Antonio Campos-Caro

Immunization-Induced Perturbation of Human Blood Plasma Cell Pool: Progressive Maturation, IL-6 Responsiveness, and High PRDI-BF1/BLIMP1 Expression Are Critical Distinctions between Antigen-Specific and Nonspecific Plasma Cells

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC+ and tetC− PC, and revealed remarkable differences between them: 1) the kinetics of tetC+ PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC− PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC+ PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC− PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC+, but not tetC−, PC subset; sequencing of tetC+ PC IgVH genes showed clear features of Ag selection. 4) The tetC+ PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC− PC. 5) The tetC− PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC+ PC contain bone marrow PC precursors, and tetC− PC probably belong to a removable compartment of aged PC.

Cutting Edge: IL-21 Derived from Human Follicular Helper T Cells Acts as a Survival Factor for Secondary Lymphoid Organ, but Not for Bone Marrow, Plasma Cells

IL-21 induces the differentiation of activated B lymphocytes into plasma cells (PC), but its direct effect on PC remains uncertain. This study analyzes the role of IL-21 on human in vivo-generated PC. IL-21R was clearly expressed on PC from the human tonsil, the lymph node, and the spleen (secondary lymphoid organs [SLO]) but barely on terminally mature bone marrow PC. IL-21 enhanced Ig secretion by isolated SLO PC but not bone marrow PC. Tonsillar T follicular helper (Tfh) lymphocytes are known to secrete IL-21. Purified Tfh cells induced a marked increase of Ig production by tonsillar PC, and this effect was impaired when endogenous IL-21 production was blocked. IL-21 provoked a rapid and transient phosphorylation of STAT3 in tonsillar PC. Tfh cells or exogenous IL-21 reduce tonsillar PC apoptosis and increases PC recovery but does not modify their nonproliferating status. These results suggest that IL-21 derived from Tfh cells acts as a survival factor for SLO PC in vivo.

Identification of Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for Antibody Secretion

Plasma cells (PC) are B-lymphocytes terminally differentiated in a postmitotic state, with the unique purpose of manufacturing and exporting Igs. Despite the importance of this process in the survival of vertebrates, no studies have been made to understand the molecular events that regulate Ig exocytosis by PC. The present study explores the possible presence of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) system in human PC, and examines its functional role in Ig secretion. Syntaxin-2, Syntaxin-3, Syntaxin-4, vesicle-associated membrane protein (VAMP)-2, VAMP-3, and synaptosome-associated protein (SNAP)-23 could be readily detected in normal human PC obtained from intestinal lamina propria and blood, as well as in human PC lines. Because SNAP-23 plays a central role in SNAREs complex formation, it was chosen to examine possible functional implications of the SNARE system in PC Ig secretion. When recombinant SNAP-23 fusion protein was introduced into the cells, a complete abolishment of Ig production was observed in the culture supernatants of PC lines, as well as in those of normal PC. These results provide insights, for the first time, into the molecular machinery of constitutive vesicular trafficking in human PC Ig secretion and present evidence indicating that at least SNAP-23 is essential for Ab production.

Persistent polyclonal B lymphocytosis: an expansion of cells showing IgVH gene mutations and phenotypic features of normal lymphocytes from the CD27+ marginal zone B-cell compartment.

Summary.  Persistent polyclonal B‐cell lymphocytosis (PPBL) is an unusual and benign lymphoproliferation characterized by a polyclonal expansion of B lymphocytes, whose nature remains undetermined. The phenotypic analysis of three cases revealed that these cells were CD27+ IgMhigh CD21high CD5low and CD23low, a phenotype associated with the normal marginal zone (MZ) B‐cell compartment. As MZ B cells have initiated immunoglobulin (Ig)V gene somatic mutations, PPBL IgVH genes were sequenced. An average of 73% of these sequences were mutated. The mean number of mutation per sequence was 6·9, a number similar to those observed in the MZ B‐cell compartment.

