Modification of Predicted Inhibitor Risk in Non-Severe Hemophilia-a By in silico Analysis of Human Proteome Homology with Wild-Type, FVIII-Derived Peptides

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 290-290
Author(s):  
Adrian Shepherd ◽  
Stuart Skelton ◽  
David Moss ◽  
Dan Hart
Keyword(s):  
T Cell ◽  
Cell Response ◽  
In Silico ◽  
Hemophilia A ◽  
T Cell Response ◽  
Human Proteome ◽  
Wild Type ◽  
Mhc Ii ◽  
Class Ii Mhc ◽  
Factor Concentrate

Abstract Neutralising antibodies (inhibitors) are increasingly recognized to be a life-time risk in non-severe hemophilia A patients exposed to factor VIII concentrates. It is currently not possible to reliably identify the variable inhibitor risk between individuals. Risk appears to differ between F8 genotypes but also for individuals living with the same F8 genotype. As a T cell dependent process, the wild-type, factor concentrate-derived peptide sequences spanning the F8 mutation position are presented by class II MHC and responsible for driving the T helper response and subsequent B cell response. We hypothesize that the primary sequences of the 20,469 proteins in the human proteome will, coincidentally, contain short primary sequences homologous to key immunogenic peptides derived from the therapeutic factor VIII. We present in silico data and correlation with published registry inhibitor data (Fisher's exact test) to demonstrate the potential impact of such "proteome protection" and future potential to more reliably stratify individuals between low/negligible risk and more significant risk of inhibitor formation. We utilize a well-validated, computational tool, NetMHC-II, to enable large scale, computational comparison of predicted antigen presentation between endogenous, mutated FVIII derived peptides and factor-concentrate derived, wild-type FVIII peptides spanning all 520 F8 missense mutations listed on www.hadb.org. NetMHC-II analyses peptide presentation by 14 class II MHC HLA-DR alleles, resulting in analysis of 7,280 (520 x 14) permutations of F8 -MHC-II. We identify 56% (n=4,077) of these permutations to be at low/negligible risk of inhibitor formation, at a binding threshold of 500nM, defined as absence of a novel peptide-MHC surface capable of driving a helper T cell response (p=0.005). When cross referenced with potential homologous sequences buried anywhere in the human proteome (http://www.ebi.ac.uk/reference_proteomes), a further 1,237 F8 -MHC-II combinations are afforded "proteome protection" due to direct sequence homology between FVIII-derived peptide and peptide(s) derived from other proteins. This increases the total number of F8 -MHC-II combinations predicted to be unable to drive a T cell response to 73% (n=5,314). The residual 1,966 (27%) F8 -MHC-II combinations are predicted to retain the ability to present novel-interface FVIII-derived peptides to T cells with an IC50<500nM (higher affinity). Previous work exploring in silico prediction of FVIII-derived peptide presentation may have overestimated the number of F8 -MHC II combinations deemed to be at risk of contributing to inhibitor formation. Our data suggests an additional mechanism "protecting" a larger proportion of those living with non-severe HA from inhibitor formation. The contribution of "proteome protection" further reduces the "at risk" F8 -MHC II permutations to be more representative of clinically observed inhibitor rates. We identify a potential novel tolerance mechanism and provide key data for future in vitro validation strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes

2019 ◽  
Vol 3 (11) ◽  
pp. 1761-1773 ◽  
Author(s):  
Sanne M. Meinderts ◽  
Gabriella Baker ◽  
Stan van Wijk ◽  
Boukje M. Beuger ◽  
Judy Geissler ◽  
...  
Keyword(s):  
T Cell ◽  
Respiratory Burst ◽  
Cell Response ◽  
T Cell Response ◽  
Human Neutrophils ◽  
Antigen Presenting Cell ◽  
Mhc Ii ◽  
Class Ii Mhc ◽  
Antimicrobial Function ◽  
Antigen Presenting

Abstract Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4+ T-cell response. Lastly, although the “don’t eat me” signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.

