scholarly journals The MRC OX-22- CD4+ T cells that help B cells in secondary immune responses derive from naive precursors with the MRC OX-22+ CD4+ phenotype.

1989 ◽  
Vol 169 (3) ◽  
pp. 653-662 ◽  
Author(s):  
F Powrie ◽  
D Mason
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd4 T Cells ◽  
Antibody Responses ◽  
Cell Reactivity ◽  
Helper Activity ◽  
T Cell Reactivity ◽  
Paper Address

CD4+ T cells in the rat can be divided into two nonoverlapping subsets by their reactivity with the mAb MRC OX-22, which binds some of the high molecular weight forms of the CD45 antigen. The lineage relationship between subsets of CD4+ T cells expression different forms of CD45 has been a controversial issue for some time. Experiments described in this paper address this question using in vivo assays of T cell reactivity. Analysis of primary antibody responses in vivo show that it is MRC OX-22+ CD4+ T cells that are active in these assays, whereas antigen-primed T cells that provide helper activity for secondary antibody responses in vivo have the MRC OX-22- CD4+ phenotype. It is demonstrated that these memory T cells derive from MRC OX-22+ CD4+ T cell precursors and not from a putative separate lineage. It is concluded that with respect to the provision of help for B cells, MRC OX-22+ CD4+ T cells are precursors of memory cells with the phenotype MRC OX-22- CD4+.

scholarly journals Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1071-1071
Author(s):  
Yingyu Chen ◽  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Cd4 T Cell ◽  
Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

scholarly journals Lyt-23+ cyclophosphamide-sensitive T cells regulate the activity of an interleukin 2 inhibitor in vivo.

1981 ◽  
Vol 154 (2) ◽  
pp. 262-274 ◽  
Author(s):  
C Hardt ◽  
M Röllinghoff ◽  
K Pfizenmaier ◽  
H Mosmann ◽  
H Wagner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory Mechanism ◽  
Interleukin 2 ◽  
Inhibitor Activity ◽  
Cell Reactivity ◽  
Close Proximity ◽  
Producer Cell ◽  
T Cell Reactivity

Sera of thymus-bearing normal mice contain high levels of Interleukin 2 (II-2) inhibitor, whereas sera of athymic nu/nu mice do not. Evidence is presented that cyclophosphamide-sensitive Lyt-23+ T cells induce high II-2 inhibitor activity in the recipient nu/nu mice in the course of a graft-vs.-host reaction. The II-2 inhibitor has an approximately 50,000 mol wt. Its function is neither antigen specific nor H-2 restricted. During ontogeny, its activity parallels the development of T cell reactivity, i.e., it is absent both in the amniotic fluid and in sera of unborn mice, but increases to high levels during the early postnatal phase. The II-2 inhibitor described is viewed as an example of a T cell-dependent, in vivo regulatory mechanism able to effectively counteract the nonspecific activity of the Lyt-1+ helper T cell-derived II-2. Because the II-2 inhibitor activity is rather high in vivo, II-2 activity will exist only in close proximity to its producer cell, thereby maintaining specificity during the in vivo induction of cytotoxic T lymphocytes

scholarly journals B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

2021 ◽  
Author(s):  
Darryl Hayward ◽  
Lesley Vanes ◽  
Stefanie Wissmann ◽  
Sujana Sivapatham ◽  
Harald Hartweger ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Antibody Responses ◽  
B Cell Activation

AbstractMigration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5 and CD40, and using intravital imaging we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion and T cell-dependent activation.SummaryThe WNK1 kinase is essential in B cells for T-dependent antibody responses because it is activated by signaling from BCR, CXCR5 and CD40 and regulates B cell migration, adhesion, T-dependent activation, and differentiation into germinal center B cells and plasma cells.

scholarly journals Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis

2019 ◽  
Vol 10 ◽  
Author(s):  
Wolfgang Faigle ◽  
Carolina Cruciani ◽  
Witold Wolski ◽  
Bernd Roschitzki ◽  
Marco Puthenparampil ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
Cerebrospinal Fluid ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Reactivity ◽  
T Cell Reactivity

Expansion of immunoglobulin autoreactive T-helper cells in multiple myeloma

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2725-2732 ◽  
Author(s):  
Masih Ostad ◽  
Margareta Andersson ◽  
Astrid Gruber ◽  
Anne Sundblad
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Single Cell Analysis ◽  
T Helper ◽  
Cell Reactivity ◽  
Th Cells

Activation and expansion of T helper (Th) cells followed by regulation of activation are essential to the generation of immune responses while limiting concomitant autoreactivity. In order to characterize T cells reactive towards myeloma-derived monoclonal immunoglobulin (mIg), an autologous coculture assay for single-cell analysis of mIg-responding cells was developed. When cultured with dendritic cells loaded with mIg, CD4+ Th cells from patients with progressing multiple myeloma (MM) showed a proliferative MHC class II–dependent response. CD8+ T-cell reactivity and Th1 activation were consistently low or absent, and Th2 and regulatory cytokines were expressed. The presence of such non-Th1 CD4+ T cells in peripheral blood was independent of treatment status, while the frequencies of responding cells varied between patients and reached the same order of magnitude as those measured for tetanus toxoid–specific Th memory cells. Furthermore, investigations of T-cell subpopulations indicated a possible regulatory role on the mIg responsiveness mediated by suppressive CD25highFOXP3+CD4+ T cells. It is proposed from the present results that a predominant in vivo activation of non-Th1 mIg-reactive CD4+ T cells constitute an Ig-dependent autoregulatory mechanism in human MM, with possible tumor growth supporting or permissive effects.

