Publications by authors named "Stanislaw J Urbaniak"

13 Publications

  • Page 1 of 1

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the major human platelet autoantigen glycoprotein IIb/IIIa in HLA-transgenic mice.

Haematologica 2019 05 4;104(5):1074-1082. Epub 2018 Dec 4.

Institute of Medical Sciences, Ashgrove Road West, University of Aberdeen

Platelet destruction in immune thrombocytopenia is caused by autoreactive antibody and T-cell responses, most commonly directed against platelet glycoprotein IIb/IIIa. Loss of self-tolerance in the disease is also associated with deficient activity of regulatory T cells. Having previously mapped seven major epitopes on platelet glycoprotein IIIa that are recognized by helper T cells from patients with immune thrombocytopenia, the aim was to test whether peptide therapy with any of these sequences, alone or in combination, could inhibit responses to the antigen in humanized mice expressing HLA-DR15. None of the individual peptides, delivered by a putative tolerogenic regimen, consistently suppressed the antibody response to subsequent immunization with human platelet glycoprotein IIb/IIIa. However, the combination of glycoprotein IIIa peptides aa6-20 and aa711-725, which contain the predominant helper epitopes in patients and elicited the strongest trends to suppress when used individually, did abrogate this response. The peptide combination also blunted, but did not reverse, the ongoing antibody response when given after immunization. Suppression of antibody was associated with reduced splenocyte T-cell responsiveness to the antigen, and with the induction of a regulatory T-cell population that is more responsive to the peptides than to purified platelet glycoprotein IIb/IIIa. Overall, these data demonstrate that combinations of peptides containing helper epitopes, such as platelet glycoprotein IIIa aa6-20 and aa711-725, can promote suppression of responses to the major antigen implicated in immune thrombocytopenia. The approach offers a promising therapeutic option to boost T-cell regulation, which should be taken forward to clinical trials.
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http://dx.doi.org/10.3324/haematol.2017.179424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518892PMC
May 2019

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the RhD protein in HLA-transgenic mice.

Haematologica 2014 Mar 17;99(3):588-96. Epub 2014 Jan 17.

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
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http://dx.doi.org/10.3324/haematol.2012.082081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943325PMC
March 2014

Identification, immunomodulatory activity, and immunogenicity of the major helper T-cell epitope on the K blood group antigen.

Blood 2012 Jun 5;119(23):5563-74. Epub 2012 Apr 5.

Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom.

The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3(+)CD4(+) and "memory" CD45RO(+) phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses.
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http://dx.doi.org/10.1182/blood-2012-02-410324DOI Listing
June 2012

Routine antenatal anti-D prophylaxis in women who are Rh(D) negative: meta-analyses adjusted for differences in study design and quality.

PLoS One 2012 3;7(2):e30711. Epub 2012 Feb 3.

Medical Research Council Biostatistics Unit, Institute of Public Health, Cambridge, United Kingdom.

Background: To estimate the effectiveness of routine antenatal anti-D prophylaxis for preventing sensitisation in pregnant Rhesus negative women, and to explore whether this depends on the treatment regimen adopted.

Methods: Ten studies identified in a previous systematic literature search were included. Potential sources of bias were systematically identified using bias checklists, and their impact and uncertainty were quantified using expert opinion. Study results were adjusted for biases and combined, first in a random-effects meta-analysis and then in a random-effects meta-regression analysis.

Results: In a conventional meta-analysis, the pooled odds ratio for sensitisation was estimated as 0.25 (95% CI 0.18, 0.36), comparing routine antenatal anti-D prophylaxis to control, with some heterogeneity (I²  =  19%). However, this naïve analysis ignores substantial differences in study quality and design. After adjusting for these, the pooled odds ratio for sensitisation was estimated as 0.31 (95% CI 0.17, 0.56), with no evidence of heterogeneity (I²  =  0%). A meta-regression analysis was performed, which used the data available from the ten anti-D prophylaxis studies to inform us about the relative effectiveness of three licensed treatments. This gave an 83% probability that a dose of 1250 IU at 28 and 34 weeks is most effective and a 76% probability that a single dose of 1500 IU at 28-30 weeks is least effective.

