Number Of Antibody-secreting Cells Research Articles

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma and respiratory reactions to cyclooxygenase-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody secreting cells (ASCs), plasma cells (PCs) and plasmablasts, in their polyp tissue, and nasal polyp IgE levels correlate with disease severity and recurrence of nasal polyposis. ObjectiveWe sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of nasal polyp ASCs from patients with AERD and CRSwNP. MethodsNasal polyp tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. Nasal polyp cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with/without IL-5 and analyzed by bulk RNA-sequencing. ResultsAERD polyp tissue contained significantly more ASCs and had increased ASC expression of IL-5Rα compared to CRSwNP. AERD ASCs expressed higher protein levels of B cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. AERD ASCs also expressed more IL5RA, IGHE, and cell cycle and proliferation-related transcripts (CCND2, MKI67, CDC25A and CDC25B) compared to CRSwNP. Stimulation of AERD PCs with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from CRSwNP induced few transcriptomic changes. ConclusionNasal polyp tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation compared to those from aspirin-tolerant CRSwNP patients.

Nasal polyp antibody secreting cells have features of long-lived plasma cells and frequently produce autoantibodies and IgE

Nasal polyps (NP) in Chronic rhinosinusitis with NP (CRSwNP) have an increased number of antibody secreting cells (ASCs) that produce autoantibodies against DNA and other self-antigens. Elevated tissue anti-dsDNA IgG levels are prospectively associated with recurrent NP after ESS, but the molecular characteristics of the source ASCs are unknown.

The distribution of SIgA and IgG antibody-secreting cells in the large intestine of Bactrian camels (Camelus bactrianus).

Mucosal immunoglobulin comprises mainly secretory immunoglobulin A (SIgA) which mainly participates in the intestinal mucosal pathogenspecific immune response. Immunoglobulin G (IgG) is another common immunoglobulin. Bactrian camels' gastrointestinal mucosal tissue has a special mucosal immune system. However, the distribution characteristics of these two antibody-secreting cells (ASCs) in Bactrian camel's large intestine mucosal immunity system remain largely unknown. This study was aimed to describe the distribution characteristics and density of SIgA and IgG ASCs in the mucosal immunity tissues of Bactrian camel large intestine. Tissue samples were collected from different parts of the large intestines of 10 healthy adult Chinese Alashan Bactrian camels. Immunohistochemistry technology was used to determine the distribution of SIgA and IgG ASCs in the large intestine samples and the image-Pro Plus 6.0 was employed to calculate their densities. SIgA and IgG ASCs were distributed in lamina propria of the large intestine mucosa with some ASCs aggregating around the intestinal glands. The SIgA density increased from ileocecal orifice to the caecum and decreased from the colon to the rectum. The largest number of SIgA ASCs was observed in the caecum, followed by anterior colonic segments, ileocecal orifice, posterior colonic segments, and rectum, and the number of SIgA ASCs in the caecum was significantly larger than that in other four positions (p < 0.05). Similarly, the number of IgG ASCs was also the largest in the caecum, which was significantly higher than that in ileocecal orifice, anterior, posterior colonic segments, and rectum (p < 0.05). Our findings suggest that SIgA and IgG ASCs are mainly distributed in intestinal mucosal immunity effector sites. These distribution characteristics provide evidence to support that SIgA and IgG supply effective protection and maintain homeostasis in the large intestinal mucosa.

Altering the mRNA-1273 dosing interval impacts the kinetics, quality, and magnitude of immune responses in mice.

For a vaccine to achieve durable immunity and optimal efficacy, many require a multi-dose primary vaccination schedule that acts to first "prime" naive immune systems and then "boost" initial immune responses by repeated immunizations (ie, prime-boost regimens). In the context of the global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 2-dose primary vaccination regimens were often selected with short intervals between doses to provide rapid protection while still inducing robust immunity. However, emerging post-authorization evidence has suggested that longer intervals between doses 1 and 2 for SARS-CoV-2 vaccines may positively impact robustness and durability of immune responses. Here, the dosing interval for mRNA-1273, a messenger RNA based SARS-CoV-2 vaccine administered on a 2-dose primary schedule with 4 weeks between doses, was evaluated in mice by varying the dose interval between 1 and 8 weeks and examining immune responses through 24 weeks after dose 2. A dosing interval of 6 to 8 weeks generated the highest level of antigen-specific serum immunoglobulin G binding antibody titers. Differences in binding antibody titers between mRNA-1273 1 µg and 10 µg decreased over time for dosing intervals of ≥4 weeks, suggesting a potential dose-sparing effect. Longer intervals (≥4 weeks) also increased antibody-dependent cellular cytotoxicity activity and numbers of antibody-secreting cells (including long-lived plasma cells) after the second dose. An interval of 6 to 8 weeks elicited the strongest CD8+ T-cell responses, while an interval of 3 weeks elicited the strongest CD4+ T-cell response. Overall, these results suggest that in a non-pandemic setting, a longer interval (≥6 weeks) between the doses of the primary series for mRNA-1273 may induce more durable immune responses.