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2330-2330
Author(s):  
Constantijn J.M. Halkes ◽  
Inge Jedema ◽  
Judith Olde Wolbers ◽  
Esther M van Egmond ◽  
Peter A. Von Dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Depletion ◽  
T Cell Depletion

Abstract Abstract 2330 In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures: No relevant conflicts of interest to declare.

Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models

Blood ◽  
2012 ◽  
Vol 119 (24) ◽  
pp. 5678-5687 ◽  
Author(s):  
Jae Hun Shin ◽  
Hyung Bae Park ◽  
Yu Mi Oh ◽  
Dong Pyo Lim ◽  
Ji Eun Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Antigen ◽  
Cd4 T Cells ◽  
Antitumor Effect ◽  
Gene Modification ◽  
Cell Reactivity ◽  
Murine Tumor ◽  
T Cell Reactivity

Abstract Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene modification of CD4 T cells significantly enhanced antitumor effect of unmodified CD8 T cells. Nonetheless, the gene modification of CD8 T cells along with CD4 T cells further maximized antitumor effect of T cells in 2 different murine tumor models. Thus, CTLA4-CD28 chimera gene modification of both tumor antigen-specific CD4 and CD8 T cells would be an ideal way of modulating CTLA4 function to enhance tumor-specific T-cell reactivity.

Allo-HLA–reactive T cells inducing graft-versus-host disease are single peptide specific

Blood ◽  
2011 ◽  
Vol 118 (26) ◽  
pp. 6733-6742 ◽  
Author(s):  
Avital L. Amir ◽  
Dirk M. van der Steen ◽  
Renate S. Hagedoorn ◽  
Michel G. D. Kester ◽  
Cornelis A. M. van Bergen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Reactivity ◽  
T Cell Clones ◽  
Biologically Relevant ◽  
Peptide Specificity ◽  
Cell Clones ◽  
T Cell Reactivity ◽  
Single Peptide

Abstract T-cell alloreactivity directed against non–self-HLA molecules has been assumed to be less peptide specific than conventional T-cell reactivity. A large variation in degree of peptide specificity has previously been reported, including single peptide specificity, polyspecificity, and peptide degeneracy. Peptide polyspecificity was illustrated using synthetic peptide-loaded target cells, but in the absence of confirmation against endogenously processed peptides this may represent low-avidity T-cell reactivity. Peptide degeneracy was concluded based on recognition of Ag-processing defective cells. In addition, because most investigated alloreactive T cells were in vitro activated and expanded, the previously determined specificities may have not been representative for alloreactivity in vivo. To study the biologically relevant peptide specificity and avidity of alloreactivity, we investigated the degree of peptide specificity of 50 different allo-HLA–reactive T-cell clones which were activated and expanded in vivo during GVHD. All but one of the alloreactive T-cell clones, including those reactive against Ag-processing defective T2 cells, recognized a single peptide allo-HLA complex, unique for each clone. Down-regulation of the expression of the recognized Ags using silencing shRNAs confirmed single peptide specificity. Based on these results, we conclude that biologically relevant alloreactivity selected during in vivo immune response is peptide specific.

scholarly journals Immune response to bacterial dextrans. II. T cell control of antibody isotypes.

1983 ◽  
Vol 158 (5) ◽  
pp. 1498-1510 ◽  
Author(s):  
F Ivars ◽  
G Nyberg ◽  
D Holmberg ◽  
A Coutinho
Keyword(s):  
T Cells ◽  
T Cell ◽  
Large Fraction ◽  
Antibody Responses ◽  
Cell Control ◽  
Cell Reactivity ◽  
Protein Carrier ◽  
Antibody Isotypes ◽  
Iga Antibodies

The isotype distribution of Dextran B 512 (Dex)-specific plaque-forming cells (PFC) and serum antibodies was studied after in vivo immunization in C57BL/6 mice. Although IgG2b and IgG3 could also be detected in most individuals, the majority of non-IgM PFC were of the IgA isotype. All classes other than IgM were T cell-dependent, as shown by their complete absence in athymic "nude" mice. This unusual isotype pattern was further investigated by studying the antibody responses to the same Dex epitope coupled to a protein carrier, and to a different hapten coupled to the carrier Dex or to a protein. The results show that IgA responses are epitope-related and selectively associated with anti-Dex antibodies: no IgA PFC are detected against a hapten coupled to Dex or proteins, while the enhanced levels of helper cell reactivity provided by protein carrier to Dex result in the appearance of IgG1 antibodies in addition to IgA. These results indicate that T cells that modulate isotype patterns in these responses can discriminate between Dex- and DNP-specific B cells in the response to the same carrier. Since the same idiotype is detected on a large fraction of the IgM and IgA anti-Dex response and antiidiotypic helper cells have previously been detected in normal C57BL/6 mice, we suggest that idiotype-specific T cells control the production of IgA antibodies upon immunization with Dex.

