Antigenic Modulation Research Articles

Immunophenotypic Shifts in Primary Cutaneous γδ T-Cell Lymphoma Suggest Antigenic Modulation: A Study of Sequential Biopsy Specimens.

Primary cutaneous γδ T-cell lymphoma (PCGD TCL), an aggressive type of lymphoma, accounts for approximately 1% of all primary cutaneous lymphomas. We have occasionally observed changes in T-cell antigen expression (immunophenotypic [IP] shift) over time, a phenomenon that is considered rare in T-cell lymphoma including cutaneous T-cell lymphoma. Therefore, we assessed sequential biopsies of PCGD TCL for possible IP shifts of the lymphoma cells. We searched for cases of PCGD TCL with consecutive biopsies to perform a comprehensive immunohistochemical analysis of paired specimens. A median of 12 markers per case was tested. We evaluated the percentage of neoplastic lymphocytes and determined the differential expression of antigens (gain, loss, increase or decrease). We identified 9 patients with PCGD TCL with consecutive biopsies. All (100%) cases had IP shifts of at least 1 antigen, whereas overall 22 pairs of markers were shifted: gain of reactivity occurred in 7 (31.8%) and loss in 3 (13.6%); increased reactivity in 4 (18.2%) and decreased in 8 (36.4%). Molecular analysis of TCRγ showed identically sized monoclonal rearrangements between biopsy pairs in 4/4 (100%) patients. There was no correlation between IP shifts and the clinical appearance of lesions, histopathologic or cytologic features, or molecular rearrangements. IP shifts are common in PCGD TCL, occurring in all patients in this study and involving a variety of antigens. IP shifts do not seem to be linked to changes in the T-cell clone and are without obvious clinical or morphologic correlates. The occurrence of IP shifts in PCGD TCL suggests that antigen modulation may be involved in pathogenesis. IP shifts are somewhat frequent in T-cell lymphoma; however, it does not suggest a second neoplasm, and molecular studies can be used to determine clonal identity.

Presence of PSA antibodies in seminal plasma of infertile men.

Immunoinfertility due to antisperm antibodies and semen hyperviscosity are among major causes of male infertility. Although the modulation of prostate-specific antigen (PSA) has been investigated in prostate abnormalities, its role and the effect of its dysfunction in male fertility/infertility have not been extensively examined. The present study was conducted to examine the presence of PSA antibodies locally in the seminal plasma of men having immunoinfertility and semen hyperviscosity. Seminal plasma samples from immunoinfertile men (n=25), men with hyperviscous semen (n=25), and normal men (n=24) were collected and analyzed for immunoreactivity with PSA in ELISA and Western blot. In the immunoinfertile group, seminal plasma from 20% of men reacted positively with PSA. In the hyperviscous group, seminal plasma from 28% of men reacted positively with PSA. None (0%) of the seminal plasma from the normal group showed immunoreactivity to PSA. This is the first study ever to indicate the presence of PSA antibodies in semen of men having immunoinfertility or hyperviscosity. These findings may have clinical significance in the specific diagnosis and treatment of infertility in men and contraceptive vaccine development.

MicroRNA Expression Profile of Spleen CD4+ T Cells from Thioredoxin Primed Lung Transplanted Rats

We recently reported that priming donor lungs with thioredoxin-1 (Trx) prolongs allograft survival associated with inhibition of host spleen T cell response to donor antigen and cytokine modulation in a rat model of lung transplantation. The molecular events associated with Trx primed allograft survival and modulation of spleen T cell responses remained to be determined. Since micro-RNAs (miRNAs) play a critical role in the regulation of physiological and immunological responses via modulation of target gene and protein expression, we examined the impact of Trx-priming of donor lungs on miRNA expression profile of spleen CD4+T cells isolated from transplanted rats. Donor lungs were primed with Trx for 4 h prior to transplantation. Four days post transplantation recipient spleen CD4+T cells were isolated to identify miRNA profile by microarray analysis using 498 detectable rat miRNAs. Expression of the six mRNA (miR-342-3p, miR-128, miR-30b, miR-92a, miR-25, miR-146a) were significantly up-regulated whereas twelve miRNA (mir-301b, miR-33, miR-210, miR-206, miR-494, miR-29b, miR-196c, miR-362, miR-466b-2, miR-758, miR-466c, miR-101a) were significantly down-regulated in Trx-primed donor lung recipient’s spleen CD4+T cells. These sets of miRNAs are associated with modulation of multiple pathways that appeared to play critical role in the regulation of inflammatory responses and T cell proliferation after transplant injury and allograft dysfunction. The impact of Trx-priming on selective miRNA expression with protective role may provide novel therapeutic strategy to limit or prevent injury and promote allograft survival.

Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice

Red blood cell (RBC) alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of Rh immune globulin to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild-type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγRs), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild-type and single-knockout (KO) mice lacking FcγRs or C3, double-KO mice lacking both FcγRs and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild-type and single-KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double-KO mice despite treatment with immunoprophylaxis. Further, in vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double-KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγRs and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.

C3 Modulates RBC Antigen to Negatively Regulate Antibody Response

Background:Red blood cell (RBC) transfusion can result in the development of alloantibodies that can make it difficult to find compatible RBCs for future transfusions and increase the risk of hemolytic transfusion reactions. Despite the consequences of RBC alloimmunization, the factors that regulate this process remain relatively unknown. Recent studies suggest that complement deposition on an antigen surface can significantly enhance the immune response to foreign antigen. As many anti-RBC alloantibodies fix complement and RBCs otherwise lack known adjuvants, early antibody-mediated complement deposition may serve as a key regulator that enhances antibody production. To test this, we employed the KEL RBC model system, which employs RBCs that transgenically express the human KEL antigen specifically on RBCs (KEL RBCs). Using this system, we examined the immunological consequence of KEL RBC exposure following transfusion into C57BL/6 wild-type (WT) or complement component 3 (C3) knockout (KO) recipients.Methods: KEL RBCs were transfused into WT or C3 KO recipients, followed by serum collection on days 3, 5, 7, 14, and 21 post-transfusion. Antibody development in WT or C3 KO recipients was examined by flow crossmatch, where serum was incubated with KEL RBCs followed by antibody detection with fluorescently-tagged secondary anti-IgM and anti-IgG antibodies using flow cytometry. To determine the impact of complement deposition on the level of detectable antigen on the RBC surface, RBCs were labeled with the lipophilic dye, DiI, prior to transfusion and then sampled 1, 2, 3, 5, 7 and 9 days post-transfusion. The level of detectable KEL antigen, complement deposition, KEL RBC survival and antibody bound to the RBC surface was measured by flow cytometry. To examine the effect of complement deposition on the level of KEL protein in the RBC membrane post-transfusion, RBCs stroma was isolated at various time points post transfusion, followed by western blot analysis for the KEL protein.Results: While KEL RBCs induced robust anti-KEL antibody formation and C3 deposition in WT recipients, similar exposure to KEL RBCs in C3 KO recipients actually resulted in an unexpected increase in IgM and IgG anti-KEL antibodies when compared to WT recipients. To determine the consequence of C3 deposition, we examined the potential impact of antibody engagement and complement fixation on KEL antigen levels. Consistent with a potential role for complement in directly impacting KEL antigen availability to the immune system, KEL RBCs transferred into WT recipients experienced a decrease in the level of detectable KEL antigen over time that paralleled the development of anti-KEL antibodies and C3 deposition. In contrast, C3 KO recipients failed to experience the same degree of KEL antigen reduction despite the development of significant anti-KEL antibodies over this same time period. Western blot analysis of RBCs post-transfusion revealed that loss of detectable KEL antigen on the RBC surface paralleled a complete lack of detectable KEL antigen in RBC membranes, indicating that C3 may actually facilitate the removal of KEL from the RBC surface.Conclusion: These results suggest an unexpected role for C3 in negatively regulating antibody responses following RBC transfusion. The impact of C3 on the developing alloantibody response strongly suggests that C3-mediated loss of antigen over time likely reduces antigen availability to the immune system, thereby facilitating the inhibition of antibody production over time. These results not only provide novel insight into potential impact of antigen modulation on the development of an immune response to a RBC alloantigen, but also suggest a completely unexpected role for complement in negatively regulating alloantibody production. In doing so, these results suggest that unique differences in complement activity and overall activation following RBC alloantigen exposure between individuals may represent a previously unrecognized factor that influences alloantibody formation following RBC transfusion. DisclosuresNo relevant conflicts of interest to declare.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

Evaluation of new markers for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia: CD73 and CD86 are the most relevant new markers to increase the efficacy of MRD 2016; 00B: 000-000.

