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.

Reduced Immunogenicity of FVIII Peptides by Rational Modifications of An Immunodominant T-Cell Epitope.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 220-220
Author(s):  
Ruth A. Ettinger ◽  
Joseph A. Liberman ◽  
Douglas C. Bolgiano ◽  
Arthur R. Thompson ◽  
Kathleen P. Pratt
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Mhc Class Ii ◽  
Hemophilia A ◽  
Peptide Binding ◽  
Cell Epitope ◽  
Class Ii ◽  
Amino Acid Substitutions ◽  
T Cell Epitope

Abstract Abstract 220 Introduction. The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages. Aims: (1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles. Methods. The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined. Results. Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 > 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency). Conclusions. MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential. Disclosures: Pratt: CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.

Regulatory CD4+CD25+ T Cells Modulated the Immune Response of a Mild Hemophilia A Subject to an Immunodominant FVIII Epitope within a Distinct Time Window.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 764-764
Author(s):  
Eddie A. James ◽  
William W. Kwok ◽  
Arthur R. Thompson ◽  
Kathleen P. Pratt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Factor Viii ◽  
Mhc Class Ii ◽  
Cell Epitope ◽  
C2 Domain ◽  
T Cell Epitope ◽  
Wild Type ◽  
Cell Responses ◽  
Proliferation Assays

Abstract A mild hemophilia A subject with a missense mutation, A2201P, developed high titer neutralizing antibodies against infused factor VIII (FVIII) following post−operative FVIII administration. This missense mutation is in the C2 domain of FVIII. His baseline clotting activity fell from 10% to 3% of normal; FVIII infusions were discontinued, and his factor VIII clotting activity improved to its baseline within 2 mo. His inhibitor titer to FVIII declined over the ensuing year during which 12 samples were studied for T−cell epitopes, antibody titer, or both. His HLA haplotype was DR0101/DR1503, allowing use of fluorescent−labeled MHC Class II tetramers specific for DR0101. These were loaded with synthetic 20−mer peptides spanning the FVIII C2 domain to identify specific T cells responding to epitopes on FVIII by FACS. Proliferation assays were also carried out stimulating T cells with FVIII, the recombinant wild−type FVIII C2 domain, the C2 domain with mutation A2201P, and peptides. Tetramer staining and proliferation assays of total CD4+ and CD4+CD25− fractions indicated that CD4+CD25+ regulatory T cells modulated his immune response to FVIII between 10 and 19 weeks following the initial bleed. Peptides containing the wild type A2201, but not P2201, elicited strong T−cell responses. Markedly decreased staining of the total CD4 fractions compared to CD4+CD25− fractions in response to a peptide containing A2201 was observed between weeks 10 and 19 (see Figure showing staining at week 10). T−cell proliferation assays were carried out for cloned, expanded T cells that were selected using the tetramers. These T cell clones proliferated in response to FVIII, wild−type C2 protein, and peptides with the native A2201 sequence, whereas a peptide containing the mutation A2201P abrogated tetramer binding and proliferation. The T−cell epitope corresponded to a predicted MHC Class II binding motif for DR0101, underscoring the importance of studying T−cell responses within the context of each individual’s HLA haplotype. The epitope also overlapped a B−cell epitope identified by peptide mapping of the subject’s anti−FVIII C2 domain IgG fraction. Tetramer−guided T−cell epitope mapping provides greater sensitivity and specificity and markedly less ambiguous information than traditional mapping methods based on proliferation in response to peptides. FACS sorting of stained T cells allowed cloning of single T cells that retained the original specificity for the factor VIII peptides comprising this immunodominant T−cell epitope. Response to peptide 2194-2213 10 weeks after inhibitor first appeared Response to peptide 2194-2213 10 weeks after inhibitor first appeared

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.

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

Peptides ◽  
2016 ◽  
Vol 78 ◽  
pp. 68-76 ◽  
Author(s):  
Paola Lasso ◽  
Constanza Cárdenas ◽  
Fanny Guzmán ◽  
Fernando Rosas ◽  
María Carmen Thomas ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Trypanosoma Cruzi ◽  
Cell Epitope ◽  
Amino Acid Substitutions ◽  
T Cell Epitope ◽  
Immunogenic Properties

scholarly journals Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast.

