Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigens

Veterinary

immunology and immunopathology

Veterinary Immunology and Immunopathology 59 (1997) 191-204

Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigens A. Corato, C-R. Shen, G. Mazza, R.N. Barker, M.J. Day Department

ofPathology

and Microbiology.

UrGersi@

ofBristol.

Langford

BS18 7DU

*

UK

Accepted 12 February 1997

Abstract

Autoimmune haemolytic anaemia (AIHA), one of the most common autoimmune diseases of the dog, is characterised by binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. Failure of self-tolerance with activation of autoreactive T-lymphocytes is thought to play a key role in the initiation of such autoimmune events, Peripheral blood mononuclear cells (PBMC) were obtained from 11 clinically normal dogs, six clinically normal relatives of two littermate dogs which died from AIHA, and four dogs which had recovered from primary AIHA. Cells were stimulated in vitro with a panel of canine RBC-derived antigens (RBC membranes, glycophorin, spectrin, five 1.5-mer glycophorin peptides), the non-recall antigen keyhole limpet haemocyanin (KLH), and the mitogen concanavalin A (Con A). The kinetics of the proliferative responses to specific antigens were assessed by serially sampling the cultures from days 4 to 10. PBMC from all dogs responded strongly to Con A (day 21 and to KLH (maximal response on days 7 to 10) under appropriate culture conditions. Two of 11 normal dogs responded weakly to RBC membranes (mean stimulation index = 4.25). In contrast, PBMC from all dogs recovered from AIHA responded to RBC membranes (mean SI = 9.2 + 2.51 and occasionally to other erythrocyte antigens. Similar responses were recorded with PBMC from dogs related to AIHA cases. It is considered that although normal individuals harbour erythrocyte-reactive lymphocytes, such cells are primed in dogs with AIHA or a genetic susceptibility to this disease. 0 1997 Elsevier Science B.V. Keywords:

Autoimmune;

ITCorresponding

Anaemia;

Dog

author. Tel.: +44

117 92X 9521; fax: +44

117 928 9588; e-mail: [email protected].

0165-2427/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SO165-2427(97)00032-9

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1. Introduction Autoimmune haemolytic anaemia (AIHA) is characterised by the binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. The antibody-coated RBC may be phagocytosed by cells of the reticuloendothelial system (extravascular haemolysisl or may undergo complement-mediated lysis (intravascular haemolysis). AIHA occurs spontaneously in man (Dacie, 1953) and other species including the dog (Miller et al., 1954), cat (Day, 1996a), and New Zealand black (NZB) mouse (DeHeer and Edgington, 1976). There is a well recognised genetic influence on the development of canine AIHA. Breeds such as the old English sheepdog, cocker spaniel and poodle are predisposed to AIHA and familial disease has been documented (Stewart and Feldman, 1993b). There are numerous similarities in the clinical and immunological features of AIHA in man and the dog, and a range of subtypes of disease are recognised in both species (Stewart and Feldman, 1993a). Most dogs with AIHA rapidly recover from the initial anaemia with immunosuppressive therapy, but erythrocyte autoantibodies may persist for many weeks and clinical relapse is not uncommon (Day, 1996b). In humans, the specificity of the erythrocyte autoantibodies is for molecules bearing the Rhesus (Rh) blood group system (Weiner and Vos, 1963). A panel of synthetic peptides derived from the amino acid sequence of the two 30-kD Rh polypeptides associated with the expression of the D and Cc/Ee Rhesus blood groups has been used in studies of T-lymphocyte specificity in human AIHA. Splenic mononuclear cells (SMC) and peripheral blood mononuclear cells (PBMC) from patients with AIHA and Rh-specific autoantibodies, proliferated in vitro when stimulated by peptides containing cryptic epitopes of the Rh molecule (Barker et al., unpublished data), and primary in vitro Rhesus-peptide specific T-cell responses have also been demonstrated in normal individuals (Barker and Elson, 1994). Similarly, the integral RBC membrane protein Band 3 has recently been shown to be the major target antigen for pathogenic autoantibodies in the NZB mouse (Barker et al., 1993). Culture of splenic T-cells from NZB mice with purified Band 3 resulted in marked proliferation of that subpopulation expressing CD4 (Perry et al., 1996). Examination of the cytokine profile of the proliferating T-cells, and of the isotype of the RBC-bound autoantibody has provided evidence that cells of the Th 1 type dominate this autoimmune response (Shen et al., 1996). In canine AIHA, erythrocyte autoantibodies have been well characterised by the Coombs’ test (Mills et al., 1985) and the direct enzyme-linked antiglobulin test (DELAT; Jones et al., 1987). The IgG subclass distribution (Day, 1996~) and specificity (Tsuchida et al., 1991) of the autoantibodies has been examined, and in one study, the autoantibodies eluted from the RBC of dogs with AIHA were shown to react predominantly with erythrocyte glycophorin (in four of five cases), or less frequently with Band 3 (one of five cases) (Barker et al., 1991). In all species studied (man, mouse and dog), antibodies reactive with the cytoskeletal molecule spectrin have been demonstrated in AIHA and in clinically normal individuals, where such antibodies are likely involved in the physiological clearance of senescent erythrocytes (Lutz and Wipf, 1982). One preliminary study has been performed to examine T-cell responses in canine