Multiparametric flow cytometry (MFC) is a popular technique for minimal residual disease (MRD) analysis. However, its applicability is still limited to 90% of B-cell precursor acute lymphoblastic leukemia (BCPALL) due to two major issues, i.e. a proportion of cases do not express adequate leukemia associated immunophenotype (LAIPs) with currently used markers and drug-induced antigen modulation. Hence, the incorporation of additional reliable markers is required for the further improvement of MFC-based MRD evaluation. We studied the utility of new markers in improvising MFC-based MRD detection in BCPALL. Expression-patterns of six new markers, i.e. CD24, CD44, CD72, CD73, CD86, and CD200 were studied in leukemic-blasts from ninety childhood BCPALL patients and in hematogones from 20 uninvolved staging bone marrow (BM) and ten postinduction non-BCPALL BM samples using eight-color MFC. The utility of these new markers in the day 35 postinduction MRD evaluation was determined. Frequencies of LAIPs of CD73, CD86, CD72, CD44, CD200, and CD24 in diagnostic samples were 76.7, 56.7, 55.6, 50, 28.9, and 20%, respectively. Differential expression of all new markers was highly significant (P<0.01) between early (CD10+ CD19+ CD34+) hematogones, late (CD10+ CD19+ CD34-) hematogones and BCPALL blasts except between early hematogones and BCPALL blasts for CD200 (P=0.1). In MRD-positive samples, CD73 showed the maximum (83%) frequency of LAIP and CD86 showed the highest (100%) stability of aberrant expression. Inclusion of CD73 and CD86 increased the applicability of MFC-MRD assay to 98.9% MRD samples. CD73 and CD86 are the most relevant markers to incorporate in the routine MRD evaluation of BCPALL. © 2016 International Clinical Cytometry Society.

Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.

Clinical characteristics and frequency of TLR4 polymorphisms in Brazilian patients with ankylosing spondylitis

ObjectivesInnate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram-negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll-like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. MethodsA cross-sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA-B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p-values less than 0.05 considered significant. ResultsMean age and disease duration were 43.1±12.7 and 16.6±9.2 years, respectively. The sample was predominantly male (71%) and non-Caucasian (52%). A total of 66% of the group of patients were positive for HLA-B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. ConclusionsTLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms.

Características clínicas e frequência de polimorfismos em TLR4 em pacientes brasileiros com espondilite anquilosante

ObjectivesInnate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram‐negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll‐like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. MethodsA cross‐sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA‐B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p‐values less than 0.05 considered significant. ResultsMean age and disease duration were 43.1±12.7 and 16.6±9.2 years, respectively. The sample was predominantly male (71%) and non‐Caucasian (52%). A total of 66% of the group of patients were positive for HLA‐B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. ConclusionsTLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms.

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle.

Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co-expression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.

Hemolytic Disease of the Fetus and Newborn: Modern Practice and Future Investigations.

Red blood cell (RBC) sensitization occurs in some women in response to exposure to paternally derived RBC antigens during pregnancy or to nonself antigens on transfused RBCs during their lifetime. Once sensitized, future pregnancies may be at risk for hemolytic disease of the fetus and newborn. Although great strides have been made over the past few decades in terms of identifying blood group antigens and in predicting fetal anemia through the use of noninvasive monitoring, many questions remain in terms of understanding RBC alloimmunization risk factors, preventative therapies, and treatment strategies. At the present time, there is room for improvement in these areas in both developed and developing countries. Evidence-based, universal guidelines describing recommended RBC antigen matching transfusion strategies for girls or women, before pregnancy or during intrauterine transfusions, would be welcomed. A better understanding of the mechanism(s) of action of Rh immunoglobulin, first introduced more than half of a century ago and one of the most successful immunoprophylaxis therapies in existence today, would also be a large step forward. For example, answers to questions of the role(s) that fetal RBC clearance, antigen masking, antigen modulation, and immune suppression play in the effectiveness of Rh immunoglobulin may help to guide the development of novel preventative therapies during pregnancy for immunization to RhD and non-RhD antigens. Furthermore, a better understanding of the importance of anti-RhD or other alloantibody glycosylation patterns may be beneficial not only in developing such novel immunoprophylaxis therapies but also in predicting the clinical significance of existing maternal alloantibodies. One other area of need includes the development of therapies beyond intrauterine transfusions to mitigate the dangers of maternal alloantibodies to developing fetuses. We challenge physicians, scientists, and funding agencies to prioritize studies of RBC alloimmunization and hemolytic disease of the fetus and newborn and to invest in the children of our future.