1991 ◽  
Vol 174 (2) ◽  
pp. 425-434 ◽  
Author(s):  
K Falk ◽  
O Rötzschke ◽  
K Deres ◽  
J Metzger ◽  
G Jung ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Synthetic Peptides ◽  
Cell Epitope ◽  
Reversed Phase ◽  
T Cell Epitope ◽  
Amino Acid Residues ◽  
Allele Specific ◽  
Infected Cells ◽  
Natural Peptides

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

scholarly journals Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

1989 ◽  
Vol 170 (3) ◽  
pp. 1027-1032 ◽  
Author(s):  
J Gorski ◽  
C Irle ◽  
E M Mickelson ◽  
M J Sheehy ◽  
A Termijtelen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Theoretical Model ◽  
T Cell ◽  
Gene Conversion ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Allelic Series ◽  
T Cell Clones ◽  
Cell Responses ◽  
Cell Clones

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

scholarly journals Pocket 4 of the HLA-DR(α,β 1*0401) molecule is a major determinant of T cells recognition of peptide.

1995 ◽  
Vol 181 (3) ◽  
pp. 915-926 ◽  
Author(s):  
X T Fu ◽  
C P Bono ◽  
S L Woulfe ◽  
C Swearingen ◽  
N L Summers ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Peptide Binding ◽  
Alpha Helix ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Cell Recognition ◽  
T Cell Recognition ◽  
Hla Dr ◽  
Alpha Beta

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.

scholarly journals Pathogenic Potential of Borna Disease Virus Lacking the Immunodominant CD8 T-Cell Epitope

2007 ◽  
Vol 81 (20) ◽  
pp. 11187-11194 ◽  
Author(s):  
Kirsten Richter ◽  
Karen Baur ◽  
Andreas Ackermann ◽  
Urs Schneider ◽  
Jürgen Hausmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Neurological Disease ◽  
Cell Epitope ◽  
Cd8 T Cell ◽  
T Cell Epitope ◽  
Wild Type ◽  
Antiviral T Cells ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2k mice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2k mice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.

scholarly journals Peptide HER2(776–788) represents a naturally processed broad MHC class II-restricted T cell epitope

2001 ◽  
Vol 85 (10) ◽  
pp. 1527-1534 ◽  
Author(s):  
R Sotiriadou ◽  
S A Perez ◽  
A D Gritzapis ◽  
P A Sotiropoulou ◽  
H Echner ◽  
...  
Keyword(s):  
T Cell ◽  
Mhc Class Ii ◽  
Cell Epitope ◽  
Class Ii ◽  
T Cell Epitope

Breaking Immune Tolerance to Granule Proteases with Full-Length Antigen Vaccine in Humanized Transgenic Mice Reveals Alternative Antigen Processing and Immunodominance Heirarchy Applicable to Clinical Immunotherapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2054-2054
Author(s):  
J. Diamond Don ◽  
Wendi Zhou ◽  
Tumul Srivastava ◽  
Ravindra Rawal ◽  
Katharine Hagan ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Peptide Vaccine ◽  
Cell Epitope ◽  
Peptide Library ◽  
Full Length ◽  
T Cell Epitope ◽  
Computer Prediction ◽  
Mouse Splenocytes ◽  
Ctl Epitope

