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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-05-1383.
IMMUNOBIOLOGY
From the Department of Medicine and Therapeutics,
Institute of Medical Sciences, University of Aberdeen Foresterhill,
Aberdeen, United Kingdom; and the Academic Transfusion
Medicine Unit, Regional Transfusion Centre, 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- Current treatments for patients with autoimmune
diseases rely on inducing generalized immunosuppression and are
associated with serious side effects. The rational design of more
specific, effective approaches will depend on a better understanding of the responses to the relevant autoantigens, which are poorly
characterized in most human autoimmune diseases.
Autoantigen-specific CD4+ helper T (Th) cells are pivotal
in the development of animal models of autoimmunity and therefore provide a target for novel forms of immunotherapy.1,2 Th cell clones can be classified into different functional types on the
basis of the cytokines they secrete,3 and it has become clear that the pathogenicity of autoimmune responses can be determined by mutual antagonism between these helper
subpopulations.1,2 Initially, attention focused on Th1 and
Th2 cells, which produce interferon- Although important to the design of future therapy,4,19
the subsets of autoantigen-specific Th cells in most human autoimmune diseases have not been defined and, in particular, virtually nothing is
known about secretion of the regulatory cytokines IL-10 and TGF- Examples of murine AIHA are helper dependent,23-27 with
the spontaneous form of the disease affecting NZB mice being mediated by a Th1-biased autoreactive response.2,28 In human AIHA, Th cells have been studied29 in disease caused by warm RBC
antibodies. The Rh complex has been shown to carry autoreactive helper
determinants, since Rh peptide-specific Th cells that have been
activated in vivo can be detected in the peripheral blood of all
patients with anti-Rh autoantibodies.29 These autoreactive
T cells were identified by their ability to proliferate in vitro, but
their patterns of cytokine production, and thus the contribution of
different Th subpopulations to the responses, have not yet been
reported. The first aim was therefore to establish whether Rh-specific
Th cells secreting the respective Th1 and Th2 cytokines IFN- Patients
Determination of autoantibody specificity
Antigens and mitogens A complete panel of 42 15-mer peptides, with 5 amino acid overlaps, was synthesized29 (Department of Biochemistry, University of Bristol, United Kingdom), corresponding to the sequence of the 30-kDa Rh protein associated with expression of the D blood group antigen.30 To ensure purity, peptides were synthesized by fluorenylmethoxycarbonyl chemistry31 on resin using a base-labile linker, rather than by pin technology, and screened by high-performance liquid chromatography (HPLC) and amino acid analysis. As previously optimized,29,32 the peptides were used to stimulate cultures at 20 µg/mL.RhD protein was purified from RBCs by immunoprecipitation using a monoclonal anti-D (T19, Scottish National Blood Transfusion Service), which is specific for an epitope on loop 6.33 The immunoprecipitation method was adapted from that previously published.21 Briefly, 0.4 mL packed D-positive RBCs were incubated with 0.5 mL 250 µg/mL T19 for 1 hour at 37°C, washed, lysed in hypotonic solution at 4°C, and solubilized in 2% Triton X100 (Sigma, Dorset, United Kingdom). After removing insoluble debris by centrifugation, immune complexes were immobilized by incubation with magnetic beads (Biomag, PerSeptive Biosystems, MA). RhD protein was added to cultures at an estimated concentration of 5 µg/mL, still complexed to the beads, since antigen-presenting cells (APCs) efficiently take up and process proteins bound to an insoluble matrix.27 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blotting of the preparation21 revealed a major band estimated at 30 kDa, corresponding to the migration of RhD protein, but no evidence of the Rh-associated glycoprotein. In all experiments, control cultures were stimulated with magnetic beads coated with T19 antibody alone. The control antigen Mycobacterium tuberculosis purified protein derivative (PPD) (Statens Seruminstitut, Denmark) was added to cultures at 20 µg/mL. PPD readily provokes recall T-cell responses in vitro,34 since most United Kingdom citizens have been immunized with Bacillus Calmette-Guérin (BCG). Proliferation assays As previously described,29,32 peripheral blood mononuclear cells (PBMCs) were separated from fresh blood samples by density gradient centrifugation and cultured at a concentration of 1.25 × 106 cells/mL. Cell proliferation was estimated from the incorporation of 3H-thymidine in triplicate microtiter wells 5 days after stimulation with antigen. Results are presented either as mean cpm = +/ SD of the triplicate
sample, or as a stimulation index (SI), expressing the ratio of mean
CPM in stimulated versus unstimulated control cultures. An SI > 3 is interpreted as representing a significant positive
response.35
Measurement of cytokine production The production of IFN- , IL-4, IL-10, and TGF- in cultures
was measured by a highly sensitive cellular enzyme-linked immunosorbent assay (ELISA).35 Briefly, 5 days after stimulation
with peptide, PBMC cultures were transferred into duplicate wells in
microtiter plates (Nunc, Roskilde, Denmark) coated with monoclonal
anti-cytokine capture antibody (Pharmingen, Oxon, United Kingdom).
