Memory B-cell Populations Research Articles

Reduced serological response to COVID-19 booster vaccine is associated with reduced B cell memory in patients with Inflammatory Bowel Disease; VARIATION (VAriability in Response in IBD AgainsT SARS-COV-2 ImmunisatiON).

Patients with inflammatory bowel disease (IBD) have an attenuated response to initial COVID-19 vaccination. We sought to characterise the impact of IBD and its treatment on responses after third vaccine against SARS-CoV-2. Prospective multicentre observational study of patients with IBD (n=202) and healthy controls (HC, n=92). Serological response to vaccination was assessed by quantification of anti-spike protein (SP) immunoglobulin (Ig)G levels (anti-SPIgG) and in-vitro neutralisation of binding to Angiotensin-Converting-Enzyme (ACE2). Peripheral blood B-cell phenotype populations were assessed by flow cytometry. SARS-CoV-2 antigen specific B-cell responses were assessed in ex-vivo culture. Median anti-SP IgG post-third vaccination in our IBD cohort is significantly lower than HCs (7,862 versus 19,622 AU/mL, p <0.001) as is ACE2 binding inhibition (p <0.001). IBD patients previously infected with COVID-19 (30%) have similar quantitative antibody response as HCs previously infected with COVID-19 (p = 0.12). Lowest anti-SP IgG titres and neutralisation are seen in IBD patients on anti-TNF agents, without prior COVID-19 infection but all IBD patients show attenuated vaccine response compared to HCs. Patients with IBD have reduced memory B-cell populations and attenuated B-cell responses to SARS-CoV-2 antigens if not previously infected with COVID-19 (p = 0.01). Higher anti-TNF drug levels and zinc levels < 65ng/ml are associated with significantly lower serologic response. Patients with IBD have an attenuated response to three doses of SARS-CoV-2 vaccine. Physicians should consider patients with higher anti-TNF drug levels and/or zinc deficiency as potentially at higher risk of attenuated response to vaccination.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Hierarchical Clustering and Trajectory Analyses Reveal Viremia-Independent B-Cell Perturbations in HIV-2 Infection.

Time to AIDS in HIV-2 infection is approximately twice as long compared to in HIV-1 infection. Despite reduced viremia, HIV-2-infected individuals display signs of chronic immune activation. In HIV-1-infected individuals, B-cell hyperactivation is driven by continuous antigen exposure. However, the contribution of viremia to B-cell perturbations in HIV-2-infected individuals remains largely unexplored. Here, we used polychromatic flow cytometry, consensus hierarchical clustering and pseudotime trajectory inference to characterize B-cells in HIV-1- or HIV-2-infected and in HIV seronegative individuals. We observed increased frequencies of clusters containing hyperactivated T-bethighCD95highCD27int and proliferating T-bet+CD95highCD27+CD71+ memory B-cells in viremic HIV-1 (p < 0.001 and p < 0.001, respectively), viremic HIV-2 (p < 0.001 and p = 0.014, respectively) and in treatment-naïve aviremic HIV-2 (p = 0.004 and p = 0.020, respectively)-infected individuals, compared to seronegative individuals. In contrast, these expansions were not observed in successfully treated HIV-1-infected individuals. Finally, pseudotime trajectory inference showed that T-bet-expressing hyperactivated and proliferating memory B-cell populations were located at the terminal end of two trajectories, in both HIV-1 and HIV-2 infections. As the treatment-naïve aviremic HIV-2-infected individuals, but not the successfully ART-treated HIV-1-infected individuals, showed B-cell perturbations, our data suggest that aviremic HIV-2-infected individuals would also benefit from antiretroviral treatment.

Germinal center activity and B cell maturation are associated with protective antibody responses against Plasmodium pre-erythrocytic infection.

Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. However, the lack of well-defined features within vaccine-elicited antibody responses that correlate with protection represents a major roadblock to improving on current generation vaccines. We vaccinated mice (BALB/cJ and C57BL/6J) with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated vaccine-elicited humoral immunity and identified immunological factors associated with protection after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice. In contrast, all C57BL/6J mice were infected similar to controls. Protection was mediated by antibodies and could be passively transferred from immunized BALB/cJ mice into naïve C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer CSP-specific germinal center experienced B cells and class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG γ chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that the development of protective antibody responses in BALB/cJ mice in response to vaccination with PyCSP was associated with increased germinal center activity and somatic mutation compared to C57BL/6J mice, highlighting the key role B cell maturation may have in the development of vaccine-elicited protective antibodies against CSP.

Early human B cell signatures of the primary antibody response to mRNA vaccination

Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2-naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

Seroconversion following COVID-19 vaccination: can we optimize protective response in CD20-treated individuals?

Although there is an ever-increasing number of disease-modifying treatments for relapsing multiple sclerosis (MS), few appear to influence coronavirus disease 2019 (COVID-19) severity. There is concern about the use of anti-CD20-depleting monoclonal antibodies, due to the apparent increased risk of severe disease following severe acute respiratory syndrome corona virus two (SARS-CoV-2) infection and inhibition of protective anti-COVID-19 vaccine responses. These antibodies are given as maintenance infusions/injections and cause persistent depletion of CD20+ B cells, notably memory B-cell populations that may be instrumental in the control of relapsing MS. However, they also continuously deplete immature and mature/naïve B cells that form the precursors for infection-protective antibody responses, thus blunting vaccine responses. Seroconversion and maintained SARS-CoV-2 neutralizing antibody levels provide protection from COVID-19. However, it is evident that poor seroconversion occurs in the majority of individuals following initial and booster COVID-19 vaccinations, based on standard 6 monthly dosing intervals. Seroconversion may be optimized in the anti-CD20-treated population by vaccinating prior to treatment onset or using extended/delayed interval dosing (3-6 month extension to dosing interval) in those established on therapy, with B-cell monitoring until (1-3%) B-cell repopulation occurs prior to vaccination. Some people will take more than a year to replete and therefore protection may depend on either the vaccine-induced T-cell responses that typically occur or may require prophylactic, or rapid post-infection therapeutic, antibody or small-molecule antiviral treatment to optimize protection against COVID-19. Further studies are warranted to demonstrate the safety and efficacy of such approaches and whether or not immunity wanes prematurely as has been observed in the other populations.

High-affinity memory B cells induced by SARS-CoV-2 infection produce more plasmablasts and atypical memory B cells than those primed by mRNA vaccines.

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A+ subset and secondary MBCs that are mostly IgG+ and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses.

Vaccine against SARS-CoV-2 in previously infected health care workers.

A growing amount of data have demonstrated, after either severe, mild or asymptomatic infection by SARS-CoV-2, a decline of neutralizing antibody titers (NAbT) over time, although the persistence of NAb as long as 10-13 months was reported [1–2]. The significance and the level of NAbT as correlate of protection from reinfection is still to be defined; nevertheless memory B-cell population and T-cell mediated immunity appear to be maintained [3].

Walking down the memory lane with SARS-CoV-2 B cells.

The B-cell response to COVID-19 vaccines in convalescent individuals.

B-Cell Memory Responses to Variant Viral Antigens.

A central feature of vertebrate immune systems is the ability to form antigen-specific immune memory in response to microbial challenge and so provide protection against future infection. In conflict with this process is the ability that many viruses have to mutate their antigens to escape infection- or vaccine-induced antibody memory responses. Mutable viruses such as dengue virus, influenza virus and of course coronavirus have a major global health impact, exacerbated by this ability to evade immune responses through mutation. There have been several outstanding recent studies on B-cell memory that also shed light on the potential and limitations of antibody memory to protect against viral antigen variation, and so promise to inform new strategies for vaccine design. For the purposes of this review, the current understanding of the different memory B-cell (MBC) populations, and their potential to recognize mutant antigens, will be described prior to some examples from antibody responses against the highly mutable RNA based flaviviruses, influenza virus and SARS-CoV-2.

Analysis of Antigen-Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation.

Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells Basic Protocol 2: ELISpot assay for enumeration of memory B cell-derived antibody-secreting cells.

Higher frequencies of functional HIV-envelope-specific memory B cells are associated with nonprogressive HIV infection in Indian population.

The HIV-1-specific antibodies are being considered for prevention and therapy in HIV infection. For effective antibody response, presence of functionally competent memory B cells (MEBs) is important; however, HIV-infection is known to alter the B-cell functionality. Very limited data are available on the HIV-specific memory B-cell population in HIV-infected Indian population. In this study, the frequencies of HIV-gp140-specific MEBs were measured in individuals with nonprogressive [long-term-nonprogressors (LTNPs), N = 20] and progressive (N = 19) HIV infection using multicolor flow cytometry. The activation and functional status of these MEBs were assessed as frequencies and mean fluorescence intensity (MFI) of the CD38 and CD40 expression, respectively. The percentages of gp140 + MEBs were higher in LTNPs than seen in progressors (P = 0.0475) and associated with higher CD4 cell count (P = 0.0312, r = 0.2833). As compared with the progressors, LTNPs also showed higher functional (CD40+) gp140 + MEBs both frequencies (P < 0.0001) and CD40 MFI (P = 0.0222), whereas the frequencies (<0.0001) and the MFI (P = 0.0047) of CD38 expression was significantly lower. Higher CD4 cell counts and lower plasma viral load values were associated with higher frequencies of CD40+ gp140 + MEBs (P < 0.0001, r = 0.4962) (P = 0.0036, r = -0.4202) and lower frequencies (P = 0.0008, r = -0.4231) and CD38 expression (MFI) (P = 0.004, r = -0.3719) (P = 0.0066, r = 0.4033). Our study suggests that LTNPs have functional HIV-specific memory B-cell compartment with reduced activation that may lead to effective HIV-specific humoral immune responses contributing to their nondisease progression status. These findings would help in better understanding of the characteristics of the HIV-specific memory B-cell population in nonprogressive HIV infection.

Longitudinal dynamics of the human B cell response to the yellow fever 17D vaccine

A comprehensive understanding of the development and evolution of human B cell responses induced by pathogen exposure will facilitate the design of next-generation vaccines. Here, we utilized a high-throughput single B cell cloning technology to longitudinally track the human B cell response to the yellow fever virus 17D (YFV-17D) vaccine. The early memory B cell (MBC) response was mediated by both classical immunoglobulin M (IgM) (IgM+CD27+) and switched immunoglobulin (swIg+) MBC populations; however, classical IgM MBCs waned rapidly, whereas swIg+ and atypical IgM+ and IgD+ MBCs were stable over time. Affinity maturation continued for 6 to 9 mo following vaccination, providing evidence for the persistence of germinal center activity long after the period of active viral replication in peripheral blood. Finally, a substantial fraction of the neutralizing antibody response was mediated by public clones that recognize a fusion loop-proximal antigenic site within domain II of the viral envelope glycoprotein. Overall, our findings provide a framework for understanding the dynamics and complexity of human B cell responses elicited by infection and vaccination.

Effect of biological agents on B-lymphocyte subpopulations in patients with systemic lupus erythematosus

