lived PCs are preferentially derived from memory B cells, and acquire a CD138high phenotype analogous to that of human bone marrow PCs. Analysis of gene expression across the system defines clusters of genes with related dynamics and linked functional characteristics. Importantly, genes in these differentiation clusters demonstrate a similar overall pattern of expression for in vitro and ex vivo PCs. In vitro PCs are fully reprogrammed to a secretory state and are adapted to their secretory load, maintaining IgG secretion of 120 pg/cell/day in the absence of XBP1 mRNA splicing. By establishing a set of conditions sufficient to allow the development and persistence of mature human PCs in vitro, to our knowledge, we provide the first platform with which to sequentially explore and manipulate each stage of human PC differentiation.

PRDM1/BLIMP-1 Modulates IFN-γ-Dependent Control of the MHC Class I Antigen-Processing and Peptide-Loading Pathway

A diverse spectrum of unique peptide-MHC class I complexes guides CD8 T cell responses toward viral or stress-induced Ags. Multiple components are required to process Ag and facilitate peptide loading in the endoplasmic reticulum. IFN-γ, a potent proinflammatory cytokine, markedly up-regulates transcription of genes involved in MHC class I assembly. Physiological mechanisms which counteract this response are poorly defined. We demonstrate that promoters of functionally linked genes on this pathway contain conserved regulatory elements that allow antagonistic regulation by IFN-γ and the transcription factor B lymphocyte-induced maturation protein-1 (also known as PR domain-containing 1, with ZNF domain (PRDM1)). Repression of ERAP1, TAPASIN, MECL1, and LMP7 by PRDM1 results in failure to up-regulate surface MHC class I in response to IFN-γ in human cell lines. Using the sea urchin prdm1 ortholog, we demonstrate that the capacity of PRDM1 to repress the IFN response of such promoters is evolutionarily ancient and that dependence on the precise IFN regulatory factor element sequence is highly conserved. This indicates that the functional interaction between PRDM1 and IFN-regulated pathways antedates the evolution of the adaptive immune system and the MHC, and identifies a unique role for PRDM1 as a key regulator of Ag presentation by MHC class I.

Repression of IFN-γ Induction of Class II Transactivator: A Role for PRDM1/Blimp-1 in Regulation of Cytokine Signaling

MHC class II is expressed in restricted lineages and is modulated in response to pathogens and inflammatory stimuli. This expression is controlled by MHC CIITA, which is transcribed from multiple promoters. Although factors required for induction of CIITA are well characterized, less is known about the mechanisms leading to repression of this gene. During plasma cell differentiation, B lymphocyte-induced maturation protein-1 (PRDM1/Blimp-1) represses promoter (p)III of CIITA, responsible for constitutive expression in B cells. pIV is inducible by IFN-γ in epithelia, macrophages and B cells. An IFN regulatory factor-element (IRF-E) in CIITA-pIV, which is bound by IRF-1 and IRF-2, is necessary for this response. This site matches the PRDM1/Blimp-1 consensus binding site, and PRDM1/Blimp-1 is expressed in cell lineages in which this promoter is operative. We, therefore, investigated whether PRDM1 regulates CIITA-pIV and found that PRDM1 bound to CIITA-pIV in vivo and the IRF-E in vitro. PRDM1 repressed IFN-γ-mediated induction of a CIITA-pIV luciferase reporter in a fashion dependent on an intact consensus sequence and competes with IRF-1/IRF-2 for binding to the IRF-E and promoter activation. In human myeloma cell lines that express IRFs, PRDM1 occupancy of CIITA-pIV was associated with resistance to IFN-γ stimulation, while short interfering RNA knockdown of PRDM1 led to up-regulation of CIITA. Our data indicate that PRDM1 is a repressor of CIITA-pIV, identifying a target of particular relevance to macrophages and epithelia. These findings support a model in which PRDM1/Blimp-1 can modulate the cellular response to IFN-γ by competing with IRF-1/IRF-2 dependent activation of target promoters.

BLIMP-1 and STAT3 Counterregulate MicroRNA-21 during Plasma Cell Differentiation

During cellular differentiation, mRNA transcription and translation require precise coordination. The mechanisms controlling this are not well defined. IL-21 is an important regulator of plasma cell differentiation, and it controls the master regulator of plasma cell differentiation, B lymphocyte-induced maturation protein-1 (BLIMP-1), via STAT3 and IRF4. Among the other targets of STAT3 is microRNA-21 (miR-21). miR-21 is the most frequently deregulated microRNA in malignancy, including B cell lymphomas, and it has oncogenic potential downstream of STAT3. However, the regulation and function of miR-21 during plasma cell differentiation are not characterized. In contrast to the induction of miR-21 observed in response to STAT3 activation in other systems, we demonstrate that miR-21 is repressed during IL-21–driven plasma cell differentiation. We explored the molecular basis for this repression and identify primary miR-21 transcription as a direct target of BLIMP-1–dependent repression, despite continued STAT3 activation and phospho-STAT3 binding to the primary miR-21 promoter. Thus, STAT3 and BLIMP-1 constitute an incoherent feed-forward loop downstream of IL-21 that can coordinate microRNA with mRNA expression during plasma cell differentiation.

Network Analysis Identifies Proinflammatory Plasma Cell Polarization for Secretion of ISG15 in Human Autoimmunity.