After incubation of PBMC for 24 hours at 37°C, the plates were
developed with the appropriate biotinylated monoclonal detection
antibody (Pharmingen), ExtraAvidin-alkaline phosphatase conjugate
(Sigma), and p-nitrophenyl phosphate substrate (Sigma). The absorbance
at 405 nm was measured using a multiscan plate reader (Labsystems,
Basingstoke, United Kingdom). Cytokine secretion was calculated by
interpolation from a standard curve generated by incubating duplicate
wells with doubling dilutions of recombinant human, IFN-, IL-4,
IL-10, and TGF- (Pharmingen). Results are presented either as the
mean cytokine concentration in duplicate wells, or as SI, expressing
the ratio of mean concentration in stimulated versus unstimulated
control cultures. An SI > 2.0 is interpreted as representing a
significant positive response.35
Blocking antibodies To inhibit the effects of IL-10, a neutralizing antibody specific for this cytokine (Pharmingen) was added to newly established cultures at 1 ng/mL. As reported by others,16,36 costimulation via cytotonic T lymphocyte-associated antigen (CTLA-4) was prevented by titrating a blocking antibody F(ab')2 fragment (Alexis Biochemicals, Nottingham, United Kingdom) into newly established cultures.Characterization of responding cells The phenotypes of cultured cells that secrete cytokine in response to antigen were determined by flow cytometry. PBMCs from responding cultures were stained with anti-CD3 phycoerythrin-Texas Red-X (Beckman Coulter, Bucks, United Kingdom) and anti-CD4 fluorescein isothiocyanate (Beckman Coulter). Cells synthesizing IL-10 were labeled by incubating with anti-IL-10 phycoerythrin (Pharmingen) after inhibition of protein secretion with Brefeldin A (Sigma) and permeabilization with Intraprep (Beckman Coulter). Stained cells were analyzed using an EPICS XL cytometer (Beckman Coulter) and Expo v2 analysis software (Applied Cytometry Systems, Yorks, United Kingdom).Statistical analysis A nonparametric test, Spearman rank correlation, was used with the level for significance taken as P < .05 (2-tailed).
Cytokine responses to RhD protein by Th cells from AIHA patients PBMCs from a panel of AIHA patients were tested for the ability to respond to purified RhD protein by proliferating or secreting the respective Th1 and Th2 cytokines IFN- and IL-4, or the regulatory Tr1 and Th3 cytokines IL-10 and TGF-1 (Figure
1). The principal cytokine produced in
response to the autoantigen by each PBMC sample was either IFN- or
IL-10, with no IL-4 or TGF-1 detectable in any of the stimulated
cultures despite the use of well-validated, highly sensitive
assays35 capable of measuring these cytokines in
allo-responses to blood group antigens.37 Whether IFN-
or IL-10 production predominated was related to the magnitude of the
proliferative response. Thus, high levels of IFN- were associated with strong proliferation (Rs = 0.63,
P < .01), while IL-10 predominated when proliferation was
weak (Rs = 0.58, P < .005). Serial
results from individual patients demonstrated that this bias toward
proliferative or IL-10 responses was unstable over time and could
reverse in subsequent samples. Such deviations in response type were
specific to the autoantigen, since the control recall antigen PPD
elicited strong proliferation, with IFN- the predominant cytokine
produced, from all PBMC samples tested (results not shown).
