US20090075303A1

US20090075303A1 - Allo and Auto-Reactive T-Cell Epitopes

Info

Publication number: US20090075303A1
Application number: US11/874,977
Authority: US
Inventors: Stanislaw Joseph Urbaniak; Robert Norman Barker
Original assignee: University of Aberdeen; Common Services Agency for Scottish Health Service
Current assignee: University of Aberdeen; Common Services Agency for Scottish Health Service
Priority date: 1998-12-01
Filing date: 2007-10-19
Publication date: 2009-03-19
Also published as: JP4988090B2; WO2000032632A2; US7285524B1; EP1135414B1; ATE292145T1; HK1040719B; AU1400700A; WO2000032632A3; EP1135414A2; DE69924519D1; DE69924519T2; CA2353523C; WO2000032632A9; JP2004508273A; ES2239483T3; GB9826378D0; HK1040719A1; CA2353523A1

Abstract

The present invention relates to a pharmaceutical composition for the prevention of alloimmunisation of a subject or the immunosuppression of a response elicited by alloimmunisation of a subject or an autoimmune haemolytic disease for said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.

Description

The present invention relates to the mapping of allo-reactive T-cell epitopes on the rhesus (RhD and RhCc/Ee) proteins and to the use of such epitopes to modulate the corresponding immune responses to these antigens.
Human blood contains a genetically complex rhesus (Rh) blood group system. For example, humans are either RhD positive or negative and this can lead to problems during transfusions or pregnancy when RhD negative individuals are exposed to RhD positive blood and become immunised to produce anti-D.
The most important allele in the RhD blood group system is the D antigen. The RhD antigen is carried by the RhD protein which is a transmembrane protein consisting of 417 amino acids with 12 putative transmembrane domains and 6 extracellular loops. A series of peptides have been constructed in the present invention based on the RhD protein each being 15 amino acids (AA) long, and tested in vitro against T-lymphocytes from normal individuals, donors who have been alloimmunised to produce anti-D, and patients with warm type autoimmune haemolytic anaemia.
The full amino acid sequence of the RhCE polypeptide and the differences in sequence for c, e and D polypeptides is shown in FIG. 1 hereinafter (Reference: The Blood Group Antigen Facts Book, p 94, Editors; M E Reid & C Lomas-Francis, Academic Press London).
The complexity of the blood system can cause problems during pregnancy when a woman who is RhD negative is carrying a RhD positive fetus, as the woman is at risk of being immunized by the RhD positive blood cells of her own baby. This immunisation can take place during situations when the mother's and baby's blood can become mixed, for example during amniocentesis, antepartum haemorrhage but mainly at parturition.
Once the mother's immune system has been exposed to RhD positive blood cells, she will produce anti-D antibodies which can cross the placenta and cause Rh haemolytic disease in any subsequent RhD positive pregnancies. Such haemolytic disease can be fatal for the neonate.
Currently, purified anti-D immunoglobulin is injected whenever a mother is exposed to fetal RhD positive red blood cells which may occur during e.g., amniocentesis, antepartum haemorrhage but mainly at parturition. About 17% of Caucasian women are RhD negative so that most industrialized countries have RhD prevention programmes wherein all RhD negative women receive prophylaxis with anti-D immunoglobulin at delivery or in association with the other high risk events alluded to above. Further in many countries, routine antepartum prophylaxis to minimize the incidence of Rh haemolytic disease is practised.
There are a number of problems with this approach. In the first place efficacy is never entirely complete since events can be missed or undeclared or a fetal haemorrhage can be larger than the anti-D can neutralize. Secondly, current anti-D immunoglobulin comes from deliberately immunised donors, which puts volunteers, often male (paid or not) at some small risk. In addition it takes at least 12 months to accredit the donors during which time their blood products are not available. For these reasons there is a worldwide shortage of anti-D immunoglobulin. Finally, there are also concerns about the safety of recipients who may be exposed to transfusion transmitted infections such as by inadvertent infection with agents, for example variant Creutzfeld-Jacob Disease (vCJD) for which there is no satisfactory test.
Other groups that can be at risk from alloimmunisation are those who are regular recipients of bloods products, for example those suffering from haemological malignant disease, sickle cell disease or thalassemia.
Certain RhD peptides have been found to specifically stimulate the helper T-cells of alloimmunised individuals. Conversely, certain RhD peptides have been found to stimulate the production of immunosuppressive cytokines by helper T-cells. There is furthermore some correlation between the HLA-DR type of allo- and auto-immunised donors and the peptides which stimulate helper T-cell responses.
An object of the present invention is to provide an effective treatment for subjects that have become alloimmunised or have an autoimmune disease against red blood cells.
A further objective of the invention is to provide an effective prophylactic to prevent alloimmunisation.
In a first embodiment of the invention there is provided a pharmaceutical composition for the prevention of alloimmunisation of a subject, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.
We have mapped helper T-cell epitopes on the RhD protein. The characterization of a helper epitope that is targeted in most alloimmunised donors and the identification of correlations between HLA-DR type and particular dominant epitopes opens the way for the evaluation of peptide immunotherapy as a novel way to regulate the immune response to RhD and to prevent Rh haemolytic disease and anti-D related transfusion problems.
Currently, anti-D which is given to pregnant women during significant events in pregnancy may be considered as a passive form of immunotherapy because it has the effect of blocking the effects of immune events on a temporary basis.
The replacement of passive with active peptide immunotherapy in RhD negative women is an attractive option since safe synthetic tolerogens can be developed and given before pregnancy thus avoiding foetal exposure. Suppression throughout pregnancy would mean that only one injection was necessary, considerably simplifying management of RhD negative women and also it may be possible for the first time to reverse rather than prevent alloimmunisation by administration of tolerogenic peptides to individuals who already have produced anti-D with the objective of “switching-off” the immune response to RhD.
Tolerogenic peptides to other Rh antigens, as determined by the current invention, would be of equivalent value in preventing, or modifying the production of alloantibodies by the respective antigens, including (but not exclusively) RhC, Rhc, RhE and Rhe; and Rh50 (peptides are shown in Table 4) in autoimmune haemolytic anaemia.
Accordingly the categories of individual in whom prior immunization would be considered are as follows: —