Objective : to evaluate the effect of different biological agents (BAs), including rituximab (RTM) and belimumab (BLM) combination therapy, on B-lymphocyte subpopulations during a follow-up of patients with systemic lupus erythematosus (SLE). Patients and methods . The investigation enrolled 64 patients with a verified diagnosis of SLE with high and moderate disease activities according to the Systemic Lupus Erythromatosus Disease Activity Index (SLEDAI)-2K scores; 47patients of them took RTM, 10 used BLM, and 7 received RTM + BLM combination therapy. Peripheral blood B-lymphocyte subpopulations were measured by multicolor flow cytofluorom-etry, using a panel of monoclonal antibodies to B-lymphocyte surface membrane markers. The results were assessed using the SLEDAI-2K scores and the British Isles Lupus Assessment Group (BILAG) index. Results and discussion . RTM therapy led to a marked decrease in major B-lymphocyte populations, the residual cells being naXve B-cells and different memory B cell populations, the percentage of which depended on the degree of depletion after a RTM cycle. Incomplete B-lymphocyte depletion was associated with the large baseline numbers of plasma cells (PCs) (&gt;0.2%). One year after initiation of therapy, the percentage ratio of B-lymphocyte subpopulations returned almost completely to baseline values, except the whole memory B-cellpopulation. BLM therapy resulted in a decrease in PCs and plasmablasts (PBs) to the point of their complete depletion. There were reductions in total CD19+ B-lym-phocytes and naive B lymphocytes. The use of the combination of BAs permitted the monitoring of the total B-lymphocyte population; its slower recovery was seen in patients with its complete depletion after a rituximab cycle. The therapy promoted maintenance of low concentrations of PCs and PBs, total memory B-cell and naive B-cell populations. Conclusion. In patients with SLE, all the three therapy with BAs demonstrated a good efficiency manifested by a decrease in clinical and laboratory disease activity. The found time course of changes in B-lymphocyte subpopulations can be used for the selection of therapy and for the evaluation of its efficacy.

Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.

Preparing for re-entry: is sequential switching the result of recurrent secondary responses?

Preparing for re-entry: is sequential switching the result of recurrent secondary responses?

Distinct T helper cell dependence of memory B‐cell proliferation versus plasma cell differentiation

Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP+ memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP+ memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

Polysaccharide-Specific Memory B Cells Predict Protection against Experimental Human Pneumococcal Carriage.

We have previously demonstrated that experimental pneumococcal carriage enhances immunity and protects healthy adults against carriage reacquisition after rechallenge with a homologous strain. To investigate the role of naturally acquired pneumococcal protein and polysaccharide (PS)-specific immunity in protection against carriage acquisition using a heterologous challenge model. We identified healthy volunteers that were naturally colonized with pneumococcus and, after clearance of their natural carriage episode, challenged them with a heterologous 6B strain. In another cohort of volunteers we assessed 6BPS-specific, PspA-specific, and PspC-specific IgG and IgA plasma and memory B-cell populations before and 7, 14, and 35 days after experimental pneumococcal inoculation. Heterologous challenge with 6B resulted in 50% carriage among volunteers with previous natural pneumococcal carriage. Protection from carriage was associated with a high number of circulating 6BPS IgG-secreting memory B cells at baseline. There were no associations between protection from carriage and baseline levels of 6BPS IgG in serum or nasal wash, PspA-specific, or PspC-specific memory B cells or plasma cells. In volunteers who did not develop carriage, the number of circulating 6BPS memory B cells decreased and the number of 6BPS plasma cells increased postinoculation. Our data indicate that naturally acquired PS-specific memory B cells, but not levels of circulating IgG at time of pneumococcal exposure, are associated with protection against carriage acquisition.

Adoptive Transfer of Purified Donor-B-Lymphocytes after Allogeneic Stem Cell Transplantation: Results from a Phase I/IIa Clinical Trial