Plasmodium falciparum Secretome in Erythrocyte and Beyond

Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies.

Mast Cell-Derived Exosomes Promote Th2 Cell Differentiation via OX40L-OX40 Ligation.

Exosomes are nanovesicles released by different cell types, such as dendritic cells (DCs), mast cells (MCs), and tumor cells. Exosomes of different origin play a role in antigen presentation and modulation of immune response to infectious disease. In this study, we demonstrate that mast cells and CD4+ T cells colocated in peritoneal lymph nodes from BALB/c mouse. Further, bone marrow-derived mast cells (BMMCs) constitutively release exosomes, which express CD63 and OX40L. BMMC-exosomes partially promoted the proliferation of CD4+ T cells. BMMC-exosomes significantly enhanced the differentiation of naive CD4+ T cells to Th2 cells in a surface contact method, and this ability was partly inhibited by the addition of anti-OX40L Ab. In conclusion, BMMC-exosomes promoted the proliferation and differentiation of Th2 cells via ligation of OX40L and OX40 between exosomes and T cells. This method represents a novel mechanism, in addition to direct cell surface contacts, soluble mediators, and synapses, to regulate T cell actions by BMMC-exosomes.

Clearance of Incompatible RBC Is Compromised during Persistent Viral Infection

Background: While immune-mediated destruction of red blood cells (RBCs) can occur following incompatible transfusion, the development of a hemolytic transfusion reaction is not the invariable outcome of an incompatible transfusion. Although a variety of factors may impact the likelihood of RBC clearance following incompatible RBC transfusion, chronic infection-induced immune complex formation may inhibit Fc gamma receptor-mediated removal of antibody coated RBCs. To examine this possibility, we tested the effect of persistent viral infection on the clearance of RBCs following incompatible transfusion.Methods: For chronic viral infection, C57BL/6J recipients were injected intravenously (iv) with 2 x 106 pfu of LCMV clone-13 strain. Total IgG, anti-LCMV specific IgG or circulating immune complex formation was assessed by ELISA against anti-mouse Ig, LCMV lysate, or human C1q respectively. At 45 days post infection, LCMV or non-infected recipients were immunized with anti-Fy3 or injected with PBS. RBCs expressing the HEL, OVA and Duffy chimeric antigen (HOD), were labeled with chloromethylbenzamido 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI ), while WT RBC were labeled with fluorescently distinct 3,3'-dihexadecyloxacarbocyanine perchlorate (DiO) to facilitate detection of antigen positive RBCs post-transfusion. HOD RBCs were then transfused into anti-Fy3 immunized or non-immunized LCMV infected or non-infected recipients, followed by analysis for RBC clearance at 10 min, 1h, 2h, 4h, 24h and 48h following transfusion. In addition, RBCs were stained for potential bound antibody, complement and possible alterations in the HOD antigen followed by flow cytometric analysis.Results: Recipients chronically infected with LCMV clone-13 displayed high levels of total and LCMV specific antibody in the serum. Furthermore, large immune complexes could be observed following the development of chronic infection, which persisted 45 days following initial LCMV inoculation. While HOD RBCs transfused into control non-infected, yet anti-Fy3-immunized C57BL/6J recipients displayed rapid clearance following incompatible transfusion, clearance of HOD RBCs failed to occur at similar levels in LCMV clone-13 infected recipients 45 days following LCMV exposure despite immunization with anti-Fy3. Regardless of the LCMV infection state, binding of anti-Fy3 to HOD RBCs was unaffected. However, HOD antigen decreased following the initial transfusion into non-infected, anti-Fy3-immunized recipients, while significant alterations in the HOD antigen failed to occur in anti-Fy3 immunized recipients also infected with LCMV or any recipients lacking anti-Fy3.Conclusion: Differences in recipient clearance capacity following incompatible RBC exposure in non-infected mice and LCMV clone-13 infected mice suggests that rapid phagocytic removal of RBCs can be blocked by the production of immune complexes secondary to chronic LCMV infection. While antibody binding to HOD cells was not decreased in chronically infected mice, antigen modulation was almost completely abrogated, suggesting that engagement of unimpeded clearance pathways may be important for antigen modulation. Taken together, these data demonstrate that chronic viral infection can lead to significantly altered outcomes of RBC clearance and antigen modulation following incompatible transfusion. DisclosuresNo relevant conflicts of interest to declare.