- (1) All women during their child bearing years; and
- (2) regular recipients of blood products; who might be exposed to blood transfusion for example haemological malignant disease, sickle cell disease and thalassemia.

Such a pharmaceutical composition can be given to expectant mothers with RhD negative blood and a RhD positive child in this respect, the composition would result in the mother not producing an immune response at any occasion when the fetuses blood comes in contact with her own immune system. In this connection, there is a reduced likelihood that any subsequent baby which is RhD positive would suffer from haemolytic disease.
The use of synthetic peptides in accordance with the present invention removes concerns about viral infection being transmitted either by anti-D immunoglobulin used for passive immunotherapy or by red blood cells given to volunteer recipients. The time consuming and expensive procedures required to validate accredited donors and donations are also important considerations.
In addition, by use of these compositions, volunteers who are often RhD negative men, can avoid the usual injection of red blood cells when they are deliberately immunised for the production of anti-D immunoglobulin.
In a second embodiment of the invention there is provided a pharmaceutical composition for the immunosuppression of a response elicited by alloimmunisation of a subject or an autoimmune haemolytic disease, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.
If the immune system of an RhD negative mother has already been in contact with the blood from a RhD positive baby, such a composition can used during subsequent pregnancies with a RhD positive baby to reduce the likelihood of the baby suffering from RhD haemolytic disease.
In addition, such a composition can be given to patients who have accidentally been given an RhD positive blood transfusion when they are RhD negative. In this connection, the availability of such a composition reduces the need for very large doses of anti-D immunoglobulin for prophylaxis and the likelihood of becoming alloimmunised thereafter.
Preferably autoimmune disease is idiopathic or secondary autoimmune haemolytic anaemia mediated by ‘warm-type’ autoantibodies. The trigger for this autoimmune disease is unknown and therefore it may occur at anytime and results in the body producing autoantibodies of broad Rh group specificity which attack the bodies own red blood cells.
Conveniently the rhesus protein is either RhD, RhC, Rhc, RhE or Rhe protein.
These determine the main Rh-specific antigens found on the surface of a red blood cell.
In a preferred embodiment an epitope selected from at least one of numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
The aforementioned are the most common epitopes recognised by T-cells of alloimmunised subjects and those suffering from autoimmune haemolytic anaemia. In autoimmune haemolytic anaemia, the preferred epitopes are 2, 5, 14, 29, 31 and 38. Therefore induced tolerance to such epitopes would stop an immune response being mounted if they appear in the blood of the subject.
Preferably the epitope is either epitope 12A or 29 since epitope 12A is the most common epitope recognised by alloreactive T-cells, epitope 29 is most commonly recognised in autoimmune haemolytic anaemia.
Conveniently any of the epitopes or immunoreactive derivatives can be synthesised.
If the epitope sequences are artificially synthesised microbial contamination is negligible.
In a third embodiment of the invention there is provided a pharmaceutical composition for the induction of alloimmunisation of a subject, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof disposed in a pharmacologically acceptable vehicle.
Preferably the rhesus protein is either RhD, RhC, Rhc, RhE or Rhe protein, conveniently an epitope selected from at least one of numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
Preferably the vehicle is selected such that the composition is in an injectable, oral, rectal, topical or spray-uptake form.
It is known that mammals may be tolerised to certain stimuli by taking in specific peptide fragments, for example from the nasal mucosa or via the gut. We propose that a good way of abolishing the immune response to RhD in recipient females prior to, during, or after pregnancy is to administer rhesus peptides via the mucosa such as the nasal, buccal, or anal mucosa or transdermally. The peptide fragments in accordance with the present invention will enter via mucosal tissues and effectively tolerise the subject without causing a full blown immune response which may well be the case should the peptide fragments of the present invention reach circulating blood system at the first instance.
In an injectable form the epitopes can be used to deliberately immunise the subject with an epitope which can for example produce IL-10 or TGF-β which have immunosuppressive effects.
The outcome of this approach is to develop a “vaccine” using Rh epitopes which will suppress the immune response to Rh proteins.