Patients after allogeneic stem cell transplantation (alloSCT) show a long lasting immune deficiency involving both T- and B-lymphocytes. Antibody responses to vaccination, as a measure of functional B-cell immunity, are insufficient in the first months after alloSCT and usually recover within 1-2 years. To improve B-cell immune reconstitution after alloSCT, we developed a novel concept of adoptive transfer of memory B-cells from the original stem cell donor (Klenovsek et al., Blood 110:3472-79, 2007). To this end, we first established the technology for the manufactoring of a GMP-qualified B-cell product and initiated a first-in-man phase I/IIa clinical trial in patients after alloSCT to evaluate safety and tolerability of adoptively transferred donor B-cells.B-cells were manufactured under GMP conditions from 16 unstimulated leukapheresis products derived from the original stem cell donor. The separation protocol consists of two subsequent separation steps on the CliniMACS® System (Miltenyi Biotec) including depletion of CD3+ T-cells and subsequent positive separation of CD19+ B-cells by magnetic beads. In all of the 16 cell products high purities of CD20+ B-cells (average 96%, range 85-99%) with only low frequencies of contaminating T-cells (average 0.10 %, range 0.01-0.82%) were achieved. After manufacturing, B-cell products were cryopreserved for single administration to 4 groups of patients with escalating doses. To prevent GvH reactions the absolute number of CD3+ T lymphocytes in the B-cell product had to be below 4 x 104 CD3+ cells/kg BW.The clinical trial was designed as an open label dose escalating study using the traditional 3+3 design with 4 dose levels of 0.5-1-2-4x106 B-cells/kg BW. Patients were enrolled on day +140 ± 20 after alloSCT after tapering of immunosuppression. Patients with acute GVHD grade III/IV, chronic GVHD with intermediate to high risk, EBV reactivation (>10,000 EBV copies/ml) or after therapy with B-cell antibodies (i.e. rituximab) were not eligible. Up to now, 13 patients after alloSCT received donor B-cells in 4 different B-cell dosages (average day +147, range from day +130 to d +160). Three patients received 0.5x106 B-cells per kg BW, 3 patients 1.0x106 B-cells/kg BW, 5 patients 2.0x106 B-cells per kg BW, and 2 patients 4.0x106 B-cells/kg BW, respectively. The median number of infused T cells was 0.06x104 T-cells per kg BW (range 0.01-0.13 x104/kg BW). Regarding primary safety data, the B-cell transfer was well tolerated without any acute adverse reactions. Importantly, neither significant EBV-viremia nor acute or chronic GvHD reactions were observed during the observation period of 4 months after the transfer of B-cells.In addition to the analysis of the primary safety endpoints of the trial, the activity of infused donor memory B-cells in vivo were tested. A characteristic memory B-cell response is defined as a mobilization of plasmablasts (PBs) 7 days after booster vaccination into the peripheral blood (Odendahl et al., Blood 105: 1614-21, 2005). Preliminary results obtained from patients that received the donor derived B-cells demonstrated significant mobilization of PBs in some of the patients after re-vaccination with a pentavalent vaccine (Pentavac®), suggestive for a memory B-cell response. A detailed analysis of the vaccination data from the study patients in comparison to a control group of transplanted patients without B-cell transfer after routine re-vaccination will be presented. Furthermore, the analysis of immunoglobulin VH-sequences by next-generation-sequencing in memory B-cell populations of two individual patients after B-cell transfer revealed that at least 50% of all memory B-cell clones identified in the recipient are unambiguously contained within the memory B-cell population of the infused B-cell product.In summary, our data show that the manufacturing of a B-cell product from the original stem cell donor under GMP conditions without significant T-cell contamination is feasible. The transfer of donor-derived B-cells into patients after alloSCT is safe with regard to EBV reactivation and induction of acute or chronic GvHD. Future clinical trials will evaluate whether the adoptive transfer of memory B-cells from the donor can significantly reduce the frequency of infections after alloSCT.(Supported by the Deutsche Forschungsgemeinschaft through SFB 643 "Strategies of cellular immune intervention") DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-155 influences B-cell function through PU.1 in rheumatoid arthritis.

MicroRNA-155 (miR-155) is an important regulator of B cells in mice. B cells have a critical role in the pathogenesis of rheumatoid arthritis (RA). Here we show that miR-155 is highly expressed in peripheral blood B cells from RA patients compared with healthy individuals, particularly in the IgD-CD27- memory B-cell population in ACPA+ RA. MiR-155 is highly expressed in RA B cells from patients with synovial tissue containing ectopic germinal centres compared with diffuse synovial tissue. MiR-155 expression is associated reciprocally with lower expression of PU.1 at B-cell level in the synovial compartment. Stimulation of healthy donor B cells with CD40L, anti-IgM, IL-21, CpG, IFN-α, IL-6 or BAFF induces miR-155 and decreases PU.1 expression. Finally, inhibition of endogenous miR-155 in B cells of RA patients restores PU.1 and reduces production of antibodies. Our data suggest that miR-155 is an important regulator of B-cell activation in RA.