Prolactin promotes proliferation of germinal center B cells, formation of plasma cells, and elevated levels of IgG3 anti-dsDNA autoantibodies.

Systemic lupus erythematosus (SLE) mainly affects females at reproductive age, which has been associated with hormones, such as prolactin (PRL). Different studies suggest that PRL exacerbates the clinical manifestations of SLE both in patients and in mouse models (e.g., the MRL/lpr strain), increasing the production of autoantibodies, which can be deposited as immune complexes and trigger inflammation and damage to different tissues. The objective of this work was to explore the potential mechanisms by which PRL increases the concentration of self-reactive antibodies in the MRL/lpr SLE model. To this end, we determined the role of PRL on the activation and proliferation of germinal center B cells (B-GCs) and their differentiation into antibody-secreting cells (ASCs). We show that the absolute number and percentage of B-GCs were significantly increased by PRL in vivo or upon in vitro treatment with anti-IgM and anti-CD40 antibodies and PRL. The augmented B-GC numbers correlated with enhanced proliferation, but we did not observe enhanced expression of CD80 and CD86 activation markers or the BCL6 transcription factor, arguing against a more effective differentiation. Nevertheless, we observed enhanced phosphorylation of STAT1, secretion of IL-6, expression of IRF4, numbers of ASCs, and levels of IgG3 antibodies directed against dsDNA. Altogether, these results support the hypothesis that a PRL-mediated expansion of B-GCs yields more self-reactive ASCs, potentially explaining the pathogenic immune complexes that steadily lead to tissue damage during SLE.

Impaired Circulating Antibody-Secreting Cells Generation Predicts the Dismal Outcome in the Elderly Septic Shock Patients.

PurposeSepsis is a condition that derives from a dysregulated host response to infection. Although B lymphocytes play a pivotal role in immune response, little is known about status of their terminally differentiated cells, antibody-secreting cells (ASCs) during immunosuppressive phase of sepsis, especially in elderly patients. Our aim was to extensively characterize the immune functions of ASCs in elderly septic patients.Patients and MethodsClinical and laboratory data were collected on days 1, 3, and 7 of hospitalization. Circulating ASCs were evaluated by flow cytometry from fresh whole blood in elderly septic patients at the onset of disease. RNA sequencing analyzed ASCs gene expression profile. Receiver operating characteristic (ROC) curve analysis and logistic regression predicted the survival rate of 28-day mortality.ResultsA total of 103 septic patients were enrolled. The number and proportion of ASCs among total lymphocytes dramatically increased in septic patients, and RNA sequencing analysis showed that ASCs from septic patients exhibited a different gene expression profile. Furthermore, we found these ASCs could promote the function of T cells. Logistic regression analysis showed ASCs population was an independent outcome predictor in septic shock patients.ConclusionOur study revealed the complex nature of immune disorders in sepsis and identified circulating ASCs population as a useful biomarker for predicting mortality in elderly septic patients, which provided a novel clue to combat this severe disease.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Antibody in Lymphocyte Supernatant (ALS) responses after oral vaccination with live Shigella sonnei vaccine candidates WRSs2 and WRSs3 and correlation with serum antibodies, ASCs, fecal IgA and shedding.