The Impact of VWF on FVIII Immune Responses in Hemophilia a Mice with Pre-Existing Anti-FVIII Immunity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 84-84
Author(s):  
Juan Chen ◽  
Jocelyn A. Schroeder ◽  
Xiaofeng Luo ◽  
Robert R. Montgomery ◽  
Qizhen Shi
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Specific Memory ◽  
The Impact

Abstract Development of inhibitory antibodies (inhibitors) against FVIII is the significant complication in protein replacement therapy for hemophilia A (HA). Currently, immune tolerance induction (ITI) with aggressive infusion of high-dose FVIII represents the only effective therapeutic approach for eradication of FVIII inhibitors and results in restoration of normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing VWF will affect the efficacy of the ITI is still a debated issue in the treatment of inhibitor patients. In this study, we explored the impact of VWF on FVIII immune responses in HA with pre-existing anti-F8 immunity using both in vitro and in vivomodels. Since the FVIII immune response is CD4+ T cell dependent, we first investigated how VWF affects FVIII-primed CD4+ T cells in response to FVIII restimulation. To address this question, we used a T cell proliferation assay. FVIIInull (F8null) mice were immunized with recombinant human FVIII (rhF8) to induce inhibitor development. Splenocytes from primed mice were labeled with CellTrace™ Violet and cultured with rhF8 with or without rhVWF. Four days later, cells were analyzed by FACS to assess the daughter (proliferated) cell population. The percentage of daughter CD4+ T cells (14.0±7.5%) in the condition cultured with 1 U/ml of rhF8 was significantly higher than without rhF8 (3.7±1.7%, n=6). The daughter cells further increased to 21.5±10.3% when cells were incubated with 10 U/ml of rhF8. However, when rhVWF was added to the culture media in addition to rhF8, percentages of daughter CD4+ T cells were significantly decreased in both the 1 U/ml and 10 U/ml rhF8 treatment groups (10.4±7.1% and 15. 8±8.4%, respectively). To further explore how VWF affects the FVIII immune response, we analyzed cytokine profiles in T cell culture supernants using a multiplex ELISA assay. The levels of IFNg and IL10 in the groups cultured with rhF8 in the presence of rhVWF were significantly lower than in the groups cultured with rhF8 only. The levels of TNFa, IL4, IL5, and IL12 in the groups cultured with rhF8 together with rhVWF were not significantly different than those in rhF8 groups without VWF. These results demonstrated that VWF significantly suppresses rhF8-primed CD4+ T cell proliferation in response to rhF8 restimulation and the inhibition is via the Th1 pathway. In a setting of pre-existing anti-F8 immunity, how FVIII-specific memory B cells respond to FVIII-restimulation and mature to antibody secreting cells (ASCs) is the critical pathway in terms of the clinical efficacy of FVIII infusion. To investigate how VWF affects memory B cell maturation upon FVIII restimulation, we used ELISPOT-based assay. Splenocytes from rhF8-primed HA mice were used as the source to prepare F8-specific memory B cell pools. CD138+ cells were depleted and the remaining cells were used as a pool of memory B cells. To stimulate the maturation of F8-specific memory B cells into ASCs, memory B cell pools from primed F8null mice were cultured with rhF8 with or without rhVWF for 6 days. After culture, newly formed ASCs were assessed by the ELISPOT assay. There were 54.4±19.5 ASCs/106 cells when cells from memory B cell pool were cultured with 0.05 U/ml rhF8. In contrast, there was only 15.6±1.6 ASCs/106cells after the cells were cultured with rhF8 together with rhVWF, indicating that memory B cell maturation is suppressed in the presence of rhVWF. We then used an in vivo model to further evaluate the impact of VWF on the immunogenicity of FVIII in HA with pre-existing immunity. Since we are unable to mimic the human ITI in F8null mice, we transferred memory B cells from rhF8-primed F8null splenocytes into immunocompromised F8null mice followed by rhF8 immunization in the presence or absence of rhVWF. Blood samples were collected one week after immunization for analysis. The inhibitor titer in animals that received rhF8-primed memory B cell pool followed by rhF8 immunization was 45.9±63.0 BU/ml (n=11), which was significantly higher than the titer in animals immunized with rhF8 together with rhVWF (23.9±38.4, P<.01). These results demonstrate that VWF suppressed the anti-F8 memory response in vivo. In summary, our ex vivo and in vivo data demonstrated that VWF attenuates F8-primed CD4+T cells and memory B cells in response to rhF8 restimulation, suggesting that infusion of FVIII together with VWF might reduce anti-F8 memory responses in HA with inhibitors. Disclosures No relevant conflicts of interest to declare.

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