Antigen Density Impacts RBC Survival and Antigen Modulation Following Incompatible RBC Transfusion

Background: While chronic transfusion therapy can signficantly reduce morbidity and mortality in patients with hemoglonbinopathies, transfusion is not without risk. Differences in red blood cell (RBC) antigen distributions between donors and recipients can result in the development of RBC alloantibodies, which can make it difficult, if not impossible, to find compatible blood. As a result, heavily alloimmunized individuals experience greater morbidity and mortality than those who do not possess RBC-induced alloantibodies. While incompatible RBC transfusion can result in fatal hemolytic transfusion reactions (HTRs), these adverse events are not the invariable outcome of incompatible transfusion. Although a variety of factors likely contribute to the likilihood of developing a HTR following incompatible RBC transfusion, variability in the density of the donor antigen may contribute to different outcomes. Using a novel model that utilizes RBC donors with different levels of the KEL antigen, we sought to directly examine the impact of antigen density on RBC clearance following incompatible RBC transfusion.Methods: Wild type C57BL/6 mice (WT) were passively immunized with anti-KEL serum, generated following immunization of WT recipients with KEL RBCS, 2 hours prior to incompatible KEL RBC transfusion. RBC collected from previously generated KEL transgenic mice that express high (KELhi), intermediate (KELmed), and low (KELlo) levels of human KEL glycoprotein antigen, were labeled with chloromethylbenzamido 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), while WT RBC were labeled with fluorescently distinct 3,3'-dihexadecyloxacarbocyanine perchlorate (DiO) to facilitate detection post-transfusion. Passively immunized recipient WT mice were transfused with labeled KEL RBC to WT RBC. Percent KEL RBC survival was measured using flow cytometric analysis at various time points post transfusion by comparing the DiI to DiO ratio. Bound antibody, complement and the KEL antigen were assessed by staining cells with anti-Ig, anti-C3 and anti-KEL followed by flow cytometric analysis.Results: Transfusion of RBCs expressing low, intermediate or high levels of the KEL antigen into non-immunized mice failed to result in any detectable alteration in RBC survival or KEL expression. However, transfusion of KELlo, KELmed and KELhi RBCs into anti-KEL immunized recipients resulted in differential clearance of each population, with KELhi experiencing the most clearance, followed by KELmed and KELlo. Similarly, the level of detectable C3 and bound antibody on KEL positive cells likewise correlated with the initial level of KEL antigen expression, with more C3 and IgG being detected on the surface of KELhi following transfusion into immunized recipients, than KELmed or KELlo. However, KELhi RBCs also experience the most significant alterations in KEL antigen levels following antibody engagement. Regardless of the KEL positive RBC transfused, each population developed a relatively rapid state of resistance to additional anti-KEL-mediated RBC removal.Conclusions: Our results indicate that lower antigen densities may be less efficient in not only facilitating rapid clearance of RBCs following incompatible transfusion, but also antibody-induced antigen modulation. While KELhi RBCs displayed the greatest degree of clearance and antigen modulation, each population developed resistance to antibody-mediate removal, suggesting that once RBC antigen levels fall below a certain threshold, clearance discontinues, regardless of the initial levels of antigen prior to incompatible transfusion. Taken together, these results suggest that antigen density may provide an additional feature of RBC transfusion that may impact the outcome of incompatible transfusion in heavily alloimmunized recipients. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic Modulation and Neuroprotective Effects of Olfactory Ensheathing Cells: a Promising Tool for Cell Therapy.

Olfactory Ensheathing Cells (OECs), exhibiting phenotypic characteristics of both astrocytes and Schwann Cells, show peculiar plasticity. In vitro, OECs promote axonal growth, while in vivo they promote remyelination of damaged axons. We decided to further investigate OEC potential for regeneration and functional recovery of the damaged Central Nervous System (CNS). To study OEC antigen modulation, OECs prepared from postnatal mouse olfactory bulbs were grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs) and analyzed for different neural specific markers. OEC functional characterizations were also achieved. Resistance of OECs to the neurotoxin 6-hydroxydopamine (6-OHDA) was analyzed by evaluating apoptosis and death. OEC neuroprotective properties were investigated by in vitro co-cultures or by addition of OEC conditioned medium to the neuroblastoma SH-SY5Y cells exposed to 6-OHDA. We observed: 1) modification of OEC morphology, reduced cell survival and marker expression in serum-free medium; 2) GF addition to serum-free medium condition influenced positively survival and restored basal marker expression; 3) no OEC apoptosis after a prolonged exposition to 6-OHDA; 4) a clear OEC neuroprotective tendency, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them potential clinical agents able to support injured CNS.