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19.3

AIHA. Peripheral blood T-cells from a dog with AIHA were cultured with RBC membrane components fractionated by SDS-PAGE, transferred to nitrocellulose by western blotting and solubilised in DMSO (Barker and Elson. 1995). After four days of culture, proliferation was observed in response to the fractions corresponding to the erythrocyte glycophorins, to Band 3 and to spectrin. In contrast, PBL from three healthy dogs failed to respond, with the exception of one individual that responded to the fraction containing spectrin. The aim of the present study was to extend these observations by defining the kinetics and specificity of the T-cell response to RBC antigens in normal dogs and dogs with AIHA, in order to better understand the immunological mechanisms underlying the disease.

2. Materials and methods 2.1. Samples

Four groups of dogs were used as the source of peripheral blood mononuclear cells (PBMC) in this study. Group 1 (Table 1) comprised four dogs in which primary AIHA was diagnosed on the basis of history, clinical signs, results of haematological tests and a positive Coombs’ test. Samples were taken from these dogs during a period of remission of clinical signs and Coombs’ negativity (3 to 7 months after the initial diagnosis) and suspension of immunosuppressive therapy (prednisolone 2 mg/kg daily; withdrawn at least 8 weeks before sampling). Only dog 3 had received a blood transfusion (500 ml whole blood) as part of the initial therapy for AIHA. The donor blood was cross-matched and was from a dog that was negative for expression of the blood group antigens DEA 1.1, 1.2 and 7. By sampling at this time, any potential effect of immunosuppressive therapy on cellular activity was minimised, and the proliferative assays would be most likely to reflect restimulation of lymphocytes primed in vivo during the disease phase. Group 2 consisted of two clinically normal siblings of two male border collie littermate dogs that each died from AIHA at the age of 6 and 7 years, respectively; and four dogs from a second litter following a repeated mating of the same dam and sire. The two dogs from the same litter (both males) were 7.5 years of age, and the four dogs from the second litter (two males and two females) were 6.5 years old. Group 3 comprised six healthy dogs, admitted for routine screening or surgery. These dogs were aged 1 to 14 years (mean = 8.5 + 4.5) and included 3 males (1 neutered) and 3 females. Group 4 comprised five clinically normal laboratory beagles (1 female and 4 male), aged 3 months. Dogs in all groups had received a routine course of vaccination for viral diseases. Blood samples (15-40 ml) were collected by cephalic or jugular veinepuncture into hepatin anticoagulant, and a smaller sample was taken into EDTA for haematological analysis.