The regulatory effects of the IL-10 produced in response to the RhD protein were tested by adding a neutralizing antibody specific for this cytokine to cultures of AIHA patients' PBMCs stimulated with the autoantigen. In each experiment, the blocking antibody resulted in markedly higher proliferative responses (mean increase in SI = 203% SD = 116, n = 6). These results lead us to propose that the patients are capable of mounting IL-10-mediated regulatory responses to the autoantigen, but that these are inconsistent and unable permanently to control the pathogenic Th1 cells in vivo. A close correlation between the cytokine responses and the clinical course of AIHA would not be expected because multiple factors influence the severity of the disease,20,38 and the anemia in most patients was well controlled during this study. Even so, analysis of cytokine responses by 17 samples from 5 patients, together with the corresponding blood hemoglobin levels, reveals that higher levels of IL-10 elicited by the RhD protein are associated with less severe anemia (Rs = 0.57, P < .02). Mapping epitopes on the RhD protein that induce cytokine responses Having demonstrated that the RhD protein is capable of eliciting different types of response in human AIHA, we next mapped the fine specificity of the respective Th cells. To determine the breadth of the responses and to avoid selection pressures inherent in cloning, we took advantage of techniques successfully developed for studying polyclonal T cells.27-29,32,34,35 Proliferation and the secretion of IFN-, IL-4, IL-10, and TGF-1 were measured when PBMCs from the
AIHA patients were stimulated with an overlapping panel of 15-mer
peptides spanning the RhD protein sequence. Representative results from
3 of the 7 patients tested illustrate that multiple peptides were
stimulatory, with the production of IFN- or IL-10 being the
predominant cytokine responses (Figure
2A-C). By contrast, TGF-1 induced by
peptides was unusual, and IL-4 very rare. In 4 of the 7 patients
tested, proliferative responses against peptides were positively
associated with secretion of the Th1 cytokine IFN-
(P < .05, Spearman rank correlation). It should be noted that, despite the complexity of the response profiles, particular peptides could be identified in each patient that selectively and
reproducibly elicited IL-10, but not proliferation. These IL-10-inducing peptides include 652-66,
16152-166, 24232-246, and 34332-346
for patient 1 and peptides 11102-116 and
24232-246 for patient 2, which were chosen for
later investigation. In healthy control donors (n = 7, representative
results shown in Figure 2D), proliferative, but not IL-10, responses to
the RhD peptide panel were uncommon. In previous studies, recall T-cell
proliferation in response to Rh peptides was also found to be
rare.29,34
Regulatory activity by the Tr1 cytokine IL-10 secreted in response to RhD peptides Since IL-10-secreting Th cells play an important role controlling animal models of immune-mediated disease,7-12 the production of this cytokine in response to the RhD protein and peptides was considered in detail. The first step was to determine the phenotype of the IL-10-secreting cells. Flow cytometry confirmed that most of the cells producing IL-10 when PBMCs from AIHA patients (n = 4) were stimulated with RhD protein (mean = 92%, SD = 11) or RhD peptides (mean = 93%, SD = 3, n = 36) were of the CD4+ helper phenotype (Figure 3). We next examined the relationship between the IL-10 responses to the RhD protein and to the peptide panel. In 19 samples from 7 patients, a significant correlation (Rs = 0.46, P < .05) was found between the level of IL-10 production induced by the purified autoantigen and the number of peptides that elicited this cytokine.