Endogenously produced TNF-α contributes to the expression of CXCL10/IP-10 in IFN-λ3-activated plasmacytoid dendritic cells.

The interplay between IFN-λs and dendritic cells is becoming increasingly relevant, particularly in light of their key role in inducing the antiviral state, including in hepatitis C virus infection. In this work, we have analyzed extensively how human plasmacytoid dendritic cells respond to IFN-λ3. We report that plasmacytoid dendritic cells incubated with IFN-λ3 prolong their survival; alter their expression pattern of surface HLA-DRα, CD123, CD86, and CD303; and time dependently produce IFN-α, CXCL10/IFN-γ-induced protein 10, and even modest quantities of TNF-α. Nevertheless, endogenously produced TNF-α, but not IFN-α, was found to be essential for driving the expression of CXCL10/IFN-γ-induced protein 10 in IFN-λ3-treated plasmacytoid dendritic cells, as revealed by neutralizing experiments by use of adalimumab, etanercept, and infliximab. We also observed that based on the kinetics and levels of IFN-α and CXCL10/IFN-γ-induced protein 10 produced by their IFN-λ3-treated plasmacytoid dendritic cells, healthy donors could be categorized into 2 and 3 groups, respectively. In particular, we identified a group of donors whose plasmacytoid dendritic cells produced modest quantities of CXCL10/IFN-γ-induced protein 10; another one whose plasmacytoid dendritic cells produced elevated CXCL10/IFN-γ-induced protein 10 levels, already after 18 h, declining thereafter; and a 3rd group characterized by plasmacytoid dendritic cells releasing very high CXCL10/IFN-γ-induced protein 10 levels after 42 h only. Finally, we report that in plasmacytoid dendritic cells, equivalent concentrations of IFN-λ3 and IFN-λ1 promote survival, antigen modulation, and cytokine production in a comparable manner and without acting additively/synergistically. Altogether, data not only extend the knowledge on the biologic effects that IFN-λs exert on plasmacytoid dendritic cells but also add novel light to the networking between IFN-λs and plasmacytoid dendritic cells in fighting viral diseases.

A versatile, non genetically modified organism (GMO)-based strategy for controlling low-producer mutants in Bordetella pertussis cultures using antigenic modulation.

The uncontrolled presence of non-producer mutants negatively affects bioprocesses. In Bordetella pertussis cultures, avirulent mutants emerge spontaneously and accumulate. We characterized the dynamics of accumulation using high-throughput growth assays and competition experiments between virulent and avirulent (bvg(-) ) isolates. A fitness advantage of bvg(-) cells was identified as the main driver for bvg(-) accumulation under conditions of high virulence factor production. Conversely, under conditions that reduce their expression (antigenic modulation), bvg(-) takeover could be avoided. A control strategy was derived, which consists in applying modulating conditions whenever virulence factor production is not required. It has a wide range of applications, from routine laboratory operations to vaccine manufacturing, where pertussis toxin yields were increased 1.4-fold by performing early pre-culture steps in modulating conditions. Because it only requires subtle modifications of the culture medium and does not involve genetic modifications, this strategy is applicable to any B. pertussis isolate, and should facilitate regulatory acceptance of process changes for vaccine production. Strategies based on the same concept, could be derived for other industrially relevant micro-organisms. This study illustrates how a sound scientific understanding of physiological principles can be turned into a practical application for the bioprocess industry, in alignment with Quality by Design principles.

Biotechnology and Bioengineering: Volume 112, Number 6, June 2015

Cover Legend Modular virus‐like particle (VLP) engineering. Antigenic modules are integrated, at the DNA level (bottom), with VLP subunit protein (middle). Modular subunits self‐assemble into VLPs (top). See related article by Chuan et al. Volume 110, Issue 9, pages 2343–2351, September 2013 (Image courtesy of Dr. Yap Pang Chuan and co‐workers.) image