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2.2. Antigens Canine RBC membranes were prepared by hypotonic lysis (Dodge et al., 1963), resuspended in a minimum amount of sterile PBS and stored at -20°C until used. Glycophorin was prepared from canine RBC membranes according to the method of Marchesi and Andrews (1971) by extraction with lithium diiodosalicylate and partition in aqueous phenol. After dialysis against phosphate buffered saline (PBS, pH 7.4, 0.01 M), the glycophorin preparation was concentrated (Minicon-B, Amicon, Stonehouse, UK) and analysed by SDS-PAGE and PAS staining (Barker and Elson, 1995). The glycophorin preparation used here was distinct from the major canine blood group antigens that migrated within a different molecular weight range after immunoprecipitation from the erythrocyte surface (Corato et al., 1997 (in press)). Spectrin was extracted from dog RBC membranes using a low ionic strength solution according to the method of Ungewickell and Gratzer (1978), and checked for purity by SDS-PAGE. A panel of five 15-mer peptides overlapping by 5 amino acids (Table 2) were derived from the published sequence for canine glycophorin (Murayama et al., 1983) and synthesised by a multiple-peptide solid-phase synthetic method using Fluorenylmethoxycarbonyl-polyamide (Fmoc) chemistry (Peptide Synthesis Service, MR Centre, Bristol, UK). Following synthesis, completed peptides were cleaved from the resin and side chain protection was removed by using trifluoroacetic acid (TFA) in the presence of the appropriate scavenger. Peptides were precipitated with diethylether and extracted. The peptide thus obtained was either stored as a powder at -70°C or dissolved in PBS at a Purity of peptides was checked concentration of 4 mg/ml and stored at -70°C. randomly by high-pressure liquid chromatography (HPLC). Concanavalin A (Con A) was obtained from Sigma (Dorset, UK) and keyhole limpet haemocyanin (KLH) was obtained from Calbiochem-Behring (CA, USA). Canine Distemper virus, Parvovirus and Parainfluenza virus vaccines were a kind gift from Intervet (Cambridge, UK). 2.3. Coombs’ test The Coombs’ test was performed in duplicate at 37°C and at 4°C using a microtitre system. Erythrocytes from each dog were washed once in normal saline and twice in

Table 3 Peptides derived from the amino-terminal Peptide

sequence of dog glycophorin

Amino acid sequence EDVTEIIPHQISSKL

2

HQISSKLPTQAGFIS

3

AGFISTEDPSFNTPS FNTPSTREDPSGTMY

4 5 Areas of overlap are indicated

SGTMYQHLPDGGQK by underlining

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PBS and resuspended at 2% in PBS. The test was performed using a polyvalent Coombs’ reagent with specificity for canine IgG, IgM and C3 (ICN Pharmaceuticals, Thame, Oxfordshire, UK), rabbit anti-dog IgG (Fc), rabbit anti-dog IgM (Fcl and goat anti-dog C3 (all from Nordic Laboratories; Tilburg, Netherlands). All reagents were absorbed against a pool of normal canine erythrocytes and titrated in PBS from l/5 across the rows of round-bottomed microtitre trays. Equal volumes of the 2% erythrocyte suspension were added to each well and to control wells containing PBS alone. The plates were incubated at the appropriate temperature until erythrocytes in the control wells had settled. Agglutination was assessed visually and the titre recorded. 2.4. Isolation of peripheral

blood mononuclear

cells and cell proliferation

assays

Peripheral blood mononuclear cells (PBMC) were obtained by centrifugation of fresh whole blood diluted 1 in 2 in Hanks’ balanced salt solution without calcium and magnesium (Life-Technologies, Gibco BRL, Scotland UK) on Histopaque- 1077 (Sigma), at 1800 rpm for 40 min. The viability of cells was judged by trypan blue exclusion (> 95% viability in all cases). PBMC were cultured in l- or 2-ml volumes, in 48 or 24 well flat-bottomed plates respectively (Life-Technologies, Gibco BRL, Scotland UK), at a concentration of 1.25 X lo6 cells/ml in the Alpha Modification of Eagle’s Medium (ICN Flow, Bucks, UK) supplemented with 5% decomplemented dog serum, 4 mM L-glutamine (Gibco, Paisley, UK), 100 U/ml sodium benzylpenicillin G (Sigma), 100 pug/ml streptomycin sulphate (Sigma), 5 X 10e5 M 2-mercaptoethanol (Sigma) and 20 mM HEPES, pH 7.2 (Sigma). All plates were incubated at 37°C in a humid atmosphere of 5% CO,/95% air. RBC membranes were used at a concentration of 100 pg/ml; spectrin and glycophorin at 10 pug/ml, Con A at 2 pg/ml, KLH at 10 pug/ml, and each peptide at 10 pg/ml. Distemper virus was used at 3 X lo3 TClD,,, Parvovirus at 8 X lo”.’ TCID,, and Parainfluenza virus at 3 X 103.5 TCID,, to stimulate cells from dogs in Group 1 and Group 2 (Distemper virus only). The decision to culture in l- or 2-ml volumes, and the number of antigens included in the panel for each dog was determined by the volume of the initial blood sample provided by the attending veterinary surgeon, and the yield of viable lymphocytes obtained. Duplicate 100 ~1 samples were withdrawn from the wells over a period of 4-10 days after establishing the cultures. The samples were pulsed for 6 h with 0.5 &i/well of 3H-thymidine (Amersham, Bucks, UK) after which the cells were harvested onto glass-fibre mats (Whatman Labsales, Maidstone, Kent, UK) using a multisample harvester (Skatron, Lier, Norway). The radioactivity incorporated into newly synthesised deoxyribonucleic acid (DNA) was measured with a beta counter (Rackbeta, LKB Wallac, Turku, Finland), Proliferation data are presented as maximum mean counts per minute (cpm) in stimulated and unstimulated control cultures, as well as the stimulation index @I), expressing the ratio of mean counts per minute (CPM) in stimulated, versus unstimulated cultures. A SI > 3 was interpreted as representing a positive response.