The dependence of the IL-10 response to the RhD protein on the breadth of specificities of the IL-10-secreting Th cells that are recruited, together with the identification of peptides that selectively stimulated IL-10 production, suggested a means to manipulate the type of response to the autoantigen. We tested whether peptides that induce only IL-10 production were able to alter the balance of the response to the RhD protein in vitro at times when this was dominated by proliferation. Cultures of PBMC from AIHA patients were stimulated to proliferate with the purified RhD protein, and the effect of adding RhD peptides that elicit IL-10 responses was determined. The results of 2 representative experiments demonstrate that IL-10-inducing peptides strongly inhibit proliferative responses to the purified RhD autoantigen (Figure 4A-B). Similar results were obtained in a total of 8 experiments, using samples from 3 patients that were obtained when the RhD protein elicited proliferation, and testing at least 2 IL-10-inducing peptides on each occasion. The dominant inhibition appears to be specific to the RhD protein, since no such effect was observed on Th cells stimulated to proliferate with the control recall antigen PPD. An alternative explanation for this difference between the 2 antigens is that PPD-induced proliferation is more resistant to the suppressive effects of IL-10. However, proliferative responses elicited by either RhD or PPD alone were equally inhibited by addition of the cytokine to cultures (> 90% inhibition by > 100 pg/mL IL-10 in experiments using PBMCs from 2 patients). To determine whether the suppression of proliferative responses to the
RhD protein mediated by peptides is IL-10 dependent, a neutralizing
antibody specific for this cytokine was added to cultures. In each
experiment (a total of 4 experiments in 3 patients, testing up to 3 peptides on each occasion), the inhibitory effects were entirely or
largely blocked by this treatment (Figure
5A). Furthermore, RhD peptides that
failed to elicit IL-10 did not inhibit proliferative responses to the
RhD protein (results not shown).
Evidence from animal models indicates that costimulation via ligation of CTLA-4 can be important in the induction of cytokine-mediated Tr responses.39 We therefore tested whether the regulatory responses elicited by the IL-10-inducing RhD peptides could be inhibited by a blocking antibody F(ab')2 fragment specific for CTLA-4.16,36 Representative results (from a total of 4 experiments in 2 patients) demonstrate that anti-CTLA-4 antibody virtually abrogated the suppressive effect of these responses on proliferation stimulated by the purified RhD protein (Figure 5B), a reversal that was accompanied by a marked reduction in the level of IL-10 induced by the RhD peptides (Figure 5C). Characterization of IL-10-inducing RhD peptides The ability of particular RhD peptides to induce IL-10-mediated Tr responses in vitro raises the questions of whether it would be practical to exploit them therapeutically, and why these sequences have such a property. To examine these issues, we analyzed the results across the patient panel to determine whether the same peptides can elicit IL-10 responses in different individuals. Figure 6 demonstrates that certain peptides preferentially induced IL-10 in many patients, despite variation in HLA type (Table 1). For example, peptides 322-36, 652-66, and 34332-346 each stimulated IL-10 responses in more than 50% of the patients and are therefore candidates to test for the ability to enhance autoantigen-specific regulation in vivo. Such peptides are predicted to bind to a number of different HLA-DR molecules (http://imtech.res.in/raghava/propred/index.html, http://www.csd.abdn.ac.uk/~gjlk/MHC-Thread).
The criteria that determine the specificities of both pathogenic and regulatory autoreactive Th cells remain unresolved and, in particular, it is not clear whether they recognize epitopes that are normally naturally processed and presented. We therefore took advantage of a previous study32 of healthy RhD-negative blood donors who lack the RhD protein.40 These volunteers had been alloimmunized with RhD-positive RBCs, and we mapped the naturally processed RhD peptides that elicit proliferative responses by Th cells from their blood.32 Figure 6 demonstrates a positive correlation (Rs = 0.37, P < .02) between the abilities of each RhD peptide to elicit autoreactive IL-10 responses in the AIHA patients, and alloreactive Th proliferation in the group of immunized healthy donors. For example, peptide 652-66 is the most dominant sequence that stimulates autoimmune IL-10 production and also alloimmune proliferation. Our interpretation of these results is that naturally processed RhD peptides, some of which are presented promiscuously by a wide variety of HLA class II molecules, preferentially drive the selection of Tr cells capable of mounting IL-10 responses when the protein is expressed as an autoantigen.