In a fourth embodiment of the invention there is provided a tolerising peptide fragment disposed in a pharmacologically effective vehicle, said vehicle being adapted for injection, oral, rectal via a suppository, topical or spray-uptake administration to the subject wherein the tolerising peptide fragment is selected from an epitope of either a RhD, RhC, Rhc, RhE or Rhe protein. Preferably the epitope is selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
Thus the pharmaceutically acceptable vehicle may be adapted for transdermal or transmucosal administration or wherein said vehicle may be a formulation with an enteric coating for oral administration.
In a fifth embodiment of the present invention there is provided a method of tolerizing a subject which comprises administering to said subject a tolerising peptide fragment.
In a sixth embodiment of the present invention there is provided an epitope from a RhD, RhC, Rhc, RhE or Rhe protein selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39.
In a seventh embodiment of the present invention there is provided the use in the manufacture of a medicament for the tolerisation of a patient who may become alloimmunised comprising an epitope selected from a RhD, RhC, Rhc, RhE or Rhe protein or selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 disposed in a pharmaceutically acceptable vehicle therefor.
In an eighth embodiment of the invention there is provided the use in the manufacture of a medicament for the immunosuppression of an alloimmunised patient or a patient with warm-type autoimmune haemolytic anaemia comprising an epitope selected from a RhD, RhC, Rhc, RhE or Rhe protein or selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 disposed in a pharmaceutically acceptable vehicle therefor.
In a ninth embodiment of the invention there is provided a method for determining the effect of an epitope from a rhesus protein on a human lymphocyte, in vitro, comprising the steps of:—
a) stimulating the lymphocyte with one or more epitope of a rhesus protein;
b) between 4 and 7 days later resuspending the cultures and transferring aliquots into plates prepared in the following manner;
c) washing the plate at least once with Hanks Buffered Salt Solution (HBSS);
d) coating each well in the plate with monoclonal anti-cytokine capture antibody;
e) blocking any non-specific binding using an appropriate solution;
f) incubating the plates with the lymphocyte culture for 12-36 hours at 30-40° C. in an atmosphere of substantially 5% CO₂and substantially 95% air;
g) washing the plates at least once with Tween/PBS;
h) introducing an appropriate biotinylated monoclonal detection antibody to each well and incubating for 30-60 min at room temperature;
I) washing the plates at least once with Tween/PBS;
j) introducing ExtrAvidin-alkaline phosphatase conjugate and incubating for 15-45 mins;
k) washing the plates again at least once with Tween/PBS;
l) developing the plates with p-nitrophenyl phosphate in 0.05M carbonate alkaline buffer pH9.6 added to each well; and
m) reading the absorbance at 405 nm.
Traditionally, among other techniques, researchers have used a captive assay called ELISPOT to determine the amount of cytokines produced by a cell. This assay produces a colour spot for each cytokine producing cell. A crude calculation based on the number of coloured spots is then used to estimate the amount of cytokines produced. The use of p-nitrophenyl phosphate in the present assay allows the amount of cytokine captured by the antibody in the wall to be established on the basis of the colour change produced which can be measured by the more accurate method of spectrophotometry.
Accordingly, this method is very sensitive and therefore can identify that a particular RhD protein is capable of stimulating human T-cells to produce potentially immunosuppressive cytokines rather than to proliferate. This is important for the determination of the method of delivery of an epitope. An epitope which leads to T-cell proliferation may be given as a tolerogen through the nasal or mucosal route whereas an epitope which leads to immunosuppressive cytokines may be injected.
In a tenth embodiment of the present invention there is provided a method for the determination of the propensity of a RhD negative subject to produce anti-D antibodies after exposure to RhD positive blood comprising ascertaining the tissue type of the subject and determining if they are HLA-DRB1*15.
If the subject has a tissue type of HLA-DRB1*15 they are more likely to raise anti-D antibodies therefore they should be given treatment before being put at risk of exposure to RhD positive red blood cells.

The invention will now be described, by way of illustration only, with reference to the following examples and the accompanying figures.

FIG. 1 shows the full amino acid sequence of RhCE polypeptide; differences in the sequence for Rhc, Rhe and RhD polypeptides are also shown (Reference: The Blood Group Antigen Facts Book P94, Editor; M E Reid & C Lomas-Francis, Academic Press London).

FIG. 2 shows the distribution of stimulatory RhD peptides in donors alloimmunised with RhD antigen from peptides 1 to 42 and 6A to 40A as per Tables 1, 2 and 3; x—RhD peptide added to culture; y—percentage of subjects responding to specific RhD peptides.