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et al.

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3. Results 3.1. Proliferative

responses

of PBMC from Group I dogs

PBMC from four dogs in which primary AIHA was diagnosed were cultured in 2-ml volumes with the panel of RBC-derived antigens, KLH and a range of vaccine antigens (Table 3). PBMC from dog 1 died on day 7 of culture. All dogs in this group made strong proliferative responses to Con A, with a SI ranging between 17- 187 (mean = 73.8 + 76.81 after 2 days of culture. Cells from all dogs made proliferative responses to RBC membranes (mean SI = 9.2 f 2.51 and to KLH (mean SI = 13.7 + 15.6) (Fig. Il. PBMC from three dogs proliferated in response to at least one vaccine antigen. PBMC from dog 4 proliferated in response to glycophorin @I = 11 at day 81 and cells from dog 3 responded to spectrin @I = 6.7 at day 91. 3.2. Proliferative

responses

of PBMC from Group 2 dogs

PBMC from two border collie dogs from the same litter as two animals that died from AIHA were cultured in l-ml volumes with the panel of RBC-derived antigens and Distemper virus vaccine (but not with KLH) and the results are summarised in Table 4. Cells from both dogs responded strongly to Con A. RBC membranes, glycophorin spectrin, peptide and Distemper virus elicited a strong proliferative response in one dog, whereas PBMC from the second dog responded only to spectrin (Table 4). PBMC from four border collie dogs from a subsequent litter (same sire and dam) were cultured in l-ml volumes with the panel of RBC-derived antigens, except in one

Table 3 Proliferative

responses

Case Con A I

2

3

4

12462 (254) SI = 49h day 2’ 19073 (102) SI = 187 day 2 5101(300) Sl= 17 day 2 6816(161) Sl = 42 day 3

of PBMC from Group

RBC membranes 648 (106) SI = 6 day 6 7350 (622) SI=11.8 day 9 1772 (198) s1=9 day 9 1920 (190) SI = 10 day 8

Glycophorin

Sl < 3

SI<3

SI < 3 2070 (190) SI=ll day 8

1 dogs Spectrin

KLH

745 (106) SI = 7 day 6 846 (220) SI = 3.8 SI < 3 day 8 1338 (198) 7413 (198) SI = 37 SI = 6.7 day 9 day 9 1370(190) SI = 7 SI < 3 day 8

Parainfluenza virus

Parvovirus

Distemper virus

n.d.

n.d.

n.d.

SI < 3

SI < 3 2816(198) SI= 14 day 9 7500(190) SI = 30 day 8

2360 (220) SI = 10.7 day 8 1355(198) SI = 6.X day 9 n.d.

SI < 3

SI < 3 nd.

“For each dog, the maximum counts per minute is given and the background cpm is recorded in brackets. ‘The stimulation index @I) obtained after culture with each antigen is also shown. ‘The day of maximal response is given for those cultures in which proliferation occurred. No dog responded any of the glycophorin peptides. n.d. = not done.

to

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h

10

59 (1997) 191-204

krvovirus

--A--spectrin

6

a6 )

4 2 \

9.

