CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1351-1358 ◽  
Author(s):  
Marc Jacquemin ◽  
Valérie Vantomme ◽  
Cécile Buhot ◽  
Renaud Lavend'homme ◽  
Wivine Burny ◽  
...  
Keyword(s):  
T Cell ◽  
Factor Viii ◽  
Mhc Class Ii ◽  
Hemophilia A ◽  
Cd4 T Cell ◽  
Wild Type ◽  
T Cell Clones ◽  
Mhc Molecules ◽  
Cell Clones ◽  
C1 Domain

Mild/moderate hemophilia A patients carrying certain mutations in the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor occurrence. To analyze the mechanisms responsible for inhibitor development in such patients, we characterized FVIII-specific CD4+ T-cell clones derived from a mild hemophilia A patient carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All T-cell clones recognized synthetic peptides encompassing Arg2150. The peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of inhibitor formation. Peptide I2144-T2161 also bound to other DR molecules such as DRB1*0101 and DRB1*0701, indicating that the peptide binds to major histocompatibility complex (MHC) class II molecules expressed in more than 60% of the population. None of the T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as the native peptide. These observations demonstrate that T cells of this patient with mutation Arg2150His distinguish between self- and wild-type FVIII and provide a plausible mechanism for the frequent occurrence of an inhibitor in patients carrying this substitution. A similar phenomenon may occur with other mutations associated to an increased incidence of inhibitor formation.

Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1330-1330
Author(s):  
Sanja Stevanovic ◽  
Bart Nijmeijer ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
Marieke Griffioen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones ◽  
Human T Cells

Abstract Abstract 1330 Poster Board I-352 Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses

Blood ◽  
2009 ◽  
Vol 114 (7) ◽  
pp. 1423-1428 ◽  
Author(s):  
Ruth A. Ettinger ◽  
Eddie A. James ◽  
William W. Kwok ◽  
Arthur R. Thompson ◽  
Kathleen P. Pratt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Hemophilia A ◽  
T Helper ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
Polarized Cells

AbstractThe development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (TH)17/TH1- or TH1/TH2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were TH2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were TH1-polarized, indicating that tolerance to FVIII can be maintained even with circulating TH1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that TH17/TH1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human TH17/TH1 clones using standard culture conditions.

Rupture of T Cell Tolerance to Self Factor VIII in Mild/Moderate Hemophilia A.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3076-3076 ◽  
Author(s):  
Marc G. Jacquemin ◽  
Renaud Lavend’homme ◽  
Benhida Abdellah ◽  
Kathelijne Peerlinck ◽  
Jos Vermylen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Factor Viii ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
T Cell Tolerance ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ ◽  
Recombinant Fviii

Abstract Some patients with mild/moderate hemophilia A develop anti-Factor VIII (FVIII) antibodies following treatment with FVIII. In rare cases, the patient antibodies neutralize normal FVIII but do not recognize the patient FVIII. In most cases, the antibodies cross-react with the patient FVIII thereby changing the patient’s bleeding phenotype into that of a severe hemophilia A patient. We recently investigated the CD4+ T cells of a patient with mild hemophilia A, who produced antibodies recognizing only exogenous normal FVIII. Likewise, the patient CD4+ T cells recognized normal FVIII but not the patient FVIII. These observations raised the question of the specificity of FVIII-specific T cells occurring in mild/moderate hemophilia A patients and who developed an immune response towards both exogenous and self FVIII. We therefore investigated the specificity of CD4+ T lymphocytes in a mild hemophilia A patient (Ba), carrying a substitution Pro2292His in the FVIII gene, who produced high titer inhibitor antibodies recognizing both self and normal FVIII. The patient’s antibodies exclusively recognized the FVIII C2 domain, as determined in immunoprecipitation experiments using recombinant FVIII fragments produced in reticulocyte lysate. Eradication of the inhibitor was not successful despite using several therapeutic options (plasma exchanges, cyclophosphamide, low dose (25 U/kg) immune tolerance induction with plasma-derived Factor VIII and eventually anti-CD20 monoclonal antibody). Patient Ba FVIII specific T cells were expanded using dendritic cells. Out of 20 microcultures initiated with a total of 2 x 106 T cells, 3 cell lines specifically recognised FVIII. The frequency of FVIII-specific T cells in blood of this patient is therefore at least 1/700.000 CD4+ T cells. Patient Ba T cells were cloned using as antigen presenting cells an autologous lymphoblastoid cell line producing a non inhibitory anti-FVIII IgG4 antibody. Two FVIII-specific T cell clones were successfully derived. In control experiments, no FVIII-specific T cell clones could be derived from normal individuals. Both Ba T cell clones were activated by recombinant FVIII fragments encompassing the C1 and C2 domains. T cell activation was compared in presence of normal and His2292 recombinant FVIII. In the presence of 1 IU/ml normal or His2292 FVIII, clone 4E1 produced 0,793 ± 0,024 ng/ml and 0,277 ± 0,087 ng/ml IFN-γ, respectively, whereas clone 3F9 secreted 0,208 ± 0,07 and 0,238 ± 0,021 ng/ml, respectively. The concentration of normal FVIII inducing the same IFN-γ secretion as 1 IU/ml His2292 FVIII was only 0,45 IU/ml for clone 4E1 whereas it was 1,3 IU/ml for clone 3F9. Accordingly, one T cell clone recognizes patient His2292 FVIII at least as well as normal FVIII. These observations demonstrate that in a patient with mild/moderate hemophilia A who develops an immune response to his own FVIII, the T cell tolerance to self FVIII can also be broken. Such a cellular response to self FVIII may render restoration of tolerance to self and exogenous FVIII more difficult.