The levels of antigen-specific Antibodies in Lymphocyte Supernatant (ALS) using an ELISA are being used to evaluate mucosal immune responses as an alternate to measuring the number of Antibody Secreting Cells (ASCs) using an ELISpot assay. A recently completed trial of two novel S. sonnei live oral vaccine candidates WRSs2 and WRSs3 established that both candidates were safe, well tolerated and immunogenic in a vaccine dose-dependent manner. Previously, mucosal immune responses were measured by assaying IgA- and IgG-ASC in peripheral blood mononuclear cells (PBMCs). In this report, the magnitude of the S. sonnei antigen-specific IgA- and IgG-ALS responses was measured and correlated with previously described ASCs, serum antibodies, fecal IgA and vaccine shedding. Overall, the magnitude of S. sonnei anti-Invaplex50 ALS was higher than that of LPS or IpaB, and both vaccines demonstrated a more robust IgA-ALS response than IgG; however, compared to WRSs3, the magnitude and percentage of responders were higher among WRSs2 recipients for IgA- or IgG-ALS. All WRSs2 vaccinees at the two highest doses responded for LPS and Invaplex50-specific IgA-ALS and 63–100% for WRSs3 vaccinees responded. Regardless of the vaccine candidate, vaccine dose or detecting antigen, the kinetics of ALS responses were similar peaking on days 7 to 9 and returning to baseline by day 14. The ALS responses were vaccine-specific since no responses were detected among placebo recipients at any time. A strong correlation and agreement between responders/non-responders were noted between ALS and other mucosal (ASC and fecal IgA) and systemic (serum antibody) immune responses. These data indicate that the ALS assay can be a useful tool to evaluate mucosal responses to oral vaccination, an observation noted with trials of other bacterial diarrheal pathogens. Furthermore, this data will guide the list of immunological assays to be conducted for efficacy trials in different populations. It is hoped that an antigen-specific-ALS titer may be a key mucosal correlate of protection, a feature not currently available for any Shigella vaccines candidates. https://clinicaltrials.gov/show/NCT01336699.

Preclinical to clinical translation of cenerimod, a novel S1P1 receptor modulator, in systemic lupus erythematosus

Objectives:SLE is an autoimmune disease characterised by aberrant lymphocyte activation and autoantibody production. This study provides an in-depth preclinical and clinical characterisation of the treatment effect of cenerimod, a sphingosine-1-phosphate...

Detection of allergen-specific antibody-secreting cells in dogs by ELISPOT

Current laboratory tests are unable to distinguish healthy from allergic dogs. Unlike serum antibody responses, circulating antibody-secreting cells (ASC) are temporarily induced after each contact with the antigen. These ASC can be identified using ELISPOT and the observation of allergen-specific ASC might correlate with the causative allergens in dogs with an allergic dermatitis. In this study, blood was sampled from six privately-owned allergic dogs and six non-allergic laboratory beagles to determine the frequency of circulating allergen-specific ASC for common allergens. Blood IgE+, IgA + and IgG + cells were magnetically isolated to determine the number of allergen-specific ASC with ELISPOT for Dermatophagoides farinae, Dermatophagoides pteronyssinus, Alternaria alternata, birch, timothy grass, wheat, cow’s milk, bovine, chicken and lamb meat. For IgA and IgG, allergen-specific spots were observed, however for IgE, no spots were detected for any of the allergens. ELISPOT could not differentiate allergic from non-allergic dogs. When the responses to the different allergens were compared, more IgA ASC for D. pteronyssinus were observed compared to some of the other allergens which was statistically significant for the non-allergic dogs and approached significance in the allergic dogs. These findings indicate that ELISPOT can be used to identify circulating allergen-specific IgA- and IgG-secreting cells. The technique did however not detect allergen-specific IgE ASC and was unable to distinguish allergic from non-allergic dogs. Only a small number of studies have studied allergen-specific IgA in dogs. The finding that dogs have higher numbers of D. pteronyssinus-specific IgA ASC points out that apart from IgE and IgG, it might be interesting to include IgA measurements for certain allergens to analyse the complete spectrum of both the protective and pro-allergic antibody responses.

Association of Antibody-Secreting Cells With Allograft Rejection After Renal Transplantation

BackgroundDonor-specific antibodies (DSA) are measured at the time of transplantation to predict renal allograft outcome, but pretransplantation DSA are sometimes not adequate to predict antibody-mediated rejection (AMR). We previously developed a flow cytometric assay that could measure the number of antibody-secreting cells (ASCs) instead of DSA. Here, we evaluated the performance of the flow cytometric ASC assay in predicting renal allograft rejection and compared it with that of the enzyme-linked immunospot (ELISpot) assay. MethodsWe enrolled 25 patients who received renal transplantation between May 2017 and August 2017 at Seoul National University Hospital. Mononuclear cells separated from patient peripheral blood obtained on pretransplantation day 1 were incubated with CpG 2006, human CD40L, interleukin-21, and donor or autologous lymphocyte lysates for 6 days. Flow cytometry and ELISpot assay (Mabtech) were performed to measure the ASCs and their association with graft rejection. ResultsThe number of donor-reactive ASCs, as measured by flow cytometry, was higher in the rejection group than in the nonrejection group (mean ± standard deviation [SD], 3688.9 ± 3875.3 vs 257.9 ± 297.3, P = .014), and no significant difference was observed in the ELISpot assay. Multiple linear regression analysis showed that the number of donor-reactive ASCs measured by flow cytometry was independently and negatively associated with the number of rejection-free days (P = .008, partial R2 = 0.368, adjusted R2 = 0.496). ConclusionAfter renal transplantation, an increased number of donor-reactive ASCs, as measured by flow cytometry, was associated with allograft rejection. This may be useful to predict renal allograft rejection by measuring the sensitization status of patients who are awaiting renal transplantation.