0

day 4

day 6

day 7

day 8

day 9

1 day IO

PI 12 IO

t

6 a

6 4

-_-_---_--2 OI-

~~

day 6

_ ._-_,

1

day 7

--

day 6

,

-I

day 9

day 10

Fig. 1. (a) Proliferative response of a dog with AIHA (case 2, Group 1) to RBC membranes, spectrin and parvovirus vaccine antigen. (b) Proliferative response of a dog with AIHA (case 3, Group 1) to RBC membranes, spectrin and parvovirus vaccine antigen.

case where PBMC were cultured in a single 300 ~1 well with glycophorin only, and tested for proliferation at day 6 (due to the small volume of the initial blood sample). Glycophorin elicited a strong proliferative response with cells from three dogs, and responds to glycophorin peptides, RBC membranes and to spectrin were observed (Table 4; Fig. 2). PBMC from only one of these dogs were challenged with canine Distemper virus, and these made a strong proliferative response. KLH was not used in any of these cultures. 3.3. Proliferative

responses

of PBMC from Croup 3 dogs

PBMC from six clinically normal dogs were cultured in l-ml volumes with the complete panel of antigens. All dogs in this group made strong proliferative responses to Con A with a SI ranging from U-150 (mean = 112 + 26) after 2 days of culture. There was no response to any of the erythrocyte antigens, or to KLH. PBMC from one of these dogs were cultured on a second occasion in 2-ml volumes and proliferated to KLH (maximum SI = 4.8 at day IO)

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200 160 140 120

&

qXXztr1n

&

glycophonn

100 B

80 60

40 7.0 0 kY7

kb

day8

day9

day10

Fig. 2. Proliferative responses of a border collie dog (dog 5, Group 2) related to animals which died of AIHA. to RBC membranes. spectrin and glycophorin.

3.4. Prolijbatiue

responses

of PBMC from Group 4 dogs

PBMC from five clinically normal laboratory beagles were cultured in 2-ml volumes with the panel of antigens and the data are summarised in Table 5. All dogs responded to Con A with a SI ranging from 25 to 278 (mean = 116 + 971, and four of five dogs to KLH (SI = 28 to 68, mean = 46 f 17) with maximal response on days 7 to 10 (Fig. 3).

Table 5 Proliferative Dog

responses

of PBMC from Group 4 dogs

Con A

KLH

22589 (811 SI = 278b

6140 (1051 SI = 58 day 10 4649 (681 SI = 68 day 7 8700 (287) SI = 30 day 7 1026 (180) SI = 5.7 day 6 I3 352 (476) SI = 28 dav 8

day 2’ 20742 (323) SI = 64 day 2 10550 (591 SI = 178 day 2 3580 (1401 SI = 25 day 2 I904 (501 SI = 38 dav 2

RBC membranes

SI < 3

SI < 3 1800 (514) Sl= 3.5 day 8 1218 (2431 SI = 5 day 8 Sl < 3

aFor each dog. the maximum counts per minute is given and the background cpm is recorded ‘The stimulation index (SI) obtained after culture with each antigen is also shown. ‘The day of maximal response is given for those cultures in which proliferation occurred. response to glycophorin, spectrin or the five peptides derived from glycophorin.

in brackets. There was no

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10 0 day5

day6

Fig. 3. Proliferative

bY7

responses of clinically

day 8

day 10

day 11

da! 12

normal beagle dogs (Group 4) to KLH.

The cells from two of the dogs made a weak proliferative (SI = 3.5 and 5, respectively, at day 8).