Sequence-Modified Factor VIII Variants Having Reduced Immunogenicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 39-39
Author(s):  
Ruth A. Ettinger ◽  
Melinda S. Epstein ◽  
Komal Puranik ◽  
Richard J. Hughes ◽  
Joseph A Liberman ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Factor Viii ◽  
Mhc Class Ii ◽  
Peptide Binding ◽  
Cell Epitope ◽  
Amino Acid Substitutions ◽  
T Cell Epitope ◽  
Wild Type ◽  
Cell Clones

Abstract Abstract 39 Neutralizing anti-factor VIII (FVIII) antibodies, or “inhibitors”, interfere with FVIII pro-coagulant activity, and persistent inhibitors can result in significant morbidity and mortality in hemophilia A (HA) patients and in individuals who develop autoantibodies to their endogenous FVIII. Inhibitor production follows stimulation of helper T cells by linear amino acid sequences in FVIII corresponding to HLA-restricted T-cell epitopes. An immunodominant HLA-DRB1*01:01-restricted T-cell epitope within a peptide corresponding to FVIII residues 2194–2213 was identified previously using blood samples from two mild HA subjects with hemophilic mutation A2201P. This same immunodominant epitope was found recently in inhibitor subjects with (a) a large F8 gene deletion and b) an F8 nonsense mutation in exon 12. The present study aims to identify amino acid substitutions in FVIII that will neutralize this T-cell epitope while preserving the pro-coagulant activity of the modified FVIII protein. MHC class II - peptide binding assays were carried out using truncated FVIII peptides to determine the shortest sequence with full binding affinity for a recombinant HLA-DR0101 protein, and subsequently using peptides having systematic arginine substitutions at each position to identify the specific residues that confer this binding affinity. The results indicated that FVIII2194–2205 is the minimal binding epitope and that residues F2196, M2199, A2201 and S2204 interact with the peptide-binding groove of HLA-DR0101. Next, four T-cell clones that all proliferate in response to this epitope but have different T-cell receptors were stimulated with 12 peptides having systematic alanine substitutions at each position of FVIII2194–2205. The F2196A substitution abrogated proliferation of all four clones. The M2199A-substituted peptide stimulated three of the clones more weakly than the wild-type peptide. Peptide binding and T-cell assays were next carried out with FVIII2194–2205 peptides in which the 19 common non-phenylalanine amino acids were substituted at position 2196. These results identified 12 different amino acid substitutions that decreased both MHC binding and T-cell proliferation more than 10-fold. The binding of FVIII2194–2205 and FVIII2194–2205, F2196A to 10 common HLA-DRB1 proteins was measured to determine the potential promiscuity of this epitope. Moderate or low affinity binding of FVIII2194–2205 (IC50 < 50 mM) to DR0401, DR0404, DR0901, DR1001, and DR1501 was observed. FVIII2194–2205, 2196A did not bind to any of the HLA-DRB1 proteins, suggesting that this substitution would not introduce a neo-epitope recognized by these other common MHC class II receptors. A recombinant FVIII-C2 domain protein with substitution F2196A was generated in E. coli and purified to homogeneity following a procedure that removes endotoxin. This FVIII-C2 mutein failed to stimulate the same four T-cell clones, all of which showed a strong, dose-dependent response to wild-type FVIII-C2. Recombinant B-domain-deleted FVIII (BDD-FVIII) proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W and M2199R were expressed in BHK-M cell lines. Multiple cell lines were generated to express wild-type BDD-FVIII and each of these mutant proteins. Expression levels of the muteins were similar to that of wild-type BDD-FVIII except for the M2199W and F2196A variants, which had expression levels ∼30 and 10% that of wild-type BDD-FVIII, respectively. Specific activities of the muteins, measured using chromogenic and clotting assays, were similar to that of wild-type BDD-FVIII. Binding of these muteins to plasma-derived von Willebrand factor was evaluated by ELISAs, as a surrogate assay to indicate possible effects of specific mutations on FVIII half-life in the circulation. Their affinities for VWF ranged from ∼40–100% that of wild-type BDD-FVIII. Our results suggest that FVIII muteins with amino acid substitutions that abolish binding to DR0101 and retain reasonable FVIII functionality could be developed as less immunogenic therapeutic proteins, in order to avoid HLA-DRB1*01:01-restricted immune responses in HA patients with this common allele. The immunogenicity of this T-cell epitope and of the sequence-modified peptides and proteins in HA subjects with other HLA-DRB1 alleles is currently under investigation. Disclosures: Pratt: Puget Sound Blood Center: Employment, patent describing design of novel factor VIII proteins Other.