FIG. 3A shows the distribution of stimulating RhD peptides in autoimmune haemolytic anaemia patients; x—RhD peptide stimulus; y—percentage of subjects responding to specific RhD peptides.

FIG. 3B shows the distribution of stimulating RhD peptides in normal controls; x—RhD peptide stimulus; y—percentage of subjects responding to specific RhD peptides.

FIG. 4 shows the correlation between Rh epitopes recognised in donors sharing a tissue type. X and Y axes represent the stimulation indices for

donors

1 and 2 respectively. Each square represents the response to a peptide. Correlation co-efficient (R)=0.774, p value 9.57E-015

FIG. 5A shows the response pattern to the induction of TGF-β production of T-cells after incubation with Rh peptides; x—RhD peptide stimulus; y—TGF-β1 secretion (pg/ml). Value D=none.

FIG. 5B shows the response pattern to the induction of IL-10 production of T-cells after incubation with Rh peptides; x—RhD peptide stimulus; y—IL-10 secretion (ng/ml). Value D=none.

FIG. 5C shows the response pattern to the induction of IFN-γ production of T-cells after incubation with Rh peptides; x—RhD peptide stimulus; y—IFN-γ secretion (ng/ml). Value D=none.

FIG. 5D shows the amount of incorporation of ³H-Thymidine into T-cells after incubation with Rh peptides; x—RhD peptide stimulus; y—³H-Thymidine incorporation (mean CPM×10⁻³±SD) SI=3. Value D=none.

FIG. 6 shows the inhibition of T-cells that respond to RhD protein by peptides that generate an immunosuppressive cytokine response; x—RhD peptide stimulus; y—³H-Thymidine incorporation (mean CPM×10⁻³±SD). A—none; B—control (−); C—RhD; D—RhD & 16; E—RhD & 22; F—RhD & 24; G—none; H—PPD; I—PPD & 16; J—PPD & 22; K—PPD & 24.