response to RBC membranes

4. Discussion The present study has demonstrated that canine PBMC established in long-term cultures are able to proliferate in response to erythrocyte autoantigens as well as to foreign recall (vaccine antigens) and non-recall (KLH) antigens. To our knowledge, the time course of proliferative responses has not been previously documented for the dog, where most antigen-driven proliferative responses have been measured at a single time point around days 4 to 6 of culture (Pinelli et al., 1994). Our laboratory routinely uses these culture conditions for studies of the kinetics of murine or human T-cell proliferative responses. When fractionated T-lymphocytes are cultured with an optimal number of antigen presenting cells (APC) in 2-ml volumes, primary proliferative responses to non-recall antigens peak on day 8 of culture, whereas responses to recall antigens peak on day 6 (Plebanski and Burtles, 1994). By contrast, cultures of a smaller volume, containing the same ratio of T-lymphocytes to APC are unable to support primary responses (Plebanski and Burtles, 1994). The data presented here were derived using whole PBMC cultured in either l- or 2-ml volumes, and as the ratio of T-lymphocytes to APC was unknown, it is not possible to clearly define whether the responses obtained are primary or secondary in nature. Despite this, responses to both recall (vaccine) and non-recall (KLH) antigens were observed. Vaccine antigens (Distemper virus, Parvovirus, Parainfluenza virus) were used to stimulate PBMC from dogs in Group 1 (2-ml cultures) and Group 2 (l-ml volumes, Distemper virus only) and responses were obtained in each case. The observed proliferation likely reflects secondary stimulation of antigen-primed lymphocytes in vaccinated dogs. Responses to the non-recall antigen KLH were also supported by the culture

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system used, but only when cells were cultured in 2-ml (Group 1 and 4, one dog of Group 3) as opposed to l-ml volumes. These results would be consistent with the generation of primary in vitro responses. Of greater interest are the proliferative responses observed to the panel of erythrocyte antigens used in this study. PBMC from 2/5 clinically normal dogs (Group 4) made weak responses to entire RBC membranes under culture conditions that would be expected to support primary responses. This finding suggests that, like normal mice (Hooper et al., 1987) and humans (Barker and Elson, 19941, erythrocyte autoreactive T-lymphocytes are found within clinically normal dogs. Such cells are maintained in a tolerant state in normal individuals, as they are specific for epitopes of autoantigens which are not normally generated by the processing of self proteins by APC (Elson et al., 199.5). By contrast, in dogs which had recovered from AIHA, proliferative responses to RBC antigens were consistently demonstrated. Although these cultures were conducted in 2 volumes, it is likely that the responses reflect secondary stimulation of primed autoreactive T-cells in such animals. Secondary in vitro stimulation of RBC-reactive T-lymphocytes has been similarly demonstrated in human AIHA patients (Barker et al., unpublished data) and NZB mice with spontaneously arising AIHA (Perry et al., 19961. Finally, proliferative responses of PBMC from a group of dogs closely related to two littermates that died from AIHA were examined (Group 2). These animals made strong responses to a range of the RBC membrane antigens, including on occasion, the glycophorin peptides. As these responses occurred under I-ml culture conditions, it would suggest that autoreactive T-cells have been primed in these genetically susceptible dogs. None of the dogs in Group 2 was anaemic or had a positive Coombs’ test (data not shown) at the time of testing. A spectrum of erythrocyte autoreactivity has, therefore, been demonstrated in the dogs of this study. Clinically normal dogs appear to harbour quiescent RBC-reactive T-cells, but such cells can become primed in dogs with AIHA or a genetic susceptibility to the disease. AIHA in the dog is likely to have a multifactorial aetiology, with a number of predisposing factors interacting to trigger clinical disease. One such factor is likely to be molecular mimicry with infectious agents or vaccine antigens. Recent studies in our laboratory have characterised the induction of autoreactivity to erythrocyte Band 3 following infection of mice of the C3HeB/FeJ strain with Lymphocytic Choriomeningitis Virus (Mazza et al., 1997). Similarly, it has been suggested that canine AIHA may often be associated with prior exposure to vaccine antigens (Duval and Giger, 1996). The studies reported here suggest that the way forward for research into canine AIHA lies with further characterisation of the specificity and function of erythrocyte-reactive T-lymphocytes. Such knowledge may then be applied to the development of novel immunomodulatory therapies. Acknowledgements Anna Corato is a postgraduate student registered with the University of Parma, Italy. These studies were funded by the British Small Animal Veterinary Association, Clinical Studies Trust Fund and the Holly Blood Donor Appeal. G. Mazza and R. Barker were

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supported by the Wellcome Trust. The authors gratefully acknowledge the technical advice of Dr. Khai Sew and the advice and support of Professor Chris Elson.

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