Bacteria-specific CD4+ T-cell clones induce colitis in helicobacter hepaticus-infected T-cell-deficient RAG-2 KO mice

2001 ◽  
Vol 120 (5) ◽  
pp. A519-A520
Author(s):  
Marika C. Kullberg ◽  
Dragana Jankovic ◽  
Patricia Caspar ◽  
Peter L. Gorelick ◽  
Allen Cheever ◽  
...  
Keyword(s):  
T Cell ◽  
Cd4 T Cell ◽  
Helicobacter Hepaticus ◽  
T Cell Clones ◽  
Cell Clones

Th0 to Th1 switch of CD4 T cell clones specific from the 16-kDa antigen of Mycobacterium tuberculosis after successful therapy: lack of involvement of epitope repertoire and HLA-DR

2005 ◽  
Vol 98 (2) ◽  
pp. 253-258 ◽  
Author(s):  
Nadia Caccamo ◽  
Serena Meraviglia ◽  
Francesco Dieli ◽  
Amelia Romano ◽  
Lucina Titone ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Cd4 T Cell ◽  
Successful Therapy ◽  
T Cell Clones ◽  
Hla Dr ◽  
Cell Clones

scholarly journals Allelic Variation of MHC Structure Alters Peptide Ligands to Induce Atypical Partial Agonistic CD8+ T Cell Function

2003 ◽  
Vol 198 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Dong-Gyun Lim ◽  
Jacqueline M. Slavik ◽  
Katarzyna Bourcier ◽  
Kathrine J. Smith ◽  
David A. Hafler
Keyword(s):  
T Cell ◽  
Intracellular Signaling ◽  
Cell Function ◽  
Peptide Ligands ◽  
T Cell Clones ◽  
Altered Peptide Ligands ◽  
Mhc Molecules ◽  
Cell Functions ◽  
T Cell Function ◽  
Cell Clones

T cell receptors recognize small changes in peptide ligands leading to different T cell responses. Here, we analyzed a panel of HLA-A2–Tax11-19 reactive T cell clones to examine how small allelic variations of MHC molecules could alter the functional outcome of antigen recognition. Similar to the effects induced by antigenic altered peptide ligands, weak or partial agonistic T cell functions were identified in individual T cell clones with the recognition of MHC-altered peptide ligands (MAPLs). Interestingly, one subtype of HLA-A2 molecules induced an unusual type of partial agonistic function; proliferation without cytotoxicity. Modeling of crystallographic data indicated that polymorphic amino acids in the HLA-A2 peptide binding groove, especially the D-pocket, were responsible for this partial agonism. Reciprocal mutations of the Tax peptide side chain engaging the D-pocket indeed restored the agonist functions of the MHC–peptide complex. Whereas early intracellular signaling events were not efficiently induced by these MAPLs, phosphorylated c-Jun slowly accumulated with sustained long-term expression. These data indicate that MAPLs can induce atypical partial agonistic T cell function through structural and biochemical mechanisms similar to altered peptide ligands.

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