Rainbow trout mount a robust specific immune response upon anal administration of thymus-independent antigens

Despite the strong demand for orally-delivered fish vaccines and the deficient response of those currently available in the market, little is known about how teleost B cells differentiate to antibody secreting cells (ASCs) in response to antigens delivered to the intestinal mucosa. To fill this gap, in the current study, we have studied the dynamics of B cell differentiation in spleen and kidney of rainbow trout (Oncorhynchus mykiss) anally immunized with antigens catalogued in mammals as thymus dependent (TD) or thymus-independent (TI). Our results show that, in the absence of additional adjuvants, rainbow trout preferentially responded to a model TI antigen such as TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The anal administration of TNP-LPS elicited TNP-specific serum antibodies, and a significant increase in the number of total and TNP-specific ASCs in both spleen and kidney, being the kidney the site where most ASCs are found at later time points. In the spleen, a proliferative response of both IgM+ B and T cells was also clearly visible, while the proliferative response was weaker in the kidney. Finally, TNP-LPS also provoked a transcriptional regulation of some immune genes in the spleen and the intestine, including a decreased transcription of foxp3a and foxp3b in intestine that suggests a breach in tolerogenic responses in response to TI stimulation. These results contribute to a better understanding of how intestinal immunity is regulated in teleost and will aid in the future design of effective oral strategies for aquaculture.

Flavivirus-Mediating B Cell Differentiation Into Antibody-Secreting Cells in Humans Is Associated With the Activation of the Tryptophan Metabolism.

Patients infected with the Dengue virus (DENV) often present with a massive generation of DENV-specific antibody-secreting cells (ASCs) in the blood. In some cases, these ASCs represent more than 50% of the circulating B cells, a higher magnitude than those induced by other infections, vaccinations, and plasma cell lymphomas. However, it remains unclear how the DENV infection elicits this colossal response. To address this issue, we utilised an in vitro strategy to induce human PBMCs of healthy individuals incubated with DENV particles (DENV4 TVP/360) to differentiate into ASCs. As controls, PBMCs were incubated with a mitogen cocktail or supernatants of uninfected C6/36 cells (mock). The ASC phenotype and function were increasingly detected in the DENV and mitogen-cultured PBMCs as compared to mock-treated cells. In contrast to the in vivo condition, secreted IgG derived from the PBMC-DENV culture was not DENV-specific. Lower ASC numbers were observed when inactivated viral particles or purified B cells were added to the cultures. The physical contact was essential between B cells and the remaining PBMCs for the DENV-mediated ASC response. Considering the evidence for the activation of the tryptophan metabolism detected in the serum of Dengue patients, we assessed its relevance in the DENV-mediated ASC differentiation. For this, tryptophan and its respective metabolites were quantified in the supernatants of cell cultures through mass spectrophotometry. Tryptophan depletion and kynurenine accumulation were found in the supernatants of PBMC-DENV cultures, which presented enhanced detection of indoleamine 2,3-dioxygenase 1 and 2 transcripts as compared to controls. In PBMC-DENV cultures, tryptophan and kynurenine levels strongly correlated to the respective ASC numbers, while the kynurenine levels were directly proportional to the secreted IgG titers. Contrastingly, PBMCs incubated with Zika or attenuated Yellow Fever viruses showed no correlation between their kynurenine concentrations and ASC numbers. Therefore, our data revealed the existence of distinct pathways for the DENV-mediated ASC differentiation and suggest the involvement of the tryptophan metabolism in this cellular process triggered by flavivirus infections.