This report describes, for the first time, human autoantigen-specific regulatory cells. Although compelling evidence has accumulated that Tr cells secreting inhibitory cytokine are important in the control of pathogenic immune responses in animal models,7-14 the cells' existence and character in human autoimmune disease previously have been unclear. The work was facilitated in AIHA because, unlike many other human autoimmune conditions, the unequivocal identification of the major RBC autoantigens21,22,29 has enabled the relevant Th-cell responses to be studied in detail. The major findings are that IL-10-secreting Tr cells responsive to a major RBC autoantigen, the RhD protein, are detectable in the blood of patients with AIHA, and they specifically suppress proliferative Th1 responses elicited by the antigen in vitro, and they predominantly recognize peptides containing naturally processed epitopes. Such peptides therefore represent a new type of immunotherapeutic agent for AIHA and other autoimmune diseases. PBMCs from AIHA patients would be predicted to secrete a wide variety
of cytokines when stimulated with RBC antigens,41,42 and
those measured here were selected to be characteristic of particular
CD4+ Th subsets. The RhD protein was chosen as the
autoantigen studied, since Th cells specific for epitopes from this
protein are activated in AIHA patients with autoantibodies specific for
the Rh complex,29 and it affords a unique comparison with
alloimmune responses in RhD-negative healthy control donors who had
been deliberately immunized with RhD-positive RBCs.32 The
current results reveal complex patterns of proliferation and cytokine
production by AIHA patients' CD4+ T cells when stimulated
in vitro with the purified RhD protein or the panel of peptides
spanning this RBC autoantigen. However, the predominant responses
either were of the Th1 type, with proliferation accompanied by IFN- Although Th cells specific for at least some autoantigens are
eliminated centrally in the thymus,11 it is nevertheless
clear that peripheral mechanisms are critical in preventing
autoaggressive responses, including those against
RBCs.23,24,29,34,37,44-46 The current work supports the
view2,46 that the balance between autoantigen-specific
Th-cell subsets secreting different patterns of cytokines is an
important mechanism for regulating autoimmune responses. In many
examples of rodent autoimmunity, including AIHA, Th1 responses appear
to be pathogenic, while Th2 responses can mediate protection from
disease.1,2,28,46 Here, a Th1 bias is demonstrated in
human AIHA patients whose Th cells commonly secreted IFN- Antagonism between Th1 and Th2 responses may modulate the
pathogenicity of autoantibody by providing help for different isotypes, rather than determine whether it is produced.2 However,
autoantigen-specific regulatory Th cells secreting inhibitory cytokines
such as IL-10 or TGF- The Tr cells identified in this study have a number of characteristics that are relevant both to the understanding of the pathogenesis of autoimmune disease and to the design of future therapy. First, they include distinct sets of specificities from the Th1 cells that proliferate in response to the RhD protein, since particular peptides from the autoantigen sequence, for example, peptide 652-66, selectively stimulate Th IL-10 production in most of the AIHA patients. The notion that regulatory and pathogenic Th cells may recognize different epitopes is supported by recent analysis of T-cell responses to islet cell autoantigen in nonobese diabetic mice.47 A comparison with the results of a parallel study of healthy RhD-negative donors who have been immunized with the RhD protein as a foreign antigen32 revealed that the regulatory responses in the AIHA patients are focused on peptides that contain naturally processed epitopes, including those such as peptide 652-66 that are presented promiscuously by a variety of HLA class II molecules. This analysis confirms that at least some Th cells specific for naturally processed self-peptides attain a regulatory phenotype5 rather than being deleted in vivo. The data also support the belief that the pathogenic Th1 response in diseases such as AIHA2,28,46 targets epitopes that are normally cryptic or subdominant.48,49 Since