MOG extracellular domain (p1–125) triggers elevated frequency of CXCR3+ CD4+ Th1 cells in the CNS of mice and induces greater incidence of severe EAE

2014 ◽  
Vol 20 (10) ◽  
pp. 1312-1321 ◽  
Author(s):  
Jyothi T Mony ◽  
Reza Khorooshi ◽  
Trevor Owens
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Interferon Γ ◽  
T Cell Response ◽  
Receptor Expression ◽  
Myelin Proteins ◽  
Mhc Ii

Background: Myelin-specific T cells are implicated in multiple sclerosis (MS) and drive experimental autoimmune encephalomyelitis (EAE). EAE is commonly induced with short peptides, whereas in MS, whole myelin proteins are available for immune response. We asked whether immunization with the immunoglobulin-like domain of myelin oligodendrocyte glycoprotein (MOGIgd, residues 1–125) might induce distinct CD4+ T-cell response and/or a stronger CD8+ T-cell response, compared to the 21 amino acid immunodominant MHC II-associating peptide (p35–55). Objectives: Compare both EAE and T-cell responses in C57BL/6 mice immunized with MOGIgd and MOG p35–55. Methods: Cytokine production, and chemokine receptor expression by CD4+ and CD8+ T cells in the mouse central nervous system (CNS), were analyzed by flow cytometry. Results: MOGIgd triggered progression to more severe EAE than MOG p35–55, despite similar time of onset and overall incidence. EAE in MOGIgd-immunized mice was characterized by an increased percentage of CXCR3+ interferon-γ-producing CD4+ T cells in CNS. The CD8+ T-cell response to both immunogens was similar. Conclusions: Increased incidence of severe disease following MOGIgd immunization, accompanied by an increased percentage of CD4+ T cells in the CNS expressing CXCR3 and producing interferon-γ, identifies a pathogenic role for interferon-γ that is not seen when disease is induced with a single Major Histocompatibility Complex (MHC) II-associating epitope.

scholarly journals The SA85-1.1 Protein of the Trypanosoma cruzi trans-Sialidase Superfamily Is a Dominant T-Cell Antigen

2000 ◽  
Vol 68 (6) ◽  
pp. 3574-3580 ◽  
Author(s):  
Amanda E. Millar ◽  
Stuart J. Kahn
Keyword(s):  
T Cell ◽  
Trypanosoma Cruzi ◽  
Surface Protein ◽  
T Cell Response ◽  
Cd4 Cells ◽  
Protective Response ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Class Ii Mhc ◽  
Ifn Γ

ABSTRACT Trypanosoma cruzi currently infects 18 million people, and 30% of those infected develop a chronic inflammatory process that causes significant morbidity or mortality. The major histocompatibility complex class II (MHC-II)-restricted T-cell response is critical to the control of the infection and to the ensuing inflammatory pathology. The specific epitopes or major antigens of this response have not been identified. The parasite simultaneously expresses variant members of the trans-sialidase superfamily. To begin to analyze the MHC-II response to these variant proteins, the response to a single surface protein, SA85-1.1, was initiated. These studies have demonstrated that a biased gamma interferon (IFN-γ) response to the SA85-1.1 protein develops during T. cruzi infection. In addition, adoptive transfer of a CD4 clone that recognizes an SA85-1.1 epitope, named epitope 1, and immunization with a peptide encoding epitope 1 were protective and suggested that epitope 1 may be immunodominant. In this report IFN-γ intracellular staining demonstrated that splenocytes from acutely and chronically infected mice, incubated with SA85-1.1 protein or peptides that encode epitope 1, result in IFN-γ synthesis by 4 to 6% of the splenic CD4 cells. These data indicate that during T. cruzi infection epitope 1 is a major epitope and that 4 to 6% of the CD4 cells are stimulated by a single trans-sialidase superfamily epitope and suggest that a combination of trans-sialidase superfamily proteins combines to stimulate a majority of CD4 cells. These data suggest that during T. cruzi infection the CD4 response to thetrans-sialidase superfamily is critical to the protective response and to the ensuing chronic inflammatory pathology.

scholarly journals Lack of CD4+ T Cells Does Not Affect Induction of CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