Antibody responses to merozoite antigens after natural Plasmodium falciparum infection: kinetics and longevity in absence of re-exposure

BackgroundAntibodies against merozoite antigens are key components of malaria immunity. The naturally acquired antibody response to these antigens is generally considered short-lived; however, the underlying mechanisms remain unclear. Prospective studies of travellers with different levels of prior exposure, returning to malaria-free countries with Plasmodium infection, offer a unique opportunity to investigate the kinetics and composition of the antibody response after natural infection.MethodsAdults diagnosed with P. falciparum malaria in Stockholm, Sweden (20 likely malaria naïve and 41 with repeated previous exposure during residency in sub-Saharan Africa) were sampled at diagnosis and 10 days and 1, 3, 6, and 12 months after treatment. Total and subclass-specific IgG responses to P. falciparum merozoite antigens (AMA-1, MSP-119, MSP-2, MSP-3, and RH5) and tetanus toxoid were measured by multiplex bead-based immunoassays and ELISA. Mathematical modelling was used to estimate the exposure-dependent longevity of antibodies and antibody-secreting cells (ASCs).ResultsA majority of individuals mounted detectable antibody responses towards P. falciparum merozoite antigens at diagnosis; however, the magnitude and breadth were greater in individuals with prior exposure. In both exposure groups, antibody levels increased rapidly for 2 weeks and decayed thereafter. Previously exposed individuals maintained two- to ninefold greater antibody levels throughout the 1-year follow-up. The half-lives of malaria-specific long-lived ASCs, responsible for maintaining circulating antibodies, ranged from 1.8 to 3.7 years for merozoite antigens and were considerably short compared to tetanus-specific ASCs. Primary infected individuals did acquire a long-lived component of the antibody response; however, the total proportion of long-lived ASCs generated in response to infection was estimated not to exceed 10%. In contrast, previously exposed individuals maintained substantially larger numbers of long-lived ASCs (10–56% of total ASCs).ConclusionThe short-lived nature of the naturally acquired antibody response, to all tested merozoite antigens, following primary malaria infection can be attributed to a combination of a poor acquisition and short half-life of long-lived ASCs. Greater longevity is acquired with repeated infections and can be explained by the maintenance of larger numbers of long-lived ASCs. These insights advance our understanding of naturally acquired malaria immunity and will guide strategies for further development of both vaccines and serological tools to monitor exposure.

Elimination of factor VIII‐specific B cells by immunotoxins composed of a single factor VIII domain fused to Pseudomonas exotoxin A

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA exvivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested exvivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors.

CXCR4-CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice.

Antibody-secreting cells (ASCs), including short-lived plasmablasts and long-lived memory plasma cells (LLPCs), contribute to autoimmune pathology. ASCs, particularly LLPCs, refractory to conventional immunosuppressive drugs pose a major therapeutic challenge. Since stromal cells expressing C-X-C motif chemokine-12 (CXCL12) organize survival niches for LLPCs in the bone marrow, we investigated the effects of CXCL12 and its ligand CXCR4 (C-X-C chemokine receptor 4) on ASCs in lupus mice (NZB/W). Fewer adoptively transferred splenic ASCs were retrieved from the bone marrow of recipient immunodeficient Rag1-/- mice when the ASCs were pretreated with the CXCR4 blocker AMD3100. CXCR4 blockade also significantly reduced anti-OVA ASCs in the bone marrow after secondary immunization with OVA. In this study, AMD3100 efficiently depleted ASCs, including LLPCs. After two weeks, it decreased the total number of ASCs in the spleen and bone marrow by more than 60%. Combination with the proteasome inhibitor bortezomib significantly enhanced the depletion effect of AMD3100. Continuous long-term (five-month) CXCR4 blockade with AMD3100 after effective short-term LLPCs depletion kept the number of LLPCs in the bone marrow low, delayed proteinuria development and prolonged the survival of the mice. These findings identify the CXCR4-CXCL12 axis as a potential therapeutic target likely due to its importance for ASC homing and survival.

Plasma cell and serum antibody responses to influenza vaccine in preterm and full-term infants.