Conclusion: There is strong evidence for the effectiveness of routine antenatal anti-D prophylaxis for prevention of sensitisation, in support of the policy of offering routine prophylaxis to all non-sensitised pregnant Rhesus negative women. All three licensed dose regimens are expected to be effective.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030711PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272015PMC
September 2012

The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia.

Transfus Apher Sci 2011 Aug 25;45(1):53-9. Epub 2011 Jun 25.

Laboratory Medicine, University Hospital of North Norway, Tromsø, Norway.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibodies that cross the placenta in connection with pregnancy and destroy fetal platelets. Recently, maternal T cell responses associated with FNAIT have been studied at the clonal level. These T cell clones recognize an integrin β3 epitope, which is anchored to the HLA-DRB3∗0101-encoded MHC molecule DR52a. The same MHC allele is strongly associated with FNAIT. As the production of pathological antibodies reactive with fetal platelets is likely dependent on these T cell responses, there exists a potential for preventing FNAIT by targeting these T cells.
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http://dx.doi.org/10.1016/j.transci.2011.06.003DOI Listing
August 2011

Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells.

Blood 2009 Aug 3;114(9):1954-7. Epub 2009 Jun 3.

Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, United Kingdom.

In neonatal alloimmune thrombocytopenia, almost all human platelet antigen (HPA)-1b1b mothers who produce anti-HPA-1a antibody through carrying an HPA-1a fetus are human histocompatibility leukocyte antigen (HLA)-DRB3*0101 positive. It is predicted that the HPA-1a Leu(33) polymorphism forms part of an HLA-DRB3*0101-restricted T-helper epitope, and acts as an anchor residue for binding this class II molecule. However, it is not known whether any corresponding peptides are naturally processed and presented from platelet glycoprotein. In this study, peptides displayed by a homozygous HLA-DRB3*0101 antigen-presenting cell line were identified after pulsing with recombinant HPA-1a (Leu(33) plexin-semaphorin-integrin domain). The peptides were eluted from HLA-DR molecules, fractionated by high performance liquid chromatography, and analyzed by tandem mass spectrometry. A "nested set" of naturally presented HPA-1a-derived peptides, each containing the Trp(25)-Leu(33) core epitope, was identified, with the most abundant member being the 16-mer Met(22)-Arg(37). These peptides may provide the basis for novel treatments to tolerize the corresponding T-helper response in women at risk of neonatal alloimmune thrombocytopenia.
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http://dx.doi.org/10.1182/blood-2009-04-211839DOI Listing
August 2009

The relationship of anti-HPA-1a amount to severity of neonatal alloimmune thrombocytopenia - Where does it stand?

Transfus Apher Sci 2009 Apr 14;40(2):75-8. Epub 2009 Feb 14.

Research and Development Directorate, Scottish National Blood Transfusion Service, Edinburgh and Aberdeen, Scotland, UK.

The issue of whether or not antibody quantity during pregnancy is related to severity of neonatal alloimmune thrombocytopenia remains unresolved. In this article we cite studies in support of both sides of the argument and highlight some of the reasons that may lie behind the observed differences amongst those studies. It may well be that some of the reasons for the discrepant results could be due to the type of study carried out (eg retrospective versus prospective), the sample size, the timing of antibody sampling, and possibly the type or protocol of assay used. Another major reason is the absence, until recently, of an international anti-HPA-1a standard.
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http://dx.doi.org/10.1016/j.transci.2009.01.010DOI Listing
April 2009

Deletion of the dominant autoantigen in NZB mice with autoimmune hemolytic anemia: effects on autoantibody and T-helper responses.

Blood 2007 Dec 4;110(13):4511-7. Epub 2007 Sep 4.

Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK.

