Nicholas A. Barnes

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.

Blimp‐1 homolog Hobit identifies effector‐type lymphocytes in humans

Human cytomegalovirus (CMV) induces the formation of effector CD8+ T cells that are maintained for decades during the latent stage of infection. Effector CD8+ T cells appear quiescent, but maintain constitutive cytolytic capacity and can immediately produce inflammatory cytokines such as IFN‐γ after stimulation. It is unclear how effector CD8+ T cells can be constitutively maintained in a terminal stage of effector differentiation in the absence of overt viral replication. We have recently described the zinc finger protein Homolog of Blimp‐1 in T cells (Hobit) in murine NKT cells. Here, we show that human Hobit was uniformly expressed in effector‐type CD8+ T cells, but not in naive or in most memory CD8+ T cells. Human CMV‐specific but not influenza‐specific CD8+ T cells expressed high levels of Hobit. Consistent with the high homology between the DNA‐binding Zinc Finger domains of Hobit and Blimp‐1, Hobit displayed transcriptional activity at Blimp‐1 target sites. Expression of Hobit strongly correlated with T‐bet and IFN‐γ expression within the CD8+ T‐cell population. Furthermore, Hobit was both necessary and sufficient for the production of IFN‐γ. These data implicate Hobit as a novel transcriptional regulator in quiescent human effector‐type CD8+ T cells that regulates their immediate effector functions.

Recent advances in fluorovinyl-containing compounds

Abstract The reaction of the HFC-replacements CF3CH2F (HFC-134a) and CF3CH2Cl (HCFC-133a) with two equivalents of butyllithium generates, in high yield, the fluorovinyllithium reagents CF2CFLi and CF2CClLi, respectively. These lithium reagents have been used to synthesise fluorovinyl-containing transition metal and main-group compounds in good yields. The resulting compounds may themselves then be used as stable transfer reagents. Here we report the synthesis of new fluorovinyl-containing organometallic complexes, fluorovinyl-containing phosphine ligands of the type RnP(CXCF2)3−n (n=1, 2; X=Cl, F) and their complexes. The single crystal X-ray structures of [Fe(η5-C5H5)(CO)2(CClCF2)], cis-[PtBr2{PEt2(CFCF2)}2] and trans-[PtCl2{PPh2(CClCF2)}2] are reported.

CFC replacement HCFC-133a (CF3CH2Cl) as a convenient precursor for the synthesis of chlorodifluorovinyl-metal derivatives of main group and transition metal elements: the first X-ray structural characterisation of chlorodifluorovinyl-containing organometallic complexes

Abstract The one-pot reaction of the CFC replacement 1-chloro-2,2,2-trifluoroethane (CF3CH2Cl, HCFC-133a) with two equivalents of butyllithium in diethylether at −78°C followed by the addition of main group or transition metal halides results in good yields of the metal-chlorodifluorovinyl-containing compounds R3Sn(CClCF2) {R=Me, Et, Bu}, Sn(CClCF2)4, Sb(CClCF2)3, Hg(CClCF2)nCl(2−n) (n=1,2), trans-[Ni(CClCF2)2(PBu3)2], trans-[Pd(CClCF2)2(PBu3)2] and [Au(CClCF2)(PPh3)]. The molecular structures of Hg(CClCF2)Cl, trans-[Pd(CClCF2)2(Bu3P)2] and [Au(CClCF2)(PPh3)] have been obtained from single crystal data; these are the first such structural data to be reported for any chlorodifluorovinyl-containing organometallic complexes. The molecular structure of [Au(CFCF2)(PPh3)], which was prepared using a similar method based on CF3CH2F (HFC-134a), is also reported and compared with that of the chlorodifluorovinyl analogue.

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.

Structural relationships between o-, m- and p-tolyl substituted R3EI2 (E = As, P) and [(R3E)AuX] (E = As, P; X = Cl, Br, I)

The compounds R3EI2 (R = o-tolyl, E = As, 1a; R = m-tolyl, E = P 1c; R = p-tolyl, E = As, 1d, P, 1e), which display the charge transfer spoke structure, and [(o-tolyl3As)AuCl] 2 have been synthesised and their solid state structures compared to the related complexes [(R3P)AuX] (R = o-tolyl, X = Cl, I, Ia; Br, II; I, III; R = m-tolyl, X = Cl, IV; R = p-tolyl, X = Cl, V, Va; Br, VI; I, VII) on the basis of a similarity of their molecular shape and volume. All of the new compounds 1a, 1c–1e and 2 have been fully spectroscopically characterised and by single crystal X-ray crystallography. The sterically demanding exo3o-tolyl ring conformation is observed for 1a, which is comparable to that reported for o-tolyl3PI21b, with a long As–I bond 2.7351(14) A and short I⋯I distance 2.9528(11) A. The exo3o-tolyl ring conformation is maintained on complexation to gold(I) in 2, but has no significant impact on the expected bond lengths, with As–Au 2.3443(15) A and Au–Cl 2.284(4) A. The exo3 conformation appears to be stabilised in both cases by the formation of a six-fold edge-to-face (EF)6 embrace. It is found that in some cases the structures of the dihalogen adducts and the gold(I) complexes are isomorphous indicating that ligand packing requirements are most significant i.e. for 1c and IV. Where the structures digress this is due either to the greater ability of the dihalogen adduct to engage in hydrogen bonding 1a, b and I–III; or subtle changes in the nature of the tolyl ring embraces 1d, e and V–VII. Subtle changes in the nature of the tolyl ring embraces also account for the different polymorphs I and Ia and V and Va. There is no credible evidence to suggest that the aurophilic contact, seen in only one polymorph Va, exerts any influence on the overall crystal packing. The structural comparisons presented here add further to the applicability of the recently recognised structural mimicking ability of the R3PX2 systems and [R3PAuX] complexes, and that the aurophilic contact is a poor supramolecular synthon.

The reactions of alkylamino substituted phosphines with I2 and (Ph2Se2I2)2: structural features of alkylamino phosphonium cations

The reactions of the tris-dialkylamino phosphines (Et2N)3P and (nPr2N)3P, and the pyrrolidinyl substituted phosphines (C4H8N)3P and tBuP(NC4H8)2, with I2 and (Ph2Se2I2)2, have been reported. The reactions with diiodine lead to the formation of [R3PI]I adducts, which are essentially ionic, but show a tendency to display long, soft-soft, II interactions in the solid state. The crystal structures of [(Et2N)3PI]I, (1), [(nPr2N)3PI]I, (2), and [(C4H8N)3PI]I, (3), have all been determined, and display II interactions varying between 3.5170(6) and 3.6389(14) A. The analogous reactions with (Ph2Se2I2)2 lead to the formation of phenylseleno-phosphonium salts, [R3PSePh]I. The structures of [(C4H8N)3PSePh]I, (6) and [(C4H8N)2tBuPSePh] I, (7), have been determined and do not display any soft-soft interactions between the selenium and iodine atoms. All of the phosphonium salts represent examples of structures containing tris-dialkylamino phosphine fragments which show no special nitrogen atom, i.e. all three nitrogen atoms are planar. This type of arrangement is usually observed when a C3 symmetric conformation is observed, (which is the case for 1 and 2), but not for the (C4H8N)3P adducts 3 and 6, where the conformation is closer to Cs, although the nitrogen atoms are still essentially planar. The P-N bonds in all the compounds reported herein are short, ranging between 1.599(12) A and 1.643(12) A, and are consistent with the previously reported short P-N bonds in phosphonium salts featuring tris-dialkylamino substituted phosphines.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

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.