the size of the IL-10 response to the RhD protein varies with the number of sequences that elicit IL-10, we now hypothesize that the progress of disease is determined by changes in antigen presentation that alter the relative abundance with which the different sets of epitopes are processed and presented. The second characteristic of the Tr cells is that their ability to inhibit proliferative responses against the RhD protein is largely dependent on the secretion of IL-10. Such a population of autoreactive cells, here identified for the first time ex vivo, therefore resembles the murine and human Tr1 cells that can be driven to differentiate in vitro, which also mediate suppression via IL-10.12 Furthermore, both the secretion of IL-10 in response to RhD peptides and the inhibition of proliferation were shown to be dependent on costimulation via CTLA-4, a requirement shared by other Tr cytokine responses.39 Despite the dependence on IL-10, the inhibitory effect appeared to be a form of linked suppression that was specific for proliferative responses induced by the RhD autoantigen, but not by a control recall antigen. Such specificity would be advantageous in the exploitation of this effect therapeutically and suggests that clustering of IL-10-secreting and -proliferating Th cells on the same APCs may be necessary for inhibition. Overall, the Tr responses characterized here differ from those recently shown to be mediated by CD4+CD25+ human peripheral blood cells, the effects of which are not antigen specific and neither IL-10 nor CTLA-4 dependent.15-18 This study opens the way for the evaluation of a novel form of treatment for AIHA and other autoimmune diseases. The identification of autoantigen-specific Tr cells secreting IL-10 raises the possibility that selective stimulation of this regulatory population could restore tolerance and lead to the resolution of disease. Since particular epitopes on the RhD protein preferentially induce such regulatory responses, the balance between Th subsets in autoimmune disease could be controlled therapeutically by administering autoantigenic peptides that induce only inhibitory cytokine production. The promiscuity of some such peptides for presentation by many different class II molecules raises the prospect that such treatments need not be individually matched to the HLA type of the patient.
The authors are grateful to Mr D. Wilson (Scottish National Blood Transfusion Service) for technical assistance with flow cytometry, to Mr John Duncan (Scottish National Blood Transfusion Service) for serological testing, and to Drs D. Culligan, J. Tighe, and H. Watson (Aberdeen Royal Infirmary) for help with patient recruitment.
Submitted May 13, 2002; accepted July 17, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-05-1383.
Supported by grants from the Wellcome Trust (United Kingdom), the Scottish National Blood Transfusion Service, and the Grampian University Hospitals Trust (United Kingdom).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: R. N. Barker, Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom; e-mail: r.n.barker{at}abdn.ac.uk.
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  G. Serafini, M. Andreani, M. Testi, M. Battarra, A. Bontadini, E. Biral, K. Fleischhauer, S. Marktel, G. Lucarelli, M. G. Roncarolo, et al. Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia Haematologica, October 1, 2009; 94(10): 1415 - 1426. [Abstract] [Full Text] [PDF]
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 A. D'Ambrosio, M. Colucci, O. Pugliese, F. Quintieri, and M. Boirivant Cholera toxin B subunit promotes the induction of regulatory T cells by preventing human dendritic cell maturation J. Leukoc. Biol., September 1, 2008; 84(3): 661 - 668. [Abstract] [Full Text] [PDF]
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 P. Szodoray, B. Nakken, S. Barath, J. Gaal, M. Aleksza, M. Zeher, S. Sipka, A. Szilagyi, E. Zold, G. Szegedi, et al. Progressive divergent shifts in natural and induced T-regulatory cells signify the transition from undifferentiated to definitive connective tissue disease Int. Immunol., August 1, 2008; 20(8): 971 - 979. [Abstract] [Full Text] [PDF]