The mechanisms underlying apparently spontaneous autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, are unknown. Here, we determine the contribution of the dominant red blood cell (RBC) autoantigen, the anion exchanger protein Band 3, to the development of NZB autoimmune responses. The approach was to prevent Band 3 expression in NZB mice by disrupting the AE1 gene. AE1(-/-) NZB mice produced RBC autoantibodies at the same levels as the wild-type strain, but they differed in recognizing antigens that correspond to glycophorins, rather than Band 3. Splenic T-helper (Th) cells from wild-type NZB mice proliferated strongly against multiple Band 3 peptides, particularly the dominant epitope within aa861-874. This helper response was severely attenuated in AE1(-/-) animals, leaving only weak proliferation to peptide aa861-874. The results demonstrate that the defect in self-tolerance in NZB AIHA is directed to the RBC type, and is not specific for, or dependent on, Band 3. However, the predisposition to RBC autoimmunity may be focused onto Band 3 by weak Th cell cross-reactivity between the helper dominant epitope and an exogenous antigen. The redundancy of the major autoantigen illustrates the requirement for specific therapy to induce dominant forms of tolerance, such as T-cell regulation.
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http://dx.doi.org/10.1182/blood-2007-06-094383DOI Listing
December 2007

Clonal regulatory T cells specific for a red blood cell autoantigen in human autoimmune hemolytic anemia.

Blood 2008 Jan 30;111(2):680-7. Epub 2007 Aug 30.

Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, UK.

Regulatory T (Tr) cells have the potential to treat immune-mediated disease, but cloning such cells for study from patients with autoimmune disease has proven difficult. Here, we describe autoantigen-specific, interleukin-10 (IL-10)-secreting Tr cell clones recovered ex vivo from a patient with autoimmune hemolytic anemia (AIHA) and characterize their phenotype, origin, and regulatory function. These IL-10+ Tr cells recognized a peptide, 72H-86L, derived from the Rh red blood cell autoantigen and shared phenotypic characteristics with both natural and inducible Tr cells. The clones also expressed different Tr markers depending on activation state: high levels of CD25 and LAG-3 when expanding nonspecifically, but FoxP3 after activation by the autoantigen they recognize. Despite a discrete Tr phenotype, these cells stably expressed the T helper 1 (Th1) signature transcription factor T-bet, suggesting they derive from Th1 T cells. Finally, the contribution of CTLA-4 in activating these IL-10+ Tr cells was confirmed by analyzing responses to transgenic B7.1-like molecules that preferentially bind either CD28 or CTLA-4. Overall, these Tr cells have a functional phenotype different from those described in previous studies of human Tr populations, which have not taken account of antigen specificity, and understanding their properties will enable them to be exploited therapeutically in AIHA.
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http://dx.doi.org/10.1182/blood-2007-07-101345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575838PMC
January 2008

Mapping helper T-cell epitopes on platelet membrane glycoprotein IIIa in chronic autoimmune thrombocytopenic purpura.

Blood 2007 May 1;109(10):4528-38. Epub 2007 Feb 1.

Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, United Kingdom.

Chronic autoimmune thrombocytopenic purpura (AITP) is associated with autoantibodies specific for platelet membrane components, often including glycoprotein GPIIIa. T helper (Th) cells reactive with GPIIIa, which are capable of driving the autoantibody response, are activated in AITP, and the aim here was to map the epitopes that they recognize. Peripheral blood mononuclear cells (PBMCs) were obtained from 31 patients with AITP and 30 control donors and stimulated with a panel of 86 overlapping synthetic 15-mer peptides spanning the complete sequence of GPIIIa. One or more peptides elicited recall proliferation by PBMCs from 28 of the patients, and, typically, multiple sequences were stimulatory. In contrast, responses in healthy control donors were rare (chi-square test = 115.967; P
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http://dx.doi.org/10.1182/blood-2006-09-044388DOI Listing
May 2007

Characterization of the alloreactive helper T-cell response to the platelet membrane glycoprotein IIIa (integrin-beta3) in human platelet antigen-1a alloimmunized human platelet antigen-1b1b women.

Transfusion 2005 Jul;45(7):1165-77

Academic Transfusion Medicine Unit and the Institute of Medical Sciences, Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, United Kingdom.

Background: The aims were to characterize the helper T-cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)-1a, to identify immunodominant epitopes and to examine the HLA Class II associations.

Study Design And Methods: Peripheral blood mononuclear cells (PBMNCs) were obtained from 21 HPA-1b1b women who had an HPA-1a-mismatched pregnancy, 14 of whom developed anti-HPA-1a, and 11 control donors. PBMNCs were stimulated with two panels of 15-mer peptides corresponding to the HPA-1a/1b polymorphic region, with either Leu33 (-1a) or Pro33 (-1b) at each possible position, and the proliferative responses were measured. HLA Class II and HPA genotyping was by conventional polymerase chain reaction-sequence-specific priming.