2000 ◽  
Vol 68 (11) ◽  
pp. 6223-6232 ◽  
Author(s):  
Magali Moretto ◽  
Lori Casciotti ◽  
Brigit Durell ◽  
Imtiaz A. Khan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Cell Mediated Immunity ◽  
Wild Type ◽  
Encephalitozoon Cuniculi ◽  
Cell Immunity ◽  
Mouse Tissues

ABSTRACT Cell-mediated immunity has been reported to play an important role in defense against Encephalitozoon cuniculi infection. Previous studies from our laboratory have underlined the importance of cytotoxic CD8+ T lymphocytes (CTL) in survival of mice infected with E. cuniculi. In the present study, immune response against E. cuniculi infection in CD4+T-cell-deficient mice was evaluated. Similar to resistant wild-type animals, CD4−/− mice were able to resolve E. cuniculi infection even at a very high challenge dose (5 × 107 spores/mouse). Tissues from infected CD4−/− mice did not exhibit higher parasite loads in comparison to the parental wild-type mice. Conversely, at day 21 postinfection, susceptible CD8−/− mice had 1014 times more parasites in the liver compared to control wild-type mice. Induction of the CD8+ T-cell response in CD4−/− mice against E. cuniculi infection was studied. Interestingly, a normal antigen-specific CD8+T-cell response to E. cuniculi infection was observed in CD4−/− mice (precursor proliferation frequency, 1/2.5 × 104 versus 1/104 in wild-type controls). Lack of CD4+ T cells did not alter the magnitude of the antigen-specific CTL response (precursor CTL frequency; 1/1.4 × 104 in CD4−/− mice versus 1/3 × 104 in control mice). Adoptive transfer of immune CD8+ T cells from both CD4−/− and wild-type animals prevented the mortality in CD8−/− mice.E. cuniculi infection thus offers an example of an intracellular parasitic infection where CD8+ T-cell immunity can be induced in the absence of CD4+ T cells.

scholarly journals Hydroxyethylene isosteres introduced in type II collagen fragments substantially alter the structure and dynamics of class II MHC Aq/glycopeptide complexes

2015 ◽  
Vol 13 (22) ◽  
pp. 6203-6216 ◽  
Author(s):  
Cecilia Lindgren ◽  
Ida E. Andersson ◽  
Lotta Berg ◽  
Doreen Dobritzsch ◽  
Changrong Ge ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
T Cell ◽  
Cell Response ◽  
Type Ii Collagen ◽  
Class Ii ◽  
T Cell Response ◽  
Type Ii ◽  
Structure And Dynamics ◽  
Class Ii Mhc ◽  
Hydrogen Bond Networks

Introduction of hydroxyethylene isosteres into glycopeptides led to loss of Aq affinity and subsequent T cell response due to disruption of hydrogen bond networks.

scholarly journals Growth, Virulence, and Immunogenicity of Listeria monocytogenes aro Mutants

2004 ◽  
Vol 72 (10) ◽  
pp. 5622-5629 ◽  
Author(s):  
Jochen Stritzker ◽  
Jozef Janda ◽  
Christoph Schoen ◽  
Marcus Taupp ◽  
Sabine Pilgrim ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
T Cell ◽  
Cell Response ◽  
Culture Media ◽  
Lethal Dose ◽  
T Cell Response ◽  
Wild Type ◽  
Protein Antigens ◽  
Cell Immunity ◽  
Virulence Attenuation

ABSTRACT Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 104-fold for the aroA, aroB, and aroA/B mutants and >105-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 × 106 aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 × 108 aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

Prevention Of Murine FVIII-Specific Memory B Cells Due To FcγR2B (CD32) Blockade Is Associated With Distinctive Alterations In The FVIII-Specific Cytokine Profile

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 572-572
Author(s):  
Nadine Vollack ◽  
Arne Trummer ◽  
Andreas Tiede ◽  
Sonja Werwitzke
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Response ◽  
Research Funding ◽  
Hemophilia A ◽  
T Cell Response ◽  
Memory B Cells ◽  
Ifn Γ ◽  
High Doses ◽  
Very High