Abstract Abstract 2054 Poster Board II-31 The serine proteinases, human neutrophil elastase (HNE) and proteinase 3 (PR3) are degradative enzymes stored in cytoplasmic azurophilic granules of neutrophils. Both proteins are aberrantly expressed in human myeloid leukemias. Over-expression of PR3 is important in maintaining a leukemia phenotype, while HNE may suppress hematopoietic progenitors. PR3 and HNE contain a conserved nonameric HLA-A*02 restricted T-cell epitope called PR1. The presence of PR1-specific CTL has been correlated with molecular remission in CML. A PR1 nonameric peptide vaccine has been evaluated clinically, with promising results. A limitation of the PR1 peptide vaccine is its restriction to patients with the HLA-A*02 allele. A vaccine comprising a larger portion of the PR3 and/or HNE protein and capable of inducing broader T-cell responses restricted by a wider range of HLA alleles is a potential alternative. We have generated numerous recombinant vaccinia viruses (rVV) expressing forms of PR3 and HNE and have evaluated CTL responses in an HLA-A*02 transgenic mouse model (Tg-A2): (1) rVV-EGFP-PR3, contains a gene encoding EGFP fused to human PR3 lacking the N-terminal signal peptide, while retaining both pro-dipeptide and C-terminal propeptide sequences; (2) EGFP fused to C-terminal truncated PR3 (rVV-EGFP-PR3-T) deleted of PR2 CTL epitope, but maintaining PR1 and; (3) rVV-EGFP-HNE. Tg-A2 were immunized with rVV-EGFP-PR3, and 2 weeks later immune splenocytes were expanded by in vitrostimulation (IVS) with syngeneic naïve mouse splenocytes loaded with an overlapping PR3 peptide library. Intracellular cytokine (ICC) assays using a PR3 peptide library or PR1 9-mer epitope peptide demonstrated a robust PR3-specific CD8+ T-cell response, surprisingly without a detectable response to the PR1 peptide. However, PR2, a decamer, is immunodominant in mice following this immunization. This result was unexpected, since the PR2 epitope was identified by computer prediction as a potential HLA-A*02-restricted epitope within PR3 but, unlike the HLA-A*02-restricted 9-mer epitope PR1, has not been extensively studied as a T-cell epitope in humans. We tested whether the immunodominant PR2-specific response in rVV-EGFP-PR3 immunized mice is also found when a naturally-processed PR3 protein is used as the stimulating antigen, expressed from the cell line K562-A2-PR3, a derivative of K562-A2 endogenously expressing human PR3. Splenocytes from mice immunized with rVV-EGFP-PR3 were expanded by co-culture with irradiated K562-A2-PR3 cells, then tested in ICC assays using PR1 and PR2 CTL epitope peptides. We observed a robust response to PR2 but not PR1. While the PR1 epitope is absolutely conserved between human and murine versions of PR3 and HNE, the PR2 epitope is unique to human PR3, diverging from murine PR3 by 3 amino acids, and by 5 amino acid from human and murine HNE. We speculated that Tg-A2 mice may be tolerant to PR1 due to the presence of this epitope within murine PR3 and HNE, which may account for dominance of PR2-specific responses. To investigate the possibility of inducing PR1-specific CTL in Tg-A2, mice were immunized with rVV-EGFP-HNE, and we detected PR1-specific responses by IVS followed by ICC with the PR1 peptide. These results indicate that tolerance to PR1 can likely be broken by immunization with rVV expressing HNE protein that contains the PR1 epitope. These observations can be explained by either the PR2>PR1 competition hypothesis, or by different amino acid sequences flanking the PR1 epitope in PR3 and HNE proteins affecting processing of the protein to generate the PR1 peptide. To test this question, we used rVV-PR3-T to immunize Tg-A2 mice, then conducted IVS followed by ICC with the PR1 peptide to detect PR1-specific CTL. Results showed that immunization with rVV expressing truncated PR3 reliably induced PR1-specific CTL, unlike immunization with rVV expressing the full-length PR3 antigen. We concluded that PR2 epitope prevented the recognition of PR1. Therefore in Tg-A2 mice, vaccination with poxvirus vaccines expressing PR3 will induce an HLA-A2-restricted CTL response almost entirely focused on PR2, whereas immunization with rVV-HNE will induce PR1-specific CTL. If this were replicated in humans, the majority of PR1-specific CTL detected in CML patients could be derived from HNE rather than PR3 antigen presentation, and that PR2-specific CTL derived from PR3 should be equally frequent as PR1 and likely contribute to clinical GVL and tumor regression. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document