Results: Peptides with Leu33 at, or near, the C-terminus contained an immunodominant epitope, stimulating proliferation by helper T cells from all nine women who had anti-HPA-1a at the time of testing; peptide L1 (Val19-Leu33) stimulated a response in 50 percent of these women. Their T cells did not respond to the corresponding HPA-1b Pro33 peptides, and responses to either peptide panel were rare in unimmunized women and controls. HLA-DRB3*01+ was significantly overrepresented (p = 0.014) in alloimmunized women whose T cells responded to the major HPA-1a Leu33-containing epitope. Conversely, HLA-DRB1*15 was negatively associated (p = 0.014) with this response.

Conclusions: The HPA-1a polymorphic region of GPIIIa contains both the linear T-cell and the conformational B-cell epitopes. The immunodominant T-cell epitope is constrained by HLA-DRB3*01+, and if presented by a tolerogenic route, a peptide containing this epitope may form the basis for the prevention or reversal of the alloimmune response to HPA-1a.
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http://dx.doi.org/10.1111/j.1537-2995.2005.00188.xDOI Listing
July 2005

Immune responses and tolerance to the RhD blood group protein in HLA-transgenic mice.

Blood 2005 Mar 21;105(5):2175-9. Epub 2004 Sep 21.

Institute of Medical Sciences and Academic Transfusion Medicine Unit, Department of Medicine and Therapeutics, Regional Transfusion Center, Foresterhill Road, Aberdeen AB25 2ZW, United Kingdom.

RhD is a major blood group and the most important target antigen in hemolytic disease of the newborn (HDN). The aims of this study were to establish a humanized mouse model of responses to the RhD protein and to test whether these could be prevented by the induction of immune tolerance. HLA-DR15 is a major restricting element for human T-helper (Th) cells specific for RhD protein, and expression of this HLA-DR transgene was found to confer on mice the ability to respond to immunization with purified RhD protein. Synthetic peptides containing dominant Th cell epitopes, previously identified from studies of human alloimmunized donors, were administered to the nasal mucosa of transgenic mice before immunization with purified RhD protein. Treatment with each of the 4 dominant peptides, RhD52-66, RhD97-111, RhD117-131, and RhD177-191, inhibited T-cell priming and prevented antibody responses to the RhD protein. The ability to induce such active tolerance offers the prospect of peptide immunotherapy as a replacement for passive immune globulin in the prophylaxis of HDN.
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http://dx.doi.org/10.1182/blood-2004-04-1554DOI Listing
March 2005

Interleukin-10-mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen.

Blood 2002 Dec 1;100(13):4529-36. Epub 2002 Aug 1.

Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen Foresterhill, Aberdeen, United Kingdom.

Regulatory T cells have been shown to control animal models of immune-mediated pathology by inhibitory cytokine production, but little is known about such cells in human disease. Here we characterize regulatory T-cell responses specific for a human red blood cell autoantigen in patients with warm-type autoimmune hemolytic anemia. Peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia were found either to proliferate and produce interferon-gamma or to secrete the regulatory cytokine interleukin 10 when stimulated in vitro with a major red blood cell autoantigen, the RhD protein. Flow cytometric analysis confirmed that the majority of the responding cells were of the CD4(+) phenotype. Serial results from individual patients demonstrated that this bias toward proliferative or interleukin-10 responses was unstable over time and could reverse in subsequent samples. Epitope mapping studies identified peptides from the sequence of the autoantigen that preferentially induced interleukin-10 production, rather than proliferation, and demonstrated that many contain naturally processed epitopes. Responses to such peptides suppressed T-cell proliferation against the RhD protein, an inhibition that was mediated largely by interleukin 10 and dependent on cytotonic T lymphocyte-associated antigen (CTLA-4) costimulation. Antigenic peptides with the ability to stimulate specific regulatory cells may represent a new class of therapeutic agents for immune-mediated disease.
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http://dx.doi.org/10.1182/blood-2002-05-1383DOI Listing
December 2002