EXAMPLE 1

Two complete panels of 68 15-mer peptides, with 5 or 10 amino acid overlaps, were synthesized (Multiple Peptide Service, Cambridge Research Biochemicals, Cheshire, UK and Dept. Of Biochemistry, University of Bristol, UK), corresponding to the sequences of the 30 kD Rh proteins associated with expression of the RhD or RhCc/Ee blood group antigens respectively. The amino acid sequences for each of these proteins were deduced independently from cDNA analyses by 2 laboratories. Since the two polypeptide sequences show 92% homology, 16 of the synthetic peptides were shared between the panels (numbering from the amino terminus, peptides 1-5, 8, 9, 14, 21, 28, 29, 37-39, 41 and 42). In order to ensure purity, each panel was synthesized by fluorenylmethoxycarbonyl chemistry on resin using a base-labile linker, rather than by conventional pin technology, and randomly selected peptides were screened for purity by HPLC and amino acid analysis. The peptides were used to stimulate cultures at 20 μg/ml, although it should be noted that the responses of the cultures had previously been shown to be similar in magnitude and kinetics at peptide concentration between 5-20 μg/ml.
The control antigens Mycobacterium tuberculosis purified protein derivative (PPD) (Statens Seruminstut, Denmark) and keyhole limpet hemocyanin (KLH) (Calbiochem-Behring, La Jolla, Calif., USA) were dialysed extensively against phosphate buffered saline pH 7.4 (PBS) and filter sterilized before addition to cultures at 50 μg/ml, PPD, but not KLH, readily provokes recall T-cell responses in vitro, since most UK citizens have been immunised with BCG. Concanavalin A (Con A) was obtained from Sigma, Poole, Dorset, UK, and used to stimulate cultures at 10 μg/ml.
Antibodies
FITC- or phycoerythrin-conjugated in Abs against human CD3, CD19, CD45 or CD14 were obtained from Dako UK Ltd. Blocking mAbs specific for HLA-DP, -DQ, or -DR supplied by Becton Dickinson (Oxford, UK) were dialysed thoroughly against PBS before addition to cultures at the previously determined optimum concentration of 2.5 μg/ml.
Isolation of Peripheral Blood Mononuclear Cells and T-Cells
Peripheral blood mononuclear cells (PBMC) from donors or patients were separated from fresh blood samples using Ficoll-Hypaque. The donors and patients had become alloimmunised with RhD positive blood either through pregnancy, a blood transfusion or through immunization with the relevant blood.
The viability of PBMC was greater than 90% in all experiments, as judged by trypan blue exclusion. T-cells were isolated from PBMC by passage through glass bead affinity columns coated with human IgG/sheep anti-human IgG immune complexes. Flow cytometry (Becton Dickinson FACScan) demonstrated that typical preparations contained more than 95% T-cells.
Cell Proliferation Assays
PBMC were cultured in 100 μl volumes in microtitre plates at a concentration of 1.25×10⁶cells/ml in an Alpha Modification of Eagle's Medium (ICN Flow, Bucks UK) supplemented with 5% autologous serum, 4 mM L-Glutamine (Gibco, Paisley, UK), 100 U/ml sodium benzylpenicillin G (Sigma), 100 μg/ml streptomycin sulphate (Sigma), 5×10⁻⁵M 2-mercaptoethanol (Sigma) and 20 mM HEPES pH7.2 (Sigma). All plates were incubated at 37° C. in a humidified atmosphere of 5% CO₂/95% air. The cell proliferation in cultures was estimated from the incorporation of ³H-Thymidine in triplicate wells 5 days after stimulation with antigen as described previously. Proliferation results are presented either as the mean CPM+/−SD of the triplicate samples, or as a stimulation index (SI), expressing the ratio of mean CPM in stimulated versus unstimulated control cultures. An S1>3 with CPM>1000 is interpreted as representing a positive response.
Activation Assay
The aforementioned experiments were designed to minimise the response by quiescent or naive T-cells that can recognise RhD protein, but which are not activated by immunisation. To validate the experiments, the T-cells proliferated in the aforementioned experiment were tested using a modification of the method set out in European Journal of Immunology (1994) 24: 1578-1582 to identify if they had been activated in vivo. In this connection, the T-cells were screened to ascertain if they were from the subset bearing CD45RO which is a marker of previous activation or “memory”, rather than from the subset bearing CD45RA which is the marker of quiescent or “naive” T-cells.
As shown in FIG. 2 various peptide fragments have been selected in accordance with their particular peptide sequences. These are given in Tables 1, 2 and 3 which follow and the results achieved by means of the foregoing example are shown in FIG. 2.
Accordingly we have shown that helper T-cells from all donors deliberately immunised against RhD can be stimulated in vitro by RhD peptides.
Further there is a variation between alloimmune donors in the T-cell response profile to the RhD peptides. Despite these variations, RhD peptides Nos. 2, 6, 6A, 10A, 11, 11A, 12, 12A, 15A, 18A, 28 and 39 are most commonly targeted and a proliferative response was elicited by peptide 12A in 70% of donors. However significantly related profiles are found in donors sharing HLA-DR alleles. It is predicted that alloreactive T-cell epitopes on the RhD protein would comprise sequences that are foreign to RhD-negative individuals, and would thus not be carried on the related RhCc/Ee protein that is expressed on the erythrocytes of such individuals. With the exception of peptide 28, all of the fragments identified are sequences that fulfil this prediction. It is therefore considered that such peptides, or derived sequences, could be used to stimulate either T-cell response or tolerance in vivo as desired, depending on the route of administration and/or the dose and formulation of the preparation.
The T-cells which were proliferated were in fact drawn from those that have been previously activated. This is important because it is these cells which will drive anti-D antibody production in RhD-negative donors immunised with RhD.
It follows that the characterisation of the putative helper T-cell epitopes we have identified is a key step in the development of safe immunogens for anti-immunoglobulin donors and opens the way to the evaluation of peptide immunotherapy as a novel approach to the prevention of haemolytic disease inter alia in neonates.
These experiments can be carried out using other rhesus proteins, such as RhC, Rhc, RhE or Rhe protein.
The aforementioned experiments were repeated using blood from subjects suffering from autoimmune haemolytic anaemia. It was therefore established that the T-cells of the subjects exhibited a proliferative response to peptides 2, 5, 14, 29, 31 and 38 (see FIG. 3) and 65% of patients responded to peptide 29. The results also showed a correlation between patients suffering from autoimmune haemolytic anaemia and having tissue type HLA-DR15. With the exception of peptide 31 all of the peptides are shared in common between the RhD and RhCe/Ee proteins.

EXAMPLE 2

The HLA class II tissue type of the donors tested in Example 1 was ascertained by standard SSP-PCR methods. This was carried out because the molecules that determine tissue type select and bind antigenic peptide fragments for display to T-cells therefore they are important in this investigation.
The techniques described in Barker et al (1997) Blood 90:2701-2715 were used to determine that the HLA-D loci was more important than either the HLA-DP or HLA-DQ in the presentation of Rh D peptide fragments that stimulate T-cells in vitro.
A significant proportion of Rh D-negative donors selected for responsiveness to Rh D carry the HLA-DRB1*15 gene (56% versus approx. 29% in a control population). Thus carrying this tissue type is associated with an increase risk of producing anti-D antibodies after exposure to RhD positive erythrocytes, and there is smaller variation in HLA-DR tissue type among responders than in the general population. It has also been shown that the patterns of RhD peptides that elicit T-cell proliferation are significantly related in Rh D-negative donors who share the same HLA-DR type (see FIGS. 3A and 3B).
For warm-type autoimmune haemolytic anaemia there is also an association with HLA DR15 with 65% of patients carrying this HLA type.
Nevertheless, a statistical analysis of all the data shows that the effect of HLA-DR type on the identity of the peptides recognised is relatively weak. In other words, many of the RhD peptides stimulate T-cells regardless of tissue type.
These analyses demonstrate that the selection of RhD peptide fragments for immunisation/tolerisation regimes may not be dependent on prior tissue typing of recipients, an important practical consideration for the clinical application of this approach.