Abstract Development of neutralizing antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in hemophilia A. Long-term application of high doses of FVIII can induce tolerance in the context of immune tolerance therapy (ITT). Very high concentrations of FVIII have been shown to prevent the development of FVIII-specific antibody-secreting cells (ASCs) from memory B cells by inducing apoptosis. We have previously demonstrated that ASC differentiation from memory B cells is also abolished when CD32, an inhibitory Fc-gamma receptor expressed on B cells and dendritic cells, was genetically deleted or blocked by monoclonal antibodies (mAb). Here, we addressed the question how CD32 inhibition prevented ASC development by studying the FVIII-specific T cell response in the absence or presence of CD32 inhibition. Hemophilia A mice (B6;129S4-F8tmKaz/J) were immunized with recombinant human FVIII (rhFVIII) for 4 weeks, and spleen cells were re-stimulated with rhFVIII (0, 0.5, 1 or 10 IU/ml) ex vivo in the presence or absence of anti-CD16/CD32 antibody (2.4G2) to inhibit CD32. Formation of FVIII-specific ASCs was assessed on day 6 by ELISPOT. IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF-α were detected in culture supernatant using a cytometric bead-based multiplex assay on days 1 to 6. In line with previous findings, very high doses of rhFVIII (10 IU/ml) or blockade of CD32 with mAb 2.4G2 (at high or low doses of rhFVIII) inhibited the differentiation of FVIII-specific ASCs. We observed a FVIII-dose dependent increase in the secretion of IFN-γ, IL-2, IL-4, IL-6, and IL-10. Very high doses of rhFVIII (10 IU/ml) suppressed ACS formation, but not the formation of these cytokines indicating an intact FVIII-specific T cell response. Secretion of TNF-α appeared not to be FVIII-dose dependent and was also observed in the cultures without rhFVIII. Inhibition of CD32 with mAb 2.4G2 in the presence of ASC stimulating FVIII concentrations (e.g. 1 IU/ml) prevented the development of ASC, but also diminished the formation of IFN-γ (334.2 ± 58.0 pg/ml versus 14.9 ± 1.4 pg/ml) and significantly reduced IL-10 (297.7 ± 78.5 pg/ml versus 131.3 ± 26.8 pg/ml). IL-6 was only slightly reduced, whereas IL-4 remained unchanged. IL-2 was even increased at later time points during cell culture (day 4: 16.1 ± 3.4 pg/ml versus 24.0 ± 1.4 pg/ml, day 6: 3.1 ± 0.6 pg/ml versus 21.8 ± 3.6 pg/ml). These results indicate that very high doses of FVIII prevented ASC formation, but not FVIII-specific T cell stimulation. In contrast, inhibition of CD32 prevented ASC formation, but also changed the secretion of T cell dependent cytokines. The lack of IFN-γ and IL-10 production after re-stimulation with various doses of rhFVIII indicates a reduced stimulation of Th1 and Th2 helper cells. Similar effects have been described previously, when B7-1 co-stimulation of CD4+ T cells was prevented by anti-CD80 mAb. In conclusion, inhibition of FVIII-specific ASC formation by means of very high doses of FVIII or inhibition of CD32 appears to occur differently. High doses of FVIII induce apoptosis in FVIII-specific memory B cells, but do not prevent FVIII-specific T cell responses. In contrast, inhibition of the Fcγ receptor CD32 on B cells and dendritic cells interferes with the FVIII-specific T cell response indicating a defective antigen presentation. Both high dose FVIII treatment and CD32 blockade, alone or in combination, should be further investigated to specifically address the FVIII-specific immune response in hemophilia A, and to evaluate a potential improvement of ITT. Disclosures: Vollack: Biotest AG: Research Funding. Trummer:Biotest AG: Research Funding. Tiede:CSL Behring: Consultancy, Honoraria, Research Funding; Biogen Idec: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Werwitzke:Biotest AG: Honoraria, Research Funding.

scholarly journals An MHC class Ib–restricted CD8 T cell response confers antiviral immunity