Preterm (PT) infants are at greater risk for severe influenza infection and experience decrements in long-term antibody responses to vaccines. This may related to defects in antibody secreting cell (ASC) generation. To investigate the relationships among the frequencies of influenza-specific antibody secreting cells, ASC numbers and subsets, and antibody responses to influenza vaccines (IV) among PT and full-term (FT) infants. We enrolled 11 former PT (≤32weeks' gestation, ≤1500 g' birth weight) and 11FT infants, 6-17months of age, receiving their first influenza immunizations. Infants received two doses of inactivated trivalent (T)IV or quadrivalent (Q)IV during the 2012-2013 and 2013-2014 influenza seasons, respectively, at 0 and 28days, and blood was drawn at 0, 10, 35, and 56days and 9months. Vaccine-specific antibody was measured by hemagglutination inhibition (HAI) at 0 and 56days and 9months, vaccine-specific ASC numbers by enzyme linked immunospot (ELISPOT) at 10 and 35days, and ASC subsets by flow cytometry at 0, 10 and 35days. PT infants had post-vaccine HAI titers to all 4 vaccine strains at least equal to FT infants at 56days and 9months after beginning immunization. Influenza-specific ASC ELISPOT responses at 35days were higher among PT than FT infants (median 100 v. 30 per 106 PBMC, p=0.04). ASC numbers at 35days were positively correlated with serum HAI titers at 56days (ρ=0.50-0.80). There were no statistical differences between PT and FT infants in the frequency of five ASC subsets and no specific ASC subset correlated with durability of serum antibody titers. Influenza-specific ASC numbers in both FT and PT infants correlated with peak antibody titers, but ASC subsets did not correlate with durability of antibody response.

The LFA1 ligand, CD54, is not required for the differentiation of TFH cells or germinal center formation

Abstract In germinal centers (GC), B cells undergo cellular proliferation and affinity maturation, and ultimately differentiate into either high-affinity antibody-secreting cells (ASCs) or memory B cells. GC B cells present antigen to T follicular helper (TFH) cells in order to elicit survival and proliferation signals. The integrin, Lymphocyte Function-Associated Antigen 1 (LFA1), is required for this process, as mice lacking LFA1 or treated with LFA1 blocking antibodies fail to form GCs or TFH cells. We hypothesized that the counter-receptor for LFA1, ICAM-1 or CD54, would be similarly required for GC formation and TFH differentiation. To test this hypothesis, we infected CD54-deficient (KO) and wild-type (WT) mice with influenza virus (PR8) and enumerated ASCs and GC B cells on days 7, 9, and 14. We found that KO mice had more GC B cells than WT mice on days 9 and 14, but observed no difference in the number of ASCs or TFH cells. To determine if this result was intrinsic to the B cell lineage, we generated 50:50 (KO:WT) bone marrow chimeras. Following reconstitution, we infected the mice with PR8 and assayed donor-specific GC B cells and ASCs on days 9 and 14. On day 9, we found that GC B cells were highly enriched for KO cells, whereas the ASCs were highly enriched for WT cells. In contrast, TFH cells were represented equally in WT and KO populations. On day 14, we found that GC B cells were still highly enriched for KO cells, whereas there was no significant difference between WT and KO ASCs at this time. These results lead us to the surprising conclusion that, although the loss of LFA1 leads to the failure of TFH and GC responses, the loss of CD54 does not affect the TFH compartment and actually improves GC B cell responses.

MiR-148a controls plasma cell differentiation and survival

Abstract microRNAs (miRNAs) are critical regulators of central and Ag-driven B cell development. However, it is unknown whether miRNAs regulate the differentiation of B cells into plasma cells (PCs) and control the survival of long-lived PCs. We recently showed that miR-148a, the most abundantly expressed miRNA in PCs, is upregulated in activated B cells and promotes in vitro plasmablast differentiation and survival by targeting Bach2, MiTF, PTEN and Bim. To investigate if miR-148a supports PC differentiation in vivo, we established a conditional B cell-specific miR-148a knock-out mouse. Non-immunized mice showed a significant decrease in serum Ig, antibody-secreting cells (ASC) and splenic PCs. 28d after boost, TNP-KLH-immunized 148a-KO mice had lower serum TNP-IgG and decreased numbers of splenic ASCs and bone marrow (BM) PCs. To ask whether miR-148a controls PC survival, we generated tamoxifen-inducible miR-148a deficient mice. Tamoxifen-mediated miR-148a deletion, 28 days after boost with TNP-KLH, resulted in reduced numbers of plasmablasts and IgG-ASCs in the spleen and of resting PCs and IgG-ASCs in BM. In parallel, increased numbers of resting PCs were found in the blood, indicating a possible egress from survival niches. These findings support the hypothesis that miR-148a controls the differentiation of B cells into plasmablasts as well as the survival of long-lived PCs. We will now identify the underlying mechanism by a biosystems approach utilizing transcriptome and proteome profiles of fresh follicular B cells and BM PCs. Proteome analysis was done in collaboration with the Warscheid lab at the University of Freiburg.