EXAMPLE 3

Cultured T-cells are stimulated with each of the epitopes given in Tables 1 to 3 and after 5 days the responding cells were transferred to a flat-bottomed microtitre plates (96-well Nunc-Immuno Maxisorp) coated with 50 μl per well of monoclonal anti-cytokine capture antibody diluted in 0.05M alkaline carbonate coating buffer pH 9.6. Unbound capture antibody was removed by two washes with HBSS and non-specific binding potential blocked by incubation with 200 μl per well of phosphate buffered saline, pH 7.4 (PBS containing 3% BSA).
Five days after stimulation, lymphocyte cultures were mixed to resuspend the cells and duplicate 100 μl aliquots were transferred into wells coated with the respective capture antibody specific for IFN-γ and or IL-10 or TGF-β. The plates coated with capture antibodies and layered by lymphocytes were then incubated for a further 24 hours at 37° C. in a humidified atmosphere of 5% CO₂and 95% air. After this incubation the PBMC were removed by four washes with 0.2% Tween/PBS. One hundred microlitre aliquots of the appropriate biotinylated monoclonal detection antibody in 0.2% BSA/PBS were then added to the wells and incubated at room temperature for 45 minutes. After six washes with 0.5% Tween/PBS, 100 μl of 1:100,000 ExtrAvidin-alkaline phosphatase conjugate (Sigma) was then added to each of the wells and incubated at room temperature for 30 minutes. The ExtrAvidin conjugate was removed by eight washes with 0.2% Tween/PBS, and the plates developed using 100 μl per well of p-nitrophenyl phosphate (Sigma) 1.0 mg/ml in 0.05M carbonate alkaline buffer pH 9.6. The absorbence of 405 nm was then measured using a Multiscan plate reader (Labsystems Basingstoke UK).
Cytokine secretion was measured by interpolation from a standard curve generated by incubating duplicate wells with doubling dilutions of recombinant human IFN-γ or IL-10 or TGF-β (Pharmingen). The standard curves were modelled by a smoothed cubic spline function applied to the absorbence reading and the cytokine concentrations after a quasilogarithmic transformation, where:
quasilog_e(z)=log_e [z+√[z ²+1]).
This method is very sensitive and therefore can identify that a particular RhD peptide is capable of stimulating human T-cells to produce potentially immunosuppressive cytokines rather than to proliferate.
From FIGS. 5A and 5B it can be seen that epitopes 10, 16, 22, 24 and 34 induce IL-10 and/or TGF-β production by human T-cells. IL-10 and TGF-β molecules are known to have immunosuppressive properties. In preliminary experiments RhD peptides that induce IL-10 have been shown to inhibit T-cell proliferation in response to the entire RhD protein in vitro. Accordingly, prior administration of RhD peptides that elicit T-cell IL-10 or TGF-β production can be used to prevent RhD negative individuals from responding to RhD. It is also possible to inhibit established responses. This novel approach to manipulating the immune system has other application, including treatment of warm-type autoimmune haemolytic anaemia, in which the Rh proteins are important targets. The identification of peptides with similar properties derived from other antigens could also lead to therapy for a wide range of autoimmune diseases where the antigens/proteins are identified.
No IL-4 production was detected in any culture. In FIG. 5C it can be seen that epitopes 5, 21 and 27 stimulate IFN-γ secretion. FIG. 5D shows the level of incorporation of ³H-Thymidine into the T-cells after stimulation with the RhD peptides.
From FIG. 6 it can be seen that the addition of such peptides to T-cell cultures specifically blocks the proliferative response to the RhD protein, but not to a control antigen PPD. This result is very important since it raises the possibility that these peptides may also be able to inhibit damaging responses in vivo if given to patients, whilst not suppressing the rest of the immune system.

TABLE 1

PEPTIDE NUMBER	PEPTIDE SEQUENCE	RESIDUES

RhCE (R2 cE)