STAT-3 Activation by Differential Cytokines Is Critical for Human In Vivo–Generated Plasma Cell Survival and Ig Secretion

Maturation and survival of plasma cells (PCs) depends on extrinsic factors provided in specialized niches. In addition, B lymphocyte differentiation into PCs requires the activation of the JAK–STAT-3 pathway. However, whether STAT-3 is needed only during the transition of B lymphocytes to PC, or it is also involved in the survival and function of PCs at different stages of maturation, has not been unequivocally clarified. This study analyzes the effect of IL-10, IL-21, and IL-6 on human in vivo–generated PCs isolated from secondary lymphoid organs, blood (circulating, recently Ag-induced PCs), and bone marrow. PCs from these different organs show specific profiles of receptors for, and responsiveness to, these cytokines required for their survival and sustained Ab secretion. However, IL-10, IL-21, and IL-6 commonly induce STAT-3 phosphorylation in the three PC subsets, and all of their effects are exerted strictly through the STAT-3 activation. The inhibition or nonactivation of this pathway in the three PC populations impairs not only the effect of STAT-3–activating cytokines, but also the action of other cytokines important at the PC level, including a proliferation-induced ligand, BAFF, insulin-like growth factor 1, vascular endothelial growth factor, and stromal cell–derived factor-1α. These results indicate that STAT-3 activation is critical for human PCs throughout their maturation.

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR-1 GC-box.

The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC‐box as an essential element for PRDM1 expression. This GC‐box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC‐box.

Higher maturity and connective tissue association distinguish resident from recently generated human tonsil plasma cells

Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PCMECH). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PCCOLL). Moreover, PCMECH were present in suspensions highly enriched in germinal center cells whereas PCCOLL contained most of the IgA‐secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue‐rich zones such as tonsil subepithelium, respectively. Tonsil PCMECH and PCCOLL shared the phenotype CD38high CD19+ CD20low CD45high, expressed equivalent amounts of PRDI BF1/Blimp‐1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PCMECH still expressed the early B cell transcription factor BSAP and were HLA‐DRhigh; in contrast, PCCOLL were BSAP−and HLA‐DRlow. 2) PCMECH were CD95+ and Bcl‐2+/− whereas PCCOLL showed CD95+/− and Bcl‐2+ expression; in addition, PCMECH exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PCCOLL expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PCMECH. These results suggest that PCMECH are recently generated, short‐living PC, and PCCOLL constitutes a subset with higher maturity and survival, which resides in connective tissue‐rich areas.

Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells

PCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody‐secretion process are not well‐known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane‐fusion processes in the cell. We show here that freshly isolated human colon LP‐PCs and T‐PCs from MM‐PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP‐PCs.

Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells

Systemic antigen‐induced PCs are generated in inductive lymphoid tissues. Some of them are selected to travel through the circulation and finally, to home onto BM niches. BM PCs show prolonged survival and secrete high‐affinity antibodies. In this study, human PCs were isolated from tonsil, blood, and BM, their IGHV3 and IGHV6 genes were sequenced, and their SHM were evaluated. The SHM analysis reveals the existence of a maturational gradient in these genes, as demonstrated by a progressive increase in the frequency of total and R mutations and total and NC aa changes following the direction: tonsil → blood → BM. The ratio of R to S mutations in the CDR1 and ‐2, but not in the FRs, increases from tonsil to blood and BM; this parameter reaches a maximum threshold when more than 10 mutations/sequence occur. Further analyses indicate that CDR1 and CDR2 SHM followed different strategies to provide appropriate amino acid changes, but both exhibited maximal resistance to incorporating drastic molecular alterations in the BM PCs. Finally, all of the findings are similar in IGHV3 and IGHV6 sequences, indicating that they reflect general rules imposed by in vivo antigen selection.

Survival of Human Circulating Antigen-Induced Plasma Cells Is Supported by Plasma Cell–Niche Cytokines and T Follicular Helper Lymphocytes

Human circulating Ag-induced plasma cells (PCs) contain a high proportion of cycling cells. This study reveals that these PCs spontaneously proliferate in culture during 72 h, as determined by BrdU-uptake detection. Transcriptome analysis indicates that, in comparison with tonsil and bone marrow (BM) PCs, these PCs distinctively upregulate genes involved in cell division. Blood PC proliferation occurs simultaneously with increasing apoptosis rates, and is associated with PC survival. In addition, the proliferating activity of these PCs is enhanced by the addition of cytokines present in PC survival niches. Moreover, blood Ag-induced, but not BM, PCs exhibit the expression of molecules involved in the interaction between memory B cells and T follicular helper (Tfh) cells. In fact, purified circulating and tonsil Tfh cells increased IgG secretion by blood Ag-induced, but not by BM, PCs. This effect is exerted by augmenting blood PC survival through a mechanism partly dependent on cell contact. These results strongly suggest that the proliferating capacity of circulating Ag-induced PCs contributes to their competitive migration to survival niches, either to long-living PC niches or to temporal niches present in reactive lymphoid organs and inflamed tissues, structures where Tfh cells appear to participate.