Plasma cells (PCs) as effectors of humoral immunity produce Igs to match pathogenic insult. Emerging data suggest more diverse roles exist for PCs as regulators of immune and inflammatory responses via secretion of factors other than Igs. The extent to which such responses are preprogrammed in B-lineage cells or can be induced in PCs by the microenvironment is unknown. In this study, we dissect the impact of IFNs on the regulatory networks of human PCs. We show that core PC programs are unaffected, whereas PCs respond to IFNs with distinctive transcriptional responses. The IFN-stimulated gene 15 (ISG15) system emerges as a major transcriptional output induced in a sustained fashion by IFN-α in PCs and linked both to intracellular conjugation and ISG15 secretion. This leads to the identification of ISG15-secreting plasmablasts/PCs in patients with active systemic lupus erythematosus. Thus, ISG15-secreting PCs represent a distinct proinflammatory PC subset providing an Ig-independent mechanism of PC action in human autoimmunity.

Amino Acid Deprivation Links BLIMP-1 to the Immunomodulatory Enzyme Indoleamine 2,3-Dioxygenase

Catabolism of tryptophan by IDO1 plays an important role in the control of immune responses. Activation of the eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible-2 (GCN2) following tryptophan depletion is a major pathway mediating this effect. However, immunomodulatory target genes of GCN2 activation are poorly defined. The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP-1) is a target of the eIF2α kinase1, protein kinase-like ER kinase (PERK) during the unfolded protein response of the endoplasmic reticulum. Thus, BLIMP-1 might also be a mediator of the GCN2 stress response pathway activated by IDO1 and tryptophan depletion. Indeed, in human monocytes BLIMP-1 mRNA and protein are up-regulated in response to both a pharmacological activator of GCN2 and tryptophan-depletion generated by IDO1-transfected cells. This suggests a functional role for BLIMP-1 in the immunomodulatory effects of the IDO1-GCN2 axis. BLIMP-1 has been shown to repress IFN-γ-regulated promoters. As IDO1 is itself highly responsive to IFN-γ, we hypothesized that BLIMP-1 functions in a feedback loop to regulate IDO1 expression. We found that BLIMP-1 binds to IFN-responsive sites in the IDO1 promoter and represses IFN-dependent IDO1 activation. We propose that BLIMP-1 acts in a negative feedback loop to successfully balance the outcome of tolerance vs inflammation.

Early Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell Transition

Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19neg human PCs. However, whether human CD19neg PCs are necessarily aged cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19neg plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19pos, CD19low, and CD19neg ASCs secrete vaccine-specific Abs and show linked IGHV repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19neg state can be established at the plasmablast to PC transition, that CD19neg PCs increase as a percentage of surviving PCs in vitro, and that CD19neg and CD19pos PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19neg state to be established.

OP0202 Long-Lived CD19-Negative Human Plasma Cells Are Generated at The Plasmablast To Plasma Cell Transition

Background B-cells are key mediators in a wide variety of autoimmune disorders as evidenced by the clinical response to B-cell depletion. Lack of response in a proportion of patients could be due to long-lived plasma cells (PCs) that are resistant to B-cell depletion therapy. Thus long-lived PCs in addition to their central role in humoral immunity are also involved in autoimmunity. Absence of CD19 expression, a feature of neoplastic PCs, identifies a minor subpopulation of normal human bone marrow PCs potentially linked to long-lived cells [1]. Whether CD19neg PCs are “aged” cells that have gradually lost CD19 expression, or derive from a distinct differentiation decision is not established. Objectives We sought to ascertain whether human CD19neg PCs are derived at the plasmablast to PC transition or are established by gradual loss of CD19 expression with increasing PC age. Methods We performed a sensitive flow cytometric analysis of CD19 expression during the human acute immune responses to flu vaccination, and tracked the generation and maintenance of CD19-negative PCs using CD40:CD40L and TLR mediated in vitro human PC differentiation systems [2]. Results Using intracellular staining for IRF4 alongside conventional surface markers such as CD27, CD38, CD95 and CD20, we robustly identify a CD19neg plasmablast population present in steady state peripheral blood. The CD19neg plasmablasts expanded during the acute plasmablast response to flu vaccination (p=0.063) but the degree of expansion was smaller compared to CD19pos plasmablasts. Influenza specific CD19neg plasmablasts were detectable 6 days after vaccination in the peripheral blood by ELISpot. CD19neg state was established at the plasmablast to PC transition in both CD40:CD40L and TLR mediated B-cell differentiation in vitro. CD19neg PCs increase as a percentage of surviving PCs with time in vitro at all the three subsequent time points compared to the previous time point (p=0.0009, 0.0015 and 0.0179). Flow-cytometrically sorted CD19neg PCs can be maintained independent of CD19pos PCs. Conclusions CD19neg PCs are independently established as an early event during human PC differentiation and predominantly do not derive by age-associated conversion from CD19pos PCs. Absence of CD19 expression does not provide evidence of PC age, but may identify PCs with greater potential for longevity in humans. Understanding the plasmablast to PC transition is vital in determining strategies for depletion of long-lived PCs and stages of immune response at which they can be employed for treatment of autoimmune disorders. References Mei HE, et al. Blood 2015, 125(11):1739–1748. Cocco M, et al. Journal of Immunology 2012, 189(12):5773–5785. Disclosure of Interest None declared