1	SSKYPRSVRRCLPLW	2-16

2	CLPLWALTLEAALIL	12-26

3	AALILLFYFFTHYDA	22-36

4	THYDASLEDQKGLVA	32-46

5	KGLVASYQVGQDLTV	42-56

6	QDLTVMAALGLGFLT	52-66

7	LGFLTSNFRRHSWSS	62-76

8	HSWSSVAFNLFMLAL	72-86

9	FMLALGVQWAILLDG	82-96

10	ILLDGFLSQFPPGKV	92-106

11	PPGKVVITLFSIRLA	102-116

12	SIRLATMSAMSVLIS	112-126

13	SVLISAGAVLGKVNL	122-136

14	GKVNLAQLVVMVLVE	132-146

15	MVLVEVTALGTLRMV	142-156

16	TLRMVISNIFNTDYH	152-166

17	NTDYHMNLRHFYVFA	162-176

18	FYVFAAYFGLTVAWC	172-186

19	TVAWCLPKPLPKGTE	182-196

20	PKGTEDNDQRATIPS	192-206

21	ATIPSLSAMLGALFL	202-216

22	GALFLWMFWPSVNSP	212-226

23	SVNSPLLRSPIQRKN	222-236

24	IQRKNAMFNTYYALA	232-246

25	YYALAVSVVTAISGS	242-256

26	AISGSSLAHPQRKIS	252-266

27	QRKISMTYVHSAVLA	262-276

28	SAVLAGGVAVGTSCH	272-286

29	GTSCHLIPSPWLAMV	282-296

30	WLAMVLGLVAGLISI	292-306

31	GLISIGGAKCLPVCC	302-316

32	LPVCCNRVLGIHHIS	312-326

33	IHHISVMHSIFSLLG	322-336

34	FSLLGLLGEITYIVL	332-346

35	TYIVLLVLHTVWNGN	342-356

36	VWNGNGMIGFQVLLS	352-366

37	QVLLSIGELSLAIVI	362-376

38	LAIVIALTSGLLTGL	372-386

39	LLTGLLLNLKIWKAP	382-396

40	IWKAPHVAKYFDDQV	392-406

41	FDDQVFWKFPHLAVG	402-416

42	DDQVFWKFPHLAVGF	403-417

TABLE 2

PEPTIDE NUMBER	PEPTIDE SEQUENCE	RESIDUES

RhCE (R1 Ce)

1 (C)	SSKYPRSVRRCLPLC	2-16

2 (C)	CLPLCALTLEAALIL	12-26

22 (e)	GALFLWMFWPSVNSA	212-226

23 (e)	SVNSALLRSPIQRKN	222-236

RhD
6 (also C)	QDLTVMAAIGLGFLT	52-66

7 (also C)	LGFLTSSFRRHSWSS	62-76

10 (also C)	ILLDGFLSQFPSGKV	92-106

11 (also C)	PSGKVVITLFSIRLA	102-116

12	SIRLATMSALSVLIS	112-126

13	SVLISVDAVLGKVNL	122-136

15	MVLVEVTALGNLRMV	142-156

16	NLRMVISNIFNTDYH	152-166

17	NTDYHMNMMHIYVFA	162-176

18	IYVFAAYFGLSVAWC	172-186

19	SVAWCLPKPLPEGTE	182-196

20	PEGTEDKDQTATIPS	192-206

22	GALFLWIFWPSFNSA	212-226

23	SFNSALLRSPIERKN	222-236

24	IERKNAVFNTYYAVA	232-246

25	YYAVAVSVVTAISGS	242-256

26	AISGSSLAHPQGKIS	252-266

27	QGKISKTYVHSAVLA	262-276

30	WLAMVLGLVAGLISV	292-306

31	GLISVGGAKYLPGCC	302-316

32	LPGCCNRVLGIPHSS	312-326

33	IPHSSINGYNFSLLG	322-336

34	FSLLGLLGEIIYIVL	332-346

35	IYIVLLVLDTVGAGN	342-356

36	VGAGNGMIGFQVLLS	352-366

40	IWKAPHEAKYFDDQV	392-406

TABLE 3

PEPTIDE NUMBER	PEPTIDE SEQUENCE	RESIDUES

RhCE (R1 Ce)