2008 ◽  
Vol 205 (7) ◽  
pp. 1647-1657 ◽  
Author(s):  
Phillip A. Swanson ◽  
Christopher D. Pack ◽  
Annette Hadley ◽  
Chyung-Ru Wang ◽  
Iwona Stroynowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Mhc Class Ib ◽  
Wild Type ◽  
Mhc Class Ia ◽  
Class Ib

Although immunity against intracellular pathogens is primarily provided by CD8 T lymphocytes that recognize pathogen-derived peptides presented by major histocompatibility complex (MHC) class Ia molecules, MHC class Ib–restricted CD8 T cells have been implicated in antiviral immunity. Using mouse polyoma virus (PyV), we found that MHC class Ia–deficient (Kb−/−Db−/−) mice efficiently control this persistently infecting mouse pathogen. CD8 T cell depletion mitigates clearance of PyV in Kb−/−Db−/− mice. We identified the ligand for PyV-specific CD8 T cells in Kb−/−Db−/− mice as a nonamer peptide from the VP2 capsid protein presented by Q9, a member of the β2 microglobulin–associated Qa-2 family. Using Q9-VP2 tetramers, we monitored delayed but progressive expansion of these antigen-specific CD8αβ T cells in Kb−/−Db−/− mice. Importantly, we demonstrate that Q9-VP2–specific CD8 T cells more effectively clear wild-type PyV than a VP2 epitopenull mutant PyV. Finally, we show that wild-type mice also generate Q9-restricted VP2 epitope–specific CD8 T cells to PyV infection. To our knowledge, this is the first evidence for a defined MHC class Ib–restricted antiviral CD8 T cell response that contributes to host defense. This study motivates efforts to uncover MHC class Ib–restricted CD8 T cell responses in other viral infections, and given the limited polymorphism of MHC class Ib molecules, it raises the possibility of developing peptide-based viral vaccines having broad coverage across MHC haplotypes.

scholarly journals Deletion of cationic amino acid transporter 2 exacerbates dextran sulfate sodium colitis and leads to an IL-17-predominant T cell response

2013 ◽  
Vol 305 (3) ◽  
pp. G225-G240 ◽  
Author(s):  
Kshipra Singh ◽  
Lori A. Coburn ◽  
Daniel P. Barry ◽  
Mohammad Asim ◽  
Brooks P. Scull ◽  
...  
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Cell Response ◽  
Dextran Sulfate ◽  
Dextran Sulfate Sodium ◽  
T Cell Response ◽  
Cationic Amino Acid Transporter ◽  
Wild Type ◽  
Cationic Amino Acid

l-Arginine (l-Arg) is a semiessential amino acid that has altered availability in human ulcerative colitis (UC), a form of inflammatory bowel disease, and is beneficial in murine colitis induced by dextran sulfate sodium (DSS), a model with similarity to UC. We assessed the role of cationic amino acid transporter 2 (CAT2), the inducible transporter of l-Arg, in DSS colitis. Expression of CAT2 was upregulated in tissues from colitic mice and localized predominantly to colonic macrophages. CAT2-deficient (CAT2−/−) mice exposed to DSS exhibited worsening of survival, body weight loss, colon weight, and histological injury. These effects were associated with increased serum l-Arg and decreased tissue l-Arg uptake and inducible nitric oxide synthase protein expression. Clinical benefits of l-Arg supplementation in wild-type mice were lost in CAT2−/− mice. There was increased infiltration of macrophages, dendritic cells, granulocytes, and T cells in colitic CAT2−/− compared with wild-type mice. Cytokine profiling revealed increases in proinflammatory granulocyte colony-stimulating factor, macrophage inflammatory protein-1α, IL-15, and regulated and normal T cell-expressed and -secreted and a shift from an IFN-γ- to an IL-17-predominant T cell response, as well as an increase in IL-13, in tissues from colitic CAT2−/− mice. However, there were no increases in other T helper cell type 2 cytokines, nor was there a global increase in macrophage-derived proinflammatory cytokines. The increase in IL-17 derived from both CD4 and γδ T cells and was associated with colonic IL-6 expression. Thus CAT2 plays an important role in controlling inflammation and IL-17 activation in an injury model of colitis, and impaired l-Arg availability may contribute to UC pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document