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 J. Zou, S. Hannier, L. S. Cairns, R. N. Barker, A. J. Rees, A. N. Turner, and R. G. Phelps Healthy Individuals Have Goodpasture Autoantigen-Reactive T Cells J. Am. Soc. Nephrol., February 1, 2008; 19(2): 396 - 404. [Abstract] [Full Text] [PDF]
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 F. J. Ward, A. M. Hall, L. S. Cairns, A. S. Leggat, S. J. Urbaniak, M. A. Vickers, and R. N. Barker Clonal regulatory T cells specific for a red blood cell autoantigen in human autoimmune hemolytic anemia Blood, January 15, 2008; 111(2): 680 - 687. [Abstract] [Full Text] [PDF]
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A. M. Hall, F. J. Ward, C.-R. Shen, C. Rowe, L. Bowie, A. Devine, S. J. Urbaniak, C. J. Elson, and R. N. Barker Deletion of the dominant autoantigen in NZB mice with autoimmune hemolytic anemia: effects on autoantibody and T-helper responses Blood, December 15, 2007; 110(13): 4511 - 4517. [Abstract] [Full Text] [PDF]
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 W. J. Pickford, A. J.M. Watson, and R. N. Barker Different Forms of Helper Tolerance to Carcinoembryonic Antigen: Ignorance and Regulation Clin. Cancer Res., August 1, 2007; 13(15): 4528 - 4537. [Abstract] [Full Text] [PDF]
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 F. Monneaux, J. Hoebeke, C. Sordet, C. Nonn, J.-P. Briand, B. Maillere, J. Sibillia, and S. Muller Selective Modulation of CD4+ T Cells from Lupus Patients by a Promiscuous, Protective Peptide Analog J. Immunol., November 1, 2005; 175(9): 5839 - 5847. [Abstract] [Full Text] [PDF]
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A. M. Hall, M. A. Vickers, E. McLeod, and R. N. Barker Rh autoantigen presentation to helper T cells in chronic lymphocytic leukemia by malignant B cells Blood, March 1, 2005; 105(5): 2007 - 2015. [Abstract] [Full Text] [PDF]
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A. M. Hall, L. S. Cairns, D. M. Altmann, R. N. Barker, and S. J. Urbaniak Immune responses and tolerance to the RhD blood group protein in HLA-transgenic mice Blood, March 1, 2005; 105(5): 2175 - 2179. [Abstract] [Full Text] [PDF]
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H. Ansart-Pirenne, D. Zeliszewski, K. Lee, S. Martin-Blanc, P. Rouger, and F. Noizat-Pirenne Identification of immunodominant alloreactive T-cell epitopes on the Jka red blood cell protein inducing either Th1 or Th2 cytokine expression Blood, November 15, 2004; 104(10): 3409 - 3410. [Full Text] [PDF]
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N. A. Marshall, L. E. Christie, L. R. Munro, D. J. Culligan, P. W. Johnston, R. N. Barker, and M. A. Vickers Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma Blood, March 1, 2004; 103(5): 1755 - 1762. [Abstract] [Full Text] [PDF]
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C.-R. Shen, A.-R. Youssef, A. Devine, L. Bowie, A. M. Hall, D. C. Wraith, C. J. Elson, and R. N. Barker Peptides containing a dominant T-cell epitope from red cell band 3 have in vivo immunomodulatory properties in NZB mice with autoimmune hemolytic anemia Blood, November 15, 2003; 102(10): 3800 - 3806. [Abstract] [Full Text] [PDF]
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L. S. Cairns, R. G. Phelps, L. Bowie, A. M. Hall, W. W.M. Saweirs, A. J. Rees, and R. N. Barker The Fine Specificity and Cytokine Profile of T-Helper Cells Responsive to the {alpha}3 Chain of Type IV Collagen in Goodpasture's Disease J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2801 - 2812. [Abstract] [Full Text] [PDF]
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N. A. Marshall, M. A. Vickers, and R. N. Barker Regulatory T Cells Secreting IL-10 Dominate the Immune Response to EBV Latent Membrane Protein 1 J. Immunol., June 15, 2003; 170(12): 6183 - 6189. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, Mia
), 2 (
), 9 (
), 10 (
), and 11 (
) were
stimulated in vitro with purified RhD protein. Results from serial
samples from each patient, taken one month to one year apart, are
included. The proliferative and IL-10 responses are inversely
correlated (Rs = 
), or (B)
blocking anti-CTLA-4 F(ab')2 0.5 µg/mL (
). The
anti-CTLA-4 F(ab')2 also inhibits IL-10 production by the
PBMCs responding to the RhD peptides alone (
lessons from characterising helper T-cell responses to the RhD protein.
Trans Med.
2000;10:331.