1A (C)	RSVRRCLPLCALTLE	7-21

22A (e)	WMFWPSVNSALLRSP	217-231

RhD
6A (also C)	MAAIGLGFLTSSFRR	57-71

7A (also C)	SSFRRHSWSSVAFNL	67-81

10A (also C)	FLSQFPSGKVVITLF	97-111

11A (also C)	VITLFSIRLATMSAL	107-121

12A	TMSALSVLISVDAVL	117-131

13A	VDAVLGKVNLAQLVV	127-141

15A	VTALGNLRMVISNIF	147-161

16A	ISNIFNTDYHMNMMH	157-171

17A	MNMMHIYVFAAYFGL	167-181

18A	AYFGLSVAWCLPKPL	177-191

19A	LPKPLPEGTEDKDQT	187-201

20A	DKDQTATIPSLSAML	197-211

21A	LSAMLGALFLWIFWP	207-221

22A	WIFWPSFNSALLRSP	217-231

23A	LLRSPIERKNAVFNT	227-241

24A	AVFNTYYAVAVSVVT	237-251

26A	SLAHPQGKISKTYVH	257-271

27A	KTYVKSAVLAGGVAV	267-281

30A	LGLVAGLISVGGAKY	297-311

31A	GGAKYLPGCCNRVLG	307-321

32A	NRVLGIPHSSIMGYN	317-331

33A	IMGYNFSLLGLLGEI	327-341

34A	LLGEIIYIVLLVLDT	337-351

35A	LVLDTVGAGNGMIGF	347-361

39A	LLNLKIWKAPHEAKY	387-401

40A	HEAKYFDDQVFWKFP	397-411

TABLE 4

PEPTIDE NUMBER	PEPTIDE SEQUENCE	RESIDUES

Rh50 GP

1	MRFTFPLMAIVLEIA	1-15

2	VLEIAMIVLFGLFVE	11-25

3	GLFVEYETDQTVLEQ	21-35

4	TVLEQLNITKPTDMG	31-45

5	PTDMGIFFELYPLFQ	41-55

6	YPLFQDVHVMIFVGF	51-65

7	IFVGFGFLMTFLKKY	61-75

8	FLKKYGFSSVGINLL	71-85

9	GINLLVAALGLQWGT	81-95

10	LQWGTIVQGILQSQG	91-105

11	LQSQGQKFNIGIKNM	101-115

12	GIKNMINADFSAATV	111-125

13	SAATVLISFGAVLGK	121-135

14	AVLGKTSPTQMLIMT	131-145

15	MLIMTILEIVFFAHN	141-155

16	FFAHNEYLVSEIFKA	151-165

17	EIFKASDIGASMTIH	161-175

18	SMTIHAFGAYFGLAV	171-185

19	FGLAVAGILYRSGLR	181-195

20	RSGLRKGHENEESAY	191-205

21	EESAYYSDLFAMIGT	201-215

22	AMIGTLFLWMFWPSF	211-225

23	FWPSFNSAIAEPGDK	221-235

24	EPGDKQCRAIVDTYF	231-245

25	VDTYFSLAACVLTAF	241-255

26	VLTAFAFSSLVEHRG	251-265

27	VEHRGKLNMVHIQNA	261-275

28	HIQNATLAGGVAVGT	271-285

29	VAVGTCADMAIHPFG	281-295

30	IHPFGSMIIGSIAGM	291-305

31	SIAGMVSVLGYKFLT	301-315

32	YKFLTPLFTTKLRIH	311-325

33	KLRIHDTCGVHNLHG	321-335

34	HNLHGLPGVVGGLAG	331-345

35	GGLAGIVAVAMGASN	341-355

36	MGASNTSMAMQAAAL	351-365

37	QAAALGSSIGTAVVG	361-375

38	TAVVGGLMTGLILKL	371-385

39	LILKLPLWGQPSDQN	381-395

40	PSDQNCYDDSVYWKV	391-405

41	NCYDDSVYWKVPKTR	395-409

Other Peptides

	BR	SKYPNCAYKTTQANKH

	AV2	TIPEQSFQGSPSADT

	AV4	TVKADFEFSSAPAPD

	AV6	TVEERQQFGELPVSE

	P23	ELKIISRCQVCMKKRH

	HA	PKYVKQNTLKLAT

Claims

1-23. (canceled)

24: A method for determining effect of one or more epitopes from a rhesus protein on a human lymphocyte, in vitro, comprising:

(a) stimulating the lymphocyte with one or more epitope/peptide of a rhesus protein;

(b) between 4 to 7 days later resuspending the cultures and transferring aliquots into plates prepared in the following manner:

(i) coating each well in the plate with monoclonal anticytokine capture antibody;

(ii) washing the plate at least once with Hanks Buffered Salt Solution (HBSS);

(iii) blocking any non-specific binding using an appropriate solution;

(c) incubating the plates with lymphocyte culture for 12-36 hours at 30-40° C. in an atmosphere of substantially 5% CO₂and substantially 95% air;

(d) washing the plates at least once with Tween/PBS to remove unbound lymphocytes;

(e) introducing an appropriate biotinylated monoclonal detection antibody to each well and incubating for 30-60 minutes at room temperature;

(f) washing the plates at least once with Tween/PBS to remove unbound detection antibody;

(g) introducing ExtrAvidin-alkaline phosphatase conjugate and incubating for 15-45 minutes;

(h) washing the plates at least once with Tween/PBS to remove unbound ExtrAvidin-alkaline phosphatase conjugate;

(i) developing the plates with p-nitrophenyl phosphate in 0.05M carbonate alkaline buffer pH 9.6 added to each well; and

(j) reading the absorbance at 405 nm.

25: The method according to claim 24 wherein the rhesus protein is selected from the group consisting of RhD, RhC, Rhc, RhE, Rhe and Rh50 protein.

26: The method according to claim 24 wherein the epitope/peptide of a rhesus protein is selected from at lease one of SEQ ID Numbers 2, 5, 6, 11, 12, 14, 28, 29, 31, 38, 39, 44, 47, 50, 51, 66, 75, 77, 78, 79, 81 and 84.

27: The method according to claim 24 wherein the epitope/peptide is artificially synthesized.

28: The method according to claim 24 wherein the monoclonal anti-cytokine capture antibody is specific for IFN-γ, IL-10 or TGF-β.